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Sample records for repeat protein gene

  1. Polymorphic CAG Repeat and Protein Expression of Androgen Receptor Gene in Colorectal Cancer.

    Science.gov (United States)

    Huang, Rui; Wang, Guiyu; Song, Yanni; Wang, Feng; Zhu, Bing; Tang, Qingchao; Liu, Zheng; Chen, Yinggang; Zhang, Qian; Muhammad, Shan; Wang, Xishan

    2015-04-01

    Although somatic alterations in CAG repeats in the androgen receptor (AR) gene have been suggested to predispose to colorectal cancer, less is known about AR in colorectal cancer carcinogenesis. Because of lack of relevant analysis on CAG repeat length and AR expression in colorectal cancer, we aimed to investigate the prognostic value of polymorphic CAG and protein expression of the AR gene in patients with colorectal cancer. A case-control study was carried out on 550 patients with colorectal cancer and 540 healthy controls to investigate whether polymorphic CAG within the AR gene is linked to increased risk for colorectal cancer. Polymorphic CAG and AR expression were analyzed to clarify their relationship with clinicopathologic and prognostic factors in patients with colorectal cancer. The study showed that the AR gene in patients with colorectal cancer had a longer CAG repeat sequence than those in the control group, as well as increased risk for colorectal cancer among females (P = 0.013), males (P = 0.002), and total colorectal cancer population (P CAG repeat sequence among males (P CAG repeat sequence and negative AR expression were associated with a short 5-year overall survival (OS) rate in colorectal cancer. Long CAG repeat sequences and the absence of AR expression were closely related to the development of colorectal cancer. Both long CAG and decreased AR expression were correlated with the poor 5-year OS in patients with colorectal cancer.

  2. Extended gene expression by medium exchange and repeated transient transfection for recombinant protein production enhancement.

    Science.gov (United States)

    Cervera, Laura; Gutiérrez-Granados, Sonia; Berrow, Nicholas Simon; Segura, Maria Mercedes; Gòdia, Francesc

    2015-05-01

    Production of recombinant products in mammalian cell cultures can be achieved by stable gene expression (SGE) or transient gene expression (TGE). The former is based on the integration of a plasmid DNA into the host cell genome allowing continuous gene expression. The latter is based on episomal plasmid DNA expression. Conventional TGE is limited to a short production period of usually about 96 h, therefore limiting productivity. A novel gene expression approach termed extended gene expression (EGE) is explored in this study. The aim of EGE is to prolong the production period by the combination of medium exchange and repeated transfection of cell cultures with plasmid DNA to improve overall protein production. The benefit of this methodology was evaluated for the production of three model recombinant products: intracellular GFP, secreted GFP, and a Gag-GFP virus-like particles (VLPs). Productions were carried out in HEK 293 cell suspension cultures grown in animal-derived component free media using polyethylenimine (PEI) as transfection reagent. Transfections were repeated throughout the production process using different plasmid DNA concentrations, intervals of time, and culture feeding conditions in order to identify the best approach to achieve sustained high-level gene expression. Using this novel EGE strategy, the production period was prolonged between 192 and 240 h with a 4-12-fold increase in production levels, depending on the product type considered. © 2014 Wiley Periodicals, Inc.

  3. Expansion of protein domain repeats.

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    Asa K Björklund

    2006-08-01

    Full Text Available Many proteins, especially in eukaryotes, contain tandem repeats of several domains from the same family. These repeats have a variety of binding properties and are involved in protein-protein interactions as well as binding to other ligands such as DNA and RNA. The rapid expansion of protein domain repeats is assumed to have evolved through internal tandem duplications. However, the exact mechanisms behind these tandem duplications are not well-understood. Here, we have studied the evolution, function, protein structure, gene structure, and phylogenetic distribution of domain repeats. For this purpose we have assigned Pfam-A domain families to 24 proteomes with more sensitive domain assignments in the repeat regions. These assignments confirmed previous findings that eukaryotes, and in particular vertebrates, contain a much higher fraction of proteins with repeats compared with prokaryotes. The internal sequence similarity in each protein revealed that the domain repeats are often expanded through duplications of several domains at a time, while the duplication of one domain is less common. Many of the repeats appear to have been duplicated in the middle of the repeat region. This is in strong contrast to the evolution of other proteins that mainly works through additions of single domains at either terminus. Further, we found that some domain families show distinct duplication patterns, e.g., nebulin domains have mainly been expanded with a unit of seven domains at a time, while duplications of other domain families involve varying numbers of domains. Finally, no common mechanism for the expansion of all repeats could be detected. We found that the duplication patterns show no dependence on the size of the domains. Further, repeat expansion in some families can possibly be explained by shuffling of exons. However, exon shuffling could not have created all repeats.

  4. The Pentapeptide Repeat Proteins

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    Vetting,M.; Hegde, S.; Fajardo, J.; Fiser, A.; Roderick, S.; Takiff, H.; Blanchard, J.

    2006-01-01

    The Pentapeptide Repeat Protein (PRP) family has over 500 members in the prokaryotic and eukaryotic kingdoms. These proteins are composed of, or contain domains composed of, tandemly repeated amino acid sequences with a consensus sequence of [S, T,A, V][D, N][L, F]-[S, T,R][G]. The biochemical function of the vast majority of PRP family members is unknown. The three-dimensional structure of the first member of the PRP family was determined for the fluoroquinolone resistance protein (MfpA) from Mycobacterium tuberculosis. The structure revealed that the pentapeptide repeats encode the folding of a novel right-handed quadrilateral {beta}-helix. MfpA binds to DNA gyrase and inhibits its activity. The rod-shaped, dimeric protein exhibits remarkable size, shape and electrostatic similarity to DNA.

  5. Sustained downregulation of YY1-associated protein-related protein gene expression in rat hippocampus induced by repeated electroconvulsive shock.

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    Ohtomo, Takayuki; Kanamatsu, Tomoyuki; Fujita, Mariko; Takagi, Mitsuhiro; Yamada, Junji

    2011-01-01

    YY1AP-related protein (YARP) is a structural homolog of YY1-associated protein (YY1AP), which has a YY1-binding domain. During perinatal development, YARP mRNA expression is increased at a late stage of embryonic neurogenesis. It is not known whether YARP expression is regulated during adult neurogenesis. Electroconvulsive shock (ECS), a model for a highly effective depression treatment, is known to induce hippocampal neurogenesis after repeated treatment, so we employed ECS to measure the expression of YARP mRNA. Northern blots revealed significantly decreased expression of the YARP gene after repeated ECS but not single ECS. In situ hybridization clearly demonstrated a reduction of YARP mRNA expression in the CA (CA1, CA2, and CA3) subfields. Although clonic-tonic seizure was induced not only by ECS but also by injection of kainic acid to the striatum, the regulation of YARP mRNA expression was different between ECS and kainic acid. YARP mRNA was decreased only by the ECS method, suggesting that YARP expression is different at embryonic and adult neurogenic stage.

  6. The TIS11 primary response gene is a member of a gene family that encodes proteins with a highly conserved sequence containing an unusual Cys-His repeat.

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    Varnum, B C; Ma, Q F; T. H. Chi; Fletcher, B.; Herschman, H.R.

    1991-01-01

    The TIS11 primary response gene is rapidly and transiently induced by both 12-O-tetradecanoylphorbol-13-acetate and growth factors. The predicted TIS11 protein contains a 6-amino-acid repeat, YKTELC. We cloned two additional cDNAs, TIS11b and TIS11d, that contain the YKTELC sequence. TIS11, TIS11b, and TIS11d proteins share a 67-amino-acid region of sequence similarity that includes the YKTELC repeat and two cysteine-histidine containing repeats. TIS11 gene family members are not coordinately...

  7. A New Aspergillus fumigatus Typing Method Based on Hypervariable Tandem Repeats Located within Exons of Surface Protein Coding Genes (TRESP).

    Science.gov (United States)

    Garcia-Rubio, Rocio; Gil, Horacio; Monteiro, Maria Candida; Pelaez, Teresa; Mellado, Emilia

    2016-01-01

    Aspergillus fumigatus is a saprotrophic mold fungus ubiquitously found in the environment and is the most common species causing invasive aspergillosis in immunocompromised individuals. For A. fumigatus genotyping, the short tandem repeat method (STRAf) is widely accepted as the first choice. However, difficulties associated with PCR product size and required technology have encouraged the development of novel typing techniques. In this study, a new genotyping method based on hypervariable tandem repeats within exons of surface protein coding genes (TRESP) was designed. A. fumigatus isolates were characterized by PCR amplification and sequencing with a panel of three TRESP encoding genes: cell surface protein A; MP-2 antigenic galactomannan protein; and hypothetical protein with a CFEM domain. The allele sequence repeats of each of the three targets were combined to assign a specific genotype. For the evaluation of this method, 126 unrelated A. fumigatus strains were analyzed and 96 different genotypes were identified, showing a high level of discrimination [Simpson's index of diversity (D) 0.994]. In addition, 49 azole resistant strains were analyzed identifying 26 genotypes and showing a lower D value (0.890) among them. This value could indicate that these resistant strains are closely related and share a common origin, although more studies are needed to confirm this hypothesis. In summary, a novel genotyping method for A. fumigatus has been developed which is reproducible, easy to perform, highly discriminatory and could be especially useful for studying outbreaks.

  8. A New Aspergillus fumigatus Typing Method Based on Hypervariable Tandem Repeats Located within Exons of Surface Protein Coding Genes (TRESP)

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    Garcia-Rubio, Rocio; Gil, Horacio; Monteiro, Maria Candida; Pelaez, Teresa; Mellado, Emilia

    2016-01-01

    Aspergillus fumigatus is a saprotrophic mold fungus ubiquitously found in the environment and is the most common species causing invasive aspergillosis in immunocompromised individuals. For A. fumigatus genotyping, the short tandem repeat method (STRAf) is widely accepted as the first choice. However, difficulties associated with PCR product size and required technology have encouraged the development of novel typing techniques. In this study, a new genotyping method based on hypervariable tandem repeats within exons of surface protein coding genes (TRESP) was designed. A. fumigatus isolates were characterized by PCR amplification and sequencing with a panel of three TRESP encoding genes: cell surface protein A; MP-2 antigenic galactomannan protein; and hypothetical protein with a CFEM domain. The allele sequence repeats of each of the three targets were combined to assign a specific genotype. For the evaluation of this method, 126 unrelated A. fumigatus strains were analyzed and 96 different genotypes were identified, showing a high level of discrimination [Simpson’s index of diversity (D) 0.994]. In addition, 49 azole resistant strains were analyzed identifying 26 genotypes and showing a lower D value (0.890) among them. This value could indicate that these resistant strains are closely related and share a common origin, although more studies are needed to confirm this hypothesis. In summary, a novel genotyping method for A. fumigatus has been developed which is reproducible, easy to perform, highly discriminatory and could be especially useful for studying outbreaks. PMID:27701437

  9. Origin and diversification of leucine-rich repeat receptor-like protein kinase (LRR-RLK) genes in plants.

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    Liu, Ping-Li; Du, Liang; Huang, Yuan; Gao, Shu-Min; Yu, Meng

    2017-02-07

    Leucine-rich repeat receptor-like protein kinases (LRR-RLKs) are the largest group of receptor-like kinases in plants and play crucial roles in development and stress responses. The evolutionary relationships among LRR-RLK genes have been investigated in flowering plants; however, no comprehensive studies have been performed for these genes in more ancestral groups. The subfamily classification of LRR-RLK genes in plants, the evolutionary history and driving force for the evolution of each LRR-RLK subfamily remain to be understood. We identified 119 LRR-RLK genes in the Physcomitrella patens moss genome, 67 LRR-RLK genes in the Selaginella moellendorffii lycophyte genome, and no LRR-RLK genes in five green algae genomes. Furthermore, these LRR-RLK sequences, along with previously reported LRR-RLK sequences from Arabidopsis thaliana and Oryza sativa, were subjected to evolutionary analyses. Phylogenetic analyses revealed that plant LRR-RLKs belong to 19 subfamilies, eighteen of which were established in early land plants, and one of which evolved in flowering plants. More importantly, we found that the basic structures of LRR-RLK genes for most subfamilies are established in early land plants and conserved within subfamilies and across different plant lineages, but divergent among subfamilies. In addition, most members of the same subfamily had common protein motif compositions, whereas members of different subfamilies showed variations in protein motif compositions. The unique gene structure and protein motif compositions of each subfamily differentiate the subfamily classifications and, more importantly, provide evidence for functional divergence among LRR-RLK subfamilies. Maximum likelihood analyses showed that some sites within four subfamilies were under positive selection. Much of the diversity of plant LRR-RLK genes was established in early land plants. Positive selection contributed to the evolution of a few LRR-RLK subfamilies.

  10. Complete gene sequence of spider attachment silk protein (PySp1) reveals novel linker regions and extreme repeat homogenization.

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    Chaw, Ro Crystal; Saski, Christopher A; Hayashi, Cheryl Y

    2017-02-01

    Spiders use a myriad of silk types for daily survival, and each silk type has a unique suite of task-specific mechanical properties. Of all spider silk types, pyriform silk is distinct because it is a combination of a dry protein fiber and wet glue. Pyriform silk fibers are coated with wet cement and extruded into "attachment discs" that adhere silks to each other and to substrates. The mechanical properties of spider silk types are linked to the primary and higher-level structures of spider silk proteins (spidroins). Spidroins are often enormous molecules (>250 kDa) and have a lengthy repetitive region that is flanked by relatively short (∼100 amino acids), non-repetitive amino- and carboxyl-terminal regions. The amino acid sequence motifs in the repetitive region vary greatly between spidroin type, while motif length and number underlie the remarkable mechanical properties of spider silk fibers. Existing knowledge of pyriform spidroins is fragmented, making it difficult to define links between the structure and function of pyriform spidroins. Here, we present the full-length sequence of the gene encoding pyriform spidroin 1 (PySp1) from the silver garden spider Argiope argentata. The predicted protein is similar to previously reported PySp1 sequences but the A. argentata PySp1 has a uniquely long and repetitive "linker", which bridges the amino-terminal and repetitive regions. Predictions of the hydrophobicity and secondary structure of A. argentata PySp1 identify regions important to protein self-assembly. Analysis of the full complement of A. argentata PySp1 repeats reveals extreme intragenic homogenization, and comparison of A. argentata PySp1 repeats with other PySp1 sequences identifies variability in two sub-repetitive expansion regions. Overall, the full-length A. argentata PySp1 sequence provides new evidence for understanding how pyriform spidroins contribute to the properties of pyriform silk fibers. Copyright © 2017 The Authors. Published by

  11. The carriage of the serine-aspartate repeat protein-encoding sdr genes among Staphylococcus aureus lineages.

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    Liu, Huanle; Lv, Jingnan; Qi, Xiuqin; Ding, Yu; Li, Dan; Hu, Longhua; Wang, Liangxing; Yu, Fangyou

    2015-01-01

    The serine-aspartate repeat proteins (Sdr) are members of a family of surface proteins and contribute to the pathogenicity of Staphylococcus aureus. Among 288 S. aureus isolates including 158 and 130 associated with skin and soft tissue infections and bloodstream infection, respectively; 275 (95.5%) were positive for at least one of three sdr genes tested. The positivity rates for sdrC, sdrD, and sdrE among S. aureus isolates were 87.8% (253/288), 63.9% (184/288), and 68.1% (196/288), respectively. 224 (77.8%) of 288 isolates were concomitantly positive for two or three sdr genes. There was an association between carriage of sdrE and methicillin-resistant S. aureus (MRSA) isolates, while the carriage rates of sdrC and sdrD in MRSA isolates were similar to those in methicillin-sensitive S. aureus (MSSA) isolates. The prevalence of co-existence of sdrC and sdrE among MRSA isolates was significantly higher than that among MSSA isolates (p<0.05). All ST1, ST5, ST7, and ST25 isolates were positive for sdrD. While all ST121 and ST398 isolates were negative for sdrD. All ST59 and ST88 isolates were positive for sdrE. All ST1 isolates were concomitantly positive for sdrC and sdrD. Concomitant carriage of sdrC, sdrD, and sdrE was found among all ST5, 75.0% (9/12) of ST1, 69.2% (9/13) of ST6, 78.6% (11/14) of ST25, and 90.9% (20/22) of ST88 isolates. sdrD was linked to CC5, CC7 and CC88 isolates, especially CC88 isolates. There was a strong association between the presence of sdrE and CC59, CC88, and CC5 isolates. A significant correlation between concomitant carriage of sdrC, sdrD, and sdrE and CC88 isolates was found. sdrC-positive, sdrD-positive and sdrE-negative gene profile was significantly associated with CC7 clone. There was an association between sdrC-positive, sdrD-negative, and sdrE-positive gene profile and CC59 isolates. A correlation between sdrC-positive, sdrD-negative, and sdrE-negative gene profile and CC121 clone was found. More CC59 isolates carried sdr

  12. Polymorphic GGC repeat differentially regulates human reelin gene expression levels.

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    Persico, A M; Levitt, P; Pimenta, A F

    2006-10-01

    The human gene encoding Reelin (RELN), a pivotal protein in neurodevelopment, includes a polymorphic GGC repeat in its 5' untranslated region (UTR). CHO cells transfected with constructs encompassing the RELN 5'UTR with 4-to-13 GGC repeats upstream of the luciferase reporter gene show declining luciferase activity with increasing GGC repeat number (P autism.

  13. Pentatricopeptide repeat proteins in plants.

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    Barkan, Alice; Small, Ian

    2014-01-01

    Pentatricopeptide repeat (PPR) proteins constitute one of the largest protein families in land plants, with more than 400 members in most species. Over the past decade, much has been learned about the molecular functions of these proteins, where they act in the cell, and what physiological roles they play during plant growth and development. A typical PPR protein is targeted to mitochondria or chloroplasts, binds one or several organellar transcripts, and influences their expression by altering RNA sequence, turnover, processing, or translation. Their combined action has profound effects on organelle biogenesis and function and, consequently, on photosynthesis, respiration, plant development, and environmental responses. Recent breakthroughs in understanding how PPR proteins recognize RNA sequences through modular base-specific contacts will help match proteins to potential binding sites and provide a pathway toward designing synthetic RNA-binding proteins aimed at desired targets.

  14. A conserved gene family encodes transmembrane proteins with fibronectin, immunoglobulin and leucine-rich repeat domains (FIGLER

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    Haga Christopher L

    2007-09-01

    Full Text Available Abstract Background In mouse the cytokine interleukin-7 (IL-7 is required for generation of B lymphocytes, but human IL-7 does not appear to have this function. A bioinformatics approach was therefore used to identify IL-7 receptor related genes in the hope of identifying the elusive human cytokine. Results Our database search identified a family of nine gene candidates, which we have provisionally named fibronectin immunoglobulin leucine-rich repeat (FIGLER. The FIGLER 1–9 genes are predicted to encode type I transmembrane glycoproteins with 6–12 leucine-rich repeats (LRR, a C2 type Ig domain, a fibronectin type III domain, a hydrophobic transmembrane domain, and a cytoplasmic domain containing one to four tyrosine residues. Members of this multichromosomal gene family possess 20–47% overall amino acid identity and are differentially expressed in cell lines and primary hematopoietic lineage cells. Genes for FIGLER homologs were identified in macaque, orangutan, chimpanzee, mouse, rat, dog, chicken, toad, and puffer fish databases. The non-human FIGLER homologs share 38–99% overall amino acid identity with their human counterpart. Conclusion The extracellular domain structure and absence of recognizable cytoplasmic signaling motifs in members of the highly conserved FIGLER gene family suggest a trophic or cell adhesion function for these molecules.

  15. Instability of the expanded (CTG){sub n} repeats in the myotonin protein kinase gene in cultured lymphoblastoid cell lines from patients with myotonic dystrophy

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    Ashizawa, Tetsuo; Patel, B.J.; Monckton, D.G. [Baylor College of Medicine, Houston, TX (United States)] [and other

    1996-08-15

    The mutation associated with myotonic dystrophy (DM) is the expansion of an unstable trinucleotide repeat, (CTG){sub n}, in the 3{prime}-untranslated region of the myotonin protein kinase gene. Although expanded repeats show both germline and somatic instability, the mechanisms of the instability are poorly understood. To establish a model system in which somatic instability of the DM repeat could be studied in more detail, we established lymphoblastoid cell lines (LBCL) from DM patients. Analysis of the DNA from DM LBCL using Southern blotting showed that the (CTG). repeats were apparently stable up to 29 passages in culture. To study infrequent repeat size mutations that are undetectable due to the size heterogeneity, we established LBCL of single-cell origins by cloning using multiple steps of limiting dilution. After expansion to approximately 10{sup 6} cells (equivalent to approximately 20 cell cycles), the DNAs of these cell lines were analyzed by the small pool PCR technique using primers flanking the (CTG), repeat region. Two types of mutations of the expanded (CTG){sub n} repeat alleles were detected: (1) frequent mutations that show small changes of the (CTG){sub n} repeat size, resulting in alleles in a normal distribution around the progenitor allele, and (2) relatively rare mutations with large changes of the (CTG){sub n} repeat size, with a bias toward contraction. The former may represent the mechanism responsible for the so matic heterogeneity of the (CTG), repeat size observe in blood cells of DM patients. This in vitro experimental system will be useful for further studies on mechanisms involved in the regulation of the somatic stability of the (CTG). repeats in DM. 24 refs., 4 figs.

  16. Rhoptry-associated protein (rap-1) genes in the sheep pathogen Babesia sp. Xinjiang: Multiple transcribed copies differing by 3' end repeated sequences.

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    Niu, Qingli; Marchand, Jordan; Yang, Congshan; Bonsergent, Claire; Guan, Guiquan; Yin, Hong; Malandrin, Laurence

    2015-07-30

    Sheep babesiosis occurs mainly in tropical and subtropical areas. The sheep parasite Babesia sp. Xinjiang is widespread in China, and our goal is to characterize rap-1 (rhoptry-associated protein 1) gene diversity and expression as a first step of a long term goal aiming at developing a recombinant subunit vaccine. Seven different rap-1a genes were amplified in Babesia sp. Xinjiang, using degenerate primers designed from conserved motifs. Rap-1b and rap-1c gene types could not be identified. In all seven rap-1a genes, the 5' regions exhibited identical sequences over 936 nt, and the 3' regions differed at 28 positions over 147 nt, defining two types of genes designated α and β. The remaining 3' part varied from 72 to 360 nt in length, depending on the gene. This region consists of a succession of two to ten 36 nt repeats, which explains the size differences. Even if the nucleotide sequences varied, 6 repeats encoded the same stretch of amino acids. Transcription of at least four α and two β genes was demonstrated by standard RT-PCR. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Repeats in transforming acidic coiled-coil (TACC) genes.

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    Trivedi, Seema

    2013-06-01

    Transforming acidic coiled-coil proteins (TACC1, 2, and 3) are essential proteins associated with the assembly of spindle microtubules and maintenance of bipolarity. Dysregulation of TACCs is associated with tumorigenesis, but studies of microsatellite instability in TACC genes have not been extensive. Microsatellite or simple sequence repeat instability is known to cause many types of cancer. The present in silico analysis of SSRs in human TACC gene sequences shows the presence of mono- to hexa-nucleotide repeats, with the highest densities found for mono- and di-nucleotide repeats. Density of repeats is higher in introns than in exons. Some of the repeats are present in regulatory regions and retained introns. Human TACC genes show conservation of many repeat classes. Microsatellites in TACC genes could be valuable markers for monitoring numerical chromosomal aberrations and or cancer.

  18. CAG trinucleotide RNA repeats interact with RNA-binding proteins

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    McLaughlin, B.A.; Eberwine, J.; Spencer, C. [Univ. of Pennsylvania, Philadelphia, PA (United States)

    1996-09-01

    Genes associated with several neurological diseases are characterized by the presence of an abnormally long trinucleotide repeat sequence. By way of example, Huntington`s disease (HD), is characterized by selective neuronal degeneration associated with the expansion of a polyglutamine-encoding CAG tract. Normally, this CAG tract is comprised of 11-34 repeats, but in HD it is expanded to >37 repeats in affected individuals. The mechanism by which CAG repeats cause neuronal degeneration is unknown, but it has been speculated that the expansion primarily causes abnormal protein functioning, which in turn causes HD pathology. Other mechanisms, however, have not been ruled out. Interactions between RNA and RNA-binding proteins have previously been shown to play a role in the expression of several eukaryotic genes. Herein, we report the association of cytoplasmic proteins with normal length and extended CAG repeats, using gel shift and LJV crosslinking assays. Cytoplasmic protein extracts from several rat brain regions, including the striatum and cortex, sites of neuronal degeneration in HD, contain a 63-kD RNA-binding protein that specifically interacts with these CAG-repeat sequences. These protein-RNA interactions are dependent on the length of the CAG repeat, with longer repeats binding substantially more protein. Two CAG repeat-binding proteins are present in human cortex and striatum; one comigrates with the rat protein at 63 kD, while the other migrates at 49 kD. These data suggest mechanisms by which RNA-binding proteins may be involved in the pathological course of trinucleotide repeat-associated neurological diseases. 47 refs., 5 figs.

  19. Effect of pistachio oil on gene expression of IFN-induced protein with tetratricopeptide repeats 2: a biomarker of inflammatory response.

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    Zhang, Jun; Kris-Etherton, Penny M; Thompson, Jerry T; Vanden Heuvel, John P

    2010-05-01

    When incorporated into the diet, pistachios have a beneficial effect on lipid and lipoprotein profiles. However, little is known about potential anti-inflammatory properties. This study was conducted to determine whether pistachio oil and an organic extract from pistachio oil extract (PE) regulated expression of inflammation-related genes. A mouse macrophage cell line (RAW 264.7 cells) was treated with pistachio oil and gene expression microarray analyses were performed. Pistachio oil significantly affected genes involved in immune response, defense response to bacteria, and gene silencing, of which INF-induced protein with tetratricopeptide repeats 2 (Ifit-2) was the most dramatically reduced. PE reduced the LPS-induced Ifit-2 by 78% and the bioactive molecules contained in PE, linoleic acid, and beta-sitosterol recapitulated this inhibition. Promoter analysis identified two adjacent IFN-stimulated response elements, which lie between -110 and -85bp of the 5'-flanking region of the Ifit-2 promoter, as being responsive to LPS activation and inhibition by PE. Our results indicate that pistachio oil and bioactive molecules present therein decrease Ifit-2 expressions, and due to the sensitivity of this effect, this gene is a potential biomarker for monitoring diet-induced changes in inflammation.

  20. Dynamic combinatorial libraries of artificial repeat proteins.

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    Eisenberg, Margarita; Shumacher, Inbal; Cohen-Luria, Rivka; Ashkenasy, Gonen

    2013-06-15

    Repeat proteins are found in almost all cellular systems, where they are involved in diverse molecular recognition processes. Recent studies have suggested that de novo designed repeat proteins may serve as universal binders, and might potentially be used as practical alternative to antibodies. We describe here a novel chemical methodology for producing small libraries of repeat proteins, and screening in parallel the ligand binding of library members. The first stage of this research involved the total synthesis of a consensus-based three-repeat tetratricopeptide (TPR) protein (~14 kDa), via sequential attachment of the respective peptides. Despite the effectiveness of the synthesis and ligation steps, this method was found to be too demanding for the production of proteins containing variable number of repeats. Additionally, the analysis of binding of the individual proteins was time consuming. Therefore, we designed and prepared novel dynamic combinatorial libraries (DCLs), and show that their equilibration can facilitate the formation of TPR proteins containing up to eight repeating units. Interestingly, equilibration of the library building blocks in the presence of the biologically relevant ligands, Hsp90 and Hsp70, induced their oligomerization into forming more of the proteins with large recognition surfaces. We suggest that this work presents a novel simple and rapid tool for the simultaneous screening of protein mixtures with variable binding surfaces, and for identifying new binders for ligands of interest.

  1. The diversity and evolution of Wolbachia ankyrin repeat domain genes.

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    Stefanos Siozios

    Full Text Available Ankyrin repeat domain-encoding genes are common in the eukaryotic and viral domains of life, but they are rare in bacteria, the exception being a few obligate or facultative intracellular Proteobacteria species. Despite having a reduced genome, the arthropod strains of the alphaproteobacterium Wolbachia contain an unusually high number of ankyrin repeat domain-encoding genes ranging from 23 in wMel to 60 in wPip strain. This group of genes has attracted considerable attention for their astonishing large number as well as for the fact that ankyrin proteins are known to participate in protein-protein interactions, suggesting that they play a critical role in the molecular mechanism that determines host-Wolbachia symbiotic interactions. We present a comparative evolutionary analysis of the wMel-related ankyrin repeat domain-encoding genes present in different Drosophila-Wolbachia associations. Our results show that the ankyrin repeat domain-encoding genes change in size by expansion and contraction mediated by short directly repeated sequences. We provide examples of intra-genic recombination events and show that these genes are likely to be horizontally transferred between strains with the aid of bacteriophages. These results confirm previous findings that the Wolbachia genomes are evolutionary mosaics and illustrate the potential that these bacteria have to generate diversity in proteins potentially involved in the symbiotic interactions.

  2. Genome-wide cloning and sequence analysis of leucine-rich repeat receptor-like protein kinase genes in Arabidopsis thaliana

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    Yuan Tong

    2010-01-01

    Full Text Available Abstract Background Transmembrane receptor kinases play critical roles in both animal and plant signaling pathways regulating growth, development, differentiation, cell death, and pathogenic defense responses. In Arabidopsis thaliana, there are at least 223 Leucine-rich repeat receptor-like kinases (LRR-RLKs, representing one of the largest protein families. Although functional roles for a handful of LRR-RLKs have been revealed, the functions of the majority of members in this protein family have not been elucidated. Results As a resource for the in-depth analysis of this important protein family, the complementary DNA sequences (cDNAs of 194 LRR-RLKs were cloned into the GatewayR donor vector pDONR/ZeoR and analyzed by DNA sequencing. Among them, 157 clones showed sequences identical to the predictions in the Arabidopsis sequence resource, TAIR8. The other 37 cDNAs showed gene structures distinct from the predictions of TAIR8, which was mainly caused by alternative splicing of pre-mRNA. Most of the genes have been further cloned into GatewayR destination vectors with GFP or FLAG epitope tags and have been transformed into Arabidopsis for in planta functional analysis. All clones from this study have been submitted to the Arabidopsis Biological Resource Center (ABRC at Ohio State University for full accessibility by the Arabidopsis research community. Conclusions Most of the Arabidopsis LRR-RLK genes have been isolated and the sequence analysis showed a number of alternatively spliced variants. The generated resources, including cDNA entry clones, expression constructs and transgenic plants, will facilitate further functional analysis of the members of this important gene family.

  3. Genetic analysis of the leucine-rich repeat and lg domain containing Nogo receptor-interacting protein 1 gene in essential tremor.

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    Liang, Hui; Song, Zhi; Deng, Xiong; Xu, Hongbo; Zhu, Anding; Zheng, Wen; Zhao, Yongxiang; Deng, Hao

    2013-10-01

    Variants in the leucine-rich repeat and lg domain containing nogo receptor-interacting protein 1 gene (LINGO1) have been identified to be associated with the increased risk of essential tremor (ET), especially among Caucasians. To explore whether the LINGO1 gene plays a role in ET susceptibility, we performed a systematic genetic analysis of the coding region in the LINGO1 gene. Four nucleotide variants have been genotyped, including three known variants (rs2271398, rs2271397, and rs3743481), and a novel G → C transition (ss491228439). Extended analysis showed no significant difference in genotypic and allelic distributions between 151 patients and 301 control subjects for these four variants (all P > 0.05). However, further sex-stratified analysis revealed that the C allele of rs2271397 and ss491228439 contributed the risk of ET in female (P = 0.017, OR = 2.139, 95 % CI 1.135 ~ 4.030 for rs2271397 and P = 0.038, OR = 1.812, 95 % CI 1.027 ~ 3.194 for ss491228439). Haplotype analysis indicated that A465-C474-C714 haplotype was significantly associated with increased risk of ET in female (P = 0.041, OR = 1.800, 95 % CI 1.020 ~ 3.178). Our results indicate that the LINGO1 variants are associated with ET in Chinese Han female patients.

  4. The LuWD40-1 gene encoding WD repeat protein regulates growth and pollen viability in flax (Linum Usitatissimum L.).

    Science.gov (United States)

    Kumar, Santosh; Jordan, Mark C; Datla, Raju; Cloutier, Sylvie

    2013-01-01

    As a crop, flax holds significant commercial value for its omega-3 rich oilseeds and stem fibres. Canada is the largest producer of linseed but there exists scope for significant yield improvements. Implementation of mechanisms such as male sterility can permit the development of hybrids to assist in achieving this goal. Temperature sensitive male sterility has been reported in flax but the leakiness of this system in field conditions limits the production of quality hybrid seeds. Here, we characterized a 2,588 bp transcript differentially expressed in male sterile lines of flax. The twelve intron gene predicted to encode a 368 amino acid protein has five WD40 repeats which, in silico, form a propeller structure with putative nucleic acid and histone binding capabilities. The LuWD40-1 protein localized to the nucleus and its expression increased during the transition and continued through the vegetative stages (seed, etiolated seedling, stem) while the transcript levels declined during reproductive development (ovary, anthers) and embryonic morphogenesis of male fertile plants. Knockout lines for LuWD40-1 in flax failed to develop shoots while overexpression lines showed delayed growth phenotype and were male sterile. The non-viable flowers failed to open and the pollen grains from these flowers were empty. Three independent transgenic lines overexpressing the LuWD40-1 gene had ∼80% non-viable pollen, reduced branching, delayed flowering and maturity compared to male fertile genotypes. The present study provides new insights into a male sterility mechanism present in flax.

  5. The LuWD40-1 gene encoding WD repeat protein regulates growth and pollen viability in flax (Linum Usitatissimum L..

    Directory of Open Access Journals (Sweden)

    Santosh Kumar

    Full Text Available As a crop, flax holds significant commercial value for its omega-3 rich oilseeds and stem fibres. Canada is the largest producer of linseed but there exists scope for significant yield improvements. Implementation of mechanisms such as male sterility can permit the development of hybrids to assist in achieving this goal. Temperature sensitive male sterility has been reported in flax but the leakiness of this system in field conditions limits the production of quality hybrid seeds. Here, we characterized a 2,588 bp transcript differentially expressed in male sterile lines of flax. The twelve intron gene predicted to encode a 368 amino acid protein has five WD40 repeats which, in silico, form a propeller structure with putative nucleic acid and histone binding capabilities. The LuWD40-1 protein localized to the nucleus and its expression increased during the transition and continued through the vegetative stages (seed, etiolated seedling, stem while the transcript levels declined during reproductive development (ovary, anthers and embryonic morphogenesis of male fertile plants. Knockout lines for LuWD40-1 in flax failed to develop shoots while overexpression lines showed delayed growth phenotype and were male sterile. The non-viable flowers failed to open and the pollen grains from these flowers were empty. Three independent transgenic lines overexpressing the LuWD40-1 gene had ∼80% non-viable pollen, reduced branching, delayed flowering and maturity compared to male fertile genotypes. The present study provides new insights into a male sterility mechanism present in flax.

  6. Exploring the repeat protein universe through computational protein design.

    Science.gov (United States)

    Brunette, T J; Parmeggiani, Fabio; Huang, Po-Ssu; Bhabha, Gira; Ekiert, Damian C; Tsutakawa, Susan E; Hura, Greg L; Tainer, John A; Baker, David

    2015-12-24

    A central question in protein evolution is the extent to which naturally occurring proteins sample the space of folded structures accessible to the polypeptide chain. Repeat proteins composed of multiple tandem copies of a modular structure unit are widespread in nature and have critical roles in molecular recognition, signalling, and other essential biological processes. Naturally occurring repeat proteins have been re-engineered for molecular recognition and modular scaffolding applications. Here we use computational protein design to investigate the space of folded structures that can be generated by tandem repeating a simple helix-loop-helix-loop structural motif. Eighty-three designs with sequences unrelated to known repeat proteins were experimentally characterized. Of these, 53 are monomeric and stable at 95 °C, and 43 have solution X-ray scattering spectra consistent with the design models. Crystal structures of 15 designs spanning a broad range of curvatures are in close agreement with the design models with root mean square deviations ranging from 0.7 to 2.5 Å. Our results show that existing repeat proteins occupy only a small fraction of the possible repeat protein sequence and structure space and that it is possible to design novel repeat proteins with precisely specified geometries, opening up a wide array of new possibilities for biomolecular engineering.

  7. Genes encoding pentatricopeptide repeat (PPR proteins are not conserved in location in plant genomes and may be subject to diversifying selection

    Directory of Open Access Journals (Sweden)

    Brown Gregory G

    2007-05-01

    Full Text Available Abstract Background The pentatricopeptide repeat (PPR is a degenerate 35 amino acid motif that occurs in multiple tandem copies in members of a recently recognized eukaryotic gene family. Most analyzed eukaryotic genomes contain only a small number of PPR genes, but in plants the family is greatly expanded. The factors that underlie the expansion of this gene family in plants are not as yet understood. Results We show that the location of PPR genes is highly variable in comparisons between orthologous, closely related, and otherwise co-linear chromosomal regions of the Brassica rapa or radish and Arabidopsis thaliana. This observation also pertains to paralogous duplicated segments of the genomes of Arabidopsis thaliana and Brassica rapa. In addition, we show that PPR genes that seem closely linearly aligned in these comparisons are not generally found to be closely related to one another at the nucleotide and amino acid sequence level. We observe a relatively high level of non-synonomous vs synonomous changes among a group tandemly repeated radish PPR genes, suggesting that these, and possibly other PPR genes, are subject to diversifying selection. We also show that a duplicated region of the Arabidopsis genome possesses a relatively high density of PPR genes showing high similarity to restorers of fertility of cytoplasmic male sterile (CMS systems of petunia, radish and rice. The PPR genes in these regions, together with the restorer genes, are more highly similar to one another, in sequence as well as in structure, than to other PPR genes, even within the same sub-family. Conclusion Our results suggest are consistent with a model in which at least some PPR genes undergo a "birth and death" process that involves transposition to unrelated chromosomal sites. PPR genes hold certain features in common with disease resistance genes (R genes, and their "nomadic" character suggests that their evolutionary expansion in plants may have involved novel

  8. The Ancestral Gene for Transcribed, Low-Copy Repeats in the Prader-Willi/Angleman Region Encodes a Large Protein Implicated in Protein Trafficking that is Deficient in Mice with Neuromuscular and

    Energy Technology Data Exchange (ETDEWEB)

    Ji, Y.

    1999-01-01

    Transcribed, low-copy repeat elements are associated with the breakpoint regions of common deletions in Prader-Willi and Angelman syndromes. We report here the identification of the ancestral gene ( HERC2 ) and a family of duplicated, truncated copies that comprise these low-copy repeats. This gene encodes a highly conserved giant protein, HERC2, that is distantly related to p532 (HERC1), a guanine nucleotide exchange factor (GEF) implicated in vesicular trafficking. The mouse genome contains a single Herc2 locus, located in the jdf2 (juvenile development and fertility-2) interval of chromosome 7C. We have identified single nucleotide splice junction mutations in Herc2 in three independent N-ethyl-N-nitrosourea-induced jdf2 mutant alleles, each leading to exon skipping with premature termination of translation and/or deletion of conserved amino acids. Therefore, mutations in Herc2 lead to the neuromuscular secretory vesicle and sperm acrosome defects, other developmental abnormalities and juvenile lethality of jdf2 mice. Combined, these findings suggest that HERC2 is an important gene encoding a GEF involved in protein trafficking and degradation pathways in the cell.

  9. Relationship between drug resistance and the clustered, regularly interspaced, short, palindromic repeat-associated protein genes cas1 and cas2 in Shigella from giant panda dung

    Science.gov (United States)

    Ren, Lu; Deng, Lin-Hua; Zhang, Ri-Peng; Wang, Cheng-Dong; Li, De-Sheng; Xi, Li-Xin; Chen, Zhen-rong; Yang, Rui; Huang, Jie; Zeng, Yang-ru; Wu, Hong-Lin; Cao, San-Jie; Wu, Rui; Huang, Yong; Yan, Qi-Gui

    2017-01-01

    Abstract Background: To detect drug resistance in Shigella obtained from the dung of the giant panda, explore the factors leading to drug resistance in Shigella, understand the characteristics of clustered, regularly interspaced, short, palindromic repeats (CRISPR), and assess the relationship between CRISPR and drug resistance. Methods: We collected fresh feces from 27 healthy giant pandas in the Giant Panda Conservation base (Wolong, China). We identified the strains of Shigella in the samples by using nucleotide sequence analysis. Further, the Kirby-Bauer paper method was used to determine drug sensitivity of the Shigella strains. CRISPR-associated protein genes cas1 and cas2 in Shigella were detected by polymerase chain reaction (PCR), and the PCR products were sequenced and compared. Results: We isolated and identified 17 strains of Shigella from 27 samples, including 14 strains of Shigella flexneri, 2 strains of Shigella sonnei, and 1 strain of Shigella dysenteriae. Further, drug resistance to cefazolin, imipenem, and amoxicillin–clavulanic acid was identified as a serious problem, as multidrug-resistant strains were detected. Further, cas1 and cas2 showed different degrees of point mutations. Conclusion: The CRISPR system widely exists in Shigella and shares homology with that in Escherichia coli. The cas1 and cas 2 mutations contribute to the different levels of resistance. Point mutations at sites 3176455, 3176590, and 3176465 in cas1 (a); sites 3176989, 3176992, and 3176995 in cas1 (b); sites 3176156 and 3176236 in cas2 may affect the resistance of bacteria, cause emergence of multidrug resistance, and increase the types of drug resistance. PMID:28207509

  10. Relationship between drug resistance and the clustered, regularly interspaced, short, palindromic repeat-associated protein genes cas1 and cas2 in Shigella from giant panda dung.

    Science.gov (United States)

    Ren, Lu; Deng, Lin-Hua; Zhang, Ri-Peng; Wang, Cheng-Dong; Li, De-Sheng; Xi, Li-Xin; Chen, Zhen-Rong; Yang, Rui; Huang, Jie; Zeng, Yang-Ru; Wu, Hong-Lin; Cao, San-Jie; Wu, Rui; Huang, Yong; Yan, Qi-Gui

    2017-02-01

    To detect drug resistance in Shigella obtained from the dung of the giant panda, explore the factors leading to drug resistance in Shigella, understand the characteristics of clustered, regularly interspaced, short, palindromic repeats (CRISPR), and assess the relationship between CRISPR and drug resistance. We collected fresh feces from 27 healthy giant pandas in the Giant Panda Conservation base (Wolong, China). We identified the strains of Shigella in the samples by using nucleotide sequence analysis. Further, the Kirby-Bauer paper method was used to determine drug sensitivity of the Shigella strains. CRISPR-associated protein genes cas1 and cas2 in Shigella were detected by polymerase chain reaction (PCR), and the PCR products were sequenced and compared. We isolated and identified 17 strains of Shigella from 27 samples, including 14 strains of Shigella flexneri, 2 strains of Shigella sonnei, and 1 strain of Shigella dysenteriae. Further, drug resistance to cefazolin, imipenem, and amoxicillin-clavulanic acid was identified as a serious problem, as multidrug-resistant strains were detected. Further, cas1 and cas2 showed different degrees of point mutations. The CRISPR system widely exists in Shigella and shares homology with that in Escherichia coli. The cas1 and cas 2 mutations contribute to the different levels of resistance. Point mutations at sites 3176455, 3176590, and 3176465 in cas1 (a); sites 3176989, 3176992, and 3176995 in cas1 (b); sites 3176156 and 3176236 in cas2 may affect the resistance of bacteria, cause emergence of multidrug resistance, and increase the types of drug resistance.

  11. Inverted repeat of Olisthodiscus luteus chloroplast DNA contains genes for both subunits of ribulose-1,5-bisphosphate carboxylase and the 32,000-dalton QB protein: Phylogenetic implications

    Science.gov (United States)

    Reith, Michael; Cattolico, Rose Ann

    1986-01-01

    The chloroplast DNA of the chromophytic alga Olisthodiscus luteus has been physically mapped with four restriction enzymes. An inverted repeat of 22 kilobase pairs is present in this 150-kilobase-pair plastid genome. The inverted repeat contains the genes for the large and small subunit polypeptides of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) and also codes for the 32,000-dalton QB protein. These observations demonstrate that significant differences exist in chloroplast genome structure and organization among major plant taxa. Images PMID:16578794

  12. Repeat-induced gene silencing in mammals.

    Science.gov (United States)

    Garrick, D; Fiering, S; Martin, D I; Whitelaw, E

    1998-01-01

    In both plants and Drosophila melanogaster, expression from a transgenic locus may be silenced when repeated transgene copies are arranged as a concatameric array. This repeat-induced gene silencing is frequently manifested as a decrease in the proportion of cells that express the transgene, resulting in a variegated pattern of expression. There is also some indication that, in transgenic mammals, the number of transgene copies within an array can exert a repressive influence on expression, with several mouse studies reporting a decrease in the level of expression per copy as copy number increases. However, because these studies compare different sites of transgene integration as well as arrays with different numbers of copies, the expression levels observed may be subject to varying position effects as well as the influence of the multicopy array. Here we describe use of the lox/Cre system of site-specific recombination to generate transgenic mouse lines in which different numbers of a transgene are present at the same chromosomal location, thereby eliminating the contribution of position effects and allowing analysis of the effect of copy number alone on transgene silencing. Reduction in copy number results in a marked increase in expression of the transgene and is accompanied by decreased chromatin compaction and decreased methylation at the transgene locus. These findings establish that the presence of multiple homologous copies of a transgene within a concatameric array can have a repressive effect upon gene expression in mammalian systems.

  13. Cloning of full-length cDNA of Microsporum canis membrane protein PQ-loop repeat protein gene%犬小孢子菌膜蛋白PQ-LRP基因全长cDNA的克隆

    Institute of Scientific and Technical Information of China (English)

    庞娟; 祝逸平; 杨国玲

    2012-01-01

    Objective To clone the full-length cDNA of Microsporum canis membrane protein PQ-loop repeat protein (PQ-LRP) gene,so as to investigate the roles of PQ-LRP in the pathogenesis of tinea capitis.Methods A Microsporum canis strain (A518) from a patient with tinea capitis served as the experimental strain.Rapid cDNA end amplification (RACE) was performed to clone the full length cDNA sequence of PQLRP gene.Bioinformatics methods were used to make a preliminary functional analysis of the gene.Results The cDNA of PQ-LRP gene was obtained with a full length of 1522 bp,including the 5' untranslated region (49 bp),coding region (1080 bp) and 3' untranslated region (393 bp).The coding region encoded a protein precursor including 359 amino acid residues.The cloned cDNA of PQ-LRP gene shared an 81% nucleotide identity with that of Trichophyton tonsurans and a 79% nucleotide identity with that of Trichophyton rubrum.Conclusions The full-length cDNA of Microsporum canis membrane protein PQ-LRP gene has been successfully cloned,which will provide an important basis for further researches into the roles of PQ-LRP in Microsporum canis-associated diseases.%目的 克隆犬小孢子菌膜蛋白PQ-LRP(PQ-loop repeat protein)基因全长cDNA,探讨在头癣发病机制中的作用.方法 选用犬小孢子菌头癣株(A518)为实验株,采用cDNA快速末端扩增法(RACE),克隆PQ-LRP基因的全长序列.结合生物信息学方法对获得的序列进行初步功能分析.结果 获得犬小孢子菌PQ-LRP全长序列为1522 bp,拥有一个1080 bp的开放阅读框,编码359个氨基酸,5 '非编码区为49 bp,3 '非编码区为393 bp;同源性比对与断发毛癣菌的PQ-LRP同源性达到81%,与红色毛癣菌PQ-LRP同源性达到79%.结论 克隆出犬小孢子菌膜蛋白PQ-LRP cDNA全长序列,为研究膜蛋白PQ-LRP基因在犬小孢子菌病中的功能奠定基础.

  14. Severe and rapidly progressing cognitive phenotype in a SCA17-family with only marginally expanded CAG/CAA repeats in the TATA-box binding protein gene: A case report

    Directory of Open Access Journals (Sweden)

    Nielsen Troels

    2012-08-01

    Full Text Available Abstract Background The autosomal dominant spinocerebellar ataxias (SCAs confine a group of rare and heterogeneous disorders, which present with progressive ataxia and numerous other features e.g. peripheral neuropathy, macular degeneration and cognitive impairment, and a subset of these disorders is caused by CAG-repeat expansions in their respective genes. The diagnosing of the SCAs is often difficult due to the phenotypic overlap among several of the subtypes and with other neurodegenerative disorders e.g. Huntington’s disease. Case presentation We report a family in which the proband had rapidly progressing cognitive decline and only subtle cerebellar symptoms from age 42. Sequencing of the TATA-box binding protein gene revealed a modest elongation of the CAG/CAA-repeat of only two repeats above the non-pathogenic threshold of 41, confirming a diagnosis of SCA17. Normally, repeats within this range show reduced penetrance and result in a milder disease course with slower progression and later age of onset. Thus, this case presented with an unusual phenotype. Conclusions The current case highlights the diagnostic challenge of neurodegenerative disorders and the need for a thorough clinical and paraclinical examination of patients presenting with rapid cognitive decline to make a precise diagnosis on which further genetic counseling and initiation of treatment modalities can be based.

  15. Identification and expression analysis of the interferon-induced protein with tetratricopeptide repeats 5 (IFIT5 gene in duck (Anas platyrhynchos domesticus.

    Directory of Open Access Journals (Sweden)

    Bin Wang

    Full Text Available The interferon-induced proteins with tetratricopeptide repeats (IFITs protein family mediates antiviral effects by inhibiting translation initiation, cell proliferation, and migration in the interferon (IFN dependent innate immune system. Several members of this family, including IFIT1, IFIT2, IFIT3 and IFIT5, have been heavily studied in mammals. Avian species contain only one family member, IFIT5, and little is known about the role of this protein in birds. In this study, duck IFIT5 (duIFIT5 full-length mRNA was cloned by reverse transcription polymerase chain reaction (RT-PCR and rapid amplification of the cDNA ends (RACE. Based on the sequence obtained, we performed a series of bioinformatics analyses, and found that duIFIT5 was most similar to homologs in other avian species. Also, duIFIT5 contained eight conserved TPR motifs and two conserved multi-domains (TPR_11 and TPR_12. Finally, we used duck hepatitis virus type 1 (DHV-1 and polyriboinosinicpolyribocytidylic acid (poly (I:C as a pathogen or a pathogen-associated molecular pattern induction to infect three-day-old domestic ducklings. The liver and spleen were collected to detect the change in duIFIT5 transcript level upon infection by quantitative real-time PCR (qRT-PCR. DuIFIT5 expression rapidly increased after DHV-1 infection and maintained a high level, while the transcripts of duIFIT5 peaked at 8h after poly (I:C infection and then returned to normal. Taken together, these results provide a greater understanding of avian IFIT5.

  16. Mononucleotide repeats are asymmetrically distributed in fungal genes

    Directory of Open Access Journals (Sweden)

    de Graaff Leo H

    2008-12-01

    Full Text Available Abstract Background Systematic analyses of sequence features have resulted in a better characterisation of the organisation of the genome. A previous study in prokaryotes on the distribution of sequence repeats, which are notoriously variable and can disrupt the reading frame in genes, showed that these motifs are skewed towards gene termini, specifically the 5' end of genes. For eukaryotes no such intragenic analysis has been performed, though this could indicate the pervasiveness of this distribution bias, thereby helping to expose the selective pressures causing it. Results In fungal gene repertoires we find a similar 5' bias of intragenic mononucleotide repeats, most notably for Candida spp., whereas e.g. Coccidioides spp. display no such bias. With increasing repeat length, ever larger discrepancies are observed in genome repertoire fractions containing such repeats, with up to an 80-fold difference in gene fractions at repeat lengths of 10 bp and longer. This species-specific difference in gene fractions containing large repeats could be attributed to variations in intragenic repeat tolerance. Furthermore, long transcripts experience an even more prominent bias towards the gene termini, with possibly a more adaptive role for repeat-containing short transcripts. Conclusion Mononucleotide repeats are intragenically biased in numerous fungal genomes, similar to earlier studies on prokaryotes, indicative of a similar selective pressure in gene organization.

  17. CGG repeat in the FMR1 gene: Size matters

    NARCIS (Netherlands)

    R.A. Willemsen (Ralph); G.J. Levenga (Josien); B.A. Oostra (Ben)

    2011-01-01

    textabstractThe FMR1 gene contains a CGG repeat present in the 5'-untranslated region which can be unstable upon transmission to the next generation. The repeat is up to 55 CGGs long in the normal population. In patients with fragile X syndrome (FXS), a repeat length exceeding 200 CGGs (full

  18. The evolution of filamin – A protein domain repeat perspective

    OpenAIRE

    Light, Sara; Sagit, Rauan; Ithychanda, Sujay S.; Qin, Jun; Elofsson, Arne

    2012-01-01

    Particularly in higher eukaryotes, some protein domains are found in tandem repeats, performing broad functions often related to cellular organization. For instance, the eukaryotic protein filamin interacts with many proteins and is crucial for the cytoskeleton. The functional properties of long repeat domains are governed by the specific properties of each individual domain as well as by the repeat copy number. To provide better understanding of the evolutionary and functional history of rep...

  19. Seedling Lethal1, a Pentatricopeptide Repeat Protein Lacking an E/E+ or DYW Domain in Arabidopsis, Is Involved in Plastid Gene Expression and Early Chloroplast Development1[C][W

    Science.gov (United States)

    Pyo, Young Jae; Kwon, Kwang-Chul; Kim, Anna; Cho, Myeon Haeng

    2013-01-01

    Chloroplasts are the site of photosynthesis and the biosynthesis of essential metabolites, including amino acids, fatty acids, and secondary metabolites. It is known that many seedling-lethal mutants are impaired in chloroplast function or development, indicating the development of functional chloroplast is essential for plant growth and development. Here, we isolated a novel transfer DNA insertion mutant, dubbed sel1 (for seedling lethal1), that exhibited a pigment-defective and seedling-lethal phenotype with a disrupted pentatricopeptide repeat (PPR) gene. Sequence analysis revealed that SEL1 is a member of the PLS subgroup, which is lacking known E/E+ or DYW domains at the C terminus, in the PLS subfamily of the PPR protein family containing a putative N-terminal transit peptide and 14 putative PPR or PPR-like motifs. Confocal microscopic analysis showed that the SEL1-green fluorescent protein fusion protein is localized in chloroplasts. Transmission electron microscopic analysis revealed that the sel1 mutant is impaired in the etioplast, as well as in chloroplast development. In sel1 mutants, plastid-encoded proteins involved in photosynthesis were rarely detected due to the lack of the corresponding transcripts. Furthermore, transcript profiles of plastid genes revealed that, in sel1 mutants, the transcript levels of plastid-encoded RNA polymerase-dependent genes were greatly reduced, but those of nuclear-encoded RNA polymerase-dependent genes were increased or not changed. Additionally, the RNA editing of two editing sites of the acetyl-CoA carboxylase beta subunit gene transcripts in the sel1 mutant was compromised, though it is not directly connected with the sel1 mutant phenotype. Our results demonstrate that SEL1 is involved in the regulation of plastid gene expression required for normal chloroplast development. PMID:24144791

  20. STRING: a web-server to retrieve and display the repeatedly occurring neighbourhood of a gene.

    Science.gov (United States)

    Snel, B; Lehmann, G; Bork, P; Huynen, M A

    2000-09-15

    The repeated occurrence of genes in each other's neighbourhood on genomes has been shown to indicate a functional association between the proteins they encode. Here we introduce STRING (search tool for recurring instances of neighbouring genes), a tool to retrieve and display the genes a query gene repeatedly occurs with in clusters on the genome. The tool performs iterative searches and visualises the results in their genomic context. By finding the genomically associated genes for a query, it delineates a set of potentially functionally associated genes. The usefulness of STRING is illustrated with an example that suggests a functional context for an RNA methylase with unknown specificity.

  1. The evolution and function of protein tandem repeats in plants.

    Science.gov (United States)

    Schaper, Elke; Anisimova, Maria

    2015-04-01

    Sequence tandem repeats (TRs) are abundant in proteomes across all domains of life. For plants, little is known about their distribution or contribution to protein function. We exhaustively annotated TRs and studied the evolution of TR unit variations for all Ensembl plants. Using phylogenetic patterns of TR units, we detected conserved TRs with unit number and order preserved during evolution, and those TRs that have diverged via recent TR unit gains/losses. We correlated the mode of evolution of TRs to protein function. TR number was strongly correlated with proteome size, with about one-half of all TRs recognized as common protein domains. The majority of TRs have been highly conserved over long evolutionary distances, some since the separation of red algae and green plants c. 1.6 billion yr ago. Conversely, recurrent recent TR unit mutations were rare. Our results suggest that the first TRs by far predate the first plants, and that TR appearance is an ongoing process with similar rates across the plant kingdom. Interestingly, the few detected highly mutable TRs might provide a source of variation for rapid adaptation. In particular, such TRs are enriched in leucine-rich repeats (LRRs) commonly found in R genes, where TR unit gain/loss may facilitate resistance to emerging pathogens.

  2. The Rhodomonas salina mitochondrial genome: bacteria-like operons, compact gene arrangement and complex repeat region.

    Science.gov (United States)

    Hauth, Amy M; Maier, Uwe G; Lang, B Franz; Burger, Gertraud

    2005-01-01

    To gain insight into the mitochondrial genome structure and gene content of a putatively ancestral group of eukaryotes, the cryptophytes, we sequenced the complete mitochondrial DNA of Rhodomonas salina. The 48 063 bp circular-mapping molecule codes for 2 rRNAs, 27 tRNAs and 40 proteins including 23 components of oxidative phosphorylation, 15 ribosomal proteins and two subunits of tat translocase. One potential protein (ORF161) is without assigned function. Only two introns occur in the genome; both are present within cox1 belong to group II and contain RT open reading frames. Primitive genome features include bacteria-like rRNAs and tRNAs, ribosomal protein genes organized in large clusters resembling bacterial operons and the presence of the otherwise rare genes such as rps1 and tatA. The highly compact gene organization contrasts with the presence of a 4.7 kb long, repeat-containing intergenic region. Repeat motifs approximately 40-700 bp long occur up to 31 times, forming a complex repeat structure. Tandem repeats are the major arrangement but the region also includes a large, approximately 3 kb, inverted repeat and several potentially stable approximately 40-80 bp long hairpin structures. We provide evidence that the large repeat region is involved in replication and transcription initiation, predict a promoter motif that occurs in three locations and discuss two likely scenarios of how this highly structured repeat region might have evolved.

  3. RepeatsDB 2.0: improved annotation, classification, search and visualization of repeat protein structures

    Science.gov (United States)

    Paladin, Lisanna; Hirsh, Layla; Piovesan, Damiano; Andrade-Navarro, Miguel A.; Kajava, Andrey V.; Tosatto, Silvio C.E.

    2017-01-01

    RepeatsDB 2.0 (URL: http://repeatsdb.bio.unipd.it/) is an update of the database of annotated tandem repeat protein structures. Repeat proteins are a widespread class of non-globular proteins carrying heterogeneous functions involved in several diseases. Here we provide a new version of RepeatsDB with an improved classification schema including high quality annotations for ∼5400 protein structures. RepeatsDB 2.0 features information on start and end positions for the repeat regions and units for all entries. The extensive growth of repeat unit characterization was possible by applying the novel ReUPred annotation method over the entire Protein Data Bank, with data quality is guaranteed by an extensive manual validation for >60% of the entries. The updated web interface includes a new search engine for complex queries and a fully re-designed entry page for a better overview of structural data. It is now possible to compare unit positions, together with secondary structure, fold information and Pfam domains. Moreover, a new classification level has been introduced on top of the existing scheme as an independent layer for sequence similarity relationships at 40%, 60% and 90% identity. PMID:27899671

  4. Genes and pathways affected by CAG-repeat RNA-based toxicity in Drosophila

    OpenAIRE

    Shieh, Shin-Yi; Bonini, Nancy M.

    2011-01-01

    Spinocerebellar ataxia type 3 is one of the polyglutamine (polyQ) diseases, which are caused by a CAG-repeat expansion within the coding region of the associated genes. The CAG repeat specifies glutamine, and the expanded polyQ domain mutation confers dominant toxicity on the protein. Traditionally, studies have focused on protein toxicity in polyQ disease mechanisms. Recent findings, however, demonstrate that the CAG-repeat RNA, which encodes the toxic polyQ protein, also contributes to the ...

  5. Genes and pathways affected by CAG-repeat RNA-based toxicity in Drosophila.

    Science.gov (United States)

    Shieh, Shin-Yi; Bonini, Nancy M

    2011-12-15

    Spinocerebellar ataxia type 3 is one of the polyglutamine (polyQ) diseases, which are caused by a CAG-repeat expansion within the coding region of the associated genes. The CAG repeat specifies glutamine, and the expanded polyQ domain mutation confers dominant toxicity on the protein. Traditionally, studies have focused on protein toxicity in polyQ disease mechanisms. Recent findings, however, demonstrate that the CAG-repeat RNA, which encodes the toxic polyQ protein, also contributes to the disease in Drosophila. To provide insights into the nature of the RNA toxicity, we extracted brain-enriched RNA from flies expressing a toxic CAG-repeat mRNA (CAG100) and a non-toxic interrupted CAA/G mRNA repeat (CAA/G105) for microarray analysis. This approach identified 160 genes that are differentially expressed specifically in CAG100 flies. Functional annotation clustering analysis revealed several broad ontologies enriched in the CAG100 gene list, including iron ion binding and nucleotide binding. Intriguingly, transcripts for the Hsp70 genes, a powerful suppressor of polyQ and other human neurodegenerative diseases, were also upregulated. We therefore tested and showed that upregulation of heat shock protein 70 mitigates CAG-repeat RNA toxicity. We then assessed whether other modifiers of the pathogenic, expanded Ataxin-3 polyQ protein could also modify the CAG-repeat RNA toxicity. This approach identified the co-chaperone Tpr2, the transcriptional regulator Dpld, and the RNA-binding protein Orb2 as modifiers of both polyQ protein toxicity and CAG-repeat RNA-based toxicity. These findings suggest an overlap in the mechanisms of RNA and protein-based toxicity, providing insights into the pathogenicity of the RNA in polyQ disease.

  6. Ising Model Reprogramming of a Repeat Protein's Equilibrium Unfolding Pathway.

    Science.gov (United States)

    Millership, C; Phillips, J J; Main, E R G

    2016-05-08

    Repeat proteins are formed from units of 20-40 aa that stack together into quasi one-dimensional non-globular structures. This modular repetitive construction means that, unlike globular proteins, a repeat protein's equilibrium folding and thus thermodynamic stability can be analysed using linear Ising models. Typically, homozipper Ising models have been used. These treat the repeat protein as a series of identical interacting subunits (the repeated motifs) that couple together to form the folded protein. However, they cannot describe subunits of differing stabilities. Here we show that a more sophisticated heteropolymer Ising model can be constructed and fitted to two new helix deletion series of consensus tetratricopeptide repeat proteins (CTPRs). This analysis, showing an asymmetric spread of stability between helices within CTPR ensembles, coupled with the Ising model's predictive qualities was then used to guide reprogramming of the unfolding pathway of a variant CTPR protein. The designed behaviour was engineered by introducing destabilising mutations that increased the thermodynamic asymmetry within a CTPR ensemble. The asymmetry caused the terminal α-helix to thermodynamically uncouple from the rest of the protein and preferentially unfold. This produced a specific, highly populated stable intermediate with a putative dimerisation interface. As such it is the first step in designing repeat proteins with function regulated by a conformational switch. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  7. Artificial leucine rich repeats as new scaffolds for protein design.

    Science.gov (United States)

    Baabur-Cohen, Hemda; Dayalan, Subashini; Shumacher, Inbal; Cohen-Luria, Rivka; Ashkenasy, Gonen

    2011-04-15

    The leucine rich repeat (LRR) motif that participates in many biomolecular recognition events in cells was suggested as a general scaffold for producing artificial receptors. We describe here the design and first total chemical synthesis of small LRR proteins, and their structural analysis. When evaluating the tertiary structure as a function of different number of repeating units (1-3), we were able to find that the 3-repeats sequence, containing 90 amino acids, folds into the expected structure.

  8. Were protein internal repeats formed by "bricolage"?

    Science.gov (United States)

    Lavorgna, G; Patthy, L; Boncinelli, E

    2001-03-01

    Is evolution an engineer, or is it a tinkerer--a "bricoleur"--building up complex molecules in organisms by increasing and adapting the materials at hand? An analysis of completely sequenced genomes suggests the latter, showing that increasing repetition of modules within the proteins encoded by these genomes is correlated with increasing complexity of the organism.

  9. Invasive Species Management on Military Lands: Clustered Regularly Interspaced Short Palindromic Repeat/ CRISPR associated protein 9 (CRISPR/Cas9) based Gene Drives

    Science.gov (United States)

    2017-06-30

    advantage of RNA-guided gene drives. • Development time: The CRISPR-Cas9 technology has made it much easier and faster to make transgenic organisms ...environmental risk, public perception, and regulatory issues surrounding the development and field release of CRISPR-Cas9- engineered organisms . 4.7.1... ORGANIZATION NAME(S) AND ADDRESS(ES) U.S. Army Engineer Research and Development Center (ERDC) Environmental Laboratory Waterways Experiment Station, 3909

  10. Arcuate nucleus transcriptome profiling identifies ankyrin repeat and suppressor of cytokine signalling box-containing protein 4 as a gene regulated by fasting in central nervous system feeding circuits.

    Science.gov (United States)

    Li, J-Y; Kuick, R; Thompson, R C; Misek, D E; Lai, Y-M; Liu, Y-Q; Chai, B-X; Hanash, S M; Gantz, I

    2005-06-01

    The arcuate nucleus of the hypothalamus is a primary site for sensing blood borne nutrients and hormonal messengers that reflect caloric status. To identify novel energy homeostatic genes, we examined RNA extracts from the microdissected arcuate nucleus of fed and 48-h fasted rats using oligonucleotide microarrays. The relative abundance of 118 mRNA transcripts was increased and 203 mRNA transcripts was decreased during fasting. One of the down-regulated mRNAs was ankyrin-repeat and suppressor of cytokine signalling box-containing protein 4 (Asb-4). The predicted structure of Asb-4 protein suggested that it might encode an intracellular regulatory protein, and therefore its mRNA expression was investigated further. Reverse transcription quantitative polymerase chain reaction was used to validate down-regulation of Asb-4 mRNA in the arcuate nucleus of the fasted Sprague-Dawley rat (relative expression of Asb-4 mRNA: fed = 4.66 +/- 0.26; fasted = 3.96 +/- 0.23; n = 4, P regulation was also demonstrated in the obese fa/fa Zucker rat, another model of energy disequilibrium (relative expression of Asb-4 mRNA: lean Zucker = 3.91 +/- 0.32; fa/fa = 2.93 +/- 0.26; n = 5, P fasted state, the percentage of POMC neurones expressing Asb-4 mRNA drops to 73.2% (P fasted POMC neurone is markedly decreased. Conversely, expression of Asb-4 mRNA by NPY neurones in the fasted state is modestly increased to 52.7% (P < 0.05). Based on its differential expression, neuroanatomical distribution and colocalisation, we hypothesise that Asb-4 is a gene involved in energy homeostasis.

  11. MSH3 polymorphisms and protein levels affect CAG repeat instability in Huntington's disease mice.

    Directory of Open Access Journals (Sweden)

    Stéphanie Tomé

    Full Text Available Expansions of trinucleotide CAG/CTG repeats in somatic tissues are thought to contribute to ongoing disease progression through an affected individual's life with Huntington's disease or myotonic dystrophy. Broad ranges of repeat instability arise between individuals with expanded repeats, suggesting the existence of modifiers of repeat instability. Mice with expanded CAG/CTG repeats show variable levels of instability depending upon mouse strain. However, to date the genetic modifiers underlying these differences have not been identified. We show that in liver and striatum the R6/1 Huntington's disease (HD (CAG∼100 transgene, when present in a congenic C57BL/6J (B6 background, incurred expansion-biased repeat mutations, whereas the repeat was stable in a congenic BALB/cByJ (CBy background. Reciprocal congenic mice revealed the Msh3 gene as the determinant for the differences in repeat instability. Expansion bias was observed in congenic mice homozygous for the B6 Msh3 gene on a CBy background, while the CAG tract was stabilized in congenics homozygous for the CBy Msh3 gene on a B6 background. The CAG stabilization was as dramatic as genetic deficiency of Msh2. The B6 and CBy Msh3 genes had identical promoters but differed in coding regions and showed strikingly different protein levels. B6 MSH3 variant protein is highly expressed and associated with CAG expansions, while the CBy MSH3 variant protein is expressed at barely detectable levels, associating with CAG stability. The DHFR protein, which is divergently transcribed from a promoter shared by the Msh3 gene, did not show varied levels between mouse strains. Thus, naturally occurring MSH3 protein polymorphisms are modifiers of CAG repeat instability, likely through variable MSH3 protein stability. Since evidence supports that somatic CAG instability is a modifier and predictor of disease, our data are consistent with the hypothesis that variable levels of CAG instability associated with

  12. MSH3 polymorphisms and protein levels affect CAG repeat instability in Huntington's disease mice.

    Science.gov (United States)

    Tomé, Stéphanie; Manley, Kevin; Simard, Jodie P; Clark, Greg W; Slean, Meghan M; Swami, Meera; Shelbourne, Peggy F; Tillier, Elisabeth R M; Monckton, Darren G; Messer, Anne; Pearson, Christopher E

    2013-01-01

    Expansions of trinucleotide CAG/CTG repeats in somatic tissues are thought to contribute to ongoing disease progression through an affected individual's life with Huntington's disease or myotonic dystrophy. Broad ranges of repeat instability arise between individuals with expanded repeats, suggesting the existence of modifiers of repeat instability. Mice with expanded CAG/CTG repeats show variable levels of instability depending upon mouse strain. However, to date the genetic modifiers underlying these differences have not been identified. We show that in liver and striatum the R6/1 Huntington's disease (HD) (CAG)∼100 transgene, when present in a congenic C57BL/6J (B6) background, incurred expansion-biased repeat mutations, whereas the repeat was stable in a congenic BALB/cByJ (CBy) background. Reciprocal congenic mice revealed the Msh3 gene as the determinant for the differences in repeat instability. Expansion bias was observed in congenic mice homozygous for the B6 Msh3 gene on a CBy background, while the CAG tract was stabilized in congenics homozygous for the CBy Msh3 gene on a B6 background. The CAG stabilization was as dramatic as genetic deficiency of Msh2. The B6 and CBy Msh3 genes had identical promoters but differed in coding regions and showed strikingly different protein levels. B6 MSH3 variant protein is highly expressed and associated with CAG expansions, while the CBy MSH3 variant protein is expressed at barely detectable levels, associating with CAG stability. The DHFR protein, which is divergently transcribed from a promoter shared by the Msh3 gene, did not show varied levels between mouse strains. Thus, naturally occurring MSH3 protein polymorphisms are modifiers of CAG repeat instability, likely through variable MSH3 protein stability. Since evidence supports that somatic CAG instability is a modifier and predictor of disease, our data are consistent with the hypothesis that variable levels of CAG instability associated with polymorphisms of

  13. Gene conversion homogenizes the CMT1A paralogous repeats

    Directory of Open Access Journals (Sweden)

    Hurles Matthew E

    2001-12-01

    Full Text Available Abstract Background Non-allelic homologous recombination between paralogous repeats is increasingly being recognized as a major mechanism causing both pathogenic microdeletions and duplications, and structural polymorphism in the human genome. It has recently been shown empirically that gene conversion can homogenize such repeats, resulting in longer stretches of absolute identity that may increase the rate of non-allelic homologous recombination. Results Here, a statistical test to detect gene conversion between pairs of non-coding sequences is presented. It is shown that the 24 kb Charcot-Marie-Tooth type 1A paralogous repeats (CMT1A-REPs exhibit the imprint of gene conversion processes whilst control orthologous sequences do not. In addition, Monte Carlo simulations of the evolutionary divergence of the CMT1A-REPs, incorporating two alternative models for gene conversion, generate repeats that are statistically indistinguishable from the observed repeats. Bounds are placed on the rate of these conversion processes, with central values of 1.3 × 10-4 and 5.1 × 10-5 per generation for the alternative models. Conclusions This evidence presented here suggests that gene conversion may have played an important role in the evolution of the CMT1A-REP paralogous repeats. The rates of these processes are such that it is probable that homogenized CMT1A-REPs are polymorphic within modern populations. Gene conversion processes are similarly likely to play an important role in the evolution of other segmental duplications and may influence the rate of non-allelic homologous recombination between them.

  14. Gene conversion homogenizes the CMT1A paralogous repeats.

    Science.gov (United States)

    Hurles, M E

    2001-01-01

    Non-allelic homologous recombination between paralogous repeats is increasingly being recognized as a major mechanism causing both pathogenic microdeletions and duplications, and structural polymorphism in the human genome. It has recently been shown empirically that gene conversion can homogenize such repeats, resulting in longer stretches of absolute identity that may increase the rate of non-allelic homologous recombination. Here, a statistical test to detect gene conversion between pairs of non-coding sequences is presented. It is shown that the 24 kb Charcot-Marie-Tooth type 1A paralogous repeats (CMT1A-REPs) exhibit the imprint of gene conversion processes whilst control orthologous sequences do not. In addition, Monte Carlo simulations of the evolutionary divergence of the CMT1A-REPs, incorporating two alternative models for gene conversion, generate repeats that are statistically indistinguishable from the observed repeats. Bounds are placed on the rate of these conversion processes, with central values of 1.3 x 10(-4) and 5.1 x 10(-5) per generation for the alternative models. This evidence presented here suggests that gene conversion may have played an important role in the evolution of the CMT1A-REP paralogous repeats. The rates of these processes are such that it is probable that homogenized CMT1A-REPs are polymorphic within modern populations. Gene conversion processes are similarly likely to play an important role in the evolution of other segmental duplications and may influence the rate of non-allelic homologous recombination between them.

  15. feasibilty of zein proteins, simple sequence repeats and phenotypic ...

    African Journals Online (AJOL)

    journal

    Simply inherited traits such as maize streak virus disease resistance were suitable for background selection. Other traits ..... genes and when dealing with complex traits. (Stuber, 1995). ..... Mutant gene that changes the protein composition and ...

  16. Myotonin protein-kinase [AGC]n trinucleotide repeat in seven nonhuman primates

    Energy Technology Data Exchange (ETDEWEB)

    Novelli, G.; Sineo, L.; Pontieri, E. [Catholic Univ. of Rome (Italy)]|[Univ. of Milan (Italy)]|[Univ. Florence (Italy)] [and others

    1994-09-01

    Myotonic dystrophy (DM) is due to a genomic instability of a trinucleotide [AGC]n motif, located at the 3{prime} UTR region of a protein-kinase gene (myotonin protein kinase, MT-PK). The [AGC] repeat is meiotically and mitotically unstable, and it is directly related to the manifestations of the disorder. Although a gene dosage effect of the MT-PK has been demonstrated n DM muscle, the mechanism(s) by which the intragenic repeat expansion leads to disease is largely unknown. This non-standard mutational event could reflect an evolutionary mechanism widespread among animal genomes. We have isolated and sequenced the complete 3{prime}UTR region of the MT-PK gene in seven primates (macaque, orangutan, gorilla, chimpanzee, gibbon, owl monkey, saimiri), and examined by comparative sequence nucleotide analysis the [AGC]n intragenic repeat and the surrounding nucleotides. The genomic organization, including the [AGC]n repeat structure, was conserved in all examined species, excluding the gibbon (Hylobates agilis), in which the [AGC]n upstream sequence (GGAA) is replaced by a GA dinucleotide. The number of [AGC]n in the examined species ranged between 7 (gorilla) and 13 repeats (owl monkeys), with a polymorphism informative content (PIC) similar to that observed in humans. These results indicate that the 3{prime}UTR [AGC] repeat within the MT-PK gene is evolutionarily conserved, supporting that this region has important regulatory functions.

  17. The evolution of filamin – A protein domain repeat perspective

    Science.gov (United States)

    Light, Sara; Sagit, Rauan; Ithychanda, Sujay S.; Qin, Jun; Elofsson, Arne

    2013-01-01

    Particularly in higher eukaryotes, some protein domains are found in tandem repeats, performing broad functions often related to cellular organization. For instance, the eukaryotic protein filamin interacts with many proteins and is crucial for the cytoskeleton. The functional properties of long repeat domains are governed by the specific properties of each individual domain as well as by the repeat copy number. To provide better understanding of the evolutionary and functional history of repeating domains, we investigated the mode of evolution of the filamin domain in some detail. Among the domains that are common in long repeat proteins, sushi and spectrin domains evolve primarily through cassette tandem duplications while scavenger and immunoglobulin repeats appear to evolve through clustered tandem duplications. Additionally, immunoglobulin and filamin repeats exhibit a unique pattern where every other domain shows high sequence similarity. This pattern may be the result of tandem duplications, serve to avert aggregation between adjacent domains or it is the result of functional constraints. In filamin, our studies confirm the presence of interspersed integrin binding domains in vertebrates, while invertebrates exhibit more varied patterns, including more clustered integrin binding domains. The most notable case is leech filamin, which contains a 20 repeat expansion and exhibits unique dimerization topology. Clearly, invertebrate filamins are varied and contain examples of similar adjacent integrin-binding domains. Given that invertebrate integrin shows more similarity to the weaker filamin binder, integrin β3, it is possible that the distance between integrin-binding domains is not as crucial for invertebrate filamins as for vertebrates. PMID:22414427

  18. The evolution of filamin-a protein domain repeat perspective.

    Science.gov (United States)

    Light, Sara; Sagit, Rauan; Ithychanda, Sujay S; Qin, Jun; Elofsson, Arne

    2012-09-01

    Particularly in higher eukaryotes, some protein domains are found in tandem repeats, performing broad functions often related to cellular organization. For instance, the eukaryotic protein filamin interacts with many proteins and is crucial for the cytoskeleton. The functional properties of long repeat domains are governed by the specific properties of each individual domain as well as by the repeat copy number. To provide better understanding of the evolutionary and functional history of repeating domains, we investigated the mode of evolution of the filamin domain in some detail. Among the domains that are common in long repeat proteins, sushi and spectrin domains evolve primarily through cassette tandem duplications while scavenger and immunoglobulin repeats appear to evolve through clustered tandem duplications. Additionally, immunoglobulin and filamin repeats exhibit a unique pattern where every other domain shows high sequence similarity. This pattern may be the result of tandem duplications, serve to avert aggregation between adjacent domains or it is the result of functional constraints. In filamin, our studies confirm the presence of interspersed integrin binding domains in vertebrates, while invertebrates exhibit more varied patterns, including more clustered integrin binding domains. The most notable case is leech filamin, which contains a 20 repeat expansion and exhibits unique dimerization topology. Clearly, invertebrate filamins are varied and contain examples of similar adjacent integrin-binding domains. Given that invertebrate integrin shows more similarity to the weaker filamin binder, integrin β3, it is possible that the distance between integrin-binding domains is not as crucial for invertebrate filamins as for vertebrates. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Long CAG Repeat Sequence and Protein Expression of Androgen Receptor Considered as Prognostic Indicators in Male Breast Carcinoma

    OpenAIRE

    Yan-Ni Song; Jing-Shu Geng; Tong Liu; Zhen-Bin Zhong; Yang Liu; Bing-Shu Xia; Hong-Fei Ji; Xiao-Mei Li; Guo-Qiang Zhang; Yan-Lv Ren; Zhi-Gao Li; Da Pang

    2012-01-01

    BACKGROUND: The androgen receptor (AR) expression and the CAG repeat length within the AR gene appear to be involved in the carcinogenesis of male breast carcinoma (MBC). Although phenotypic differences have been observed between MBC and normal control group in AR gene, there is lack of correlation analysis between AR expression and CAG repeat length in MBC. The purpose of the study was to investigate the prognostic value of CAG repeat lengths and AR protein expression. METHODS: 81 tumor tiss...

  20. WD40-repeat proteins in plant cell wall formation: current evidence and research prospects

    Directory of Open Access Journals (Sweden)

    Gea eGuerriero

    2015-12-01

    Full Text Available The metabolic complexity of living organisms relies on supramolecular protein structures which ensure vital processes, such as signal transduction, transcription, translation and cell wall synthesis. In eukaryotes WD40-repeat (WDR proteins often function as molecular hubs mediating supramolecular interactions. WDR proteins may display a variety of interacting partners and participate in the assembly of complexes involved in distinct cellular functions. In plants, the formation of lignocellulosic biomass involves extensive synthesis of cell wall polysaccharides, a process that requires the assembly of large transmembrane enzyme complexes, intensive vesicle trafficking, interactions with the cytoskeleton, and coordinated gene expression. Because of their function as supramolecular hubs, WDR proteins could participate in each or any of these steps, although to date only few WDR proteins have been linked to the cell wall by experimental evidence. Nevertheless, several potential cell wall-related WDR proteins were recently identified using in silico aproaches, such as analyses of co-expression, interactome and conserved gene neighbourhood. Notably, some WDR genes are frequently genomic neighbours of genes coding for GT2-family polysaccharide synthases in eukaryotes, and this WDR-GT2 collinear microsynteny is detected in diverse taxa. In angiosperms, two WDR genes are collinear to cellulose synthase genes, CESAs, whereas in ascomycetous fungi several WDR genes are adjacent to chitin synthase genes, chs. In this Perspective we summarize and discuss experimental and in silico studies on the possible involvement of WDR proteins in plant cell wall formation. The prospects of biotechnological engineering for enhanced biomass production are discussed.

  1. Larger trinucleotide repeat size in the androgen receptor gene of infertile men with extremely severe oligozoospermia.

    Science.gov (United States)

    Patrizio, P; Leonard, D G; Chen, K L; Hernandez-Ayup, S; Trounson, A O

    2001-01-01

    Androgens are significant regulators of human spermatogenesis. Their action is mediated through the androgen receptor (AR), which binds to the androgen responsive element on DNA and regulates gene transcription. Men become infertile with spinobulbar muscular atrophy (Kennedy disease) caused by a trinucleotide repeat expansion, > or = 40 CAG repeats, in the AR gene located on the X chromosome. In this prospective study, we investigated whether the variable size, larger repeats, of this trinucleotide could alter AR function and result in impaired spermatogenesis. A total of 69 infertile men were studied. Clinical and laboratory analysis showed idiopathic, nonobstructive azoospermia in 16 men, extremely severe oligozoospermia in 27 men (PCR) amplification across the AR repeat region. Accurate size determination of the PCR product using an ABI 373 DNA sequencer allowed precise calculation of CAG repeat sizes. The AR gene was not analyzed for other types of mutations. The difference in CAG repeat size between infertile men and proven fertile controls was statistically significant, P = .03. Patients with extremely severe oligozoospermia had significantly longer CAG repeat tracts (mean, 25.4 +/- 4.0; P = .0005; range 20-39) than controls (mean, 22 +/- 2.8; range 12-30) or patients with severe oligozoospermia (mean, 22.2 +/- 2.3; range 18-26). None of the 26 infertile men with sperm counts CAG repeats compared with 6 out of 45 controls (13%; P = .06). This study suggests that some men with severe impairment of spermatogenesis have longer trinucleotide repeats in the AR gene. Although direct evidence is missing, lower affinity between androgen and the AR protein or decreased AR protein availability with longer repeats could be responsible for a diminished androgen effect on spermatogenesis. Two of the patients in the extremely severe oligozoospermia group had 35 and 39 CAG repeats, respectively (normal range is 11 to 33). Although not yet considered a mutation, longer

  2. Transposed genes in Arabidopsis are often associated with flanking repeats.

    Directory of Open Access Journals (Sweden)

    Margaret R Woodhouse

    2010-05-01

    Full Text Available Much of the eukaryotic genome is known to be mobile, largely due to the movement of transposons and other parasitic elements. Recent work in plants and Drosophila suggests that mobility is also a feature of many nontransposon genes and gene families. Indeed, analysis of the Arabidopsis genome suggested that as many as half of all genes had moved to unlinked positions since Arabidopsis diverged from papaya roughly 72 million years ago, and that these mobile genes tend to fall into distinct gene families. However, the mechanism by which single gene transposition occurred was not deduced. By comparing two closely related species, Arabidopsis thaliana and Arabidopsis lyrata, we sought to determine the nature of gene transposition in Arabidopsis. We found that certain categories of genes are much more likely to have transposed than others, and that many of these transposed genes are flanked by direct repeat sequence that was homologous to sequence within the orthologous target site in A. lyrata and which was predominantly genic in identity. We suggest that intrachromosomal recombination between tandemly duplicated sequences, and subsequent insertion of the circular product, is the predominant mechanism of gene transposition.

  3. Structural Studies of a Four-MBT Repeat Protein MBTD1

    Energy Technology Data Exchange (ETDEWEB)

    Eryilmaz, Jitka; Pan, Patricia; Amaya, Maria F.; Allali-Hassani, Abdellah; Dong, Aiping; Adams-Cioaba, Melanie A.; MacKenzie, Farrell; Vedadi, Masoud; Min, Jinrong; (Toronto); (Toronto)

    2010-08-17

    The Polycomb group (PcG) of proteins is a family of important developmental regulators. The respective members function as large protein complexes involved in establishment and maintenance of transcriptional repression of developmental control genes. MBTD1, Malignant Brain Tumor domain-containing protein 1, is one such PcG protein. MBTD1 contains four MBT repeats. We have determined the crystal structure of MBTD1 (residues 130-566aa covering the 4 MBT repeats) at 2.5 {angstrom} resolution by X-ray crystallography. The crystal structure of MBTD1 reveals its similarity to another four-MBT-repeat protein L3MBTL2, which binds lower methylated lysine histones. Fluorescence polarization experiments confirmed that MBTD1 preferentially binds mono- and di-methyllysine histone peptides, like L3MBTL1 and L3MBTL2. All known MBT-peptide complex structures characterized to date do not exhibit strong histone peptide sequence selectivity, and use a 'cavity insertion recognition mode' to recognize the methylated lysine with the deeply buried methyl-lysine forming extensive interactions with the protein while the peptide residues flanking methyl-lysine forming very few contacts. Nevertheless, our mutagenesis data based on L3MBTL1 suggested that the histone peptides could not bind to MBT repeats in any orientation. The four MBT repeats in MBTD1 exhibits an asymmetric rhomboid architecture. Like other MBT repeat proteins characterized so far, MBTD1 binds mono- or dimethylated lysine histones through one of its four MBT repeats utilizing a semi-aromatic cage.

  4. StaRProtein, A Web Server for Prediction of the Stability of Repeat Proteins

    Science.gov (United States)

    Xu, Yongtao; Zhou, Xu; Huang, Meilan

    2015-01-01

    Repeat proteins have become increasingly important due to their capability to bind to almost any proteins and the potential as alternative therapy to monoclonal antibodies. In the past decade repeat proteins have been designed to mediate specific protein-protein interactions. The tetratricopeptide and ankyrin repeat proteins are two classes of helical repeat proteins that form different binding pockets to accommodate various partners. It is important to understand the factors that define folding and stability of repeat proteins in order to prioritize the most stable designed repeat proteins to further explore their potential binding affinities. Here we developed distance-dependant statistical potentials using two classes of alpha-helical repeat proteins, tetratricopeptide and ankyrin repeat proteins respectively, and evaluated their efficiency in predicting the stability of repeat proteins. We demonstrated that the repeat-specific statistical potentials based on these two classes of repeat proteins showed paramount accuracy compared with non-specific statistical potentials in: 1) discriminate correct vs. incorrect models 2) rank the stability of designed repeat proteins. In particular, the statistical scores correlate closely with the equilibrium unfolding free energies of repeat proteins and therefore would serve as a novel tool in quickly prioritizing the designed repeat proteins with high stability. StaRProtein web server was developed for predicting the stability of repeat proteins. PMID:25807112

  5. Long CAG repeat sequence and protein expression of androgen receptor considered as prognostic indicators in male breast carcinoma.

    Directory of Open Access Journals (Sweden)

    Yan-Ni Song

    Full Text Available BACKGROUND: The androgen receptor (AR expression and the CAG repeat length within the AR gene appear to be involved in the carcinogenesis of male breast carcinoma (MBC. Although phenotypic differences have been observed between MBC and normal control group in AR gene, there is lack of correlation analysis between AR expression and CAG repeat length in MBC. The purpose of the study was to investigate the prognostic value of CAG repeat lengths and AR protein expression. METHODS: 81 tumor tissues were used for immunostaining for AR expression and CAG repeat length determination and 80 normal controls were analyzed with CAG repeat length in AR gene. The CAG repeat length and AR expression were analyzed in relation to clinicopathological factors and prognostic indicators. RESULTS: AR gene in many MBCs has long CAG repeat sequence compared with that in control group (P = 0.001 and controls are more likely to exhibit short CAG repeat sequence than MBCs. There was statistically significant difference in long CAG repeat sequence between AR status for MBC patients (P = 0.004. The presence of long CAG repeat sequence and AR-positive expression were associated with shorter survival of MBC patients (CAG repeat: P = 0.050 for 5y-OS; P = 0.035 for 5y-DFS AR status: P = 0.048 for 5y-OS; P = 0.029 for 5y-DFS, respectively. CONCLUSION: The CAG repeat length within the AR gene might be one useful molecular biomarker to identify males at increased risk of breast cancer development. The presence of long CAG repeat sequence and AR protein expression were in relation to survival of MBC patients. The CAG repeat length and AR expression were two independent prognostic indicators in MBC patients.

  6. Molecular evolution of Drosophila cuticular protein genes.

    Directory of Open Access Journals (Sweden)

    R Scott Cornman

    Full Text Available Several multigene families have been described that together encode scores of structural cuticular proteins in Drosophila, although the functional significance of this diversity remains to be explored. Here I investigate the evolutionary histories of several multigene families (CPR, Tweedle, CPLCG, and CPF/CPFL that vary in age, size, and sequence complexity, using sequenced Drosophila genomes and mosquito outgroups. My objective is to describe the rates and mechanisms of 'cuticle-ome' divergence, in order to identify conserved and rapidly evolving elements. I also investigate potential examples of interlocus gene conversion and concerted evolution within these families during Drosophila evolution. The absolute rate of change in gene number (per million years is an order of magnitude lower for cuticular protein families within Drosophila than it is among Drosophila and the two mosquito taxa, implying that major transitions in the cuticle proteome have occurred at higher taxonomic levels. Several hotspots of intergenic conversion and/or gene turnover were identified, e.g. some gene pairs have independently undergone intergenic conversion within different lineages. Some gene conversion hotspots were characterized by conversion tracts initiating near nucleotide repeats within coding regions, and similar repeats were found within concertedly evolving cuticular protein genes in Anopheles gambiae. Rates of amino-acid substitution were generally severalfold higher along the branch connecting the Sophophora and Drosophila species groups, and 13 genes have Ka/Ks significantly greater than one along this branch, indicating adaptive divergence. Insect cuticular proteins appear to be a source of adaptive evolution within genera and, at higher taxonomic levels, subject to periods of gene-family expansion and contraction followed by quiescence. However, this relative stasis is belied by hotspots of molecular evolution, particularly concerted evolution, during

  7. Preferential Nucleosome Assembly at DNA Triplet Repeats from the Myotonic Dystrophy Gene

    Science.gov (United States)

    Wang, Yuh-Hwa; Amirhaeri, Sorour; Kang, Seongman; Wells, Robert D.; Griffith, Jack D.

    1994-07-01

    The expansion of CTG repeats in DNA occurs in or near genes involved in several human diseases, including myotonic dystrophy and Huntington's disease. Nucleosomes, the basic structural element of chromosomes, consist of 146 base pairs of DNA coiled about an octamer of histone proteins and mediate general transcriptional repression. Electron microscopy was used to examine in vitro the nucleosome assembly of DNA containing repeating CTG triplets. The efficiency of nucleosome formation increased with expanded triplet blocks, suggesting that such blocks may repress transcription through the creation of stable nucleosomes.

  8. Tandem Repeats in Proteins : Prediction Algorithms and Biological Role

    Directory of Open Access Journals (Sweden)

    Marco ePellegrini

    2015-09-01

    Full Text Available Tandem repetitions in protein sequence and structure is a fascinatingsubject of research which has been a focus of study since the late 1990's.In this survey we give an overviewon the multi-faceted aspects of research on protein tandem repeats textcolor{red}{(PTR for short}, including prediction algorithms, databases,early classification efforts, mechanisms of PTR formation and evolution, and synthetic PTR design.We also touch on the rather open issue of the relationship between PTRand flexibility (or disorder in proteins.Detection of PTR either from protein sequence or structure data is challenging due to inherent high (biological signal-to-noise ratio that is a key feature of this problem.As early in silico analytic tools have been key enablers for starting this field of study, we expect that current and future algorithmic and statistical breakthroughs willhave a high impact on the investigations of the biological role of PTR.

  9. Genus-specific protein binding to the large clusters of DNA repeats (short regularly spaced repeats) present in Sulfolobus genomes

    DEFF Research Database (Denmark)

    Peng, Xu; Brügger, Kim; Shen, Biao

    2003-01-01

    Short regularly spaced repeats (SRSRs) occur in multiple large clusters in archaeal chromosomes and as smaller clusters in some archaeal conjugative plasmids and bacterial chromosomes. The sequence, size, and spacing of the repeats are generally constant within a cluster but vary between clusters...... that are identical in sequence to one of the repeat variants in the S. solfataricus chromosome. Repeats from the pNOB8 cluster were amplified and tested for protein binding with cell extracts from S. solfataricus. A 17.5-kDa SRSR-binding protein was purified from the cell extracts and sequenced. The protein is N...... terminally modified and corresponds to SSO454, an open reading frame of previously unassigned function. It binds specifically to DNA fragments carrying double and single repeat sequences, binding on one side of the repeat structure, and producing an opening of the opposite side of the DNA structure. It also...

  10. Repeat-mediated epigenetic dysregulation of the FMR1 gene in the Fragile X-related disorders

    Directory of Open Access Journals (Sweden)

    Karen eUsdin

    2015-06-01

    Full Text Available The Fragile X-related disorders are members of the Repeat Expansion Diseases, a group of genetic conditions resulting from an expansion in the size of a tandem repeat tract at a specific genetic locus. The repeat responsible for disease pathology in the Fragile X-related disorders is CGG/CCG and the repeat tract is located in the 5’ UTR of the FMR1 gene, whose protein product FMRP, is important for the proper translation of dendritic mRNAs in response to synaptic activation. There are two different pathological FMR1 allele classes that are distinguished only by the number of repeats. Premutation alleles have 55-200 repeats and confer risk of Fragile X-associated tremor/ataxia syndrome and Fragile X-associated primary ovarian insufficiency. Full mutation alleles on the other hand have >200 repeats and result in Fragile X syndrome, a disorder that affects learning and behavior. Different symptoms are seen in carriers of premutation and full mutation alleles because the repeat number has paradoxical effects on gene expression: Epigenetic changes increase transcription from premutation alleles and decrease transcription from full mutation alleles. This review will cover what is currently known about the mechanisms responsible for these changes in FMR1 expression and how they may relate to other Repeat Expansion Diseases that also show repeat-mediated changes in gene expression.

  11. Expression of Anaplasma marginale ankyrin repeat-containing proteins during infection of the mammalian host and tick vector

    Science.gov (United States)

    Using searches of the NCBI conserved domain database and SMART genomic architecture analysis, we identified three ankyrin repeat-containing genes in Anaplasma marginale: AM705, AM926 and AM638. Recombinant protein was used to immunize mice and generate fusion hybridomas secreting protein-specific mo...

  12. Modulation of CRISPR locus transcription by the repeat-binding protein Cbp1 in Sulfolobus

    DEFF Research Database (Denmark)

    Deng, Ling; Kenchappa, Chandra Shekar; Peng, Xu

    2012-01-01

    CRISPR loci are essential components of the adaptive immune system of archaea and bacteria. They consist of long arrays of repeats separated by DNA spacers encoding guide RNAs (crRNA), which target foreign genetic elements. Cbp1 (CRISPR DNA repeat binding protein) binds specifically to the multiple...... direct repeats of CRISPR loci of members of the acidothermophilic, crenarchaeal order Sulfolobales. cbp1 gene deletion from Sulfolobus islandicus REY15A produced a strong reduction in pre-crRNA yields from CRISPR loci but did not inhibit the foreign DNA targeting capacity of the CRISPR/Cas system....... Conversely, overexpression of Cbp1 in S. islandicus generated an increase in pre-crRNA yields while the level of reverse strand transcripts from CRISPR loci remained unchanged. It is proposed that Cbp1 modulates production of longer pre-crRNA transcripts from CRISPR loci. A possible mechanism...

  13. Not so bad after all: retroviruses and long terminal repeat retrotransposons as a source of new genes in vertebrates.

    Science.gov (United States)

    Naville, M; Warren, I A; Haftek-Terreau, Z; Chalopin, D; Brunet, F; Levin, P; Galiana, D; Volff, J-N

    2016-04-01

    Viruses and transposable elements, once considered as purely junk and selfish sequences, have repeatedly been used as a source of novel protein-coding genes during the evolution of most eukaryotic lineages, a phenomenon called 'molecular domestication'. This is exemplified perfectly in mammals and other vertebrates, where many genes derived from long terminal repeat (LTR) retroelements (retroviruses and LTR retrotransposons) have been identified through comparative genomics and functional analyses. In particular, genes derived from gag structural protein and envelope (env) genes, as well as from the integrase-coding and protease-coding sequences, have been identified in humans and other vertebrates. Retroelement-derived genes are involved in many important biological processes including placenta formation, cognitive functions in the brain and immunity against retroelements, as well as in cell proliferation, apoptosis and cancer. These observations support an important role of retroelement-derived genes in the evolution and diversification of the vertebrate lineage.

  14. TRINS: a method for gene modification by randomized tandem repeat insertions.

    Science.gov (United States)

    Kipnis, Yakov; Dellus-Gur, Eynat; Tawfik, Dan S

    2012-09-01

    In nature, the evolution of new protein functions is driven not only by side-chain substitutions (point mutations), but also by backbone modifications (insertions and deletions). The current laboratory diversification methods, however, are largely limited to point mutations. Of particular interest are short insertions-by-duplication that are frequent in nature but cannot be introduced in vitro in a library format (i.e. in random locations and lengths). Here, we describe a new procedure that allows the generation of tandem repeats of random fragments of the target gene via rolling-circle amplification, and the concurrent incorporation of these repeats into the target gene. This procedure, dubbed tandem repeat insertion, or TRINS, results in a library of genes carrying insertions-by-duplication of variable lengths (3-150 bp) at random positions. This diversification pattern allows sampling of sequence space regions that are not readily accessible by other protocols. We demonstrate this method by constructing three different gene libraries, and by selecting insertion variants of TEM-1 β-lactamase.

  15. Methylation of C9orf72 expansion reduces RNA foci formation and dipeptide-repeat proteins expression in cells.

    Science.gov (United States)

    Bauer, Peter O

    2016-01-26

    A hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), together referred to as c9FTD/ALS. It has been suggested that a loss of C9orf72 protein expression, the formation of toxic RNA foci and dipeptide-repeat proteins contribute to C9orf72-related diseases. Interestingly, it has been shown that trimethylation of histones and methylation of CpG islands near the repeat expansion may play a role in the pathogenesis c9FTD/ALS. Recently, methylation of expanded repeat itself has been reported. To further elucidate the mechanisms underlying these diseases, the influence of epigenetic modification in the repeat expansion on its pathogenic effect was assessed. Here, a reduced formation of toxic RNA foci and dipeptide-repeat proteins upon methylation of the GGGGCC repeat in a cellular model of c9FTD/ALS is shown. Additionally, a novel methylcytosine-capture DNA hybridization immunoassay for semi-quantitative detection of the repeat methylation levels is presented, potentially usable for methylation analysis in patients carrying C9orf72 repeat expansion carriers as a diagnostic tool. Presented results suggest that increased level of pathogenic GGGGCC expansion methylation may be sufficient to alleviate the molecular pathology of the C9orf72-related diseases.

  16. The association of CGG repeats in the FMR1 gene and timing of natural menopause

    NARCIS (Netherlands)

    Voorhuis, M.; Onland-Moret, N. C.; Fauser, B. C. J. M.; van Amstel, H. K. Ploos; van der Schouw, Y. T.; Broekmans, F. J.

    2013-01-01

    Is there an association between the number of CGG repeats in the FMR1 gene in the normal and intermediate range and age at natural menopause? The number of CGG repeats in the normal and intermediate range in the FMR1 gene was not associated with age at natural menopause. Excessive triple CGG repeats

  17. (TG/CAn repeats in human gene families: abundance and selective patterns of distribution according to function and gene length

    Directory of Open Access Journals (Sweden)

    Ramachandran Srinivasan

    2005-06-01

    Full Text Available Abstract Background Creation of human gene families was facilitated significantly by gene duplication and diversification. The (TG/CAn repeats exhibit length variability, display genome-wide distribution, and are abundant in the human genome. Accumulation of evidences for their multiple functional roles including regulation of transcription and stimulation of recombination and splicing elect them as functional elements. Here, we report analysis of the distribution of (TG/CAn repeats in human gene families. Results The 1,317 human gene families were classified into six functional classes. Distribution of (TG/CAn repeats were analyzed both from a global perspective and from a stratified perspective based on their biological properties. The number of genes with repeats decreased with increasing repeat length and several genes (53% had repeats of multiple types in various combinations. Repeats were positively associated with the class of Signaling and communication whereas, they were negatively associated with the classes of Immune and related functions and of Information. The proportion of genes with (TG/CAn repeats in each class was proportional to the corresponding average gene length. The repeat distribution pattern in large gene families generally mirrored the global distribution pattern but differed particularly for Collagen gene family, which was rich in repeats. The position and flanking sequences of the repeats of Collagen genes showed high conservation in the Chimpanzee genome. However the majority of these repeats displayed length polymorphism. Conclusion Positive association of repeats with genes of Signaling and communication points to their role in modulation of transcription. Negative association of repeats in genes of Information relates to the smaller gene length, higher expression and fundamental role in cellular physiology. In genes of Immune and related functions negative association of repeats perhaps relates to the smaller gene

  18. DRPLA transgenic mouse substrains carrying single copy of full-length mutant human DRPLA gene with variable sizes of expanded CAG repeats exhibit CAG repeat length- and age-dependent changes in behavioral abnormalities and gene expression profiles.

    Science.gov (United States)

    Suzuki, Kazushi; Zhou, Jiayi; Sato, Toshiya; Takao, Keizo; Miyagawa, Tsuyoshi; Oyake, Mutsuo; Yamada, Mitunori; Takahashi, Hitoshi; Takahashi, Yuji; Goto, Jun; Tsuji, Shoji

    2012-05-01

    Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant progressive neurodegenerative disorder with intellectual deterioration and various motor deficits including ataxia, choreoathetosis, and myoclonus, caused by an abnormal expansion of CAG repeats in the DRPLA gene. Longer expanded CAG repeats contribute to an earlier age of onset, faster progression, and more severe neurological symptoms in DRPLA patients. In this study, we have established DRPLA transgenic mouse lines (sublines) harboring a single copy of the full-length mutant human DRPLA gene carrying various lengths of expanded CAG repeats (Q76, Q96, Q113, and Q129), which have clearly shown motor deficits and memory disturbance whose severity increases with the length of expanded CAG repeats and age, and successfully replicated the CAG repeat length- and age-dependent features of DRPLA patients. Neuronal intranuclear accumulation of the mutant DRPLA protein has been suggested to cause transcriptional dysregulation, leading to alteration in gene expression and neuronal dysfunction. In this study, we have conducted a comprehensive analysis of gene expression profiles in the cerebrum and cerebellum of transgenic mouse lines at 4, 8, and 12 weeks using multiple microarray platforms, and demonstrated that both the number and expression levels of the altered genes are highly dependent on CAG repeat length and age in both brain regions. Specific groups of genes and their function categories were identified by further agglomerative cluster analysis and gene functional annotation analysis. Calcium signaling and neuropeptide signaling, among others, were implicated in the pathophysiology of DRPLA. Our study provides unprecedented CAG-repeat-length-dependent mouse models of DRPLA, which are highly valuable not only for elucidating the CAG-repeat-length-dependent pathophysiology of DRPLA but also for developing therapeutic strategies for DRPLA.

  19. RNA binding proteins hnRNP A2/B1 and CUGBP1 suppress Fragile X CGG premutation repeat-induced neurodegeneration in a Drosophila model of FXTAS

    OpenAIRE

    Sofola, Oyinkan A.; Jin, Peng; QIN, YUNLONG; Duan, Ranhui; LIU, Huijie; de Haro, Maria; Nelson,David L.; Botas, Juan

    2007-01-01

    Fragile X associated tremor ataxia syndrome (FXTAS) is a recently described neurodegenerative disorder of older adult carriers of premutation alleles (60-200 CGG repeats) in the fragile-X mental retardation gene (FMR1). It has been proposed that FXTAS is an RNA mediated neurodegenerative disease caused by the titration of RNA binding proteins by the CGG repeats. To test this hypothesis, we utilize a transgenic Drosophila model of FXTAS that expresses premutation length repeat (90 CGG repeats)...

  20. Expansion of tandem repeats in sea anemone Nematostella vectensis proteome: A source for gene novelty?

    Directory of Open Access Journals (Sweden)

    Linial Michal

    2009-12-01

    Full Text Available Abstract Background The complete proteome of the starlet sea anemone, Nematostella vectensis, provides insights into gene invention dating back to the Cnidarian-Bilaterian ancestor. With the addition of the complete proteomes of Hydra magnipapillata and Monosiga brevicollis, the investigation of proteins having unique features in early metazoan life has become practical. We focused on the properties and the evolutionary trends of tandem repeat (TR sequences in Cnidaria proteomes. Results We found that 11-16% of N. vectensis proteins contain tandem repeats. Most TRs cover 150 amino acid segments that are comprised of basic units of 5-20 amino acids. In total, the N. Vectensis proteome has about 3300 unique TR-units, but only a small fraction of them are shared with H. magnipapillata, M. brevicollis, or mammalian proteomes. The overall abundance of these TRs stands out relative to that of 14 proteomes representing the diversity among eukaryotes and within the metazoan world. TR-units are characterized by a unique composition of amino acids, with cysteine and histidine being over-represented. Structurally, most TR-segments are associated with coiled and disordered regions. Interestingly, 80% of the TR-segments can be read in more than one open reading frame. For over 100 of them, translation of the alternative frames would result in long proteins. Most domain families that are characterized as repeats in eukaryotes are found in the TR-proteomes from Nematostella and Hydra. Conclusions While most TR-proteins have originated from prediction tools and are still awaiting experimental validations, supportive evidence exists for hundreds of TR-units in Nematostella. The existence of TR-proteins in early metazoan life may have served as a robust mode for novel genes with previously overlooked structural and functional characteristics.

  1. Ankyrin-repeat proteins from sponge symbionts modulate amoebal phagocytosis.

    Science.gov (United States)

    Nguyen, Mary T H D; Liu, Michael; Thomas, Torsten

    2014-03-01

    Bacteria-eukaryote symbiosis occurs in all stages of evolution, from simple amoebae to mammals, and from facultative to obligate associations. Sponges are ancient metazoans that form intimate symbiotic interactions with complex communities of bacteria. The basic nutritional requirements of the sponge are in part satisfied by the phagocytosis of bacterial food particles from the surrounding water. How bacterial symbionts, which are permanently associated with the sponge, survive in the presence of phagocytic cells is largely unknown. Here, we present the discovery of a genomic fragment from an uncultured gamma-proteobacterial sponge symbiont that encodes for four proteins, whose closest known relatives are found in a sponge genome. Through recombinant approaches, we show that these four eukaryotic-like, ankyrin-repeat proteins (ARP) when expressed in Eschericha coli can modulate phagocytosis of amoebal cells and lead to accumulation of bacteria in the phagosome. Mechanistically, two ARPs appear to interfere with phagosome development in a similar way to reduced vacuole acidification, by blocking the fusion of the early phagosome with the lysosome and its digestive enzymes. Our results show that ARP from sponge symbionts can function to interfere with phagocytosis, and we postulate that this might be one mechanism by which symbionts can escape digestion in a sponge host.

  2. Bovine proteins containing poly-glutamine repeats are often polymorphic and enriched for components of transcriptional regulatory complexes

    Directory of Open Access Journals (Sweden)

    Barendse William

    2010-11-01

    Full Text Available Abstract Background About forty human diseases are caused by repeat instability mutations. A distinct subset of these diseases is the result of extreme expansions of polymorphic trinucleotide repeats; typically CAG repeats encoding poly-glutamine (poly-Q tracts in proteins. Polymorphic repeat length variation is also apparent in human poly-Q encoding genes from normal individuals. As these coding sequence repeats are subject to selection in mammals, it has been suggested that normal variations in some of these typically highly conserved genes are implicated in morphological differences between species and phenotypic variations within species. At present, poly-Q encoding genes in non-human mammalian species are poorly documented, as are their functions and propensities for polymorphic variation. Results The current investigation identified 178 bovine poly-Q encoding genes (Q ≥ 5 and within this group, 26 genes with orthologs in both human and mouse that did not contain poly-Q repeats. The bovine poly-Q encoding genes typically had ubiquitous expression patterns although there was bias towards expression in epithelia, brain and testes. They were also characterised by unusually large sizes. Analysis of gene ontology terms revealed that the encoded proteins were strongly enriched for functions associated with transcriptional regulation and many contributed to physical interaction networks in the nucleus where they presumably act cooperatively in transcriptional regulatory complexes. In addition, the coding sequence CAG repeats in some bovine genes impacted mRNA splicing thereby generating unusual transcriptional diversity, which in at least one instance was tissue-specific. The poly-Q encoding genes were prioritised using multiple criteria for their likelihood of being polymorphic and then the highest ranking group was experimentally tested for polymorphic variation within a cattle diversity panel. Extensive and meiotically stable variation was

  3. Bovine proteins containing poly-glutamine repeats are often polymorphic and enriched for components of transcriptional regulatory complexes

    LENUS (Irish Health Repository)

    Whan, Vicki

    2010-11-23

    Abstract Background About forty human diseases are caused by repeat instability mutations. A distinct subset of these diseases is the result of extreme expansions of polymorphic trinucleotide repeats; typically CAG repeats encoding poly-glutamine (poly-Q) tracts in proteins. Polymorphic repeat length variation is also apparent in human poly-Q encoding genes from normal individuals. As these coding sequence repeats are subject to selection in mammals, it has been suggested that normal variations in some of these typically highly conserved genes are implicated in morphological differences between species and phenotypic variations within species. At present, poly-Q encoding genes in non-human mammalian species are poorly documented, as are their functions and propensities for polymorphic variation. Results The current investigation identified 178 bovine poly-Q encoding genes (Q ≥ 5) and within this group, 26 genes with orthologs in both human and mouse that did not contain poly-Q repeats. The bovine poly-Q encoding genes typically had ubiquitous expression patterns although there was bias towards expression in epithelia, brain and testes. They were also characterised by unusually large sizes. Analysis of gene ontology terms revealed that the encoded proteins were strongly enriched for functions associated with transcriptional regulation and many contributed to physical interaction networks in the nucleus where they presumably act cooperatively in transcriptional regulatory complexes. In addition, the coding sequence CAG repeats in some bovine genes impacted mRNA splicing thereby generating unusual transcriptional diversity, which in at least one instance was tissue-specific. The poly-Q encoding genes were prioritised using multiple criteria for their likelihood of being polymorphic and then the highest ranking group was experimentally tested for polymorphic variation within a cattle diversity panel. Extensive and meiotically stable variation was identified

  4. Mutagenic roles of DNA "repair" proteins in antibody diversity and disease-associated trinucleotide repeat instability.

    Science.gov (United States)

    Slean, Meghan M; Panigrahi, Gagan B; Ranum, Laura P; Pearson, Christopher E

    2008-07-01

    While DNA repair proteins are generally thought to maintain the integrity of the whole genome by correctly repairing mutagenic DNA intermediates, there are cases where DNA "repair" proteins are involved in causing mutations instead. For instance, somatic hypermutation (SHM) and class switch recombination (CSR) require the contribution of various DNA repair proteins, including UNG, MSH2 and MSH6 to mutate certain regions of immunoglobulin genes in order to generate antibodies of increased antigen affinity and altered effector functions. Another instance where "repair" proteins drive mutations is the instability of gene-specific trinucleotide repeats (TNR), the causative mutations of numerous diseases including Fragile X mental retardation syndrome (FRAXA), Huntington's disease (HD), myotonic dystrophy (DM1) and several spinocerebellar ataxias (SCAs) all of which arise via various modes of pathogenesis. These healthy and deleterious mutations that are induced by repair proteins are distinct from the genome-wide mutations that arise in the absence of repair proteins: they occur at specific loci, are sensitive to cis-elements (sequence context and/or epigenetic marks) and transcription, occur in specific tissues during distinct developmental windows, and are age-dependent. Here we review and compare the mutagenic role of DNA "repair" proteins in the processes of SHM, CSR and TNR instability.

  5. Characterization of tetratricopeptide repeat-containing proteins critical for cilia formation and function.

    Directory of Open Access Journals (Sweden)

    Yanan Xu

    Full Text Available Cilia formation and function require a special set of trafficking machinery termed intraflagellar transport (IFT, consisting mainly of protein complexes IFT-A, IFT-B, BBSome, and microtubule-dependent molecular motors. Tetratricopeptide repeat-containing (TTC proteins are widely involved in protein complex formation. Nine of them are known to serve as components of the IFT or BBSome complexes. How many TTC proteins are cilia-related and how they function, however, remain unclear. Here we show that twenty TTC genes were upregulated by at least 2-fold during the differentiation of cultured mouse tracheal epithelial cells (MTECs into multiciliated cells. Our systematic screen in zebrafish identified four novel TTC genes, ttc4, -9c, -36, and -39c, that are critical for cilia formation and motility. Accordingly, their zebrafish morphants displayed typical ciliopathy-related phenotypes, including curved body, abnormal otolith, hydrocephalus, and defective left-right patterning. The morphants of ttc4 and ttc25, a known cilia-related gene, additionally showed pronephric cyst formation. Immunoprecipitation indicated associations of TTC4, -9c, -25, -36, and -39c with components or entire complexes of IFT-A, IFT-B, or BBSome, implying their participations in IFT or IFT-related activities. Our results provide a global view for the relationship between TTC proteins and cilia.

  6. An update on polygalacturonase-inhibiting protein (PGIP), a leucine-rich repeat protein that protects crop plants against pathogens.

    Science.gov (United States)

    Kalunke, Raviraj M; Tundo, Silvio; Benedetti, Manuel; Cervone, Felice; De Lorenzo, Giulia; D'Ovidio, Renato

    2015-01-01

    Polygalacturonase inhibiting proteins (PGIPs) are cell wall proteins that inhibit the pectin-depolymerizing activity of polygalacturonases secreted by microbial pathogens and insects. These ubiquitous inhibitors have a leucine-rich repeat structure that is strongly conserved in monocot and dicot plants. Previous reviews have summarized the importance of PGIP in plant defense and the structural basis of PG-PGIP interaction; here we update the current knowledge about PGIPs with the recent findings on the composition and evolution of pgip gene families, with a special emphasis on legume and cereal crops. We also update the information about the inhibition properties of single pgip gene products against microbial PGs and the results, including field tests, showing the capacity of PGIP to protect crop plants against fungal, oomycetes and bacterial pathogens.

  7. TPRpred: a tool for prediction of TPR-, PPR- and SEL1-like repeats from protein sequences

    Directory of Open Access Journals (Sweden)

    Söding Johannes

    2007-01-01

    Full Text Available Abstract Background Solenoid repeat proteins of the Tetratrico Peptide Repeat (TPR family are involved as scaffolds in a broad range of protein-protein interactions. Several resources are available for the prediction of TPRs, however, they often fail to detect divergent repeat units. Results We have developed TPRpred, a profile-based method which uses a P-value-dependent score offset to include divergent repeat units and which exploits the tendency of repeats to occur in tandem. TPRpred detects not only TPR-like repeats, but also the related Pentatrico Peptide Repeats (PPRs and SEL1-like repeats. The corresponding profiles were generated through iterative searches, by varying the threshold parameters for inclusion of repeat units into the profiles, and the best profiles were selected based on their performance on proteins of known structure. We benchmarked the performance of TPRpred in detecting TPR-containing proteins and in delineating the individual repeats therein, against currently available resources. Conclusion TPRpred performs significantly better in detecting divergent repeats in TPR-containing proteins, and finds more individual repeats than the existing methods. The web server is available at http://tprpred.tuebingen.mpg.de, and the C++ and Perl sources of TPRpred along with the profiles can be downloaded from ftp://ftp.tuebingen.mpg.de/ebio/protevo/TPRpred/.

  8. A small multifunctional pentatricopeptide repeat protein in the chloroplast of Chlamydomonas reinhardtii.

    Science.gov (United States)

    Jalal, Abdullah; Schwarz, Christian; Schmitz-Linneweber, Christian; Vallon, Olivier; Nickelsen, Jörg; Bohne, Alexandra-Viola

    2015-03-01

    Organellar biogenesis is mainly regulated by nucleus-encoded factors, which act on various steps of gene expression including RNA editing, processing, splicing, stabilization, and translation initiation. Among these regulatory factors, pentatricopeptide repeat (PPR) proteins form the largest family of RNA binding proteins, with hundreds of members in flowering plants. In striking contrast, the genome of the unicellular green alga Chlamydomonas reinhardtii encodes only 14 such proteins. In this study, we analyzed PPR7, the smallest and most highly expressed PPR protein in C. reinhardtii. Green fluorescent protein-based localization and gel-filtration analysis revealed that PPR7 forms a part of a high-molecular-weight ribonucleoprotein complex in the chloroplast stroma. RIP-chip analysis of PPR7-bound RNAs demonstrated that the protein associates with a diverse set of chloroplast transcripts in vivo, i.e. rrnS, psbH, rpoC2, rbcL, atpA, cemA-atpH, tscA, and atpI-psaJ. Furthermore, the investigation of PPR7 RNAi strains revealed that depletion of PPR7 results in a light-sensitive phenotype, accompanied by altered levels of its target RNAs that are compatible with the defects in their maturation or stabilization. PPR7 is thus an unusual type of small multifunctional PPR protein, which interacts, probably in conjunction with other RNA binding proteins, with numerous target RNAs to promote a variety of post-transcriptional events.

  9. CAG Repeat Number in the Androgen Receptor Gene and Prostate Cancer.

    Science.gov (United States)

    Madjunkova, S; Eftimov, A; Georgiev, V; Petrovski, D; Dimovski, Aj; Plaseska-Karanfilska, D

    2012-06-01

    Prostate cancer (PC) is the second leading cause of cancer deaths in men. The effects of androgens on prostatic tissue are mediated by the androgen receptor (AR) gene. The 5' end of exon 1 of the AR gene includes a polymorphic CAG triplet repeat that numbers between 10 to 36 in the normal population. The length of the CAG repeats is inversely related to the transactivation function of the AR gene. There is controversy over association between short CAG repeat numbers in the AR gene and PC. This retrospective case-control study evaluates the possible effect of short CAG repeats on the AR gene in prostate cancer risk in Macedonian males. A total of 392 male subjects, 134 PC patients, 106 patients with benign prostatic hyperplasia (BPH) and 152 males from the general Macedonian population were enrolled in this study. The CAG repeat length was determined by fluorescent polymerase chain reaction (PCR) amplification of exon1 of the AR gene followed by capillary electrophoresis (CE) on a genetic analyzer. The mean repeat length in PC patients was 21.5 ± 2.65, in controls 22.28 ± 2.86 (p = 0.009) and in BPH patients 22.1 ± 2.52 (p = 0.038). Short CAG repeats (CAG repeat (CAG repeat length. These results suggest that reduced CAG repeat length may be associated with increased prostate cancer risk in Macedonian men.

  10. Androgen insensitivity syndrome: do trinucleotide repeats in androgen receptor gene have any role?

    Institute of Scientific and Technical Information of China (English)

    Singh Rajender; Nalini J. Gupta; Baidyanath Chakravarty; Lalji Singh; Kumarasamy Thangaraj

    2008-01-01

    Aim: To investigate the role of CAG and GGN repeats as genetic background affecting androgen insensitivity syn- drome (AIS) phenotype. Methods: We analyzed lengths of androgen receptor (AR)-CAG and GGN repeats in 69 AIS cases, along with 136 unrelated normal male individuals. The lengths of repeats were analyzed using polymerase chain reaction (PCR) amplification followed by allelic genotyping to determine allele length. Results: Our study revealed significantly shorter mean lengths of CAG repeats in patients (mean 18.25 repeats, range 14-26 repeats) in comparison to the controls (mean 22.57 repeats, range 12-39 repeats) (two-tailed P < 0.0001). GGN repeats, however, did not differ significantly between patients (mean 21.48 repeats) and controls (mean 21.21 repeats) (two- tailed P = 0.474). Among patients' groups, the mean number of CAG repeats in partial androgen insensitivity cases (mean 15.83 repeats) was significantly less than in complete androgen insensitivity cases (mean 19.46 repeats) (two- tailed P < 0.0001). Conclusion: The findings suggest that shorter lengths of repeats in the AR gene might act as low penetrance genetic background in varying manifestation of androgen insensitivity. (Asian J Androl 2008 Jul; 10: 616-624)

  11. Effects of CAG repeat length, HTT protein length and protein context on cerebral metabolism measured using magnetic resonance spectroscopy in transgenic mouse models of Huntington's disease.

    Science.gov (United States)

    Jenkins, Bruce G; Andreassen, Ole A; Dedeoglu, Alpaslan; Leavitt, Blair; Hayden, Michael; Borchelt, David; Ross, Christopher A; Ferrante, Robert J; Beal, M Flint

    2005-10-01

    Huntington's disease is a neurodegenerative illness caused by expansion of CAG repeats at the N-terminal end of the protein huntingtin. We examined longitudinal changes in brain metabolite levels using in vivo magnetic resonance spectroscopy in five different mouse models. There was a large (>50%) exponential decrease in N-acetyl aspartate (NAA) with time in both striatum and cortex in mice with 150 CAG repeats (R6/2 strain). There was a linear decrease restricted to striatum in N171-82Q mice with 82 CAG repeats. Both the exponential and linear decreases of NAA were paralleled in time by decreases in neuronal area measured histologically. Yeast artificial chromosome transgenic mice with 72 CAG repeats, but low expression levels, had less striatal NAA loss than the N171-82Q mice (15% vs. 43%). We evaluated the effect of gene context in mice with an approximate 146 CAG repeat on the hypoxanthine phosphoribosyltransferase gene (HPRT). HPRT mice developed an obese phenotype in contrast to weight loss in the R6/2 and N171-82Q mice. These mice showed a small striatal NAA loss (21%), and a possible increase in brain lipids detectable by magnetic resonance (MR) spectroscopy and decreased brain water T1. Our results indicate profound metabolic defects that are strongly affected by CAG repeat length, as well as gene expression levels and protein context.

  12. RAP, the Sole Octotricopeptide Repeat Protein in Arabidopsis, Is Required for Chloroplast 16S rRNA Maturation[W

    Science.gov (United States)

    Kleinknecht, Laura; Wang, Fei; Stübe, Roland; Philippar, Katrin; Nickelsen, Jörg; Bohne, Alexandra-Viola

    2014-01-01

    The biogenesis and activity of chloroplasts in both vascular plants and algae depends on an intracellular network of nucleus-encoded, trans-acting factors that control almost all aspects of organellar gene expression. Most of these regulatory factors belong to the helical repeat protein superfamily, which includes tetratricopeptide repeat, pentatricopeptide repeat, and the recently identified octotricopeptide repeat (OPR) proteins. Whereas green algae express many different OPR proteins, only a single orthologous OPR protein is encoded in the genomes of most land plants. Here, we report the characterization of the only OPR protein in Arabidopsis thaliana, RAP, which has previously been implicated in plant pathogen defense. Loss of RAP led to a severe defect in processing of chloroplast 16S rRNA resulting in impaired chloroplast translation and photosynthesis. In vitro RNA binding and RNase protection assays revealed that RAP has an intrinsic and specific RNA binding capacity, and the RAP binding site was mapped to the 5′ region of the 16S rRNA precursor. Nucleoid localization of RAP was shown by transient green fluorescent protein import assays, implicating the nucleoid as the site of chloroplast rRNA processing. Taken together, our data indicate that the single OPR protein in Arabidopsis is important for a basic process of chloroplast biogenesis. PMID:24585838

  13. RAP, the sole octotricopeptide repeat protein in Arabidopsis, is required for chloroplast 16S rRNA maturation.

    Science.gov (United States)

    Kleinknecht, Laura; Wang, Fei; Stübe, Roland; Philippar, Katrin; Nickelsen, Jörg; Bohne, Alexandra-Viola

    2014-02-01

    The biogenesis and activity of chloroplasts in both vascular plants and algae depends on an intracellular network of nucleus-encoded, trans-acting factors that control almost all aspects of organellar gene expression. Most of these regulatory factors belong to the helical repeat protein superfamily, which includes tetratricopeptide repeat, pentatricopeptide repeat, and the recently identified octotricopeptide repeat (OPR) proteins. Whereas green algae express many different OPR proteins, only a single orthologous OPR protein is encoded in the genomes of most land plants. Here, we report the characterization of the only OPR protein in Arabidopsis thaliana, RAP, which has previously been implicated in plant pathogen defense. Loss of RAP led to a severe defect in processing of chloroplast 16S rRNA resulting in impaired chloroplast translation and photosynthesis. In vitro RNA binding and RNase protection assays revealed that RAP has an intrinsic and specific RNA binding capacity, and the RAP binding site was mapped to the 5' region of the 16S rRNA precursor. Nucleoid localization of RAP was shown by transient green fluorescent protein import assays, implicating the nucleoid as the site of chloroplast rRNA processing. Taken together, our data indicate that the single OPR protein in Arabidopsis is important for a basic process of chloroplast biogenesis.

  14. Rational design of alpha-helical tandem repeat proteins with closed architectures

    Science.gov (United States)

    Doyle, Lindsey; Hallinan, Jazmine; Bolduc, Jill; Parmeggiani, Fabio; Baker, David; Stoddard, Barry L.; Bradley, Philip

    2015-01-01

    Tandem repeat proteins, which are formed by repetition of modular units of protein sequence and structure, play important biological roles as macromolecular binding and scaffolding domains, enzymes, and building blocks for the assembly of fibrous materials1,2. The modular nature of repeat proteins enables the rapid construction and diversification of extended binding surfaces by duplication and recombination of simple building blocks3,4. The overall architecture of tandem repeat protein structures – which is dictated by the internal geometry and local packing of the repeat building blocks – is highly diverse, ranging from extended, super-helical folds that bind peptide, DNA, and RNA partners5–9, to closed and compact conformations with internal cavities suitable for small molecule binding and catalysis10. Here we report the development and validation of computational methods for de novo design of tandem repeat protein architectures driven purely by geometric criteria defining the inter-repeat geometry, without reference to the sequences and structures of existing repeat protein families. We have applied these methods to design a series of closed alpha-solenoid11 repeat structures (alpha-toroids) in which the inter-repeat packing geometry is constrained so as to juxtapose the N- and C-termini; several of these designed structures have been validated by X-ray crystallography. Unlike previous approaches to tandem repeat protein engineering12–20, our design procedure does not rely on template sequence or structural information taken from natural repeat proteins and hence can produce structures unlike those seen in nature. As an example, we have successfully designed and validated closed alpha-solenoid repeats with a left-handed helical architecture that – to our knowledge – is not yet present in the protein structure database21. PMID:26675735

  15. Rational design of α-helical tandem repeat proteins with closed architectures.

    Science.gov (United States)

    Doyle, Lindsey; Hallinan, Jazmine; Bolduc, Jill; Parmeggiani, Fabio; Baker, David; Stoddard, Barry L; Bradley, Philip

    2015-12-24

    Tandem repeat proteins, which are formed by repetition of modular units of protein sequence and structure, play important biological roles as macromolecular binding and scaffolding domains, enzymes, and building blocks for the assembly of fibrous materials. The modular nature of repeat proteins enables the rapid construction and diversification of extended binding surfaces by duplication and recombination of simple building blocks. The overall architecture of tandem repeat protein structures--which is dictated by the internal geometry and local packing of the repeat building blocks--is highly diverse, ranging from extended, super-helical folds that bind peptide, DNA, and RNA partners, to closed and compact conformations with internal cavities suitable for small molecule binding and catalysis. Here we report the development and validation of computational methods for de novo design of tandem repeat protein architectures driven purely by geometric criteria defining the inter-repeat geometry, without reference to the sequences and structures of existing repeat protein families. We have applied these methods to design a series of closed α-solenoid repeat structures (α-toroids) in which the inter-repeat packing geometry is constrained so as to juxtapose the amino (N) and carboxy (C) termini; several of these designed structures have been validated by X-ray crystallography. Unlike previous approaches to tandem repeat protein engineering, our design procedure does not rely on template sequence or structural information taken from natural repeat proteins and hence can produce structures unlike those seen in nature. As an example, we have successfully designed and validated closed α-solenoid repeats with a left-handed helical architecture that--to our knowledge--is not yet present in the protein structure database.

  16. CAG Repeat Number in the Androgen Receptor Gene and Prostate Cancer

    OpenAIRE

    Madjunkova, S.; Eftimov, A.; Georgiev, V.; Petrovski, D; Dimovski, AJ; Plaseska-Karanfilska, D

    2012-01-01

    Prostate cancer (PC) is the second leading cause of cancer deaths in men. The effects of androgens on prostatic tissue are mediated by the androgen receptor (AR) gene. The 5′ end of exon 1 of the AR gene includes a polymorphic CAG triplet repeat that numbers between 10 to 36 in the normal population. The length of the CAG repeats is inversely related to the transactivation function of the AR gene. There is controversy over association between short CAG repeat numbers in the AR gene and PC. Th...

  17. SdrI, a serine-aspartate repeat protein identified in Staphylococcus saprophyticus strain 7108, is a collagen-binding protein.

    Science.gov (United States)

    Sakinc, Türkan; Kleine, Britta; Gatermann, Sören G

    2006-08-01

    A gene encoding a serine-aspartate repeat protein of Staphylococcus saprophyticus, an important cause of urinary tract infections in young women, has been cloned and sequenced. In contrast to other SD repeat proteins, SdrI carries 21 additional N-terminal repeats with a consensus sequence of (P/A)ATKE(K/E)A(A/V)(T/I)(A/T/S)EE and has the longest SD(AD)(1-5) repetitive region (854 amino acids) described so far. This highly repetitive sequence contains only the amino acids serine, asparagine, and a distinctly greater amount of alanine (37%) than all other known SD repeat proteins (2.3 to 4.4%). In addition, it is a collagen-binding protein of S. saprophyticus and the second example in this organism of a surface protein carrying the LPXTG motif. We constructed an isogenic sdrI knockout mutant that showed decreased binding to immobilized collagen compared with wild-type S. saprophyticus strain 7108. Binding could be reconstituted by complementation. Collagen binding is specifically caused by SdrI, and the recently described UafA protein, the only LPXTG-containing protein in the genome sequence of the type strain, is not involved in this trait. Our experiments suggest that, as in other staphylococci, the presence of different LPXTG-anchored cell wall proteins is common in S. saprophyticus and support the notion that the presence of matrix-binding surface proteins is common in staphylococci.

  18. Reduced CAG repeats length in androgen receptor gene is associated with violent criminal behavior.

    Science.gov (United States)

    Rajender, Singh; Pandu, Guguluth; Sharma, J D; Gandhi, K P C; Singh, Lalji; Thangaraj, Kumarasamy

    2008-09-01

    Androgens mediate their functions through androgen receptors (AR). The two triplet repeats in the AR gene (CAG and GGN) are highly polymorphic among various populations and have been extensively studied in diverse clinical conditions and antisocial personality disorders. Several studies have reported either higher levels of testosterone among rapists or the correlation of shorter CAG repeats with criminal activities. However, to date, no study has analyzed AR gene in rapists worldwide, and no study has been conducted on criminals from Indian subcontinent. Therefore, we have analyzed the AR-CAG repeat length in 645 men, of which 241 were convicted for rape, 107 for murder, 26 for both murder and rape, and 271 were control males. The aim was to explore if there was any correlation between CAG repeat length and criminal behavior. The study revealed significantly shorter CAG repeats in the rapists (mean 18.44 repeats) and murderers (mean 17.59 repeats) compared to the control men (mean 21.19 repeats). The criminals who committed murder after rape had a far shorter mean repeat length (mean 17.31 repeats) in comparison to the controls or those convicted of rape or murder alone. In short, our study suggests that the reduced CAG repeats in the AR gene are associated with criminal behavior. This, along with other studies, would help in understanding the biological factors associated with the antisocial or criminal activities.

  19. Exaggerated phosphorylation of brain tau protein in CRH KO mice exposed to repeated immobilization stress.

    Science.gov (United States)

    Kvetnansky, Richard; Novak, Petr; Vargovic, Peter; Lejavova, Katarina; Horvathova, Lubica; Ondicova, Katarina; Manz, George; Filipcik, Peter; Novak, Michal; Mravec, Boris

    2016-07-01

    Neuroendocrine and behavioral stress responses are orchestrated by corticotropin-releasing hormone (CRH) and norepinephrine (NE) synthesizing neurons. Recent findings indicate that stress may promote development of neurofibrillary pathology in Alzheimer's disease. Therefore, we investigated relationships among stress, tau protein phosphorylation, and brain NE using wild-type (WT) and CRH-knockout (CRH KO) mice. We assessed expression of phosphorylated tau (p-tau) at the PHF-1 epitope and NE concentrations in the locus coeruleus (LC), A1/C1 and A2/C2 catecholaminergic cell groups, hippocampus, amygdala, nucleus basalis magnocellularis, and frontal cortex of unstressed, singly stressed or repeatedly stressed mice. Moreover, gene expression and protein levels of tyrosine hydroxylase (TH) and CRH receptor mRNA were determined in the LC. Plasma corticosterone levels were also measured. Exposure to a single stress increases tau phosphorylation throughout the brain in WT mice when compared to singly stressed CRH KO animals. In contrast, repeatedly stressed CRH KO mice showed exaggerated tau phosphorylation relative to WT controls. We also observed differences in extent of tau phosphorylation between investigated structures, e.g. the LC and hippocampus. Moreover, CRH deficiency leads to different responses to stress in gene expression of TH, NE concentrations, CRH receptor mRNA, and plasma corticosterone levels. Our data indicate that CRH effects on tau phosphorylation are dependent on whether stress is single or repeated, and differs between brain regions. Our findings indicate that CRH attenuates mechanisms responsible for development of stress-induced tau neuropathology, particularly in conditions of chronic stress. However, the involvement of central catecholaminergic neurons in these mechanisms remains unclear and is in need of further investigation.

  20. RRW: repeated random walks on genome-scale protein networks for local cluster discovery

    Directory of Open Access Journals (Sweden)

    Can Tolga

    2009-09-01

    Full Text Available Abstract Background We propose an efficient and biologically sensitive algorithm based on repeated random walks (RRW for discovering functional modules, e.g., complexes and pathways, within large-scale protein networks. Compared to existing cluster identification techniques, RRW implicitly makes use of network topology, edge weights, and long range interactions between proteins. Results We apply the proposed technique on a functional network of yeast genes and accurately identify statistically significant clusters of proteins. We validate the biological significance of the results using known complexes in the MIPS complex catalogue database and well-characterized biological processes. We find that 90% of the created clusters have the majority of their catalogued proteins belonging to the same MIPS complex, and about 80% have the majority of their proteins involved in the same biological process. We compare our method to various other clustering techniques, such as the Markov Clustering Algorithm (MCL, and find a significant improvement in the RRW clusters' precision and accuracy values. Conclusion RRW, which is a technique that exploits the topology of the network, is more precise and robust in finding local clusters. In addition, it has the added flexibility of being able to find multi-functional proteins by allowing overlapping clusters.

  1. Distribution of an Ankyrin-repeat Protein on the Endoplasmic Reticulum in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Liqin Wei; Yan Li

    2009-01-01

    There are many ankyrin-repeat proteins in plant cells. However, the distribution and function of these proteins are mostly unclear. By reverse transcription-polymerase chain reaction, a gene encoding an ankyrin-like protein was cloned from Arabidopsis and named AtANK1 (GenBank accession no. NM_120340). The 6-His-tagged AtAnk1-N fusion protein was affinity-purified and its rabbit polyclonal antibody was obtained. Immuno-blotting with the purified anti-AtAnk1-N polyclonal antibody revealed that the relative molecular weight of the AtANK1 protein was about 76 kDa. By immunofluorescence labeling and immuno-gold labeling with the purified anti-AtAnk1-N polyclonal antibody, coupled with confocal and transmission electron microscopy observation, AtANK1 was found to be distributed on the membrane of the endoplaamic reticulum in Arabidopsis cells. Based on these results, we suggested that AtANK1 might be involved in endoplasmic reticulum-related protein localization and sorting in plant cells.

  2. Imperfect DNA mirror repeats in E. coli TnsA and other protein-coding DNA.

    Science.gov (United States)

    Lang, Dorothy M

    2005-09-01

    DNA imperfect mirror repeats (DNA-IMRs) are ubiquitous in protein-coding DNA. However, they overlap and often have different centers of symmetry, making it difficult to evaluate their relationship to each other and to specific DNA and protein motifs and structures. This paper describes a systematic method of determining a hierarchy for DNA-IMRs and evaluates their relationship to protein structural elements (PSEs)--helices, turns and beta-sheets. DNA-IMRs are identifed by two different methods--DNA-IMRs terminated by reverse dinucleotides (rd-IMRs) and DNA-IMRs terminated by a single (mono) matching nucleotide (m-IMRs). Both rd-IMRs and m-IMRs are evaluated in 17 proteins, and illustrated in detail for TnsA. For each of the proteins, Fisher's exact test (FET) is used to measure the coincidence between the terminal dinucleotides of rd-IMRs and the terminal amino acids of individual PSEs. A significant correlation over a span of about 3 nt was found for each protein. The correlation is robust and for most genes, all rd-IMRs16 nt contain approximately 88% of the potential functional motifs. The protein translation of the longest rd- and m-IMRs span sequences important to the protein's structure and function. In all 17 proteins studied, the population of rd-IMRs is substantially less than the expected number and the population of m-IMRs greater than the expected number, indicating strong selective pressures. The association of rd-IMRs with PSEs restricts their spatial distribution, and therefore, their number. The greater than predicted number of m-IMRs indicates that DNA symmetry exists throughout the entire protein-coding region and may stabilize the sequence.

  3. Positive selection in the leucine-rich repeat domain of Gro1 genes in Solanum species

    Indian Academy of Sciences (India)

    Valentino Ruggieri; Angelina Nunziata; Amalia Barone

    2014-12-01

    In pathogen resistant plants, solvent-exposed residues in the leucine-rich repeat (LRR) proteins are thought to mediate resistance by recognizing plant pathogen elicitors. In potato, the gene Gro1-4 confers resistance to Globodera rostochiensis. The investigation of variablity in different copies of this gene represents a good model for the verification of positive selection mechanisms. Two datasets of Gro1 LRR sequences were constructed, one derived from the Gro1-4 gene, belonging to different cultivated and wild Solanum species, and the other belonging to paralogues of a resistant genotype. Analysis of non-synonymous to synonymous substitution rates $(K_{a}/K_{s})$ highlighted 14 and six amino acids with $K_{a}/K_{s} \\gt 1$ in orthologue and paralogue datasets, respectively. Selection analysis revealed that the leucine-rich regions accumulate variability in a very specific way, and we found that some combinations of amino acids in these sites might be involved in pathogen recognition. The results confirm previous studies on positive selection in the LRR domain of R protein in Arabidopsis and other model plants and extend these to wild Solanum species. Moreover, positively selected sites in the Gro1 LRR domain show that coevolution mainly occurred in two regions on the internal surface of the three-dimensional horseshoe structure of the domain, albeit with different evolutionary forces between paralogues and orthologues.

  4. Isolation and characterization of human brain genes with (CCA){sub n} trinucleotide repeats

    Energy Technology Data Exchange (ETDEWEB)

    Longshore, J.W.; Finley, W.H.; Descartes, M. [Univ. of Alabama, Birmingham, AL (United States)] [and others

    1994-09-01

    Expansion of trinucleotide repeats has been described as a new form of mutation. To date, only the expansion of (CGG){sub n} and (CAG){sub n} repeats have been associated with disease. Expansion of (CAG){sub n} repeats has been found to cause Huntington`s disease, Kennedy`s disease, myotonic dystrophy, spinocerebellar ataxia type 1, and dentatorubral pallidoluysian atrophy. (CGG){sub n} repeat expansion has been implicated in the fragile X syndrome and FRAXE mental retardation. In an effort to identify other potential repeats as candidates for expansion, a DNA linguistics approach was used to study 10 Mb of human DNA sequences in GenBank. Our study found the (CCA){sub n} repeat and the disease-associated (CGG){sub n} and (CAG){sub n} repeats to be over-represented in the human genome. The (CCA){sub n} repeat also shares other characteristics with (CGG){sub n} and (CAG){sub n}, making it a good candidate for expansion. Trinucleotide repeat numbers in disease-associated genes are normally polymorphic in a population. Therefore, by studying genes for polymorphisms, candidate genes may be identified. Twelve sequences previously deposited in GenBank with at least five tandem copies of (CCA) were studied and no polymorphisms were found. To identify other candidate genes, a human hippocampus cDNA library was screened with a (CCA){sub 8} probe. This approach identified 19 novel expressed sequences having long tandem (CCA){sub n} repeats which are currently under investigation for polymorphisms. Genes with polymorphic repeats may serve as markers for linkage studies or as candidate genes for genetic diseases showing anticipation.

  5. Tetratricopeptide repeat domain 9A is an interacting protein for tropomyosin Tm5NM-1

    Directory of Open Access Journals (Sweden)

    Ho Gay

    2008-08-01

    Full Text Available Abstract Background Tetratricopeptide repeat domain 9A (TTC9A protein is a recently identified protein which contains three tetratricopeptide repeats (TPRs on its C-terminus. In our previous studies, we have shown that TTC9A was a hormonally-regulated gene in breast cancer cells. In this study, we found that TTC9A was over-expressed in breast cancer tissues compared with the adjacent controls (P Methods Breast samples from 25 patients including the malignant breast tissues and the adjacent normal tissues were processed for Southern blot analysis. Yeast-two-hybrid assay, GST pull-down assay and co-immunoprecipitation were used to identify and verify the interaction between TTC9A and other proteins. Results Tropomyosin Tm5NM-1 was identified as one of the TTC9A partner proteins. The interaction between TTC9A and Tm5NM-1 was further confirmed by GST pull-down assay and co-immunoprecipitation in mammalian cells. TTC9A domains required for the interaction were also characterized in this study. The results suggested that the first TPR domain and the linker fragment between the first two TPR domains of TTC9A were important for the interaction with Tm5NM-1 and the second and the third TPR might play an inhibitory role. Conclusion Since the primary function of tropomyosin is to stabilize actin filament, its interaction with TTC9A may play a role in cell shape and motility. In our previous results, we have found that progesterone-induced TTC9A expression was associated with increased cell motility and cell spreading. We speculate that TTC9A acts as a chaperone protein to facilitate the function of tropomyosins in stabilizing microfilament and it may play a role in cancer cell invasion and metastasis.

  6. Toxoplasma gondii: a bradyzoite-specific DnaK-tetratricopeptide repeat (DnaK-TPR) protein interacts with p23 co-chaperone protein.

    Science.gov (United States)

    Ueno, Akio; Dautu, George; Haga, Kaori; Munyaka, Biscah; Carmen, Gabriella; Kobayashi, Yoshiyasu; Igarashi, Makoto

    2011-04-01

    The DnaK-tetratricopeptide repeat (DnaK-TPR) gene (ToxoDB ID, TGME49_002020) is expressed predominantly at the bradyzoite stage. DnaK-TPR protein has a heat shock protein (DnaK) and tetratricopeptide repeat (TPR) domains with amino acid sequence similarity to the counterparts of other organisms (40.2-43.7% to DnaK domain and 41.1-66.0% to TPR domain). These findings allowed us to infer that DnaK-TPR protein is important in the tachyzoite-to-bradyzoite development or maintenance of cyst structure although the function of this gene is still unknown. An immunofluorescence assay (IFA) revealed that DnaK-TPR protein was expressed in Toxoplasma gondii-encysted and in vitro-induced bradyzoites and distributed in the whole part of parasite cells. We conducted yeast two-hybrid screening to identify proteins interacting with DnaK-TPR protein, and demonstrated that DnaK-TPR protein interacts with p23 co-chaperone protein (Tgp23). It was expected that DnaK-TPR protein would have a function as a molecular chaperon in bradyzoite cells associated with Tgp23. Possible mechanisms for this gene are discussed.

  7. Stealing the spotlight: CUL4-DDB1 ubiquitin ligase docks WD40-repeat proteins to destroy

    Directory of Open Access Journals (Sweden)

    Zhang Hui

    2007-02-01

    Full Text Available Abstract Recent investigation of Cullin 4 (CUL4 has ushered this class of multiprotein ubiquitin E3 ligases to center stage as critical regulators of diverse processes including cell cycle regulation, developmental patterning, DNA replication, DNA damage and repair, and epigenetic control of gene expression. CUL4 associates with DNA Damage Binding protein 1 (DDB1 to assemble an ubiquitin E3 ligase that targets protein substrates for ubiquitin-dependent proteolysis. CUL4 ligase activity is also regulated by the covalent attachment of the ubiquitin-like protein NEDD8 to CUL4, or neddylation, and the COP9 signalosome complex (CSN that removes this important modification. Recently, multiple WD40-repeat proteins (WDR were found to interact with DDB1 and serve as the substrate-recognition subunits of the CUL4-DDB1 ubiquitin ligase. As more than 150–300 WDR proteins exist in the human genome, these findings impact a wide array of biological processes through CUL4 ligase-mediated proteolysis. Here, we review the recent progress in understanding the mechanism of CUL4 ubiquitin E3 ligase and discuss the architecture of CUL4-assembled E3 ubiquitin ligase complexes by comparison to CUL1-based E3s (SCF. Then, we will review several examples to highlight the critical roles of CUL4 ubiquitin ligase in genome stability, cell cycle regulation, and histone lysine methylation. Together, these studies provide insights into the mechanism of this novel ubiquitin ligase in the regulation of important biological processes.

  8. Roles of the amino terminal region and repeat region of the Plasmodium berghei circumsporozoite protein in parasite infectivity.

    Directory of Open Access Journals (Sweden)

    Cassandra Aldrich

    Full Text Available The circumsporozoite protein (CSP plays a key role in malaria sporozoite infection of both mosquito salivary glands and the vertebrate host. The conserved Regions I and II have been well studied but little is known about the immunogenic central repeat region and the N-terminal region of the protein. Rodent malaria Plasmodium berghei parasites, in which the endogenous CS gene has been replaced with the avian Plasmodium gallinaceum CS (PgCS sequence, develop normally in the A. stephensi mosquito midgut but the sporozoites are not infectious. We therefore generated P. berghei transgenic parasites carrying the PgCS gene, in which the repeat region was replaced with the homologous region of P. berghei CS (PbCS. A further line, in which both the N-terminal region and repeat region were replaced with the homologous regions of PbCS, was also generated. Introduction of the PbCS repeat region alone, into the PgCS gene, did not rescue sporozoite species-specific infectivity. However, the introduction of both the PbCS repeat region and the N-terminal region into the PgCS gene completely rescued infectivity, in both the mosquito vector and the mammalian host. Immunofluorescence experiments and western blot analysis revealed correct localization and proteolytic processing of CSP in the chimeric parasites. The results demonstrate, in vivo, that the repeat region of P. berghei CSP, alone, is unable to mediate sporozoite infectivity in either the mosquito or the mammalian host, but suggest an important role for the N-terminal region in sporozoite host cell invasion.

  9. A method for the incremental expansion of polyglutamine repeats in recombinant proteins.

    Science.gov (United States)

    Robertson, Amy L; Bottomley, Stephen P

    2013-01-01

    The polyglutamine diseases are caused by the expansion of CAG repeats. A key step in understanding the disease mechanisms, at the DNA and protein level, is the ability to produce recombinant proteins with specific length glutamine tracts which is a time-consuming first step in setting up in vitro systems to study the effects of polyglutamine expansion. Described here is a PCR-based method for the amplification of CAG repeats, which we used to incrementally extend CAG length by 3-5 repeats per cycle. This method could be translated into various contexts where amplification of repeating elements is necessary.

  10. Sequence-structure-function relations of the mosquito leucine-rich repeat immune proteins

    Directory of Open Access Journals (Sweden)

    Povelones Michael

    2010-09-01

    Full Text Available Abstract Background The discovery and characterisation of factors governing innate immune responses in insects has driven the elucidation of many immune system components in mammals and other organisms. Focusing on the immune system responses of the malaria mosquito, Anopheles gambiae, has uncovered an array of components and mechanisms involved in defence against pathogen infections. Two of these immune factors are LRIM1 and APL1C, which are leucine-rich repeat (LRR containing proteins that activate complement-like defence responses against malaria parasites. In addition to their LRR domains, these leucine-rich repeat immune (LRIM proteins share several structural features including signal peptides, patterns of cysteine residues, and coiled-coil domains. Results The identification and characterisation of genes related to LRIM1 and APL1C revealed putatively novel innate immune factors and furthered the understanding of their likely molecular functions. Genomic scans using the shared features of LRIM1 and APL1C identified more than 20 LRIM-like genes exhibiting all or most of their sequence features in each of three disease-vector mosquitoes with sequenced genomes: An. gambiae, Aedes aegypti, and Culex quinquefasciatus. Comparative sequence analyses revealed that this family of mosquito LRIM-like genes is characterised by a variable number of 6 to 14 LRRs of different lengths. The "Long" LRIM subfamily, with 10 or more LRRs, and the "Short" LRIMs, with 6 or 7 LRRs, also share the signal peptide, cysteine residue patterning, and coiled-coil sequence features of LRIM1 and APL1C. The "TM" LRIMs have a predicted C-terminal transmembrane region, and the "Coil-less" LRIMs exhibit the characteristic LRIM sequence signatures but lack the C-terminal coiled-coil domains. Conclusions The evolutionary plasticity of the LRIM LRR domains may provide templates for diverse recognition properties, while their coiled-coil domains could be involved in the formation

  11. Differential methylation of genes and repeats in land plants.

    Science.gov (United States)

    Rabinowicz, Pablo D; Citek, Robert; Budiman, Muhammad A; Nunberg, Andrew; Bedell, Joseph A; Lakey, Nathan; O'Shaughnessy, Andrew L; Nascimento, Lidia U; McCombie, W Richard; Martienssen, Robert A

    2005-10-01

    The hypomethylated fraction of plant genomes is usually enriched in genes and can be selectively cloned using methylation filtration (MF). Therefore, MF has been used as a gene enrichment technology in sorghum and maize, where gene enrichment was proportional to genome size. Here we apply MF to a broad variety of plant species spanning a wide range of genome sizes. Differential methylation of genic and non-genic sequences was observed in all species tested, from non-vascular to vascular plants, but in some cases, such as wheat and pine, a lower than expected level of enrichment was observed. Remarkably, hexaploid wheat and pine show a dramatically large number of gene-like sequences relative to other plants. In hexaploid wheat, this apparent excess of genes may reflect an abundance of methylated pseudogenes, which may thus be more prevalent in recent polyploids.

  12. Absence of association between a polymorphic GGC repeat in the 5' untranslated region of the reelin gene and autism

    OpenAIRE

    Krebs, Marie-Odile; Betancur, Catalina; Leroy, Sophie; Bourdel, Marie-Chantal; Gillberg, Christopher; Leboyer, Marion

    2002-01-01

    Autism is a complex neurodevelopmental disorder with severe cognitive and communication disabilities, that has a strong genetic predisposition predisposition.1 Reelin, a protein involved in neuronal migration during development, is encoded by a gene located on 7q22, 7q22,2 within the candidate region on 7q showing increased allele sharing in previous genome scans. 3–8 A case/control and family-based association study recently reported a positive association between a trinucleotide repeat poly...

  13. [Diversity and genetic stability of yeast flocculation caused by variation of tandem repeats in yeast flocculin genes].

    Science.gov (United States)

    Yue, Feng; Guo, Xuena; He, Xiuping; Zhang, Borun

    2013-07-01

    Yeast flocculation is described as a reversible, asexual and calcium dependent process, in which cells adhere to form flocs by interaction of specific cell surface proteins named flocculins on yeast cells with mannose residues present on the cell wall of adjacent yeast cells. Yeast flocculation provides a very economical and convenient pathway for separation of yeast cells from the fermentation broth or removal of heavy metal ions from effluent. A large number of tandem repeats have been found in genes encoding flocculins, which not only have great regulatory effect on the structure and function of flocculins, generating the diversity of flocculation characteristics, but lead to genetic instability in flocculation as well for driving slippage and recombination reactions within and between FLO genes. Here, the research progress in effect of variation of tandem repeats in FLO genes on flocculation characteristics and genetic stability were reviewed to direct and promote the controllable application of flocculation in industrial fermentation process and environmental remediation.

  14. Effects of ligand binding on the mechanical properties of ankyrin repeat protein gankyrin.

    Directory of Open Access Journals (Sweden)

    Giovanni Settanni

    Full Text Available Ankyrin repeat proteins are elastic materials that unfold and refold sequentially, repeat by repeat, under force. Herein we use atomistic molecular dynamics to compare the mechanical properties of the 7-ankyrin-repeat oncoprotein Gankyrin in isolation and in complex with its binding partner S6-C. We show that the bound S6-C greatly increases the resistance of Gankyrin to mechanical stress. The effect is specific to those repeats of Gankyrin directly in contact with S6-C, and the mechanical 'hot spots' of the interaction map to the same repeats as the thermodynamic hot spots. A consequence of stepwise nature of unfolding and the localized nature of ligand binding is that it impacts on all aspects of the protein's mechanical behavior, including the order of repeat unfolding, the diversity of unfolding pathways accessed, the nature of partially unfolded intermediates, the forces required and the work transferred to the system to unfold the whole protein and its parts. Stepwise unfolding thus provides the means to buffer repeat proteins and their binding partners from mechanical stress in the cell. Our results illustrate how ligand binding can control the mechanical response of proteins. The data also point to a cellular mechano-switching mechanism whereby binding between two partner macromolecules is regulated by mechanical stress.

  15. Effects of acute versus repeated cocaine exposure on the expression of endocannabinoid signaling-related proteins in the mouse cerebellum

    OpenAIRE

    Ana ePalomino; Francisco Javier ePavon; Eduardo eBlanco Calvo; Antonia eSerrano; Sergio eArrabal; Patricia eRivera; Antonio eVargas; Ainhoa eBilbao; Leticia eRubio; Fernando eRodriguez de Fonseca; Juan eSuarez

    2014-01-01

    Growing awareness of cerebellar involvement in addiction is based on the cerebellum’s intermediary position between motor and reward, potentially acting as an interface between motivational and cognitive functions. Here, we examined the impact of acute and repeated cocaine exposure on the two main signaling systems in the mouse cerebellum: the endocannabinoid (eCB) and glutamate systems. To this end, we investigated whether eCB signaling-related gene and protein expression {cannabinoid recept...

  16. Improved haplotype analysis of human myelin basic protein short tandem repeat loci.

    Science.gov (United States)

    Watanabe, G; Umetsu, K; Yuasa, I; Suzuki, T

    2000-06-01

    We report an improved haplotype analysis of the human myelin basic protein gene (MBP) short tandem repeat (STR) polymorphism. The polymorphic G-->A transition and 2 conventional STR polymorphisms, MBPA and MBPB, were simultaneously determined by an amplified product length polymorphism technique. After the MBPC fragments containing MBPA and MBPB were amplified, the linkage of these 2 STR loci was determined by a second amplification, using polymerase chain reaction (PCR) technique, of the isolated MBPC fragments. The present haplotype analysis dispensed with family studies for the haplotyping of MBPA and MBPB. Polymorphisms of the MBP loci studied in German and Japanese populations showed a high genomic variation. Haplotype analysis of the MBP loci showed distinct differences between the German and the Japanese populations. Consequently, haplotype analysis of the MBP loci promises to be useful in forensic identification and paternity testing.

  17. Overexpression of NOTCH-regulated Ankyrin Repeat Protein is associated with papillary thyroid carcinoma progression

    Science.gov (United States)

    Zhang, Mingdi; Qin, Yiyu; Zuo, Bin; Gong, Wei; Zhang, Shenglai; Gong, Yurong; Quan, Zhiwei; Chu, Bingfeng

    2017-01-01

    Papillary thyroid cancer (PTC) is one of the endocrine cancers with high clinical and genetic heterogeneity. NOTCH signaling and its downstream NOTCH-Regulated Ankyrin Repeat Protein (NRARP) have been implicated in oncogenesis of many cancers, but the roles in PTCs are less studied. In this study, we show that NRARP is frequently over-expressed in thyroid carcinoma. The over-activation of NRARP is highly and positively correlated with NOTCH genes. Moreover, we find that the expression of NRARP is highly associated with several epithelial mesenchymal transition (EMT) markers and contributes to poor survival outcomes. Therefore, these results indicate that NRARP is an important clinical biomarker in thyroid carcinoma and it promotes EMT induction as well as the progression of PTCs via NOTCH signaling activation. PMID:28207739

  18. Shorter CAG repeat in the AR gene is associated with atypical hyperplasia and breast carcinoma

    DEFF Research Database (Denmark)

    De Abreu, Francine Blumental; Pirolo, Leandro Júnior; Canevari, Renata de Azevedo

    2007-01-01

    -based GeneScan analysis was used to investigate the [CAG]n repeat length at exon 1 of the AR gene in 59 benign breast lesions (27 fibroadenomas, 18 atypical hyperplasias, and 14 hyperplasias without atypia) and 54 ductal breast carcinomas. Seventy-two cancer-free women were used as a control group....... In addition, [CAG]n repeats were evaluated for the presence of loss of heterozygosity (LOH) and microsatellite instability (MSI) in a subset of these samples (27 fibroadenomas, 14 hyperplasias without atypia and 22 breast carcinomas). RESULTS: Shorter [CAG]n repeat lengths were strongly correlated...

  19. Studies of the CAG repeat in the Machado-Joseph disease gene in Taiwan.

    Science.gov (United States)

    Hsieh, M; Tsai, H F; Lu, T M; Yang, C Y; Wu, H M; Li, S Y

    1997-08-01

    Machado-Joseph disease (MJD) is an autosomal dominant spinocerebellar degeneration characterized by cerebellar ataxia and pyramidal signs associated in varying degrees with a dystonic-rigid extrapyramidal syndrome or peripheral amyotrophy. Unstable CAG trinucleotide repeat expansion in the MJD gene on the long arm of chromosome 14 has been identified as the pathological mutation for MJD. While investigating the distribution of CAG repeat lengths of the MJD gene in Taiwan's population, we have identified 18 MJD-affected patients and 12 at-risk individuals in seven families. In addition, we have analyzed the range of CAG repeat lengths in 96 control individuals. The CAG repeat number ranged from 13 to 44 in the controls and 72-85 in the affected and at-risk individuals. Our results indicated that the CAG repeat number was inversely correlated with the age of onset. The differences in CAG repeat length between parent and child and between siblings are greater with paternal transmission than maternal transmission. Our data show a tendency towards the phenomenon of anticipation in the MJD families but do not support unidirectional expansion of CAG repeats during transmission. We also demonstrated that PCR amplification of the CAG repeats in the MJD gene from villous DNA was possible and might prove useful as a diagnostic tool for affected families in the future.

  20. Instability of human TATA-binding protein CAG triplet repeats during amplification by PCR.

    Science.gov (United States)

    Holstege, F C; van der Vliet, P C; Timmers, H T

    1994-09-13

    Polymerase chain reaction (PCR) of a TATA-binding protein cDNA that contains CAG triplet repeats results in heterogeneous products. This is caused by a variable loss in the number of CAG triplets. Sequence analysis of PCR products suggests that instability increases with repeat length.

  1. [Comparative analysis of internal repeating segments in proteins of species from the three kingdoms of life].

    Science.gov (United States)

    Chen, Hao; Zhu, Sheng; Chen, Liang-Biao

    2005-03-01

    In 1970's, Ohno proposed that primordial proteins might evolve from periodic amplification of oligopeptides. Internal repeating segments in proteins may play important roles in functional evolution of proteins. In this study,a new method was designed to extract internal repeating segments from proteomes of 8 modern species belong to eukaryota, bacteria and archaea, respectively. The repeating patterns and the frequencies within proteomes of each kingdom were analyzed by matrix plot. Simple repeat segments were found in eukaryotic proteins with high frequencies,but were much lower in bacteria and none in archaea. Further analysis showed that, the biased usage of amino acids in the internal repeating segments was positively related to the frequencies of individual amino acids in the proteome of a given species. The correlation coefficient was up to 0.95 in prokaryota, with the eukaryota to be lower. The high frequency of simple repeat sequences in eukaryotic proteomes, as well as the disparate relationships of amino acid compositions between the internal repeating segments and their haboring eukaryotic proteomes imply that the fast evolution of simple repeat sequences could be one force that generates the high complexity of eukarytic proteomes.

  2. Identification and characterisation of coding tandem repeat variants in incA gene of Chlamydophila pecorum.

    Science.gov (United States)

    Yousef Mohamad, Khalil; Rekiki, Abdessalem; Myers, Garry; Bavoil, Patrik M; Rodolakis, Annie

    2008-01-01

    Bacteria of the family Chlamydiaceae are obligate intracellular pathogens of human and animals. Chlamydophila pecorum is associated with different pathological conditions in ruminants, swine and koala. To characterize a coding tandem repeat (CTR) identified at the 3' end of incA gene of C. pecorum, 51 strains of different chlamydial species were examined. The CTR were observed in 18 of 18 tested C. pecorum isolates including symptomatic and asymptomatic animals from diverse geographical origins. The CTR were also found in two strains of C. abortus respectively isolated from faeces from a healthy ewe and from a goat belonging to asymptomatic herds, but were absent in C. abortus strains isolated from clinical disease specimens, and in tested strains of C. psittaci, C. caviae, C. felis and C. trachomatis. The number of CTR repeats is variable and encode several motifs that are rich in alanine and proline. The CTR-derived variable structure of incA, which encode the Chlamydiaceae-specific type III secreted inclusion membrane protein, IncA, may be involved in the adaptation of C. pecorum to its environment by allowing it to persist in the host cell.

  3. Global analysis of ankyrin repeat domain C3HC4-type RING finger gene family in plants.

    Directory of Open Access Journals (Sweden)

    Xiaowei Yuan

    Full Text Available Ankyrin repeat (ANK C3HC4-type RING finger (RF genes comprise a large family in plants and play important roles in various physiological processes of plant life. In this study, we identified 187 ANK C3HC4-type RF proteins from 29 species with complete genomes and named the ANK C3HC4-type RF proteins the XB3-like proteins because they are structurally related to the rice (Oryza sativa XB3. A phylogenetic relationship analysis suggested that the XB3-like genes originated from ferns, and the encoded proteins fell into 3 major groups. Among these groups, we found that the spacing between the metal ligand position 6 and 7, and the conserved residues, which was in addition to the metal ligand amino acids, in the C3HC4-type RF were different. Using a wide range of protein structural analyses, protein models were established, and all XB3-like proteins were found to contain two to seven ANKs and a C3HC4-type RF. The microarray data for the XB3-like genes of Arabidopsis, Oryza sative, Zea mays and Glycine max revealed that the expression of XB3-like genes was in different tissues and during different life stages. The preferential expression of XB3-like genes in specified tissues and the response to phytohormone and abiotic stress treatments of Arabidopsis and Zea mays not only confirmed the microarray analysis data but also demonstrated that the XB3-like proteins play roles in plant growth and development as well as in stress responses. Our data provide a very useful reference for the identification and functional analysis of members of this gene family and also provide a new method for the genome-wide analysis of gene families.

  4. Polycomb complexes act redundantly to repress genomic repeats and genes

    DEFF Research Database (Denmark)

    Leeb, Martin; Pasini, Diego; Novatchkova, Maria

    2010-01-01

    Polycomb complexes establish chromatin modifications for maintaining gene repression and are essential for embryonic development in mice. Here we use pluripotent embryonic stem (ES) cells to demonstrate an unexpected redundancy between Polycomb-repressive complex 1 (PRC1) and PRC2 during the form...

  5. Prevalence of Huntington's disease gene CAG repeat alleles in sporadic amyotrophic lateral sclerosis patients.

    Science.gov (United States)

    Ramos, Eliana Marisa; Keagle, Pamela; Gillis, Tammy; Lowe, Patrick; Mysore, Jayalakshmi S; Leclerc, Ashley Lyn; Ratti, Antonia; Ticozzi, Nicola; Gellera, Cinzia; Gusella, James F; Silani, Vincenzo; Alonso, Isabel; Brown, Robert H; MacDonald, Marcy E; Landers, John E

    2012-05-01

    A higher prevalence of intermediate ataxin-2 CAG repeats in amyotrophic lateral sclerosis (ALS) patients has raised the possibility that CAG expansions in other polyglutamine disease genes could contribute to ALS neurodegeneration. We sought to determine whether expansions of the CAG repeat of the HTT gene that causes Huntington's disease, are associated with ALS. We compared the HTT CAG repeat length on a total of 3144 chromosomes from 1572 sporadic ALS patients and 4007 control chromosomes, and also tested its possible effects on ALS-specific parameters, such as age and site of onset and survival rate. Our results show that the CAG repeat in the HTT gene is not a risk factor for ALS nor modifies its clinical presentation. These findings suggest that distinct neuronal degeneration processes are involved in these two different neurodegenerative disorders.

  6. Constraints and consequences of the emergence of amino acid repeats in eukaryotic proteins.

    Science.gov (United States)

    Chavali, Sreenivas; Chavali, Pavithra L; Chalancon, Guilhem; de Groot, Natalia Sanchez; Gemayel, Rita; Latysheva, Natasha S; Ing-Simmons, Elizabeth; Verstrepen, Kevin J; Balaji, Santhanam; Babu, M Madan

    2017-09-01

    Proteins with amino acid homorepeats have the potential to be detrimental to cells and are often associated with human diseases. Why, then, are homorepeats prevalent in eukaryotic proteomes? In yeast, homorepeats are enriched in proteins that are essential and pleiotropic and that buffer environmental insults. The presence of homorepeats increases the functional versatility of proteins by mediating protein interactions and facilitating spatial organization in a repeat-dependent manner. During evolution, homorepeats are preferentially retained in proteins with stringent proteostasis, which might minimize repeat-associated detrimental effects such as unregulated phase separation and protein aggregation. Their presence facilitates rapid protein divergence through accumulation of amino acid substitutions, which often affect linear motifs and post-translational-modification sites. These substitutions may result in rewiring protein interaction and signaling networks. Thus, homorepeats are distinct modules that are often retained in stringently regulated proteins. Their presence facilitates rapid exploration of the genotype-phenotype landscape of a population, thereby contributing to adaptation and fitness.

  7. De-coding and re-coding RNA recognition by PUF and PPR repeat proteins.

    Science.gov (United States)

    Hall, Traci M Tanaka

    2016-02-01

    PUF and PPR proteins are two families of α-helical repeat proteins that recognize single-stranded RNA sequences. Both protein families hold promise as scaffolds for designed RNA-binding domains. A modular protein RNA recognition code was apparent from the first crystal structures of a PUF protein in complex with RNA, and recent studies continue to advance our understanding of natural PUF protein recognition (de-coding) and our ability to engineer specificity (re-coding). Degenerate recognition motifs make de-coding specificity of individual PPR proteins challenging. Nevertheless, re-coding PPR protein specificity using a consensus recognition code has been successful.

  8. Solution properties of the archaeal CRISPR DNA repeat-binding homeodomain protein Cbp2

    DEFF Research Database (Denmark)

    Kenchappa, Chandra; Heiðarsson, Pétur Orri; Kragelund, Birthe;

    2013-01-01

    in facilitating high affinity DNA binding of Cbp2 by tethering the two domains. Structural studies on mutant proteins provide support for Cys(7) and Cys(28) enhancing high thermal stability of Cbp2(Hb) through disulphide bridge formation. Consistent with their proposed CRISPR transcriptional regulatory role, Cbp2......Clustered regularly interspaced short palindromic repeats (CRISPR) form the basis of diverse adaptive immune systems directed primarily against invading genetic elements of archaea and bacteria. Cbp1 of the crenarchaeal thermoacidophilic order Sulfolobales, carrying three imperfect repeats, binds...... specifically to CRISPR DNA repeats and has been implicated in facilitating production of long transcripts from CRISPR loci. Here, a second related class of CRISPR DNA repeat-binding protein, denoted Cbp2, is characterized that contains two imperfect repeats and is found amongst members of the crenarchaeal...

  9. Solution properties of the archaeal CRISPR DNA repeat-binding homeodomain protein Cbp2

    DEFF Research Database (Denmark)

    Kenchappa, Chandra; Heiðarsson, Pétur Orri; Kragelund, Birthe

    2013-01-01

    Clustered regularly interspaced short palindromic repeats (CRISPR) form the basis of diverse adaptive immune systems directed primarily against invading genetic elements of archaea and bacteria. Cbp1 of the crenarchaeal thermoacidophilic order Sulfolobales, carrying three imperfect repeats, binds...... specifically to CRISPR DNA repeats and has been implicated in facilitating production of long transcripts from CRISPR loci. Here, a second related class of CRISPR DNA repeat-binding protein, denoted Cbp2, is characterized that contains two imperfect repeats and is found amongst members of the crenarchaeal...... in facilitating high affinity DNA binding of Cbp2 by tethering the two domains. Structural studies on mutant proteins provide support for Cys(7) and Cys(28) enhancing high thermal stability of Cbp2(Hb) through disulphide bridge formation. Consistent with their proposed CRISPR transcriptional regulatory role, Cbp2...

  10. Gene expression profiling of R6/2 transgenic mice with different CAG repeat lengths reveals genes associated with disease onset and progression in Huntington's disease.

    Science.gov (United States)

    Tang, Bin; Seredenina, Tamara; Coppola, Giovanni; Kuhn, Alexandre; Geschwind, Daniel H; Luthi-Carter, Ruth; Thomas, Elizabeth A

    2011-06-01

    R6/2 transgenic mice with expanded CAG repeats (>300) have a surprisingly prolonged disease progression and longer lifespan than prototypical parent R6/2 mice (carrying 150 CAGs); however, the mechanism of this phenotype amelioration is unknown. We compared gene expression profiles in the striatum of R6/2 transgenic mice carrying ~300 CAG repeats (R6/2(Q300) transgenic mice) to those carrying ~150 CAG repeats (R6/2(Q150) transgenic mice) and littermate wildtype controls in order to identify genes that may play determinant roles in the time course of phenotypic expression in these mice. Of the top genes showing concordant expression changes in the striatum of both R6/2 lines, 85% were decreased in expression, while discordant expression changes were observed mostly for genes upregulated in R6/2(Q300) transgenic mice. Upregulated genes in the R6/2(Q300) mice were associated with the ubiquitin ligase complex, cell adhesion, protein folding, and establishment of protein localization. We qPCR-validated increases in expression of genes related to the latter category, including Lrsam1, Erp29, Nasp, Tap1, Rab9b, and Pfdn5 in R6/2(Q300) mice, changes that were not observed in R6/2 mice with shorter CAG repeats, even in late stages (i.e., 12 weeks of age). We further tested Lrsam1 and Erp29, the two genes showing the greatest upregulation in R6/2(Q300) transgenic mice, for potential neuroprotective effects in primary striatal cultures overexpressing a mutated human huntingtin (htt) fragment. Overexpression of Lrsam1 prevented the loss of NeuN-positive cell bodies in htt171-82Q cultures, concomitant with a reduction of nuclear htt aggregates. Erp29 showed no significant effects in this model. This is consistent with the distinct pattern of htt inclusion localization observed in R6/2(Q300) transgenic mice, in which smaller cytoplasmic inclusions represent the major form of insoluble htt in the cell, as opposed to large nuclear inclusions observed in R6/2(Q150) transgenic mice

  11. Association between a Tetranucleotide Repeat Polymorphism of SPAG16 Gene and Cataract in Male Children

    Directory of Open Access Journals (Sweden)

    Shipra Mehra

    2013-01-01

    Full Text Available Purpose. Studies involving genotyping of STR markers at 2q34 have repeatedly found the region to host the disease haplotype for pediatric cataract. Present study investigated the association of D2S2944 marker, in sperm associated antigen 16 (SPAG16 gene and rs2289917 polymorphism, in γ-crystallin B gene, with childhood cataract. Methods. 97 pediatric cataract cases and 110 children with no ocular defects were examined for tetranucleotide repeat marker/SNP using PCR-SSLP/RFLP techniques. Polymorphisms were assessed for association using contingency tables and linkage disequilibrium among alleles of the markers was estimated. Energy-optimization program predicted the secondary structure models of repeats of D2S2944. Results. Seven alleles of D2S2944, with 9–15 “GATA” repeats, were observed. Frequency of the longer allele of D2S2944, ≥(GATA13 repeats, was 0.73 in cases and 0.56 in controls (P=0.0123. Male children bearing ≥(GATA13 repeats showed >3-fold higher risk for cataract (CI95% = 1.43–7.00, P=0.0043, Pc=0.0086 as compared to female children (OR=1.19, CI95% = 0.49–2.92, P=0.70. Cases with haplotype—≥(GATA13 of D2S2944 and “C” allele rs2289917—have a higher risk for pediatric cataract (OR=2.952, CI95% = 1.595~5.463, P=0.000453. >(GATA13 repeats formed energetically more favorable stem-loop structure. Conclusion. Intragenic microsatellite repeat expansion in SPAG16 gene increases predisposition to pediatric cataract by probably interfering posttranscriptional events and affecting the expression of adjacent lens transparency gene/s in a gender bias manner.

  12. The design and structural characterization of a synthetic pentatricopeptide repeat protein.

    Science.gov (United States)

    Gully, Benjamin S; Shah, Kunal R; Lee, Mihwa; Shearston, Kate; Smith, Nicole M; Sadowska, Agata; Blythe, Amanda J; Bernath-Levin, Kalia; Stanley, Will A; Small, Ian D; Bond, Charles S

    2015-02-01

    Proteins of the pentatricopeptide repeat (PPR) superfamily are characterized by tandem arrays of a degenerate 35-amino-acid α-hairpin motif. PPR proteins are typically single-stranded RNA-binding proteins with essential roles in organelle biogenesis, RNA editing and mRNA maturation. A modular, predictable code for sequence-specific binding of RNA by PPR proteins has recently been revealed, which opens the door to the de novo design of bespoke proteins with specific RNA targets, with widespread biotechnological potential. Here, the design and production of a synthetic PPR protein based on a consensus sequence and the determination of its crystal structure to 2.2 Å resolution are described. The crystal structure displays helical disorder, resulting in electron density representing an infinite superhelical PPR protein. A structural comparison with related tetratricopeptide repeat (TPR) proteins, and with native PPR proteins, reveals key roles for conserved residues in directing the structure and function of PPR proteins. The designed proteins have high solubility and thermal stability, and can form long tracts of PPR repeats. Thus, consensus-sequence synthetic PPR proteins could provide a suitable backbone for the design of bespoke RNA-binding proteins with the potential for high specificity.

  13. Reelin gene polymorphisms in the Indian population: a possible paternal 5'UTR-CGG-repeat-allele effect on autism.

    Science.gov (United States)

    Dutta, Shruti; Guhathakurta, Subhrangshu; Sinha, Swagata; Chatterjee, Anindita; Ahmed, Shabina; Ghosh, Saurabh; Gangopadhyay, Prasanta K; Singh, Manoranjan; Usha, Rajamma

    2007-01-05

    Autism is a neurodevelopmental disorder with high heritability factor and the reelin gene, which codes for an extracellular matrix protein involved with neuronal migration and lamination is being investigated as a positional and functional candidate gene for autism. It is located on chromosome 7q22 within the autism susceptible locus (AUTS1); identified in earlier genome scans and several investigations have been carried out on various ethnic groups to assess possible association and linkage of the gene with autism. However, the findings are still inconclusive. In the present study which represents the first report of such a study on the Indian population, genotyping analyses of CGG repeat polymorphism at 5'UTR, two single nucleotide polymorphisms (SNP) at exon 6 and exon 50 were performed in 73 autistic subjects, 129 parents, and 80 controls. The allelic distributions of the repeat polymorphism and exon 50 T/C SNP were quite different from earlier reports in other populations. Allelic and genotypic distribution of the markers did not show any differences between the cases and controls. While our preliminary data on family-based association studies on 58 trios showed no preferential transmission of any allele from the parents to the affected offspring, TDT and HHRR analyses revealed significant paternal transmission distortions for 10- and > or =11-repeat alleles of CGG repeat polymorphism. Thus, the present study suggests that 5'UTR of reelin gene may have a role in the susceptibility towards autism with the paternal transmission and non-transmission respectively of 10- and > or =11-repeat alleles, to the affected offspring.

  14. Over-representation of repeats in stress response genes: a strategy to increase versatility under stressful conditions?

    Science.gov (United States)

    Rocha, Eduardo P C; Matic, Ivan; Taddei, François

    2002-05-01

    The survival of individual organisms facing stress is enhanced by the induction of a set of changes. As the intensity, duration and nature of stress is highly variable, the optimal response to stress may be unpredictable. To face such an uncertain future, it may be advantageous for a clonal population to increase its phenotypic heterogeneity (bet-hedging), ensuring that at least a subset of cells would survive the current stress. With current techniques, assessing the extent of this variability experimentally remains a challenge. Here, we use a bioinformatic approach to compare stress response genes with the rest of the genome for the presence of various kinds of repeated sequences, elements known to increase variability during the transfer of genetic information (i.e. during replication, but also during gene expression). We investigated the potential for illegitimate and homologous recombination of 296 Escherichia coli genes related to repair, recombination and physiological adaptations to different stresses. Although long repeats capable of engaging in homologous recombination are almost absent in stress response genes, we observed a significant high number of short close repeats capable of inducing phenotypic variability by slipped-mispair during DNA, RNA or protein synthesis.

  15. An ancient repeat sequence in the ATP synthase beta-subunit gene of forcipulate sea stars.

    Science.gov (United States)

    Foltz, David W

    2007-11-01

    A novel repeat sequence with a conserved secondary structure is described from two nonadjacent introns of the ATP synthase beta-subunit gene in sea stars of the order Forcipulatida (Echinodermata: Asteroidea). The repeat is present in both introns of all forcipulate sea stars examined, which suggests that it is an ancient feature of this gene (with an approximate age of 200 Mya). Both stem and loop regions show high levels of sequence constraint when compared to flanking nonrepetitive intronic regions. The repeat was also detected in (1) the family Pterasteridae, order Velatida and (2) the family Korethrasteridae, order Velatida. The repeat was not detected in (1) the family Echinasteridae, order Spinulosida, (2) the family Astropectinidae, order Paxillosida, (3) the family Solasteridae, order Velatida, or (4) the family Goniasteridae, order Valvatida. The repeat lacks similarity to published sequences in unrestricted GenBank searches, and there are no significant open reading frames in the repeat or in the flanking intron sequences. Comparison via parametric bootstrapping to a published phylogeny based on 4.2 kb of nuclear and mitochondrial sequence for a subset of these species allowed the null hypothesis of a congruent phylogeny to be rejected for each repeat, when compared separately to the published phylogeny. In contrast, the flanking nonrepetitive sequences in each intron yielded separate phylogenies that were each congruent with the published phylogeny. In four species, the repeat in one or both introns has apparently experienced gene conversion. The two introns also show a correlated pattern of nucleotide substitutions, even after excluding the putative cases of gene conversion.

  16. CAG repeat polymorphism in the androgen receptor (AR) gene of SBMA patients and a control group.

    Science.gov (United States)

    Sułek, Anna; Hoffman-Zacharska, Dorota; Krysa, Wioletta; Szirkowiec, Walentyna; Fidziańska, Elzbieta; Zaremba, Jacek

    2005-01-01

    Spinobulbar muscular atrophy (SBMA) is an X-linked form of motor neuron disease characterized by progressive atrophy of the muscles, dysphagia, dysarthria and mild androgen insensitivity. SBMA is caused by CAG repeat expansion in the androgen receptor gene. CAG repeat polymorphism was analysed in a Polish control group (n = 150) and patients suspected of SBMA (n = 60). Normal and abnormal ranges of CAG repeats were established in the control group and in 21 patients whose clinical diagnosis of SBMA was molecularly confirmed. The ranges are similar to those reported for other populations.

  17. Ternary WD40 repeat-containing protein complexes: evolution, composition and roles in plant immunity

    Directory of Open Access Journals (Sweden)

    Jimi C. Miller

    2016-01-01

    Full Text Available Plants, like mammals, rely on their innate immune system to perceive and discriminate among the majority of their microbial pathogens. Unlike mammals, plants respond to this molecular dialogue by unleashing a complex chemical arsenal of defense metabolites to resist or evade pathogen infection. In basal or non-host resistance, plants utilize signal transduction pathways to detect non-self, damaged-self and altered-self-associated molecular patterns and translate these danger signals into largely inducible chemical defenses. The WD40 repeat (WDR-containing proteins Gβ and TTG1 are constituents of two independent ternary protein complexes functioning at opposite ends of a plant immune signaling pathway. Gβ and TTG1 are also encoded by single-copy genes that are ubiquitous in higher plants, implying the limited diversity and functional conservation of their respective complexes. In this review, we summarize what is currently known about the evolutionary history of these WDR-containing ternary complexes, their repertoire and combinatorial interactions, and their downstream effectors and pathways in plant defense.

  18. Serine aspartate repeat protein D increases Staphylococcus aureus virulence and survival in blood

    NARCIS (Netherlands)

    Askarian, Fatemeh; Uchiyama, Satoshi; Valderrama, J. Andrés; Ajayi, Clement; Sollid, Johanna U E; van Sorge, Nina M.; Nizet, Victor; van Strijp, Jos A G; Johannessen, Mona

    2017-01-01

    Staphylococcus aureus expresses a panel of cell wall-anchored adhesins, including proteins belonging to the microbial surface components recognizing adhesive matrix molecule (MSCRAMM) family, exemplified by the serine-aspartate repeat protein D (SdrD), which serve key roles in colonization and

  19. Analysis of thirteen trinucleotide repeat loci as candidate genes for Schizophrenia and bipolar affective disorder

    Energy Technology Data Exchange (ETDEWEB)

    Jain, S.; Leggo, J.; Ferguson-Smith, M.A.; Rubinsztein, D.C. [Addenbrooke`s NHS Trust, Cambridge (United Kingdom)] [and others

    1996-04-09

    A group of diseases are due to abnormal expansions of trinucleotide repeats. These diseases all affect the nervous system. In addition, they manifest the phenomenon of anticipation, in which the disease tends to present at an earlier age or with greater severity in successive generations. Many additional genes with trinucleotide repeats are believed to be expressed in the human brain. As anticipation has been reported in schizophrenia and bipolar affective disorder, we have examined allele distributions of 13 trinucleotide repeat-containing genes, many novel and all expressed in the brain, in genomic DNA from schizophrenic (n = 20-97) and bipolar affective disorder patients (23-30) and controls (n = 43-146). No evidence was obtained to implicate expanded alleles in these 13 genes as causal factors in these diseases. 26 refs., 1 fig., 2 tabs.

  20. Effects of acute versus repeated cocaine exposure on the expression of endocannabinoid signaling-related proteins in the mouse cerebellum

    Directory of Open Access Journals (Sweden)

    Ana ePalomino

    2014-03-01

    Full Text Available Growing awareness of cerebellar involvement in addiction is based on the cerebellum’s intermediary position between motor and reward, potentially acting as an interface between motivational and cognitive functions. Here, we examined the impact of acute and repeated cocaine exposure on the two main signaling systems in the mouse cerebellum: the endocannabinoid (eCB and glutamate systems. To this end, we investigated whether eCB signaling-related gene and protein expression (CB1 receptors and enzymes that produce (DAGLα/β and NAPE-PLD and degrade (MAGL and FAAH eCB were altered. In addition, we analyzed the gene expression of relevant components of the glutamate signaling system (glutamate synthesizing enzymes LGA and KGA, mGluR3/5 metabotropic receptors, and NR1/2A/2B/2C-NMDA and GluR1/2/3/4-AMPA ionotropic receptor subunits and the gene expression of tyrosine hydroxylase (TH, the rate-limiting enzyme in catecholamine biosynthesis, because noradrenergic terminals innervate the cerebellar cortex. Results indicated that acute cocaine exposure decreased DAGLα expression, suggesting a down-regulation of 2-AG production, as well as gene expression of TH, KGA, mGluR3 and all ionotropic receptor subunits analyzed in the cerebellum. The acquisition of conditioned locomotion and sensitization after repeated cocaine exposure were associated with an increased NAPE-PLD/FAAH ratio, suggesting enhanced anandamide production, and a decreased DAGLβ/MAGL ratio, suggesting decreased 2-AG generation. Repeated cocaine also increased LGA gene expression but had no effect on glutamate receptors. These findings indicate that acute cocaine modulates the expression of the eCB and glutamate systems. Repeated cocaine results in normalization of glutamate receptor expression, although sustained changes in eCB is observed. We suggest that cocaine-induced alterations to cerebellar eCB should be considered when analyzing the adaptations imposed by psychostimulants that

  1. NUC-2, a component of the phosphate-regulated signal transduction pathway in Neurospora crassa, is an ankyrin repeat protein.

    Science.gov (United States)

    Poleg, Y; Aramayo, R; Kang, S; Hall, J G; Metzenberg, R L

    1996-10-28

    In response to phosphorus limitation, the fungus Neurospora crassa synthesizes a number of enzymes that function to bring more phosphate into the cell. The NUC-2 protein appears to sense the availability of phosphate and transmits the signal downstream to the regulatory pathway. The nuc-2+ gene has been cloned by its ability to restore growth of a nuc-2 mutant under restrictive conditions of high pH and low phosphate concentration. We mapped the cloned gene to the right arm of linkage group II, consistent with the chromosomal position of the nuc-2 mutation as determined by classical genetic mapping. The nuc-2' open reading frame is interrupted by five introns and codes for a protein of 1066 amino acid residues. Its predicted amino acid sequence has high similarity to that of its homolog in Saccharomyces cerevisiae, PHO81. Both proteins contain six ankyrin repeats, which have been implicated in the cyclin-dependent kinase inhibitory activity of PHO81. The phenotypes of a nuc-2 mutant generated by repeat-induced point mutation and of a strain harboring a UV-induced nuc-2 allele are indistinguishable. Both are unable to grow under the restrictive conditions, a phenotype which is to some degree temperature dependent. The nuc-2+ gene is transcriptionally regulated. A 15-fold increase in the level of the nuc-2+ transcript occurs in response to a decrease in exogenous phosphate concentration.

  2. Analysis of CAG repeats in IT15 gene in Spanish population

    Energy Technology Data Exchange (ETDEWEB)

    Sanchez, A.; Castellvi-Pel, S.; Mila, M. [Hospital Clinic i Provincial de Parcelons (Spain)] [and others

    1994-09-01

    Huntington`s disease (HD) is an autosomal dominant neurodegenerative disorder characterized by involuntary movements, and cognitive and affective changes. HD has a prevalence of 1 in 10,000 individuals in most populations of European origin. The IT15 gene is responsible for HD as it contains a highly polymorphic, unstable (CAG) repeated sequence that is abnormally expanded in HD chromosomes. The IT15 (CAG)n stretch was analyzed in 100 members (50 affected individuals, 40 asymptomatic at risk for HD, and 10 unaffected members) of 50 HD families, and 50 individuals of the general Spanish population. Expansion of the CAG repeat sequence was found in 45 affected members and 14 individuals at risk, with a repeat length of 40 to 85 repeat units. The range of the polymorphic CAG repeat in normal chromosomes was between 11 and 31 repeat units. In the families with several affected members, we found increases of the repeat length in the least generation. Inverse correlation was found between the age of onset and the length of the CAG repeat; the analysis showed also parental male bias. Presymptomatic analysis of HD has been considerably enhanced with the CAG mutation study.

  3. CAG repeat length in androgen receptor gene and male infertility in Egyptian patients.

    Science.gov (United States)

    Mosaad, Y M; Shahin, D; Elkholy, A A-M; Mosbah, A; Badawy, W

    2012-02-01

    The CAG repeat and its association with infertility has been debatable. Therefore, this study was planned to assess the distribution of CAG repeat expansion in Egyptian patients and to investigate its association with male infertility. Forty-five infertile men were eligible for the study in addition to 20 aged-matched fertile males as control. Semen analysis, scrotal sonography, assay of serum testosterone, follicle-stimulating hormone (FSH) and luteinising hormone (LH), and determination of the CAG repeat number within exon 1 of the androgen receptor (AR) gene were carried out. Statistically significant difference was found between infertile and control groups regarding sperm count, sperm motility, serum FSH level and CAG repeats (P CAG repeats (P = 1.0) was found between oligozoospermic and asthenospermic groups; negative correlation was found between CAG repeat length and sperm count, and a positive correlation was found between CAG repeat length and serum FSH (P CAG repeat may be associated with lower AR function with derangement of sperm production, and this may contribute to male infertility in Egyptian men.

  4. Large cryptic internal sequence repeats in protein structures from Homo sapiens

    Indian Academy of Sciences (India)

    R Sarani; N A Udayaprakash; R Subashini; P Mridula; T Yamane; K Sekar

    2009-03-01

    Amino acid sequences are known to constantly mutate and diverge unless there is a limiting condition that makes such a change deleterious. However, closer examination of the sequence and structure reveals that a few large, cryptic repeats are nevertheless sequentially conserved. This leads to the question of why only certain repeats are conserved at the sequence level. It would be interesting to find out if these sequences maintain their conservation at the three-dimensional structure level. They can play an active role in protein and nucleotide stability, thus not only ensuring proper functioning but also potentiating malfunction and disease. Therefore, insights into any aspect of the repeats – be it structure, function or evolution – would prove to be of some importance. This study aims to address the relationship between protein sequence and its three-dimensional structure, by examining if large cryptic sequence repeats have the same structure.

  5. Correlation of CAG repeat length between the maternal and paternal allele of the Huntingtin gene: evidence for assortative mating

    Directory of Open Access Journals (Sweden)

    Wassink Tom

    2011-10-01

    Full Text Available Abstract Triplet repeats contribute to normal variation in behavioral traits and when expanded, cause brain disorders. While Huntington's Disease is known to be caused by a CAG triplet repeat in the gene Huntingtin, the effect of CAG repeats on brain function below disease threshold has not been studied. The current study shows a significant correlation between the CAG repeat length of the maternal and paternal allele in the Huntingtin gene among healthy subjects, suggesting assortative mating.

  6. Biomimetic repeat protein derived from Xenopus tropicalis for fibrous scaffold fabrication.

    Science.gov (United States)

    Kwon, Yunkyeoung; Yang, Yun Jung; Jung, Dooyup; Hwang, Byeong Hee; Cha, Hyung Joon

    2015-12-01

    Collagen, silk, and elastin are the fibrous proteins consist of representative amino acid repeats. Because these proteins exhibited distinguishing mechanical properties, they have been utilized in diverse applications, such as fiber-based sensors, filtration membranes, supporting materials, and tissue engineering scaffolds. Despite their infinite prevalence and potential, most studies have only focused on a few repeat proteins. In this work, the hypothetical protein with a repeat motif derived from the frog Xenopus tropicalis was obtained and characterized for its potential as a novel protein-based material. The codon-optimized recombinant frog repeat protein, referred to as 'xetro', was produced at a high rate in a bacterial system, and an acid extraction-based purified xetro protein was successfully fabricated into microfibers and nanofibers using wet spinning and electrospinning, respectively. Specifically, the wet-spun xetro microfibers demonstrated about 2- and 1.5-fold higher tensile strength compared with synthetic polymer polylactic acid and cross-linked collagen, respectively. In addition, the wet-spun xetro microfibers showed about sevenfold greater stiffness than collagen. Therefore, the mass production potential and greater mechanical properties of the xetro fiber may result in these fibers becoming a new promising fiber-based material for biomedical engineering.

  7. Glutamine repeat variants in human RUNX2 associated with decreased femoral neck BMD, broadband ultrasound attenuation and target gene transactivation.

    Directory of Open Access Journals (Sweden)

    Nigel A Morrison

    Full Text Available RUNX2 is an essential transcription factor required for skeletal development and cartilage formation. Haploinsufficiency of RUNX2 leads to cleidocranial displaysia (CCD a skeletal disorder characterised by gross dysgenesis of bones particularly those derived from intramembranous bone formation. A notable feature of the RUNX2 protein is the polyglutamine and polyalanine (23Q/17A domain coded by a repeat sequence. Since none of the known mutations causing CCD characterised to date map in the glutamine repeat region, we hypothesised that Q-repeat mutations may be related to a more subtle bone phenotype. We screened subjects derived from four normal populations for Q-repeat variants. A total of 22 subjects were identified who were heterozygous for a wild type allele and a Q-repeat variant allele: (15Q, 16Q, 18Q and 30Q. Although not every subject had data for all measures, Q-repeat variants had a significant deficit in BMD with an average decrease of 0.7SD measured over 12 BMD-related parameters (p = 0.005. Femoral neck BMD was measured in all subjects (-0.6SD, p = 0.0007. The transactivation function of RUNX2 was determined for 16Q and 30Q alleles using a reporter gene assay. 16Q and 30Q alleles displayed significantly lower transactivation function compared to wild type (23Q. Our analysis has identified novel Q-repeat mutations that occur at a collective frequency of about 0.4%. These mutations significantly alter BMD and display impaired transactivation function, introducing a new class of functionally relevant RUNX2 mutants.

  8. Study of the repeatability of histone genes in the ploidy series of wheat and Aegilops

    Energy Technology Data Exchange (ETDEWEB)

    Vakhitov, V.A.; Kulikov, A.M.

    1986-10-01

    The hDNA content and number of histone genes in the genomes of different wheat and Aegilops species have been determined by molecular hybridization of DNA with /sup 125/I-histone DNA of Drosophila (L-repeat) on nitrocellulose filters. It has been demonstrated that the proportion of hDNA in the total DNA of diploid and polyploid wheat species is (1.3-7.7) x 10/sup -3/% (57-850 genes), and in the ploidy series of Aegilops species (2.0-8.0) x 10/sup -3/% (89-780 genes). The repeatability of the histone genes generally increases at each ploidy level in the species with higher DNA content. At the same time, it has been demonstrated that the DNA content is not the only factor determining repeatability of the histone genes, as some diploid and allopolyploid species have similar number of these genes. It has been concluded that genetic mechanisms are involved in the regulation of the number of histone genes.

  9. Variation of serine-aspartate repeats in membrane proteins possibly contributes to staphylococcal microevolution.

    Directory of Open Access Journals (Sweden)

    Jing Cheng

    Full Text Available Tandem repeats (either as microsatellites or minisatellites in eukaryotic and prokaryotic organisms are mutation-prone DNA. While minisatellites in prokaryotic genomes are underrepresented, the cell surface adhesins of bacteria often contain the minisatellite SD repeats, encoding the amino acid pair of serine-asparatate, especially in Staphylococcal strains. However, their relationship to biological functions is still elusive. In this study, effort was made to uncover the copy number variations of SD repeats by bioinformatic analysis and to detect changes in SD repeats during a plasmid-based assay, as a first step to understand its biological functions. The SD repeats were found to be mainly present in the cell surface proteins. The SD repeats were genetically unstable and polymorphic in terms of copy numbers and sequence compositions. Unlike SNPs, the change of its copy number was reversible, without frame shifting. More significantly, a rearrangement hot spot, the ATTC/AGRT site, was found to be mainly responsible for the instability and reversibility of SD repeats. These characteristics of SD repeats may facilitate bacteria to respond to environmental changes, with low cost, low risk and high efficiency.

  10. HHrep: de novo protein repeat detection and the origin of TIM barrels.

    Science.gov (United States)

    Söding, Johannes; Remmert, Michael; Biegert, Andreas

    2006-07-01

    HHrep is a web server for the de novo identification of repeats in protein sequences, which is based on the pairwise comparison of profile hidden Markov models (HMMs). Its main strength is its sensitivity, allowing it to detect highly divergent repeat units in protein sequences whose repeats could as yet only be detected from their structures. Examples include sequences with beta-propellor fold, ferredoxin-like fold, double psi barrels or (betaalpha)8 (TIM) barrels. We illustrate this with proteins from four superfamilies of TIM barrels by revealing a clear 4- and 8-fold symmetry, which we detect solely from their sequences. This symmetry might be the trace of an ancient origin through duplication of a betaalphabetaalpha or betaalpha unit. HHrep can be accessed at http://hhrep.tuebingen.mpg.de.

  11. Intermediates in the folding equilibrium of repeat proteins from the TPR family.

    Science.gov (United States)

    González-Charro, Vicente; Rey, Antonio

    2014-09-01

    In recent decades, advances in computational methods and experimental biophysical techniques have improved our understanding of protein folding. Although some of these advances have been remarkable, the structural variability of globular proteins usually encountered makes it difficult to extract general features of their folding processes. To overcome this difficulty, experimental and computational studies of the folding of repeat (or modular) proteins are of interest. Because their native structures can be described as linear arrays of the same, repeated, supersecondary structure unit, it is possible to seek a possibly independent behavior of the different modules without taking into account the intrinsic stability associated with different secondary structure motifs. In this work we have used a Monte Carlo-based simulation to study the folding equilibrium of four repeat proteins belonging to the tetratricopeptide repeat family. Our studies provide new insights into their energy profiles, enabling investigation about the existence of intermediate states and their relative stabilities. We have also performed structural analyses to describe the structure of these intermediates, going through the vast number of conformations obtained from the simulations. In this way, we have tried to identify the regions of each protein in which the modular structure yields a different behavior and, more specifically, regions of the proteins that can stay folded when the rest of the chain has been thermally denatured.

  12. A polymorphic GGC repeat in the NPAS2 gene and its association with melanoma.

    Science.gov (United States)

    Franzoni, Alessandra; Markova-Car, Elitza; Dević-Pavlić, Sanja; Jurišić, Davor; Puppin, Cinzia; Mio, Catia; De Luca, Marila; Petruz, Giulia; Damante, Giuseppe; Pavelić, Sandra Kraljević

    2017-09-01

    Circadian clock regulation in mammals is controlled by feedback loops of a set of circadian genes. One of these circadian genes, NPAS2, encodes for a member of the bHLH-PAS class of transcription factors and is expressed in the forebrain and in some peripheral organs such as liver and skin. Other biological processes are also regulated by circadian genes. For example, NPAS2 is involved in cell proliferation, DNA damage repair and malignant transformation. Aberrant expression of clock genes has been previously observed in melanoma which led to our effort to sequence the NPAS2 promoter region in this cancer type. The NPAS2 putative promoter and 5' untranslated region of ninety-three melanoma patients and ninety-six control subjects were sequenced and several variants were identified. Among these is a novel microsatellite comprising a GGC repeat with different alleles ranging from 7 to 13 repeats located in the 5' untranslated exon. Homozygosity of an allele with nine repeats (9/9) was more prevalent in melanoma than in control subjects (22.6% and 13.5%, respectively, P: 0.0206) suggesting that some NPAS2 variants might contribute to melanoma susceptibility. Impact statement This report describes a variable microsatellite repeat sequence located in the 5' untranslated exon of NSPAS2, a gene encoding a clock transcription factor. Significantly, this study is the first to show that a variant copy number GGC repeat sequence in the NPAS2 clock gene associates with melanoma risk and which may be useful in the assessment of melanoma predisposition.

  13. Absence of association between a polymorphic GGC repeat in the 5' untranslated region of the reelin gene and autism.

    Science.gov (United States)

    Krebs, M O; Betancur, C; Leroy, S; Bourdel, M C; Gillberg, C; Leboyer, M

    2002-01-01

    Autism is a complex neurodevelopmental disorder with severe cognitive and communication disabilities, that has a strong genetic predisposition. Reelin, a protein involved in neuronal migration during development, is encoded by a gene located on 7q22, within the candidate region on 7q showing increased allele sharing in previous genome scans. A case/control and family-based association study recently reported a positive association between a trinucleotide repeat polymorphism (GGC) located in the 5' untranslated region (UTR) of the reelin gene and autism. We performed a transmission disequilibrium test (TDT) analysis of the 5'UTR polymorphism in 167 families including 218 affected subjects (117 trios and 50 affected sib pairs) and found no evidence of linkage/association. Our results do not support previous findings and suggest that this GGC polymorphism of the reelin gene is unlikely to be a major susceptibility factor in autism and/or genetic heterogeneity.

  14. Purification of proteins specifically binding human endogenous retrovirus K long terminal repeat by affinity elution chromatography.

    Science.gov (United States)

    Trubetskoy, D O; Zavalova, L L; Akopov, S B; Nikolaev, L G

    2002-11-01

    A novel affinity elution procedure for purification of DNA-binding proteins was developed and employed to purify to near homogeneity the proteins recognizing a 21 base pair sequence within the long terminal repeat of human endogenous retroviruses K. The approach involves loading the initial protein mixture on a heparin-agarose column and elution of protein(s) of interest with a solution of double-stranded oligonucleotide containing binding sites of the protein(s). The affinity elution has several advantages over conventional DNA-affinity chromatography: (i) it is easier and faster, permitting to isolate proteins in a 1 day-one stage procedure; (ii) yield of a target protein is severalfold higher than that in DNA-affinity chromatography; (iii) it is not necessary to prepare a special affinity support for each factor to be isolated. Theaffinity elution could be a useful alternative to conventional DNA-affinity chromatography.

  15. Identification and function of leucine-rich repeat flightless-I-interacting protein 2 (LRRFIP2 in Litopenaeus vannamei.

    Directory of Open Access Journals (Sweden)

    Shuang Zhang

    Full Text Available Leucine-rich repeat flightless-I-interacting protein 2 (LRRFIP2 is a myeloid differentiation factor 88-interacting protein with a positive regulatory function in toll-like receptor signaling. In this study, seven LRRFIP2 protein variants (LvLRRFIP2A-G were identified in Litopenaeus vannamei. All the seven LvLRRFIP2 protein variants encode proteins with a DUF2051 domain. LvLRRFIP2s were upregulated in hemocytes after challenged with lipopolysaccharide, poly I:C, CpG-ODN2006, Vibrio parahaemolyticus, Staphylococcus aureus, and white spot syndrome virus (WSSV. Dual-luciferase reporter assays in Drosophila Schneider 2 cells revealed that LvLRRFIP2 activates the promoters of Drosophila and shrimp AMP genes. The knockdown of LvLRRFIP2 by RNA interference resulted in higher cumulative mortality of L. vannamei upon V. parahaemolyticus but not S. aureus and WSSV infections. The expression of L. vannamei AMP genes were reduced by dsLvLRRFIP2 interference. These results indicate that LvLRRFIP2 has an important function in antibacterials via the regulation of AMP gene expression.

  16. Shortening trinucleotide repeats using highly specific endonucleases: a possible approach to gene therapy?

    Science.gov (United States)

    Richard, Guy-Franck

    2015-04-01

    Trinucleotide repeat expansions are involved in more than two dozen neurological and developmental disorders. Conventional therapeutic approaches aimed at regulating the expression level of affected genes, which rely on drugs, oligonucleotides, and/or transgenes, have met with only limited success so far. An alternative approach is to shorten repeats to non-pathological lengths using highly specific nucleases. Here, I review early experiments using meganucleases, zinc-finger nucleases (ZFN), and transcription-activator like effector nucleases (TALENs) to contract trinucleotide repeats, and discuss the possibility of using CRISPR-Cas nucleases to the same end. Although this is a nascent field, I explore the possibility of designing nucleases and effectively delivering them in the context of gene therapy.

  17. Global expression analysis of nucleotide binding site-leucine rich repeat-encoding and related genes in Arabidopsis

    Directory of Open Access Journals (Sweden)

    St Clair Dina A

    2007-10-01

    Full Text Available Abstract Background Nucleotide binding site-leucine rich repeat (NBS-LRR-encoding genes comprise the largest class of plant disease resistance genes. The 149 NBS-LRR-encoding genes and the 58 related genes that do not encode LRRs represent approximately 0.8% of all ORFs so far annotated in Arabidopsis ecotype Col-0. Despite their prevalence in the genome and functional importance, there was little information regarding expression of these genes. Results We analyzed the expression patterns of ~170 NBS-LRR-encoding and related genes in Arabidopsis Col-0 using multiple analytical approaches: expressed sequenced tag (EST representation, massively parallel signature sequencing (MPSS, microarray analysis, rapid amplification of cDNA ends (RACE PCR, and gene trap lines. Most of these genes were expressed at low levels with a variety of tissue specificities. Expression was detected by at least one approach for all but 10 of these genes. The expression of some but not the majority of NBS-LRR-encoding and related genes was affected by salicylic acid (SA treatment; the response to SA varied among different accessions. An analysis of previously published microarray data indicated that ten NBS-LRR-encoding and related genes exhibited increased expression in wild-type Landsberg erecta (Ler after flagellin treatment. Several of these ten genes also showed altered expression after SA treatment, consistent with the regulation of R gene expression during defense responses and overlap between the basal defense response and salicylic acid signaling pathways. Enhancer trap analysis indicated that neither jasmonic acid nor benzothiadiazole (BTH, a salicylic acid analog, induced detectable expression of the five NBS-LRR-encoding genes and one TIR-NBS-encoding gene tested; however, BTH did induce detectable expression of the other TIR-NBS-encoding gene analyzed. Evidence for alternative mRNA polyadenylation sites was observed for many of the tested genes. Evidence for

  18. Polymorphic CAG and GGC repeat lengths in the androgen receptor gene and prostate cancer risk: analysis of a Brazilian population.

    Science.gov (United States)

    Silva Neto, Brasil; Koff, Walter J; Biolchi, Vanderlei; Brenner, Cleber; Biolo, Karlo D; Spritzer, Poli Mara; Brum, Ilma S

    2008-02-01

    Variations in transcriptional activity of the androgen receptor (AR) are related to polymorphic CAG and GGC repeats in exon 1 of the AR gene. We investigated the association between CAG and GGC repeat length and the risk of prostate cancer in a case-control study from a Brazilian population. We evaluated 49 patients and 51 healthy controls. DNA was extracted from peripheral leukocytes and the AR gene was analyzed by fragment analysis (GeneMapper software, Applied Biosystems, Foster City, California, USA). CAG and GGC mean lengths were not different between cases and controls. The risk for prostate cancer was higher for CAG repeats repeat lengths (CAG + GGC) repeats ( 17) were not associated with risk for prostate cancer (OR = 1.13 [95% CI 0.47-2.75]). In conclusion, fewer number of CAG repeats and total repeats (CAG + GGC) in the AR gene may be associated with increased risk for prostate cancer.

  19. Hybrid Sterility in Rice (Oryza sativa L.) Involves the Tetratricopeptide Repeat Domain Containing Protein.

    Science.gov (United States)

    Yu, Yang; Zhao, Zhigang; Shi, Yanrong; Tian, Hua; Liu, Linglong; Bian, Xiaofeng; Xu, Yang; Zheng, Xiaoming; Gan, Lu; Shen, Yumin; Wang, Chaolong; Yu, Xiaowen; Wang, Chunming; Zhang, Xin; Guo, Xiuping; Wang, Jiulin; Ikehashi, Hiroshi; Jiang, Ling; Wan, Jianmin

    2016-07-01

    Intersubspecific hybrid sterility is a common form of reproductive isolation in rice (Oryza sativa L.), which significantly hampers the utilization of heterosis between indica and japonica varieties. Here, we elucidated the mechanism of S7, which specially causes Aus-japonica/indica hybrid female sterility, through cytological and genetic analysis, map-based cloning, and transformation experiments. Abnormal positioning of polar nuclei and smaller embryo sac were observed in F1 compared with male and female parents. Female gametes carrying S7(cp) and S7(i) were aborted in S7(ai)/S7(cp) and S7(ai)/S7(i), respectively, whereas they were normal in both N22 and Dular possessing a neutral allele, S7(n) S7 was fine mapped to a 139-kb region in the centromere region on chromosome 7, where the recombination was remarkably suppressed due to aggregation of retrotransposons. Among 16 putative open reading frames (ORFs) localized in the mapping region, ORF3 encoding a tetratricopeptide repeat domain containing protein was highly expressed in the pistil. Transformation experiments demonstrated that ORF3 is the candidate gene: downregulated expression of ORF3 restored spikelet fertility and eliminated absolutely preferential transmission of S7(ai) in heterozygote S7(ai)/S7(cp); sterility occurred in the transformants Cpslo17-S7(ai) Our results may provide implications for overcoming hybrid embryo sac sterility in intersubspecific hybrid rice and utilization of hybrid heterosis for cultivated rice improvement.

  20. The CAG repeat polymorphism of androgen receptor gene and prostate cancer: a meta-analysis.

    Science.gov (United States)

    Gu, Mingliang; Dong, Xiaoqun; Zhang, Xuezhi; Niu, Wenquan

    2012-03-01

    The association between the polymorphic CAG repeat in androgen receptor gene (AR) and prostate cancer susceptibility has been studied extensively. However, the results are contradictory. The purpose of our meta-analysis was to investigate whether CAG repeat related to prostate cancer risk and had genetic heterogeneity across different geographic regions and study designs. Random-effects model was performed irrespective of between-study heterogeneity. Data and study quality were assessed in duplicate. Publication bias was assessed by the fail-safe number and Egger's test. There were 16 (patients/controls: 2972/3792), 19 (3835/4908) and 12 (3372/2631) study groups for comparisons of ≥ 20, 22 and 23 repeats of CAG sequence, respectively. Compared with CAG repeat repeats had 21% (95% CI: 0.61-1.02; P = 0.076), 5% (95% CI: 0.81-1.11; P = 0.508) and 5% (95% CI: 0.76-1.20; P = 0.681) decreased risk of prostate cancer. After classifying studies by geographic areas, carriers of ≥ 20 repeats had 11% decreased risk in populations from USA, 53% from Europe, and 20% from Asia (P > 0.05), whereas comparison of ≥ 23 repeats with others generated a significant prediction in European populations (OR = 1.17; P = 0.039). Stratification by study designs revealed no material changes in risk estimation. Meta-regression analysis found no significant sources of between-study heterogeneity for age, study design and geographic region for all comparisons. There was no identified publication bias. Taken together, our results demonstrated that AR CAG repeat polymorphism with ≥ 20 repeats might confer a protective effect among the prostate cancer patients with 45 years older but not all the prostate cancer patients.

  1. Clustered regularly interspaced short palindromic repeat associated protein genes cas1 and cas2 in Shigella%志贺菌成簇规律间隔短回文重复序列相关蛋白基因cas1和cas2研究

    Institute of Scientific and Technical Information of China (English)

    薛泽润; 王颖芳; 段广才; 王鹏飞; 王琳琳; 郭向娇; 郗园林

    2014-01-01

    Objective To detect the distribution of clustered regularly interspaced short palindromic repeat(CRISPR)associated protein genes cas1 and cas2 in Shigella and to understand the characteristics of CRISPR with relationship between CRISPR and related characteristics on drug resistance. Methods CRISPR associated protein genes cas1 and cas2 in Shigella were detected by PCR,with its products sequenced and compared.Results The CRISPR-associated protein genes cas1 and cas2 were found in all the 196 Shigella isolates which were isolated at different times and locations in China. Consistencies showed through related sequencing appepared as follows:cas2,cas1 (a) and cas1(b)were 96.44%,97.61%and 96.97%,respectively. There were two mutations including 3177129 site(C→G)and 3177126 site(G→C)of cas1(b)gene in 2003135 strain which were not found in the corresponding sites of Z23 and 2008113. Results showed that in terms of both susceptibility and antibiotic-resistance,strain 2003135 was stronger than Z23 and 2008113. Conclusion CRISPR system widely existed in Shigella,with the level of drug resistance in cas1(b) gene mutant strains higher than in wild strains. Cas1(b)gene mutation might be one of the reasons causing the different levels of resistance.%目的:研究成簇规律间隔短回文重复序列(CRISPR)相关蛋白基因cas1和cas2在志贺菌中的分布,并分析cas1和cas2基因突变与细菌耐药的关系。方法采用PCR扩增196株志贺菌cas1和cas2基因。对3株志贺菌(Z23、2003135、2008113)的cas2、cas1(a)和cas1(b)基因进行测序,分析其突变与耐药之间的关系。结果196株志贺菌均检出CRISPR相关蛋白基因cas1和cas2。测序结果显示,cas2的一致性为96.44%,cas1(a)的一致性为97.61%,cas1(b)的一致性为96.97%。菌株2003135的cas1(b)基因有2个突变位点:3177129位点(C→G)及3177126位点(G→C),Z23、2008113对应位置没有突变

  2. [Somatic hypermutagenesis in immunoglobulin genes. I. Connection of somatic mutations with repeats. A statistical weighting method].

    Science.gov (United States)

    Solov'ev, V V; Rogozin, I V; Kolchanov, N A

    1989-01-01

    Based on the analysis of a number of immunoglobulin genes' nucleotide sequences, it has been suggested, that somatic mutations emerge by means of imperfect duplexes correction, formed by mispairing of complementary regions of direct and inverted repeats. In the present work provides new data, confirming this mechanism of somatic hypermutagenesis. It has been shown that the presented sample of V- and J-segments of immunoglobulin genes is abundant in nonrandom imperfect direct repeats and complementary palindromes. To prove the connection of somatic mutations with the correction of imperfect duplexes, made up by the regions of these repeats, we have developed the method of statistical weights, permitting us to analyse the samples of mutations and repeats and to reveal the reliability of the connection between them. Using this method we have investigated the collection of 203 nucleotide substitutions in V- and J-segments and have shown a statistically reliable (P less than 10(-4) connection of these mutation positions with imperfect repeats.

  3. Analysis of the Complete Mycoplasma hominis LBD-4 Genome Sequence Reveals Strain-Variable Prophage Insertion and Distinctive Repeat-Containing Surface Protein Arrangements

    OpenAIRE

    2015-01-01

    The complete genome sequence of Mycoplasma hominis LBD-4 has been determined and the gene content ascribed. The 715,165-bp chromosome contains 620 genes, including 14 carried by a strain-variable prophage genome related to Mycoplasma fermentans MFV-1 and Mycoplasma arthritidis MAV-1. Comparative analysis with the genome of M. hominis PG21T reveals distinctive arrangements of repeat-containing surface proteins.

  4. Analysis of the Complete Mycoplasma hominis LBD-4 Genome Sequence Reveals Strain-Variable Prophage Insertion and Distinctive Repeat-Containing Surface Protein Arrangements.

    Science.gov (United States)

    Calcutt, Michael J; Foecking, Mark F

    2015-02-26

    The complete genome sequence of Mycoplasma hominis LBD-4 has been determined and the gene content ascribed. The 715,165-bp chromosome contains 620 genes, including 14 carried by a strain-variable prophage genome related to Mycoplasma fermentans MFV-1 and Mycoplasma arthritidis MAV-1. Comparative analysis with the genome of M. hominis PG21(T) reveals distinctive arrangements of repeat-containing surface proteins.

  5. Origin of a folded repeat protein from an intrinsically disordered ancestor

    Science.gov (United States)

    Zhu, Hongbo; Sepulveda, Edgardo; Hartmann, Marcus D; Kogenaru, Manjunatha; Ursinus, Astrid; Sulz, Eva; Albrecht, Reinhard; Coles, Murray; Martin, Jörg; Lupas, Andrei N

    2016-01-01

    Repetitive proteins are thought to have arisen through the amplification of subdomain-sized peptides. Many of these originated in a non-repetitive context as cofactors of RNA-based replication and catalysis, and required the RNA to assume their active conformation. In search of the origins of one of the most widespread repeat protein families, the tetratricopeptide repeat (TPR), we identified several potential homologs of its repeated helical hairpin in non-repetitive proteins, including the putatively ancient ribosomal protein S20 (RPS20), which only becomes structured in the context of the ribosome. We evaluated the ability of the RPS20 hairpin to form a TPR fold by amplification and obtained structures identical to natural TPRs for variants with 2–5 point mutations per repeat. The mutations were neutral in the parent organism, suggesting that they could have been sampled in the course of evolution. TPRs could thus have plausibly arisen by amplification from an ancestral helical hairpin. DOI: http://dx.doi.org/10.7554/eLife.16761.001 PMID:27623012

  6. Molecular cloning and long terminal repeat sequences of human endogenous retrovirus genes related to types A and B retrovirus genes

    Energy Technology Data Exchange (ETDEWEB)

    Ono, M.

    1986-06-01

    By using a DNA fragment primarily encoding the reverse transcriptase (pol) region of the Syrian hamster intracisternal A particle (IAP; type A retrovirus) gene as a probe, human endogenous retrovirus genes, tentatively termed HERV-K genes, were cloned from a fetal human liver gene library. Typical HERV-K genes were 9.1 or 9.4 kilobases in length, having long terminal repeats (LTRs) of ca. 970 base pairs. Many structural features commonly observed on the retrovirus LTRs, such as the TATAA box, polyadenylation signal, and terminal inverted repeats, were present on each LTR, and a lysine (K) tRNA having a CUU anticodon was identified as a presumed primer tRNA. The HERV-K LTR, however, had little sequence homology to either the IAP LTR or other typical oncovirus LTRs. By filter hybridization, the number of HERV-K genes was estimated to be ca. 50 copies per haploid human genome. The cloned mouse mammary tumor virus (type B) gene was found to hybridize with both the HERV-K and IAP genes to essentially the same extent.

  7. The impact of CRISPR repeat sequence on structures of a Cas6 protein-RNA complex

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Ruiying; Zheng, Han; Preamplume, Gan; Shao, Yaming; Li, Hong [FSU

    2012-03-15

    The repeat-associated mysterious proteins (RAMPs) comprise the most abundant family of proteins involved in prokaryotic immunity against invading genetic elements conferred by the clustered regularly interspaced short palindromic repeat (CRISPR) system. Cas6 is one of the first characterized RAMP proteins and is a key enzyme required for CRISPR RNA maturation. Despite a strong structural homology with other RAMP proteins that bind hairpin RNA, Cas6 distinctly recognizes single-stranded RNA. Previous structural and biochemical studies show that Cas6 captures the 5' end while cleaving the 3' end of the CRISPR RNA. Here, we describe three structures and complementary biochemical analysis of a noncatalytic Cas6 homolog from Pyrococcus horikoshii bound to CRISPR repeat RNA of different sequences. Our study confirms the specificity of the Cas6 protein for single-stranded RNA and further reveals the importance of the bases at Positions 5-7 in Cas6-RNA interactions. Substitutions of these bases result in structural changes in the protein-RNA complex including its oligomerization state.

  8. Structure of thrombospondin type 3 repeats in bacterial outer membrane protein A reveals its intra-repeat disulfide bond-dependent calcium-binding capability

    Energy Technology Data Exchange (ETDEWEB)

    Dai, Shuyan; Sun, Cancan; Tan, Kemin; Ye, Sheng; Zhang, Rongguang

    2017-09-01

    Eukaryotic thrombospondin type 3 repeat (TT3R) is an efficient calcium ion (Ca2+) binding motif only found in mammalian thrombospondin family. TT3R has also been found in prokaryotic cellulase Cel5G, which was thought to forfeit the Ca2+-binding capability due to the formation of intra-repeat disulfide bonds, instead of the inter-repeat ones possessed by eukaryotic TT3Rs. In this study, we have identified an enormous number of prokaryotic TT3R-containing proteins belonging to several different protein families, including outer membrane protein A (OmpA), an important structural protein connecting the outer membrane and the periplasmic peptidoglycan layer in gram-negative bacteria. Here, we report the crystal structure of the periplasmic region of OmpA from Capnocytophaga gingivalis, which contains a linker region comprising five consecutive TT3Rs. The structure of OmpA-TT3R exhibits a well-ordered architecture organized around two tightly-coordinated Ca2+ and confirms the presence of abnormal intra-repeat disulfide bonds. Further mutagenesis studies showed that the Ca2+-binding capability of OmpA-TT3R is indeed dependent on the proper formation of intra-repeat disulfide bonds, which help to fix a conserved glycine residue at its proper position for Ca2+ coordination. Additionally, despite lacking inter repeat disulfide bonds, the interfaces between adjacent OmpA-TT3Rs are enhanced by both hydrophobic and conserved aromatic-proline interactions.

  9. Evolutionary dynamics of leucine-rich repeat receptor-like kinases and related genes in plants:A phylogenomic approach

    Institute of Scientific and Technical Information of China (English)

    Tao Shi; Hongwen Huang; Michael J.Sanderson; Frans E.Tax

    2014-01-01

    Leucine-rich repeat (LRR) receptor-like kinases (RLKs), evolutionarily related LRR receptor-like proteins (RLPs) and receptor-like cytoplasmic kinases (RLCKs) have important roles in plant signaling, and their gene subfamilies are large with a complicated history of gene duplication and loss. In three pairs of closely related lineages, including Arabidopsis thaliana and A. lyrata (Arabidopsis), Lotus japonicus, and Medicago truncatula (Legumes), Oryza sativa ssp. japonica, and O. sativa ssp. indica (Rice), we find that LRR RLKs comprise the largest group of these LRR-related subfamilies, while the related RLCKs represent the smal est group. In addition, comparison of orthologs indicates a high frequency of reciprocal gene loss of the LRR RLK/LRR RLP/RLCK subfamilies. Furthermore, pairwise comparisons show that reciprocal gene loss is often associated with lineage-specific duplication(s) in the alternative lineage. Last, analysis of genes in A. thaliana involved in development revealed that most are highly conserved orthologs without species-specific duplication in the two Arabidopsis species and originated from older Arabidopsis-specific or rosid-specific duplications. We discuss potential pitfal s related to functional prediction for genes that have undergone frequent turnover (duplications, losses, and domain architecture changes), and conclude that prediction based on phylogenetic relationships wil likely outperform that based on sequence similarity alone.

  10. CAG repeat length in androgen receptor gene is not associated with amyotrophic lateral sclerosis.

    Science.gov (United States)

    Bruson, A; Sambataro, F; Querin, G; D'Ascenzo, C; Palmieri, A; Agostini, J; Gaiani, A; Angelini, C; Galbiati, M; Poletti, A; Pennuto, M; Pegoraro, E; Clementi, M; Soraru, G

    2012-10-01

    Epidemiological and clinical studies show higher prevalence of amyotrophic lateral sclerosis (ALS) in males than in females and more severe lesions in androgen receptor (AR)-expressing tissues. The AR gene contains a polymorphic CAG trinucleotide repeat, whose expansion over a certain threshold is toxic to motor neurons, causing spinal and bulbar muscular atrophy (SBMA). We tested the hypothesis that the AR CAG repeat linked to SBMA is a risk factor for ALS. We analyzed AR CAG expansions in 336 patients with ALS and 100 controls. We found a negative association of AR CAG expansions with ALS susceptibility, clinical presentation, and survival. Our findings do not support a role of the AR CAG repeat length in ALS. © 2012 The Author(s) European Journal of Neurology © 2012 EFNS.

  11. A novel tetratricopeptide repeat (TPR containing PP5 serine/threonine protein phosphatase in the malaria parasite, Plasmodium falciparum

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    Adams Brian

    2001-11-01

    Full Text Available Abstract Background The malarial parasite, Plasmodium falciparum (Pf, is responsible for nearly 2 million deaths worldwide. However, the mechanisms of cellular signaling in the parasite remain largely unknown. Recent discovery of a few protein kinases and phosphatases point to a thriving reversible phosphorylation system in the parasite, although their function and regulation need to be determined. Results We provide biochemical and sequence evidence for a protein serine/threonine phosphatase type PP5 in Plasmodium falciparum, and named it PfPP5. The 594-amino acid polypeptide was encoded by a 1785 nucleotide long intronless gene in the parasite. The recombinant protein, expressed in bacteria, was indistinguishable from native PfPP5. Sequencing comparison indicated that the extra-long N-terminus of PfPP5 outside the catalytic core contained four tetratricopeptide repeats (TPRs, compared to three such repeats in other PP5 phosphatases. The PfPP5 N-terminus was required for stimulation of the phosphatase activity by polyunsaturated fatty acids. Co-immunoprecipitation demonstrated an interaction between native PfPP5 and Pf heat shock protein 90 (hsp90. PfPP5 was expressed in all the asexual erythrocytic stages of the parasite, and was moderately sensitive to okadaic acid. Conclusions This is the first example of a TPR-domain protein in the Apicomplexa family of parasites. Since TPR domains play important roles in protein-protein interaction, especially relevant to the regulation of PP5 phosphatases, PfPP5 is destined to have a definitive role in parasitic growth and signaling pathways. This is exemplified by the interaction between PfPP5 and the cognate chaperone hsp90.

  12. The Influenza A Virus Non-structural Protein NS1 Upregulates The Expression of Collagen Triple Helix Repeat Containing 1 Protein.

    Science.gov (United States)

    Zhu, C; Peng, G; Yi, W; Song, H; Liu, F; Liu, X

    2016-12-01

    Influenza A virus (IAV) infection induces a strong immune response and regulates the expression of many host proteins. The collagen triple helix repeat containing 1 (CTHRC1) protein is a secreted protein that exhibits increased expression during the viral infection process. However, the regulatory function of IAV on CTHRC1 expression is obscure. In this study, we investigated the effect of IAV on CTHRC1 expression and its regulatory mechanism. A total of 106 serum specimens from healthy people and 80 serum specimens from patients infected with IAV were collected. The CTHRC1 levels in the sera from the IVA patients and healthy individuals were measured using an enzyme-linked immunosorbent assay (ELISA), and the differences were statistically analysed. A549 cells were infected with the IAV or delNS1 virus. Additionally, A549 cells were cotransfected with a eukaryotic non-structural NS1 protein gene expression plasmid and the CTHRC1 gene promoter reporter plasmid (pCTHRC1-Luc), and, the luciferase activities were assessed. The CTHRC1 mRNA and protein expression were detected using reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. The serum CTHRC1 level was significantly higher in the IAV patients than in the healthy individuals. IAV upregulated the CTHRC1 mRNA and protein expression. The non-structural NS1 protein specifically activated CTHRC1 gene promoter activity and upregulated CTHRC1 mRNA and protein expression. The activation function had a dose-dependent effect, indicating that influenza virus upregulated CTHRC1 expression through its NS1 protein.

  13. TANDEM REPEATING OF THE EXPRESSION CARTRIDGE—— A NOVEL STRATEGY TO ENHANCE THE EXPRESSION EFFICIENCY OF CLONED GENE IN ESCHERICHIA COLI

    Institute of Scientific and Technical Information of China (English)

    林缨; 卢圣栋

    1996-01-01

    A novel strategy to enhance the expression eHiciency of cloned target gene in Escher&hia colt was developed. The whole expression cartridge, consisting of promoter, SD sequence, target gene and transcription terminator, was tandem repeatedly engineered into a expression plasmld. Consequently, the copynumber of specific gene was increased substantially, leading to the improvemem of expression efficiency.Using this approach, a recombinant plasmid, designed as pLYD, was constructed and transformated into the Escherichia coti strain DHSa. Upon induction, the desired protein was synthesized in a considerable level and accumulated up to 63% of the total cell proteins, The pretent study reveated that taildem repeating of expression cartridge provided a convenient means to improve expression level efficiently.

  14. Duplication and relocation of the functional DPY19L2 gene within low copy repeats

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    Cheung Joseph

    2006-03-01

    Full Text Available Abstract Background Low copy repeats (LCRs are thought to play an important role in recent gene evolution, especially when they facilitate gene duplications. Duplicate genes are fundamental to adaptive evolution, providing substrates for the development of new or shared gene functions. Moreover, silencing of duplicate genes can have an indirect effect on adaptive evolution by causing genomic relocation of functional genes. These changes are theorized to have been a major factor in speciation. Results Here we present a novel example showing functional gene relocation within a LCR. We characterize the genomic structure and gene content of eight related LCRs on human Chromosomes 7 and 12. Two members of a novel transmembrane gene family, DPY19L, were identified in these regions, along with six transcribed pseudogenes. One of these genes, DPY19L2, is found on Chromosome 12 and is not syntenic with its mouse orthologue. Instead, the human locus syntenic to mouse Dpy19l2 contains a pseudogene, DPY19L2P1. This indicates that the ancestral copy of this gene has been silenced, while the descendant copy has remained active. Thus, the functional copy of this gene has been relocated to a new genomic locus. We then describe the expansion and evolution of the DPY19L gene family from a single gene found in invertebrate animals. Ancient duplications have led to multiple homologues in different lineages, with three in fish, frogs and birds and four in mammals. Conclusion Our results show that the DPY19L family has expanded throughout the vertebrate lineage and has undergone recent primate-specific evolution within LCRs.

  15. Exosome-bound WD repeat protein Monad inhibits breast cancer cell invasion by degrading amphiregulin mRNA.

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    Makio Saeki

    Full Text Available Increased stabilization of mRNA coding for key cancer genes can contribute to invasiveness. This is achieved by down-regulation of exosome cofactors, which bind to 3'-UTR in cancer-related genes. Here, we identified amphiregulin, an EGFR ligand, as a target of WD repeat protein Monad, a component of R2TP/prefoldin-like complex, in MDA-MB-231 breast cancer cells. Monad specifically interacted with both the 3'-UTR of amphiregulin mRNA and the RNA degrading exosome, and enhanced decay of amphiregulin transcripts. Knockdown of Monad increased invasion and this effect was abolished with anti-amphiregulin neutralizing antibody. These results suggest that Monad could prevent amphiregulin-mediated invasion by degrading amphiregulin mRNA.

  16. LRR conservation mapping to predict functional sites within protein leucine-rich repeat domains.

    Directory of Open Access Journals (Sweden)

    Laura Helft

    Full Text Available Computational prediction of protein functional sites can be a critical first step for analysis of large or complex proteins. Contemporary methods often require several homologous sequences and/or a known protein structure, but these resources are not available for many proteins. Leucine-rich repeats (LRRs are ligand interaction domains found in numerous proteins across all taxonomic kingdoms, including immune system receptors in plants and animals. We devised Repeat Conservation Mapping (RCM, a computational method that predicts functional sites of LRR domains. RCM utilizes two or more homologous sequences and a generic representation of the LRR structure to identify conserved or diversified patches of amino acids on the predicted surface of the LRR. RCM was validated using solved LRR+ligand structures from multiple taxa, identifying ligand interaction sites. RCM was then used for de novo dissection of two plant microbe-associated molecular pattern (MAMP receptors, EF-TU RECEPTOR (EFR and FLAGELLIN-SENSING 2 (FLS2. In vivo testing of Arabidopsis thaliana EFR and FLS2 receptors mutagenized at sites identified by RCM demonstrated previously unknown functional sites. The RCM predictions for EFR, FLS2 and a third plant LRR protein, PGIP, compared favorably to predictions from ODA (optimal docking area, Consurf, and PAML (positive selection analyses, but RCM also made valid functional site predictions not available from these other bioinformatic approaches. RCM analyses can be conducted with any LRR-containing proteins at www.plantpath.wisc.edu/RCM, and the approach should be modifiable for use with other types of repeat protein domains.

  17. Albino Leaf1 That Encodes the Sole Octotricopeptide Repeat Protein Is Responsible for Chloroplast Development1[OPEN

    Science.gov (United States)

    Tan, Jianjie; Xing, Yi; Liu, Changhong; Chen, Qiaoling; Zhu, Haitao; Wang, Jiang; Zhang, Jingliu; Zhang, Guiquan

    2016-01-01

    Chloroplast, the photosynthetic organelle in plants, plays a crucial role in plant development and growth through manipulating the capacity of photosynthesis. However, the regulatory mechanism of chloroplast development still remains elusive. Here, we characterized a mutant with defective chloroplasts in rice (Oryza sativa), termed albino leaf1 (al1), which exhibits a distinct albino phenotype in leaves, eventually leading to al1 seedling lethality. Electronic microscopy observation demonstrated that the number of thylakoids was reduced and the structure of thylakoids was disrupted in the al1 mutant during rice development, which eventually led to the breakdown of chloroplast. Molecular cloning revealed that AL1 encodes the sole octotricopeptide repeat protein (RAP) in rice. Genetic complementation of Arabidopsis (Arabidopsis thaliana) rap mutants indicated that the AL1 protein is a functional RAP. Further analysis illustrated that three transcript variants were present in the AL1 gene, and the altered splices occurred at the 3′ untranslated region of the AL1 transcript. In addition, our results also indicate that disruption of the AL1 gene results in an altered expression of chloroplast-associated genes. Consistently, proteomic analysis demonstrated that the abundance of photosynthesis-associated proteins is altered significantly, as is that of a group of metabolism-associated proteins. More specifically, we found that the loss of AL1 resulted in altered abundances of ribosomal proteins, suggesting that RAP likely also regulates the homeostasis of ribosomal proteins in rice in addition to the ribosomal RNA. Taken together, we propose that AL1, particularly the AL1a and AL1c isoforms, plays an essential role in chloroplast development in rice. PMID:27208287

  18. Positive Selection of a Pre-Expansion CAG Repeat of the Human SCA2 Gene.

    Directory of Open Access Journals (Sweden)

    2005-09-01

    Full Text Available A region of approximately one megabase of human Chromosome 12 shows extensive linkage disequilibrium in Utah residents with ancestry from northern and western Europe. This strikingly large linkage disequilibrium block was analyzed with statistical and experimental methods to determine whether natural selection could be implicated in shaping the current genome structure. Extended Haplotype Homozygosity and Relative Extended Haplotype Homozygosity analyses on this region mapped a core region of the strongest conserved haplotype to the exon 1 of the Spinocerebellar ataxia type 2 gene (SCA2. Direct DNA sequencing of this region of the SCA2 gene revealed a significant association between a pre-expanded allele [(CAG(8CAA(CAG(4CAA(CAG(8] of CAG repeats within exon 1 and the selected haplotype of the SCA2 gene. A significantly negative Tajima's D value (-2.20, p < 0.01 on this site consistently suggested selection on the CAG repeat. This region was also investigated in the three other populations, none of which showed signs of selection. These results suggest that a recent positive selection of the pre-expansion SCA2 CAG repeat has occurred in Utah residents with European ancestry.

  19. Positive selection of a pre-expansion CAG repeat of the human SCA2 gene.

    Directory of Open Access Journals (Sweden)

    Fuli Yu

    2005-09-01

    Full Text Available A region of approximately one megabase of human Chromosome 12 shows extensive linkage disequilibrium in Utah residents with ancestry from northern and western Europe. This strikingly large linkage disequilibrium block was analyzed with statistical and experimental methods to determine whether natural selection could be implicated in shaping the current genome structure. Extended Haplotype Homozygosity and Relative Extended Haplotype Homozygosity analyses on this region mapped a core region of the strongest conserved haplotype to the exon 1 of the Spinocerebellar ataxia type 2 gene (SCA2. Direct DNA sequencing of this region of the SCA2 gene revealed a significant association between a pre-expanded allele [(CAG8CAA(CAG4CAA(CAG8] of CAG repeats within exon 1 and the selected haplotype of the SCA2 gene. A significantly negative Tajima's D value (-2.20, p < 0.01 on this site consistently suggested selection on the CAG repeat. This region was also investigated in the three other populations, none of which showed signs of selection. These results suggest that a recent positive selection of the pre-expansion SCA2 CAG repeat has occurred in Utah residents with European ancestry.

  20. Liat1, an arginyltransferase-binding protein whose evolution among primates involved changes in the numbers of its 10-residue repeats.

    Science.gov (United States)

    Brower, Christopher S; Rosen, Connor E; Jones, Richard H; Wadas, Brandon C; Piatkov, Konstantin I; Varshavsky, Alexander

    2014-11-18

    The arginyltransferase Ate1 is a component of the N-end rule pathway, which recognizes proteins containing N-terminal degradation signals called N-degrons, polyubiquitylates these proteins, and thereby causes their degradation by the proteasome. At least six isoforms of mouse Ate1 are produced through alternative splicing of Ate1 pre-mRNA. We identified a previously uncharacterized mouse protein, termed Liat1 (ligand of Ate1), that interacts with Ate1 but does not appear to be its arginylation substrate. Liat1 has a higher affinity for the isoforms Ate1(1A7A) and Ate1(1B7A). Liat1 stimulated the in vitro N-terminal arginylation of a model substrate by Ate1. All examined vertebrate and some invertebrate genomes encode proteins sequelogous (similar in sequence) to mouse Liat1. Sequelogs of Liat1 share a highly conserved ∼30-residue region that is shown here to be required for the binding of Liat1 to Ate1. We also identified non-Ate1 proteins that interact with Liat1. In contrast to Liat1 genes of nonprimate mammals, Liat1 genes of primates are subtelomeric, a location that tends to confer evolutionary instability on a gene. Remarkably, Liat1 proteins of some primates, from macaques to humans, contain tandem repeats of a 10-residue sequence, whereas Liat1 proteins of other mammals contain a single copy of this motif. Quantities of these repeats are, in general, different in Liat1 of different primates. For example, there are 1, 4, 13, 13, 17, and 17 repeats in the gibbon, gorilla, orangutan, bonobo, neanderthal, and human Liat1, respectively, suggesting that repeat number changes in this previously uncharacterized protein may contribute to evolution of primates.

  1. Molecular phylogeny of the kelch-repeat superfamily reveals an expansion of BTB/kelch proteins in animals

    Directory of Open Access Journals (Sweden)

    Adams Josephine C

    2003-09-01

    Full Text Available Abstract Background The kelch motif is an ancient and evolutionarily-widespread sequence motif of 44–56 amino acids in length. It occurs as five to seven repeats that form a β-propeller tertiary structure. Over 28 kelch-repeat proteins have been sequenced and functionally characterised from diverse organisms spanning from viruses, plants and fungi to mammals and it is evident from expressed sequence tag, domain and genome databases that many additional hypothetical proteins contain kelch-repeats. In general, kelch-repeat β-propellers are involved in protein-protein interactions, however the modest sequence identity between kelch motifs, the diversity of domain architectures, and the partial information on this protein family in any single species, all present difficulties to developing a coherent view of the kelch-repeat domain and the kelch-repeat protein superfamily. To understand the complexity of this superfamily of proteins, we have analysed by bioinformatics the complement of kelch-repeat proteins encoded in the human genome and have made comparisons to the kelch-repeat proteins encoded in other sequenced genomes. Results We identified 71 kelch-repeat proteins encoded in the human genome, whereas 5 or 8 members were identified in yeasts and around 18 in C. elegans, D. melanogaster and A. gambiae. Multiple domain architectures were identified in each organism, including previously unrecognised forms. The vast majority of kelch-repeat domains are predicted to form six-bladed β-propellers. The most prevalent domain architecture in the metazoan animal genomes studied was the BTB/kelch domain organisation and we uncovered 3 subgroups of human BTB/kelch proteins. Sequence analysis of the kelch-repeat domains of the most robustly-related subgroups identified differences in β-propeller organisation that could provide direction for experimental study of protein-binding characteristics. Conclusion The kelch-repeat superfamily constitutes a

  2. Alternative conformations of the Tau repeat domain in complex with an engineered binding protein.

    Science.gov (United States)

    Grüning, Clara S R; Mirecka, Ewa A; Klein, Antonia N; Mandelkow, Eckhard; Willbold, Dieter; Marino, Stephen F; Stoldt, Matthias; Hoyer, Wolfgang

    2014-08-15

    The aggregation of Tau into paired helical filaments is involved in the pathogenesis of several neurodegenerative diseases, including Alzheimer disease. The aggregation reaction is characterized by conformational conversion of the repeat domain, which partially adopts a cross-β-structure in the resulting amyloid-like fibrils. Here, we report the selection and characterization of an engineered binding protein, β-wrapin TP4, targeting the Tau repeat domain. TP4 was obtained by phage display using the four-repeat Tau construct K18ΔK280 as a target. TP4 binds K18ΔK280 as well as the longest isoform of human Tau, hTau40, with nanomolar affinity. NMR spectroscopy identified two alternative TP4-binding sites in the four-repeat domain, with each including two hexapeptide motifs with high β-sheet propensity. Both binding sites contain the aggregation-determining PHF6 hexapeptide within repeat 3. In addition, one binding site includes the PHF6* hexapeptide within repeat 2, whereas the other includes the corresponding hexapeptide Tau(337-342) within repeat 4, denoted PHF6**. Comparison of TP4-binding with Tau aggregation reveals that the same regions of Tau are involved in both processes. TP4 inhibits Tau aggregation at substoichiometric concentration, demonstrating that it interferes with aggregation nucleation. This study provides residue-level insight into the interaction of Tau with an aggregation inhibitor and highlights the structural flexibility of Tau. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Target genes of microsatellite sequences in head and neck squamous cell carcinoma: mononucleotide repeats are not detected.

    Science.gov (United States)

    Wang, Yimin; Liu, Xuejuan; Li, Yulin

    2012-09-10

    Microsatellite instability (MSI) is detected in a wide variety of tumors. It is thought that mismatch repair gene mutation or inactivation is the major cause of MSI. Microsatellite sequences are predominantly distributed in intergenic or intronic DNA. However, MSI is found in the exonic sequences of some genes, causing their inactivation. In this report, we searched GenBank for candidate genes containing potential MSI sequences in exonic regions. Twenty seven target genes were selected for MSI analysis. Instability was found in 70% of these genes (14/20) with head and neck squamous cell carcinoma (HNSCC). Interestingly, no instability was detected in mononucleotide repeats in genes or in intergenic sequences. We conclude that instability of mononucleotide repeats is a rare event in HNSCC. High MSI phenotype in young HNSCC patients is limited to noncoding regions only. MSI percentage in HNSCC tumor is closely related to the repeat type, repeat location and patient's age.

  4. DNA-binding proteins from marine bacteria expand the known sequence diversity of TALE-like repeats.

    Science.gov (United States)

    de Lange, Orlando; Wolf, Christina; Thiel, Philipp; Krüger, Jens; Kleusch, Christian; Kohlbacher, Oliver; Lahaye, Thomas

    2015-11-16

    Transcription Activator-Like Effectors (TALEs) of Xanthomonas bacteria are programmable DNA binding proteins with unprecedented target specificity. Comparative studies into TALE repeat structure and function are hindered by the limited sequence variation among TALE repeats. More sequence-diverse TALE-like proteins are known from Ralstonia solanacearum (RipTALs) and Burkholderia rhizoxinica (Bats), but RipTAL and Bat repeats are conserved with those of TALEs around the DNA-binding residue. We study two novel marine-organism TALE-like proteins (MOrTL1 and MOrTL2), the first to date of non-terrestrial origin. We have assessed their DNA-binding properties and modelled repeat structures. We found that repeats from these proteins mediate sequence specific DNA binding conforming to the TALE code, despite low sequence similarity to TALE repeats, and with novel residues around the BSR. However, MOrTL1 repeats show greater sequence discriminating power than MOrTL2 repeats. Sequence alignments show that there are only three residues conserved between repeats of all TALE-like proteins including the two new additions. This conserved motif could prove useful as an identifier for future TALE-likes. Additionally, comparing MOrTL repeats with those of other TALE-likes suggests a common evolutionary origin for the TALEs, RipTALs and Bats.

  5. Effects of acute versus repeated cocaine exposure on the expression of endocannabinoid signaling-related proteins in the mouse cerebellum

    Science.gov (United States)

    Palomino, Ana; Pavón, Francisco-Javier; Blanco-Calvo, Eduardo; Serrano, Antonia; Arrabal, Sergio; Rivera, Patricia; Alén, Francisco; Vargas, Antonio; Bilbao, Ainhoa; Rubio, Leticia; Rodríguez de Fonseca, Fernando; Suárez, Juan

    2014-01-01

    Growing awareness of cerebellar involvement in addiction is based on the cerebellum’s intermediary position between motor and reward, potentially acting as an interface between motivational and cognitive functions. Here, we examined the impact of acute and repeated cocaine exposure on the two main signaling systems in the mouse cerebellum: the endocannabinoid (eCB) and glutamate systems. To this end, we investigated whether eCB signaling-related gene and protein expression {cannabinoid receptor type 1 receptors and enzymes that produce [diacylglycerol lipase alpha/beta (DAGLα/β) and N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD)] and degrade [monoacylglycerol lipase (MAGL) and fatty acid amino hydrolase (FAAH)] eCB} were altered. In addition, we analyzed the gene expression of relevant components of the glutamate signaling system [glutamate synthesizing enzymes liver-type glutaminase isoform (LGA) and kidney-type glutaminase isoform (KGA), metabotropic glutamatergic receptor (mGluR3/5), NMDA-ionotropic glutamatergic receptor (NR1/2A/2B/2C) and AMPA-ionotropic receptor subunits (GluR1/2/3/4)] and the gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, because noradrenergic terminals innervate the cerebellar cortex. Results indicated that acute cocaine exposure decreased DAGLα expression, suggesting a down-regulation of 2-arachidonylglycerol (2-AG) production, as well as gene expression of TH, KGA, mGluR3 and all ionotropic receptor subunits analyzed in the cerebellum. The acquisition of conditioned locomotion and sensitization after repeated cocaine exposure were associated with an increased NAPE-PLD/FAAH ratio, suggesting enhanced anandamide production, and a decreased DAGLβ/MAGL ratio, suggesting decreased 2-AG generation. Repeated cocaine also increased LGA gene expression but had no effect on glutamate receptors. These findings indicate that acute cocaine modulates the expression of the eCB and

  6. Repeated evolution of chimeric fusion genes in the β-globin gene family of laurasiatherian mammals.

    Science.gov (United States)

    Gaudry, Michael J; Storz, Jay F; Butts, Gary Tyler; Campbell, Kevin L; Hoffmann, Federico G

    2014-05-09

    The evolutionary fate of chimeric fusion genes may be strongly influenced by their recombinational mode of origin and the nature of functional divergence between the parental genes. In the β-globin gene family of placental mammals, the two postnatally expressed δ- and β-globin genes (HBD and HBB, respectively) have a propensity for recombinational exchange via gene conversion and unequal crossing-over. In the latter case, there are good reasons to expect differences in retention rates for the reciprocal HBB/HBD and HBD/HBB fusion genes due to thalassemia pathologies associated with the HBD/HBB "Lepore" deletion mutant in humans. Here, we report a comparative genomic analysis of the mammalian β-globin gene cluster, which revealed that chimeric HBB/HBD fusion genes originated independently in four separate lineages of laurasiatherian mammals: Eulipotyphlans (shrews, moles, and hedgehogs), carnivores, microchiropteran bats, and cetaceans. In cases where an independently derived "anti-Lepore" duplication mutant has become fixed, the parental HBD and/or HBB genes have typically been inactivated or deleted, so that the newly created HBB/HBD fusion gene is primarily responsible for synthesizing the β-type subunits of adult and fetal hemoglobin (Hb). Contrary to conventional wisdom that the HBD gene is a vestigial relict that is typically inactivated or expressed at negligible levels, we show that HBD-like genes often encode a substantial fraction (20-100%) of β-chain Hbs in laurasiatherian taxa. Our results indicate that the ascendancy or resuscitation of genes with HBD-like coding sequence requires the secondary acquisition of HBB-like promoter sequence via unequal crossing-over or interparalog gene conversion.

  7. One repeat of the cell wall binding domain is sufficient for anchoring the Lactobacillus acidophilus surface layer protein

    NARCIS (Netherlands)

    Smit, E.; Pouwels, P.H.

    2002-01-01

    The N-terminal repeat (SAC1) of the S-protein of Lactobacillus acidophilus bound efficiently and specifically to cell wall fragments (CWFs) when fused to green fluorescent protein, whereas the C-terminal repeat (SAC2) did not. Treatment of CWFs with hydrofluoric acid, but not phenol, prevented bindi

  8. The BC component of ABC toxins is an RHS-repeat-containing protein encapsulation device.

    Science.gov (United States)

    Busby, Jason N; Panjikar, Santosh; Landsberg, Michael J; Hurst, Mark R H; Lott, J Shaun

    2013-09-26

    The ABC toxin complexes produced by certain bacteria are of interest owing to their potent insecticidal activity and potential role in human disease. These complexes comprise at least three proteins (A, B and C), which must assemble to be fully toxic. The carboxy-terminal region of the C protein is the main cytotoxic component, and is poorly conserved between different toxin complexes. A general model of action has been proposed, in which the toxin complex binds to the cell surface via the A protein, is endocytosed, and subsequently forms a pH-triggered channel, allowing the translocation of C into the cytoplasm, where it can cause cytoskeletal disruption in both insect and mammalian cells. Toxin complexes have been visualized using single-particle electron microscopy, but no high-resolution structures of the components are available, and the role of the B protein in the mechanism of toxicity remains unknown. Here we report the three-dimensional structure of the complex formed between the B and C proteins, determined to 2.5 Å by X-ray crystallography. These proteins assemble to form an unprecedented, large hollow structure that encapsulates and sequesters the cytotoxic, C-terminal region of the C protein like the shell of an egg. The shell is decorated on one end by a β-propeller domain, which mediates attachment of the B-C heterodimer to the A protein in the native complex. The structure reveals how C auto-proteolyses when folded in complex with B. The C protein is the first example, to our knowledge, of a structure that contains rearrangement hotspot (RHS) repeats, and illustrates a marked structural architecture that is probably conserved across both this widely distributed bacterial protein family and the related eukaryotic tyrosine-aspartate (YD)-repeat-containing protein family, which includes the teneurins. The structure provides the first clues about the function of these protein repeat families, and suggests a generic mechanism for protein

  9. Meta-Analysis of DNA Tumor-Viral Integration Site Selection Indicates a Role for Repeats, Gene Expression and Epigenetics

    Directory of Open Access Journals (Sweden)

    Janet M. Doolittle-Hall

    2015-11-01

    Full Text Available Oncoviruses cause tremendous global cancer burden. For several DNA tumor viruses, human genome integration is consistently associated with cancer development. However, genomic features associated with tumor viral integration are poorly understood. We sought to define genomic determinants for 1897 loci prone to hosting human papillomavirus (HPV, hepatitis B virus (HBV or Merkel cell polyomavirus (MCPyV. These were compared to HIV, whose enzyme-mediated integration is well understood. A comprehensive catalog of integration sites was constructed from the literature and experimentally-determined HPV integration sites. Features were scored in eight categories (genes, expression, open chromatin, histone modifications, methylation, protein binding, chromatin segmentation and repeats and compared to random loci. Random forest models determined loci classification and feature selection. HPV and HBV integrants were not fragile site associated. MCPyV preferred integration near sensory perception genes. Unique signatures of integration-associated predictive genomic features were detected. Importantly, repeats, actively-transcribed regions and histone modifications were common tumor viral integration signatures.

  10. Comparison of serum leptin, glucose, total cholesterol and total protein levels in fertile and repeat breeder cows

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    Saime Guzel

    2014-12-01

    Full Text Available In the present study we measured serum glucose, leptin, total cholesterol and total protein concentrations in repeat breeder cows and compared them with fertile cows. For this aim, 20 repeat breeder cows and 20 fertile cows were used as material. Repeat breeder cows were found to have lower levels of leptin and glucose as compared with fertile ones. No significant differences in total cholesterol and total protein levels were observed between the two groups. No significant correlation of leptin with glucose, total cholesterol and total protein was observed in fertile and repeat breeder cows. Low concentrations of glucose and leptin can have some effects on reproductive problems as repeat breeder and help to understand potential mechanisms impairing fertility in repeat breeder cows.

  11. MNF, an ankyrin repeat protein of myxoma virus, is part of a native cellular SCF complex during viral infection.

    Science.gov (United States)

    Blanié, Sophie; Gelfi, Jacqueline; Bertagnoli, Stéphane; Camus-Bouclainville, Christelle

    2010-03-08

    Myxoma virus (MYXV), a member of the Poxviridae family, is the agent responsible for myxomatosis, a fatal disease in the European rabbit (Oryctolagus cuniculus). Like all poxviruses, MYXV is known for encoding multiple proteins that regulate cellular signaling pathways. Among them, four proteins share the same ANK/PRANC structure: M148R, M149R, MNF (Myxoma Nuclear factor) and M-T5, all of them described as virulence factors. This family of poxvirus proteins, recently identified, has drawn considerable attention for its potential role in modulating the host ubiquitin-proteasome system during viral infection. To date, many members of this novel protein family have been shown to interact with SCF components, in vitro. Here, we focus on MNF gene, which has been shown to express a nuclear protein presenting nine ANK repeats, one of which has been identified as a nuclear localization signal. In transfection, MNF has been shown to colocalise with the transcription factor NF-kappaB in the nucleus of TNFalpha-stimulated cells. Functionally, MNF is a critical virulence factor since its deletion generates an almost apathogenic virus. In this study, to pursue the investigation of proteins interacting with MNF and of its mechanism of action, we engineered a recombinant MYXV expressing a GFP-linked MNF under the control of MNF native promoter. Infection of rabbits with MYXV-GFPMNF recombinant virus provided the evidence that the GFP fusion does not disturb the main function of MNF. Hence, cells were infected with MYXV-GFPMNF and immunoprecipitation of the GFPMNF fusion protein was performed to identify MNF's partners. For the first time, endogenous components of SCF (Cullin-1 and Skp1) were co-precipitated with an ANK myxoma virus protein, expressed in an infectious context, and without over-expression of any protein.

  12. MNF, an ankyrin repeat protein of myxoma virus, is part of a native cellular SCF complex during viral infection

    Directory of Open Access Journals (Sweden)

    Gelfi Jacqueline

    2010-03-01

    Full Text Available Abstract Myxoma virus (MYXV, a member of the Poxviridae family, is the agent responsible for myxomatosis, a fatal disease in the European rabbit (Oryctolagus cuniculus. Like all poxviruses, MYXV is known for encoding multiple proteins that regulate cellular signaling pathways. Among them, four proteins share the same ANK/PRANC structure: M148R, M149R, MNF (Myxoma Nuclear factor and M-T5, all of them described as virulence factors. This family of poxvirus proteins, recently identified, has drawn considerable attention for its potential role in modulating the host ubiquitin-proteasome system during viral infection. To date, many members of this novel protein family have been shown to interact with SCF components, in vitro. Here, we focus on MNF gene, which has been shown to express a nuclear protein presenting nine ANK repeats, one of which has been identified as a nuclear localization signal. In transfection, MNF has been shown to colocalise with the transcription factor NF-κB in the nucleus of TNFα-stimulated cells. Functionally, MNF is a critical virulence factor since its deletion generates an almost apathogenic virus. In this study, to pursue the investigation of proteins interacting with MNF and of its mechanism of action, we engineered a recombinant MYXV expressing a GFP-linked MNF under the control of MNF native promoter. Infection of rabbits with MYXV-GFPMNF recombinant virus provided the evidence that the GFP fusion does not disturb the main function of MNF. Hence, cells were infected with MYXV-GFPMNF and immunoprecipitation of the GFPMNF fusion protein was performed to identify MNF's partners. For the first time, endogenous components of SCF (Cullin-1 and Skp1 were co-precipitated with an ANK myxoma virus protein, expressed in an infectious context, and without over-expression of any protein.

  13. MNF, an ankyrin repeat protein of myxoma virus, is part of a native cellular SCF complex during viral infection

    Science.gov (United States)

    2010-01-01

    Myxoma virus (MYXV), a member of the Poxviridae family, is the agent responsible for myxomatosis, a fatal disease in the European rabbit (Oryctolagus cuniculus). Like all poxviruses, MYXV is known for encoding multiple proteins that regulate cellular signaling pathways. Among them, four proteins share the same ANK/PRANC structure: M148R, M149R, MNF (Myxoma Nuclear factor) and M-T5, all of them described as virulence factors. This family of poxvirus proteins, recently identified, has drawn considerable attention for its potential role in modulating the host ubiquitin-proteasome system during viral infection. To date, many members of this novel protein family have been shown to interact with SCF components, in vitro. Here, we focus on MNF gene, which has been shown to express a nuclear protein presenting nine ANK repeats, one of which has been identified as a nuclear localization signal. In transfection, MNF has been shown to colocalise with the transcription factor NF-κB in the nucleus of TNFα-stimulated cells. Functionally, MNF is a critical virulence factor since its deletion generates an almost apathogenic virus. In this study, to pursue the investigation of proteins interacting with MNF and of its mechanism of action, we engineered a recombinant MYXV expressing a GFP-linked MNF under the control of MNF native promoter. Infection of rabbits with MYXV-GFPMNF recombinant virus provided the evidence that the GFP fusion does not disturb the main function of MNF. Hence, cells were infected with MYXV-GFPMNF and immunoprecipitation of the GFPMNF fusion protein was performed to identify MNF's partners. For the first time, endogenous components of SCF (Cullin-1 and Skp1) were co-precipitated with an ANK myxoma virus protein, expressed in an infectious context, and without over-expression of any protein. PMID:20211013

  14. Interaction between a plasma membrane-localized ankyrin-repeat protein ITN1 and a nuclear protein RTV1

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    Sakamoto, Hikaru [Department of Bioproduction, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri-shi, Hokkaido 093-2422 (Japan); Sakata, Keiko; Kusumi, Kensuke [Department of Biology, Faculty of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan); Kojima, Mikiko; Sakakibara, Hitoshi [RIKEN Plant Science Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045 (Japan); Iba, Koh, E-mail: koibascb@kyushu-u.org [Department of Biology, Faculty of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan)

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer ITN1, a plasma membrane ankyrin protein, interacts with a nuclear DNA-binding protein RTV1. Black-Right-Pointing-Pointer The nuclear transport of RTV1 is partially inhibited by interaction with ITN1. Black-Right-Pointing-Pointer RTV1 can promote the nuclear localization of ITN1. Black-Right-Pointing-Pointer Both overexpression of RTV1 and the lack of ITN1 increase salicylic acids sensitivity in plants. -- Abstract: The increased tolerance to NaCl 1 (ITN1) protein is a plasma membrane (PM)-localized protein involved in responses to NaCl stress in Arabidopsis. The predicted structure of ITN1 is composed of multiple transmembrane regions and an ankyrin-repeat domain that is known to mediate protein-protein interactions. To elucidate the molecular functions of ITN1, we searched for interacting partners using a yeast two-hybrid assay, and a nuclear-localized DNA-binding protein, RTV1, was identified as a candidate. Bimolecular fluorescence complementation analysis revealed that RTV1 interacted with ITN1 at the PM and nuclei in vivo. RTV1 tagged with red fluorescent protein localized to nuclei and ITN1 tagged with green fluorescent protein localized to PM; however, both proteins localized to both nuclei and the PM when co-expressed. These findings suggest that RTV1 and ITN1 regulate the subcellular localization of each other.

  15. HD CAGnome: a search tool for huntingtin CAG repeat length-correlated genes.

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    Ekaterina I Galkina

    Full Text Available BACKGROUND: The length of the huntingtin (HTT CAG repeat is strongly correlated with both age at onset of Huntington's disease (HD symptoms and age at death of HD patients. Dichotomous analysis comparing HD to controls is widely used to study the effects of HTT CAG repeat expansion. However, a potentially more powerful approach is a continuous analysis strategy that takes advantage of all of the different CAG lengths, to capture effects that are expected to be critical to HD pathogenesis. METHODOLOGY/PRINCIPAL FINDINGS: We used continuous and dichotomous approaches to analyze microarray gene expression data from 107 human control and HD lymphoblastoid cell lines. Of all probes found to be significant in a continuous analysis by CAG length, only 21.4% were so identified by a dichotomous comparison of HD versus controls. Moreover, of probes significant by dichotomous analysis, only 33.2% were also significant in the continuous analysis. Simulations revealed that the dichotomous approach would require substantially more than 107 samples to either detect 80% of the CAG-length correlated changes revealed by continuous analysis or to reduce the rate of significant differences that are not CAG length-correlated to 20% (n = 133 or n = 206, respectively. Given the superior power of the continuous approach, we calculated the correlation structure between HTT CAG repeat lengths and gene expression levels and created a freely available searchable website, "HD CAGnome," that allows users to examine continuous relationships between HTT CAG and expression levels of ∼20,000 human genes. CONCLUSIONS/SIGNIFICANCE: Our results reveal limitations of dichotomous approaches compared to the power of continuous analysis to study a disease where human genotype-phenotype relationships strongly support a role for a continuum of CAG length-dependent changes. The compendium of HTT CAG length-gene expression level relationships found at the HD CAGnome now provides

  16. Diversity and Plasticity of the Intracellular Plant Pathogen and Insect Symbiont “Candidatus Liberibacter asiaticus” as Revealed by Hypervariable Prophage Genes with Intragenic Tandem Repeats ▿ †

    Science.gov (United States)

    Zhou, Lijuan; Powell, Charles A.; Hoffman, Michele T.; Li, Wenbin; Fan, Guocheng; Liu, Bo; Lin, Hong; Duan, Yongping

    2011-01-01

    “Candidatus Liberibacter asiaticus” is a psyllid-transmitted, phloem-limited alphaproteobacterium and the most prevalent species of “Ca. Liberibacter” associated with a devastating worldwide citrus disease known as huanglongbing (HLB). Two related and hypervariable genes (hyvI and hyvII) were identified in the prophage regions of the Psy62 “Ca. Liberibacter asiaticus” genome. Sequence analyses of the hyvI and hyvII genes in 35 “Ca. Liberibacter asiaticus” DNA isolates collected globally revealed that the hyvI gene contains up to 12 nearly identical tandem repeats (NITRs, 132 bp) and 4 partial repeats, while hyvII contains up to 2 NITRs and 4 partial repeats and shares homology with hyvI. Frequent deletions or insertions of these repeats within the hyvI and hyvII genes were observed, none of which disrupted the open reading frames. Sequence conservation within the individual repeats but an extensive variation in repeat numbers, rearrangement, and the sequences flanking the repeat region indicate the diversity and plasticity of “Ca. Liberibacter asiaticus” bacterial populations in the world. These differences were found not only in samples of distinct geographical origins but also in samples from a single origin and even from a single “Ca. Liberibacter asiaticus”-infected sample. This is the first evidence of different “Ca. Liberibacter asiaticus” populations coexisting in a single HLB-affected sample. The Florida “Ca. Liberibacter asiaticus” isolates contain both hyvI and hyvII, while all other global “Ca. Liberibacter asiaticus” isolates contain either one or the other. Interclade assignments of the putative HyvI and HyvII proteins from Florida isolates with other global isolates in phylogenetic trees imply multiple “Ca. Liberibacter asiaticus” populations in the world and a multisource introduction of the “Ca. Liberibacter asiaticus” bacterium into Florida. PMID:21784907

  17. Assembly of neuronal connectivity by neurotrophic factors and leucine-rich repeat proteins

    Directory of Open Access Journals (Sweden)

    Fernanda Ledda

    2016-08-01

    Full Text Available Proper function of the nervous system critically relies on sophisticated neuronal networks interconnected in a highly specific pattern. The architecture of these connections arises from sequential developmental steps such as axonal growth and guidance, dendrite development, target determination, synapse formation and plasticity. Leucine-rich repeat (LRR transmembrane proteins have been involved in cell-type specific signaling pathways that underlie these developmental processes. The members of this superfamily of proteins execute their functions acting as trans-synaptic cell adhesion molecules involved in target specificity and synapse formation or working in cis as cell-intrinsic modulators of neurotrophic factor receptor trafficking and signaling. In this review, we will focus on novel physiological mechanisms through which LRR proteins regulate neurotrophic factor receptor signaling, highlighting the importance of these modulatory events for proper axonal extension and guidance, tissue innervation and dendrite morphogenesis. Additionally, we discuss few examples linking this set of LRR proteins to neurodevelopmental and psychiatric disorders.

  18. Effects of repeated psychological stress training on the spectrum of serum protein expression in special troops

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    Li ZHANG

    2011-10-01

    Full Text Available Objective To investigate the effects of repeated psychological stress training on the serum protein expression in soldiers under mental stress.Methods Ninety-six male commando soldiers were randomly assigned into the common psychological training group,the circulation psychological training group and the control group(32 each.After a 4-week training,all the soldiers were instructed to attend an one-day high-intensity simulated anti-riot exercise,and 3 days later attended another unannounced high-intensity simulated anti-riot exercise.Blood samples were collected from all the soldiers within 4 hours after each exercise,and the changes in serum protein expression were determined and statistically analyzed by using surface-enhanced laser desorption/ionization time of flight mass spectrometry(SELDI-TOF-MS combined with ProteinChip technology.Results The variance analysis showed that significant differences existed among the three groups(P < 0.05 in the relative contents of proteins with M/Z values of 6417.8,9134.2,15171.9 and 14972.7D after the first anti-riot exercise,and the relative contents of all the above mentioned proteins increased in the circulatory psychological training group;meanwhile,markedly increasing trends of the relative contents of all the proteins were observed in the three groups after the second anti-riot exercise(P < 0.05,and in control group the relative contents of the 4 above mentioned proteins were significantly higher than those after the first anti-riot exercise.Conclusion Psychological training may up-regulate the expression of serum proteins that are down-regulated after stress,and the repeated high-intensity mental training can rapidly improve the soldiers’ ability to counteract stress.

  19. Direct and accurate measurement of CAG repeat configuration in the ataxin-1 (ATXN-1) gene by "dual-fluorescence labeled PCR-restriction fragment length analysis".

    Science.gov (United States)

    Lin, Jiang X; Ishikawa, Kinya; Sakamoto, Masaki; Tsunemi, Taiji; Ishiguro, Taro; Amino, Takeshi; Toru, Shuta; Kondo, Ikuko; Mizusawa, Hidehiro

    2008-01-01

    Spinocerebellar ataxia type 1 (SCA1; OMIM: #164400) is an autosomal dominant cerebellar ataxia caused by an expansion of CAG repeat, which encodes polyglutamine, in the ataxin-1 (ATXN1) gene. Length of polyglutamine in the ATXN1 protein is the critical determinant of pathogenesis of this disease. Molecular diagnosis of SCA1 is usually undertaken by assessing the length of CAG repeat configuration using primers spanning this configuration. However, this conventional method may potentially lead to misdiagnosis in assessing polyglutamine-encoding CAG repeat length, since CAT interruptions may be present within the CAG repeat configuration, not only in normal controls but also in neurologically symptomatic subjects. We developed a new method for assessing actual CAG repeat numbers not interrupted by CAT sequences. Polymerase chain reaction using a primer pair labeled with two different fluorescences followed by restriction enzyme digestion with SfaNI which recognizes the sequence "GCATC(N)(5)", lengths of actual CAG repeats that encode polyglutamine were directly detected. We named this method "dual fluorescence labeled PCR-restriction fragment length analysis". We found that numbers of actual CAG repeat encoding polyglutamine do not overlap between our cohorts of normal chromosomes (n=385) and SCA1 chromosomes (n=5). We conclude that the present method is a useful way for molecular diagnosis of SCA1.

  20. Gene and protein nomenclature in public databases

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    Zimmer Ralf

    2006-08-01

    Full Text Available Abstract Background Frequently, several alternative names are in use for biological objects such as genes and proteins. Applications like manual literature search, automated text-mining, named entity identification, gene/protein annotation, and linking of knowledge from different information sources require the knowledge of all used names referring to a given gene or protein. Various organism-specific or general public databases aim at organizing knowledge about genes and proteins. These databases can be used for deriving gene and protein name dictionaries. So far, little is known about the differences between databases in terms of size, ambiguities and overlap. Results We compiled five gene and protein name dictionaries for each of the five model organisms (yeast, fly, mouse, rat, and human from different organism-specific and general public databases. We analyzed the degree of ambiguity of gene and protein names within and between dictionaries, to a lexicon of common English words and domain-related non-gene terms, and we compared different data sources in terms of size of extracted dictionaries and overlap of synonyms between those. The study shows that the number of genes/proteins and synonyms covered in individual databases varies significantly for a given organism, and that the degree of ambiguity of synonyms varies significantly between different organisms. Furthermore, it shows that, despite considerable efforts of co-curation, the overlap of synonyms in different data sources is rather moderate and that the degree of ambiguity of gene names with common English words and domain-related non-gene terms varies depending on the considered organism. Conclusion In conclusion, these results indicate that the combination of data contained in different databases allows the generation of gene and protein name dictionaries that contain significantly more used names than dictionaries obtained from individual data sources. Furthermore, curation of

  1. Loss of Bloom syndrome protein destabilizes human gene cluster architecture.

    Science.gov (United States)

    Killen, Michael W; Stults, Dawn M; Adachi, Noritaka; Hanakahi, Les; Pierce, Andrew J

    2009-09-15

    Bloom syndrome confers strong predisposition to malignancy in multiple tissue types. The Bloom syndrome patient (BLM) protein defective in the disease biochemically functions as a Holliday junction dissolvase and human cells lacking functional BLM show 10-fold elevated rates of sister chromatid exchange. Collectively, these phenomena suggest that dysregulated mitotic recombination drives the genomic instability underpinning the development of cancer in these individuals. Here we use physical analysis of the highly repeated, highly self-similar human ribosomal RNA gene clusters as sentinel biomarkers for dysregulated homologous recombination to demonstrate that loss of BLM protein function causes a striking increase in spontaneous molecular level genomic restructuring. Analysis of single-cell derived sub-clonal populations from wild-type human cell lines shows that gene cluster architecture is ordinarily very faithfully preserved under mitosis, but is so unstable in cell lines derived from BLMs as to make gene cluster architecture in different sub-clonal populations essentially unrecognizable one from another. Human cells defective in a different RecQ helicase, the WRN protein involved in the premature aging Werner syndrome, do not exhibit the gene cluster instability (GCI) phenotype, indicating that the BLM protein specifically, rather than RecQ helicases generally, holds back this recombination-mediated genomic instability. An ataxia-telangiectasia defective cell line also shows elevated rDNA GCI, although not to the extent of BLM defective cells. Genomic restructuring mediated by dysregulated recombination between the abundant low-copy repeats in the human genome may prove to be an important additional mechanism of genomic instability driving the initiation and progression of human cancer.

  2. Comparative semi-automated analysis of (CAG) repeats in the Huntington disease gene: use of internal standards.

    Science.gov (United States)

    Williams, L C; Hegde, M R; Herrera, G; Stapleton, P M; Love, D R

    1999-08-01

    Huntington disease (HD) belongs to the group of neurodegenerative disorders characterized by unstable expanded trinucleotide repeats. In the case of HD, the expansion of a CAG repeat occurs in the IT15 gene. The detection of the expanded CAG repeats has usually involved the electrophoretic separation of polymerase chain reaction (PCR) amplification products using conventional agarose and acrylamide gel electrophoresis. We have undertaken the comparative analysis of sizing CAG repeats of the IT15 gene using radioactive and fluorescent PCR amplification, and the subsequent separation of these products by slab gel and capillary electrophoresis. The assays have been performed on both cloned and sequenced CAG repeats, as well as genomic DNA from HD patients with a wide range of repeat lengths. The mobility of the CAG repeat amplification products of the IT15 gene is greater using capillary electrophoresis compared to slab gel electrophoresis. The analysis of 40 DNA samples from HD patients indicates that the mobility difference increases with the length of the repeat. However, we have devised an allele ladder for sizing the CAG repeats. This ladder provides a mandatory internal calibration system for diagnostic purposes and enables the confident use of either capillary or slab gel electrophoresis for sizing HD alleles.

  3. Expression of a gibberellin-induced leucine-rich repeat receptor-like protein kinase in deepwater rice and its interaction with kinase-associated protein phosphatase

    Energy Technology Data Exchange (ETDEWEB)

    Knaap, E. van der; Sauter, M.; Kende, H. (Michigan State Univ., East Lansing, MI (United States). DOE Plant Research Lab.); Song, W.Y.; Ruan, D.L.; Ronald, P.C. (Univ. of California, Davis, CA (United States). Dept. of Plant Pathology)

    1999-06-01

    The authors identified in deepwater rice (Oryza sativa L.) a gene encoding a leucine-rich repeat receptor-like transmembrane protein kinase, OsTMK (O. sativa transmembrane kinase). The transcript levels of OsTMK increased in the rice internode in response to gibberellin. Expression of OsTMK was especially high in regions undergoing cell division and elongation. The kinase domain of OsTMK was enzymatically active autophosphorylating on serine and threonine residues. A cDNA encoding a rice ortholog of a kinase-associated type 2C protein phosphatase (OsKAPP) was cloned. KAPPs are putative downstream components in kinase-mediated signal transduction pathways. The kinase interaction domain of OsKAPP was phosphorylated in vitro by the kinase domain of OsTMK. RNA gel-blot analysis indicated that the expression of OsTMK and OsKAPP was similar in different tissues of the rice plant. In protein-binding assays, OsKAPP interacted with a receptor-like protein kinase, RLK5 of Arabidopsis, but not with the protein kinase domains of the rice and maize receptor-like protein kinases Xa21 and ZmPK1, respectively.

  4. Cheetahs have 4 serum amyloid a genes evolved through repeated duplication events.

    Science.gov (United States)

    Chen, Lei; Une, Yumi; Higuchi, Keiichi; Mori, Masayuki

    2012-01-01

    Amyloid A (AA) amyloidosis is a leading cause of mortality in captive cheetahs (Acinonyx jubatus). We performed genome walking and PCR cloning and revealed that cheetahs have 4 SAA genes (provisionally named SAA1A, SAA1B, SAA3A, and SAA3B). In addition, we identified multiple nucleotide polymorphisms in the 4 SAA genes by screening 51 cheetahs. The polymorphisms defined 4, 7, 6, and 4 alleles for SAA1A, SAA3A, SAA1B, and SAA3B, respectively. Pedigree analysis of the inheritance of genotypes for the SAA genes revealed that specific combinations of alleles for the 4 SAA genes cosegregated as a unit (haplotype) in pedigrees, indicating that the 4 genes were linked on the same chromosome. Notably, cheetah SAA1A and SAA1B were highly homologous in their nucleotide sequences. Likewise, SAA3A and SAA3B genes were homologous. These observations suggested a model for the evolution of the 4 SAA genes in cheetahs in which duplication of an ancestral SAA gene first gave rise to SAA1 and SAA3. Subsequently, each gene duplicated one more time, uniquely making 4 genes in the cheetah genome. The monomorphism of the cheetah SAA1A protein might be one of the factors responsible for the high incidence of AA amyloidosis in this species.

  5. Non-target effects of repeated chlorothalonil application on soil nitrogen cycling: The key functional gene study.

    Science.gov (United States)

    Zhang, Manyun; Xu, Zhihong; Teng, Ying; Christie, Peter; Wang, Jun; Ren, Wenjie; Luo, Yongming; Li, Zhengao

    2016-02-01

    The widespread and increasing application of chlorothalonil (CTN) raises concerns about its non-target impacts, but little information is available on the effect of CTN on the key functional genes related to soil nitrogen (N) cycling, especially in the case of repeated applications. In the present study, a microcosm incubation was conducted to determine CTN residues and the impacts on the abundances of key functional genes related to N cycling after repeated CTN applications. The results demonstrated that repeated CTN applications at the recommended application rate and five times the recommended rate led to the accumulation of CTN residue in soil at concentrations of 5.59 and 78.79 mg kg(-1), respectively, by the end of incubation. Real time PCR (RT-PCR) revealed that repeated CTN applications had negative effects on the chiA and aprA gene abundances. There were significantly negative correlations between CTN residues and abundances of AOA and AOB genes. In addition, the abundances of key functional genes involved in soil denitrification were declined by repeated CTN applications with the sole exception of the nosZ gene. This study suggests that repeated CTN applications could lead to the accumulation of CTN residue and generate somewhat inconsistent and erratic effects on the abundances of key functional genes related to soil N cycling.

  6. γA gene repeats polymorphism for the analysis of haplotypes of abnormal hemoglobins

    Directory of Open Access Journals (Sweden)

    Nejat Akar

    2014-09-01

    Full Text Available Aim of this study was to analyze γ A gene repeat polymorphism for the analysis of haplotypes of hemoglobin (Hb variants such as Hb S, Hb D-Punjab, Hb O-Arab. Sickle cell cases had mainly Benin and Arab/Indian haplotype. We found three different haplotypes among Hb S, Hb O Arab and Hb D-Punjab cases. We named these three variants as Anatolian-1 and Anatolian-2 and Asian. Our data revealed that Hb O Arab may arise twice one from Asia and the other from Europe.

  7. Fuzzy tandem repeats containing p53 response elements may define species-specific p53 target genes.

    Directory of Open Access Journals (Sweden)

    Iva Simeonova

    2012-06-01

    Full Text Available Evolutionary forces that shape regulatory networks remain poorly understood. In mammals, the Rb pathway is a classic example of species-specific gene regulation, as a germline mutation in one Rb allele promotes retinoblastoma in humans, but not in mice. Here we show that p53 transactivates the Retinoblastoma-like 2 (Rbl2 gene to produce p130 in murine, but not human, cells. We found intronic fuzzy tandem repeats containing perfect p53 response elements to be important for this regulation. We next identified two other murine genes regulated by p53 via fuzzy tandem repeats: Ncoa1 and Klhl26. The repeats are poorly conserved in evolution, and the p53-dependent regulation of the murine genes is lost in humans. Our results indicate a role for the rapid evolution of tandem repeats in shaping differences in p53 regulatory networks between mammalian species.

  8. A Naturally Occurring Repeat Protein with High Internal Sequence Identity Defines a New Class of TPR-like Proteins.

    Science.gov (United States)

    Marold, Jacob D; Kavran, Jennifer M; Bowman, Gregory D; Barrick, Doug

    2015-11-01

    Linear repeat proteins often have high structural similarity and low (∼25%) pairwise sequence identities (PSI) among modules. We identified a unique P. anserina (Pa) sequence with tetratricopeptide repeat (TPR) homology, which contains longer (42 residue) repeats (42PRs) with an average PSI >91%. We determined the crystal structure of five tandem Pa 42PRs to 1.6 Å, and examined the stability and solution properties of constructs containing three to six Pa 42PRs. Compared with 34-residue TPRs (34PRs), Pa 42PRs have a one-turn extension of each helix, and bury more surface area. Unfolding transitions shift to higher denaturant concentration and become sharper as repeats are added. Fitted Ising models show Pa 42PRs to be more cooperative than consensus 34PRs, with increased magnitudes of intrinsic and interfacial free energies. These results demonstrate the tolerance of the TPR motif to length variation, and provide a basis to understand the effects of helix length on intrinsic/interfacial stability.

  9. Electrostatic effect of H1-histone protein binding on nucleosome repeat length

    Science.gov (United States)

    Cherstvy, Andrey G.; Teif, Vladimir B.

    2014-08-01

    Within a simple biophysical model we describe the effect of electrostatic binding of H1 histone proteins on the nucleosome repeat length in chromatin. The length of wrapped DNA optimizes its binding energy to the histone core and the elastic energy penalty of DNA wrapping. The magnitude of the effect predicted from our model is in agreement with the systematic experimental data on the linear variation of nucleosome repeat lengths with H1/nucleosome ratio (Woodcock C L et al 2006 Chromos. Res. 14 17-25). We compare our model to the data for different cell types and organisms, with a widely varying ratio of bound H1 histones per nucleosome. We underline the importance of this non-specific histone-DNA charge-balance mechanism in regulating the positioning of nucleosomes and the degree of compaction of chromatin fibers in eukaryotic cells.

  10. Designing genes for successful protein expression.

    Science.gov (United States)

    Welch, Mark; Villalobos, Alan; Gustafsson, Claes; Minshull, Jeremy

    2011-01-01

    DNA sequences are now far more readily available in silico than as physical DNA. De novo gene synthesis is an increasingly cost-effective method for building genetic constructs, and effectively removes the constraint of basing constructs on extant sequences. This allows scientists and engineers to experimentally test their hypotheses relating sequence to function. Molecular biologists, and now synthetic biologists, are characterizing and cataloging genetic elements with specific functions, aiming to combine them to perform complex functions. However, the most common purpose of synthetic genes is for the expression of an encoded protein. The huge number of different proteins makes it impossible to characterize and catalog each functional gene. Instead, it is necessary to abstract design principles from experimental data: data that can be generated by making predictions followed by synthesizing sequences to test those predictions. Because of the degeneracy of the genetic code, design of gene sequences to encode proteins is a high-dimensional problem, so there is no single simple formula to guarantee success. Nevertheless, there are several straightforward steps that can be taken to greatly increase the probability that a designed sequence will result in expression of the encoded protein. In this chapter, we discuss gene sequence parameters that are important for protein expression. We also describe algorithms for optimizing these parameters, and troubleshooting procedures that can be helpful when initial attempts fail. Finally, we show how many of these methods can be accomplished using the synthetic biology software tool Gene Designer.

  11. Leucine-rich repeat transmembrane proteins instruct discrete dendrite targeting in an olfactory map.

    Science.gov (United States)

    Hong, Weizhe; Zhu, Haitao; Potter, Christopher J; Barsh, Gabrielle; Kurusu, Mitsuhiko; Zinn, Kai; Luo, Liqun

    2009-12-01

    Olfactory systems utilize discrete neural pathways to process and integrate odorant information. In Drosophila, axons of first-order olfactory receptor neurons (ORNs) and dendrites of second-order projection neurons (PNs) form class-specific synaptic connections at approximately 50 glomeruli. The mechanisms underlying PN dendrite targeting to distinct glomeruli in a three-dimensional discrete neural map are unclear. We found that the leucine-rich repeat (LRR) transmembrane protein Capricious (Caps) was differentially expressed in different classes of PNs. Loss-of-function and gain-of-function studies indicated that Caps instructs the segregation of Caps-positive and Caps-negative PN dendrites to discrete glomerular targets. Moreover, Caps-mediated PN dendrite targeting was independent of presynaptic ORNs and did not involve homophilic interactions. The closely related protein Tartan was partially redundant with Caps. These LRR proteins are probably part of a combinatorial cell-surface code that instructs discrete olfactory map formation.

  12. Transcriptome analysis reveals ginsenosides biosynthetic genes, microRNAs and simple sequence repeats in Panax ginseng C. A. Meyer

    Science.gov (United States)

    2013-01-01

    Background Panax ginseng C. A. Meyer is one of the most widely used medicinal plants. Complete genome information for this species remains unavailable due to its large genome size. At present, analysis of expressed sequence tags is still the most powerful tool for large-scale gene discovery. The global expressed sequence tags from P. ginseng tissues, especially those isolated from stems, leaves and flowers, are still limited, hindering in-depth study of P. ginseng. Results Two 454 pyrosequencing runs generated a total of 2,423,076 reads from P. ginseng roots, stems, leaves and flowers. The high-quality reads from each of the tissues were independently assembled into separate and shared contigs. In the separately assembled database, 45,849, 6,172, 4,041 and 3,273 unigenes were only found in the roots, stems, leaves and flowers database, respectively. In the jointly assembled database, 178,145 unigenes were observed, including 86,609 contigs and 91,536 singletons. Among the 178,145 unigenes, 105,522 were identified for the first time, of which 65.6% were identified in the stem, leaf or flower cDNA libraries of P. ginseng. After annotation, we discovered 223 unigenes involved in ginsenoside backbone biosynthesis. Additionally, a total of 326 potential cytochrome P450 and 129 potential UDP-glycosyltransferase sequences were predicted based on the annotation results, some of which may encode enzymes responsible for ginsenoside backbone modification. A BLAST search of the obtained high-quality reads identified 14 potential microRNAs in P. ginseng, which were estimated to target 100 protein-coding genes, including transcription factors, transporters and DNA binding proteins, among others. In addition, a total of 13,044 simple sequence repeats were identified from the 178,145 unigenes. Conclusions This study provides global expressed sequence tags for P. ginseng, which will contribute significantly to further genome-wide research and analyses in this species. The novel

  13. Truncation of merozoite surface protein 3 disrupts its trafficking and that of acidic-basic repeat protein to the surface of Plasmodium falciparum merozoites.

    Science.gov (United States)

    Mills, Kerry E; Pearce, J Andrew; Crabb, Brendan S; Cowman, Alan F

    2002-03-01

    Merozoite surface protein 3 (MSP3), an important vaccine candidate, is a soluble polymorphic antigen associated with the surface of Plasmodium falciparum merozoites. The MSP3 sequence contains three blocks of heptad repeats that are consistent with the formation of an intramolecular coiled-coil. MSP3 also contains a glutamic acid-rich region and a putative leucine zipper sequence at the C-terminus. We have disrupted the msp3 gene by homologous recombination, resulting in the expression of a truncated form of MSP3 that lacks the putative leucine zipper sequence but retains the glutamic acid-rich region and the heptad repeats. Here, we show that truncated MSP3, lacking the putative leucine zipper region, does not localize to the parasitophorous vacuole or interact with the merozoite surface. Furthermore, the acidic-basic repeat antigen (ABRA), which is present on the merozoite surface, also was not localized to the merozoite surface in parasites expressing the truncated form of MSP3. The P. falciparum merozoites lacking MSP3 and ABRA on the surface show reduced invasion into erythrocytes. These results suggest that MSP3 is not absolutely essential for blood stage growth and that the putative leucine zipper region is required for the trafficking of both MSP3 and ABRA to the parasitophorous vacuole.

  14. The human protein disulfide isomerase gene family

    Directory of Open Access Journals (Sweden)

    Galligan James J

    2012-07-01

    Full Text Available Abstract Enzyme-mediated disulfide bond formation is a highly conserved process affecting over one-third of all eukaryotic proteins. The enzymes primarily responsible for facilitating thiol-disulfide exchange are members of an expanding family of proteins known as protein disulfide isomerases (PDIs. These proteins are part of a larger superfamily of proteins known as the thioredoxin protein family (TRX. As members of the PDI family of proteins, all proteins contain a TRX-like structural domain and are predominantly expressed in the endoplasmic reticulum. Subcellular localization and the presence of a TRX domain, however, comprise the short list of distinguishing features required for gene family classification. To date, the PDI gene family contains 21 members, varying in domain composition, molecular weight, tissue expression, and cellular processing. Given their vital role in protein-folding, loss of PDI activity has been associated with the pathogenesis of numerous disease states, most commonly related to the unfolded protein response (UPR. Over the past decade, UPR has become a very attractive therapeutic target for multiple pathologies including Alzheimer disease, Parkinson disease, alcoholic and non-alcoholic liver disease, and type-2 diabetes. Understanding the mechanisms of protein-folding, specifically thiol-disulfide exchange, may lead to development of a novel class of therapeutics that would help alleviate a wide range of diseases by targeting the UPR.

  15. The hypersensitive induced reaction and leucine-rich repeat proteins regulate plant cell death associated with disease and plant immunity.

    Science.gov (United States)

    Choi, Hyong Woo; Kim, Young Jin; Hwang, Byung Kook

    2011-01-01

    Pathogen-induced programmed cell death (PCD) is intimately linked with disease resistance and susceptibility. However, the molecular components regulating PCD, including hypersensitive and susceptible cell death, are largely unknown in plants. In this study, we show that pathogen-induced Capsicum annuum hypersensitive induced reaction 1 (CaHIR1) and leucine-rich repeat 1 (CaLRR1) function as distinct plant PCD regulators in pepper plants during Xanthomonas campestris pv. vesicatoria infection. Confocal microscopy and protein gel blot analyses revealed that CaLRR1 and CaHIR1 localize to the extracellular matrix and plasma membrane (PM), respectively. Bimolecular fluorescent complementation and coimmunoprecipitation assays showed that the extracellular CaLRR1 specifically binds to the PM-located CaHIR1 in pepper leaves. Overexpression of CaHIR1 triggered pathogen-independent cell death in pepper and Nicotiana benthamiana plants but not in yeast cells. Virus-induced gene silencing (VIGS) of CaLRR1 and CaHIR1 distinctly strengthened and compromised hypersensitive and susceptible cell death in pepper plants, respectively. Endogenous salicylic acid levels and pathogenesis-related gene transcripts were elevated in CaHIR1-silenced plants. VIGS of NbLRR1 and NbHIR1, the N. benthamiana orthologs of CaLRR1 and CaHIR1, regulated Bax- and avrPto-/Pto-induced PCD. Taken together, these results suggest that leucine-rich repeat and hypersensitive induced reaction proteins may act as cell-death regulators associated with plant immunity and disease.

  16. A WD40-repeat gene from Malus x domestica is a functional homologue of Arabidopsis thaliana TRANSPARENT TESTA GLABRA1.

    Science.gov (United States)

    Brueggemann, Julian; Weisshaar, Bernd; Sagasser, Martin

    2010-03-01

    The WD40 repeat protein TRANSPARENT TESTA GLABRA1 (TTG1) is involved in a multitude of developmental and biochemical reactions in Arabidopsis thaliana such as the production of seed coat colour and mucilage, pigmentation by anthocyanins as well as the formation of trichomes and root hairs. In this study, a putative TTG1 homologue was isolated from apple (Malus x domestica Borkh.) showing 80.2% identity to A. thaliana TTG1 on nucleotide and 90.7% similarity on amino acid level. The MdTTG1 candidate was able to activate the AtBAN promoter in cooperation with the A. thaliana transcription factors TT2 and TT8 in A. thaliana protoplasts. This indicates that the encoded protein can be integrated into the complex that activates BAN in A. thaliana, and that a similar complex might also be present in apple. When transformed into ttg1 mutants of A. thaliana, the apple sequence was able to restore trichome growth, anthocyanin production in young seedlings as well as proanthocyanidin production in seeds. Additionally, roots of complemented mutant plants showed root hair formation resembling wild type. These results show that the studied apple WD40 gene is a functional homologue of AtTTG1 and we refer to this gene as MdTTG1.

  17. De novo characterization of the Dialeurodes citri transcriptome: mining genes involved in stress resistance and simple sequence repeats (SSRs) discovery.

    Science.gov (United States)

    Chen, E-H; Wei, D-D; Shen, G-M; Yuan, G-R; Bai, P-P; Wang, J-J

    2014-02-01

    The citrus whitefly, Dialeurodes citri (Ashmead), is one of the three economically important whitefly species that infest citrus plants around the world; however, limited genetic research has been focused on D. citri, partly because of lack of genomic resources. In this study, we performed de novo assembly of a transcriptome using Illumina paired-end sequencing technology (Illumina Inc., San Diego, CA, USA). In total, 36,766 unigenes with a mean length of 497 bp were identified. Of these unigenes, we identified 17,788 matched known proteins in the National Center for Biotechnology Information database, as determined by Blast search, with 5731, 4850 and 14,441 unigenes assigned to clusters of orthologous groups (COG), gene ontology (GO), and SwissProt, respectively. In total, 7507 unigenes were assigned to 308 known pathways. In-depth analysis of the data showed that 117 unigenes were identified as potentially involved in the detoxification of xenobiotics and 67 heat shock protein (Hsp) genes were associated with environmental stress. In addition, these enzymes were searched against the GO and COG database, and the results showed that the three major detoxification enzymes and Hsps were classified into 18 and 3, 6, and 8 annotations, respectively. In addition, 149 simple sequence repeats were detected. The results facilitate the investigation of molecular resistance mechanisms to insecticides and environmental stress, and contribute to molecular marker development. The findings greatly improve our genetic understanding of D. citri, and lay the foundation for future functional genomics studies on this species.

  18. Sequence diversities of serine-aspartate repeat genes among Staphylococcus aureus isolates from different hosts presumably by horizontal gene transfer.

    Directory of Open Access Journals (Sweden)

    Huping Xue

    Full Text Available BACKGROUND: Horizontal gene transfer (HGT is recognized as one of the major forces for bacterial genome evolution. Many clinically important bacteria may acquire virulence factors and antibiotic resistance through HGT. The comparative genomic analysis has become an important tool for identifying HGT in emerging pathogens. In this study, the Serine-Aspartate Repeat (Sdr family has been compared among different sources of Staphylococcus aureus (S. aureus to discover sequence diversities within their genomes. METHODOLOGY/PRINCIPAL FINDINGS: Four sdr genes were analyzed for 21 different S. aureus strains and 218 mastitis-associated S. aureus isolates from Canada. Comparative genomic analyses revealed that S. aureus strains from bovine mastitis (RF122 and mastitis isolates in this study, ovine mastitis (ED133, pig (ST398, chicken (ED98, and human methicillin-resistant S. aureus (MRSA (TCH130, MRSA252, Mu3, Mu50, N315, 04-02981, JH1 and JH9 were highly associated with one another, presumably due to HGT. In addition, several types of insertion and deletion were found in sdr genes of many isolates. A new insertion sequence was found in mastitis isolates, which was presumably responsible for the HGT of sdrC gene among different strains. Moreover, the sdr genes could be used to type S. aureus. Regional difference of sdr genes distribution was also indicated among the tested S. aureus isolates. Finally, certain associations were found between sdr genes and subclinical or clinical mastitis isolates. CONCLUSIONS: Certain sdr gene sequences were shared in S. aureus strains and isolates from different species presumably due to HGT. Our results also suggest that the distributional assay of virulence factors should detect the full sequences or full functional regions of these factors. The traditional assay using short conserved regions may not be accurate or credible. These findings have important implications with regard to animal husbandry practices that may

  19. A transcription blocker isolated from a designed repeat protein combinatorial library by in vivo functional screen.

    Science.gov (United States)

    Tikhonova, Elena B; Ethayathulla, Abdul S; Su, Yue; Hariharan, Parameswaran; Xie, Shicong; Guan, Lan

    2015-01-28

    A highly diverse DNA library coding for ankyrin seven-repeat proteins (ANK-N5C) was designed and constructed by a PCR-based combinatorial assembly strategy. A bacterial melibiose fermentation assay was adapted for in vivo functional screen. We isolated a transcription blocker that completely inhibits the melibiose-dependent expression of α-galactosidase (MelA) and melibiose permease (MelB) of Escherichia coli by specifically preventing activation of the melAB operon. High-resolution crystal structural determination reveals that the designed ANK-N5C protein has a typical ankyrin fold, and the specific transcription blocker, ANK-N5C-281, forms a domain-swapped dimer. Functional tests suggest that the activity of MelR, a DNA-binding transcription activator and a member of AraC family of transcription factors, is inhibited by ANK-N5C-281 protein. All ANK-N5C proteins are expected to have a concave binding area with negative surface potential, suggesting that the designed ANK-N5C library proteins may facilitate the discovery of binders recognizing structural motifs with positive surface potential, like in DNA-binding proteins. Overall, our results show that the established library is a useful tool for the discovery of novel bioactive reagents.

  20. Lovastatin insensitive 1, a Novel pentatricopeptide repeat protein, is a potential regulatory factor of isoprenoid biosynthesis in Arabidopsis.

    Science.gov (United States)

    Kobayashi, Keiko; Suzuki, Masashi; Tang, Jianwei; Nagata, Noriko; Ohyama, Kiyoshi; Seki, Hikaru; Kiuchi, Reiko; Kaneko, Yasuko; Nakazawa, Miki; Matsui, Minami; Matsumoto, Shogo; Yoshida, Shigeo; Muranaka, Toshiya

    2007-02-01

    Higher plants have two metabolic pathways for isoprenoid biosynthesis: the cytosolic mevalonate (MVA) pathway and the plastidal non-mevalonate (MEP) pathway. Despite the compartmentalization of these two pathways, metabolic flow occurs between them. However, little is known about the mechanisms that regulate the two pathways and the metabolic cross-talk. To identify such regulatory mechanisms, we isolated and characterized the Arabidopsis T-DNA insertion mutant lovastatin insensitive 1 (loi1), which is resistant to lovastatin and clomazone, inhibitors of the MVA and MEP pathways, respectively. The accumulation of the major products of these pathways, i.e. sterols and chlorophyll, was less affected by lovastatin and clomazone, respectively, in loi1 than in the wild type. Furthermore, the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) activity analysis showed higher activity of HMGR in loi1-1 treated with lovastatin than that in the WT. We consider that the lovastatin-resistant phenotype of loi1-1 was derived from this post-transcriptional up-regulation of HMGR. The LOI1 gene encodes a novel pentatricopeptide repeat (PPR) protein. PPR proteins are thought to regulate the expression of genes encoded in organelle genomes by post-transcriptional regulation in mitochondria or plastids. Our results demonstrate that LOI1 is predicted to localize in mitochondria and has the ability to bind single-stranded nucleic acids. Our investigation revealed that the post-transcriptional regulation of mitochondrial RNA may be involved in isoprenoid biosynthesis in both the MVA and MEP pathways.

  1. Anchoring skeletal muscle development and disease: The role of ankyrin repeat domain containing proteins in muscle physiology

    NARCIS (Netherlands)

    J-M. Tee (Jin-Ming); M.P. Peppelenbosch (Maikel)

    2010-01-01

    textabstractThe ankyrin repeat is a protein module with high affinity for other ankyrin repeats based on strong Van der Waals forces. The resulting dimerization is unusually resistant to both mechanical forces and alkanization, making this module exceedingly useful for meeting the extraordinary dema

  2. Coevolution of gene expression among interacting proteins

    Energy Technology Data Exchange (ETDEWEB)

    Fraser, Hunter B.; Hirsh, Aaron E.; Wall, Dennis P.; Eisen,Michael B.

    2004-03-01

    Physically interacting proteins or parts of proteins are expected to evolve in a coordinated manner that preserves proper interactions. Such coevolution at the amino acid-sequence level is well documented and has been used to predict interacting proteins, domains, and amino acids. Interacting proteins are also often precisely coexpressed with one another, presumably to maintain proper stoichiometry among interacting components. Here, we show that the expression levels of physically interacting proteins coevolve. We estimate average expression levels of genes from four closely related fungi of the genus Saccharomyces using the codon adaptation index and show that expression levels of interacting proteins exhibit coordinated changes in these different species. We find that this coevolution of expression is a more powerful predictor of physical interaction than is coevolution of amino acid sequence. These results demonstrate previously uncharacterized coevolution of gene expression, adding a different dimension to the study of the coevolution of interacting proteins and underscoring the importance of maintaining coexpression of interacting proteins over evolutionary time. Our results also suggest that expression coevolution can be used for computational prediction of protein protein interactions.

  3. ProtRepeatsDB: a database of amino acid repeats in genomes

    Directory of Open Access Journals (Sweden)

    Chauhan Virander S

    2006-07-01

    Full Text Available Abstract Background Genome wide and cross species comparisons of amino acid repeats is an intriguing problem in biology mainly due to the highly polymorphic nature and diverse functions of amino acid repeats. Innate protein repeats constitute vital functional and structural regions in proteins. Repeats are of great consequence in evolution of proteins, as evident from analysis of repeats in different organisms. In the post genomic era, availability of protein sequences encoded in different genomes provides a unique opportunity to perform large scale comparative studies of amino acid repeats. ProtRepeatsDB http://bioinfo.icgeb.res.in/repeats/ is a relational database of perfect and mismatch repeats, access to which is designed as a resource and collection of tools for detection and cross species comparisons of different types of amino acid repeats. Description ProtRepeatsDB (v1.2 consists of perfect as well as mismatch amino acid repeats in the protein sequences of 141 organisms, the genomes of which are now available. The web interface of ProtRepeatsDB consists of different tools to perform repeat s; based on protein IDs, organism name, repeat sequences, and keywords as in FASTA headers, size, frequency, gene ontology (GO annotation IDs and regular expressions (REGEXP describing repeats. These tools also allow formulation of a variety of simple, complex and logical queries to facilitate mining and large-scale cross-species comparisons of amino acid repeats. In addition to this, the database also contains sequence analysis tools to determine repeats in user input sequences. Conclusion ProtRepeatsDB is a multi-organism database of different types of amino acid repeats present in proteins. It integrates useful tools to perform genome wide queries for rapid screening and identification of amino acid repeats and facilitates comparative and evolutionary studies of the repeats. The database is useful for identification of species or organism specific

  4. Selective serotonin re-uptake inhibitors potentiate gene blunting induced by repeated methylphenidate treatment: Zif268 versus Homer1a.

    Science.gov (United States)

    Van Waes, Vincent; Vandrevala, Malcolm; Beverley, Joel; Steiner, Heinz

    2014-11-01

    There is a growing use of psychostimulants, such as methylphenidate (Ritalin; dopamine re-uptake inhibitor), for medical treatments and as cognitive enhancers in the healthy. Methylphenidate is known to produce some addiction-related gene regulation. Recent findings in animal models show that selective serotonin re-uptake inhibitors (SSRIs), including fluoxetine, can potentiate acute induction of gene expression by methylphenidate, thus indicating an acute facilitatory role for serotonin in dopamine-induced gene regulation. We investigated whether repeated exposure to fluoxetine, in conjunction with methylphenidate, in adolescent rats facilitated a gene regulation effect well established for repeated exposure to illicit psychostimulants such as cocaine-blunting (repression) of gene inducibility. We measured, by in situ hybridization histochemistry, the effects of a 5-day repeated treatment with methylphenidate (5 mg/kg), fluoxetine (5 mg/kg) or a combination on the inducibility (by cocaine) of neuroplasticity-related genes (Zif268, Homer1a) in the striatum. Repeated methylphenidate treatment alone produced minimal gene blunting, while fluoxetine alone had no effect. In contrast, fluoxetine added to methylphenidate robustly potentiated methylphenidate-induced blunting for both genes. This potentiation was widespread throughout the striatum, but was most robust in the lateral, sensorimotor striatum, thus mimicking cocaine effects. For illicit psychostimulants, blunting of gene expression is considered part of the molecular basis of addiction. Our results thus suggest that SSRIs, such as fluoxetine, may increase the addiction liability of methylphenidate.

  5. Protein kinase C-associated kinase (PKK), a novel membrane-associated, ankyrin repeat-containing protein kinase.

    Science.gov (United States)

    Chen, L; Haider, K; Ponda, M; Cariappa, A; Rowitch, D; Pillai, S

    2001-06-15

    A novel murine membrane-associated protein kinase, PKK (protein kinase C-associated kinase), was cloned on the basis of its physical association with protein kinase Cbeta (PKCbeta). The regulated expression of PKK in mouse embryos is consistent with a role for this kinase in early embryogenesis. The human homolog of PKK has over 90% identity to its murine counterpart, has been localized to chromosome 21q22.3, and is identical to the PKCdelta-interacting kinase, DIK (Bahr, C., Rohwer, A., Stempka, L., Rincke, G., Marks, F., and Gschwendt, M. (2000) J. Biol. Chem. 275, 36350-36357). PKK comprises an N-terminal kinase domain and a C-terminal region containing 11 ankyrin repeats. PKK exhibits protein kinase activity in vitro and associates with cellular membranes. PKK exists in three discernible forms at steady state: an underphosphorylated form of 100 kDa; a soluble, cytosolic, phosphorylated form of 110 kDa; and a phosphorylated, detergent-insoluble form of 112 kDa. PKK is initially synthesized as an underphosphorylated soluble 100-kDa protein that is quantitatively converted to a detergent-soluble 110-kDa form. This conversion requires an active catalytic domain. Although PKK physically associates with PKCbeta, it does not phosphorylate this PKC isoform. However, PKK itself may be phosphorylated by PKCbeta. PKK represents a developmentally regulated protein kinase that can associate with membranes. The functional significance of its association with PKCbeta remains to be ascertained.

  6. Determination of microsatellite repeats in the human thyroid peroxidase (TPOX) gene using an automated gene analysis system with nanoscale engineered biomagnetite.

    Science.gov (United States)

    Nakagawa, Takahito; Maruyama, Kohei; Takeyama, Haruko; Matsunaga, Tadashi

    2007-04-15

    The number of repeat in the microsatellite region (AATG)(5-14) of the human thyroid peroxidase gene (TOPX) was determined using an automated DNA analysis system with nano-scale engineered biomagnetite. Thermal melting curve analysis of DNA duplexes on biomagnetite indicated that shorter repeat sequences (less than 9 repeats) were easily discriminated. However, it was difficult to determine the number of repeats at more than nine. In order to improve the selectivity of this method for the longer repeats, a "double probe hybridization assay" was performed in which an intermediate probe was used to replace a target repeat sequence having more than 9 repeats with a shorter sequence possessing less than 9 repeats. Thermal probe melting curve analyses and Tm determination confirmed that the target with 10 repeats was converted to 5 repeats, 11 repeats converted to 4 and 12 to 3, respectively. Furthermore, rapid determination of repeat numbers was possible by measuring fluorescence intensities obtained by probe dissociation at 56 and 66 degrees C, and 40, 60 and 80 degrees C for signal normalization.

  7. Crystallization of a pentapeptide-repeat protein by reductive cyclic pentylation of free amines with glutaraldehyde

    Energy Technology Data Exchange (ETDEWEB)

    Vetting, Matthew W., E-mail: vetting@aecom.yu.edu; Hegde, Subray S.; Blanchard, John S. [Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States)

    2009-05-01

    A method to modify proteins with glutaraldehyde under reducing conditions is presented. Treatment with glutaraldehyde and dimethylaminoborane was found to result in cyclic pentylation of free amines and facilitated the structural determination of a protein previously recalcitrant to the formation of diffraction quality crystals. The pentapeptide-repeat protein EfsQnr from Enterococcus faecalis protects DNA gyrase from inhibition by fluoroquinolones. EfsQnr was cloned and purified to homogeneity, but failed to produce diffraction-quality crystals in initial crystallization screens. Treatment of EfsQnr with glutaraldehyde and the strong reducing agent borane–dimethylamine resulted in a derivatized protein which produced crystals that diffracted to 1.6 Å resolution; their structure was subsequently determined by single-wavelength anomalous dispersion. Analysis of the derivatized protein using Fourier transform ion cyclotron resonance mass spectrometry indicated a mass increase of 68 Da per free amino group. Electron-density maps about a limited number of structurally ordered lysines indicated that the modification was a cyclic pentylation of free amines, producing piperidine groups.

  8. Large-scale modelling of the divergent spectrin repeats in nesprins: giant modular proteins.

    Science.gov (United States)

    Autore, Flavia; Pfuhl, Mark; Quan, Xueping; Williams, Aisling; Roberts, Roland G; Shanahan, Catherine M; Fraternali, Franca

    2013-01-01

    Nesprin-1 and nesprin-2 are nuclear envelope (NE) proteins characterized by a common structure of an SR (spectrin repeat) rod domain and a C-terminal transmembrane KASH [Klarsicht-ANC-Syne-homology] domain and display N-terminal actin-binding CH (calponin homology) domains. Mutations in these proteins have been described in Emery-Dreifuss muscular dystrophy and attributed to disruptions of interactions at the NE with nesprins binding partners, lamin A/C and emerin. Evolutionary analysis of the rod domains of the nesprins has shown that they are almost entirely composed of unbroken SR-like structures. We present a bioinformatical approach to accurate definition of the boundaries of each SR by comparison with canonical SR structures, allowing for a large-scale homology modelling of the 74 nesprin-1 and 56 nesprin-2 SRs. The exposed and evolutionary conserved residues identify important pbs for protein-protein interactions that can guide tailored binding experiments. Most importantly, the bioinformatics analyses and the 3D models have been central to the design of selected constructs for protein expression. 1D NMR and CD spectra have been performed of the expressed SRs, showing a folded, stable, high content α-helical structure, typical of SRs. Molecular Dynamics simulations have been performed to study the structural and elastic properties of consecutive SRs, revealing insights in the mechanical properties adopted by these modules in the cell.

  9. Subcellular localization of WD40 repeat 1 protein in PC12 rat pheochromocytoma cells.

    Science.gov (United States)

    Shin, Dong Hoon; Lee, Eunju; Chung, Yoon Hee; Mun, Ga Hee; Park, Ji yeong; Lomax, Margaret I; Oh, Seung Ha

    2004-09-09

    The dynamics of actin filament protein is crucial for various physiological processes of the cells. Among the proteins correlating with actin dynamics, a novel 67-kDa WD40 repeat protein 1 (WDR1) was the vertebrate homologue of actin-interacting protein 1 (Aip1). Even though previous studies have provided the clues on the function of WDR1 in specific organs under pathological conditions, the exact subcellular localization of WDR1 is not known. Therefore, in the present study, we undertook to determine the distribution of WDR1 within PC12 pheochromocytoma cells (PC12 cells) using light and electron microscopic techniques. Double immunocytochemistry clearly showed that WDR1 immunoreactivities (IRs) were co-localized with anti-actin antibody, suggesting the involvement of WDR1 in actin dynamics. WDR1 immunoreactivities (IRs) in PC12 cells showed different distribution patterns as nerve growth factor (NGF) concentrations varied. During active proliferation, the distribution of WDR1 IRs seemed to be similar to those found in cortical actin patches, whereas WDR1 IR was observed in cytoplasmic actin cables after PC12 cells were induced to differentiate by treating with NGF. Though further studies are necessary to determine the function of WDR1, the current data represents a first step towards the in vitro study of WDR1 protein.

  10. Large-scale modelling of the divergent spectrin repeats in nesprins: giant modular proteins.

    Directory of Open Access Journals (Sweden)

    Flavia Autore

    Full Text Available Nesprin-1 and nesprin-2 are nuclear envelope (NE proteins characterized by a common structure of an SR (spectrin repeat rod domain and a C-terminal transmembrane KASH [Klarsicht-ANC-Syne-homology] domain and display N-terminal actin-binding CH (calponin homology domains. Mutations in these proteins have been described in Emery-Dreifuss muscular dystrophy and attributed to disruptions of interactions at the NE with nesprins binding partners, lamin A/C and emerin. Evolutionary analysis of the rod domains of the nesprins has shown that they are almost entirely composed of unbroken SR-like structures. We present a bioinformatical approach to accurate definition of the boundaries of each SR by comparison with canonical SR structures, allowing for a large-scale homology modelling of the 74 nesprin-1 and 56 nesprin-2 SRs. The exposed and evolutionary conserved residues identify important pbs for protein-protein interactions that can guide tailored binding experiments. Most importantly, the bioinformatics analyses and the 3D models have been central to the design of selected constructs for protein expression. 1D NMR and CD spectra have been performed of the expressed SRs, showing a folded, stable, high content α-helical structure, typical of SRs. Molecular Dynamics simulations have been performed to study the structural and elastic properties of consecutive SRs, revealing insights in the mechanical properties adopted by these modules in the cell.

  11. The β-conglycinin deficiency in wild soybean is associated with the tail-to-tail inverted repeat of the α-subunit genes.

    Science.gov (United States)

    Tsubokura, Yasutaka; Hajika, Makita; Kanamori, Hiroyuki; Xia, Zhengjun; Watanabe, Satoshi; Kaga, Akito; Katayose, Yuichi; Ishimoto, Masao; Harada, Kyuya

    2012-02-01

    β-conglycinin, a major seed protein in soybean, is composed of α, α', and β subunits sharing a high homology among them. Despite its many health benefits, β-conglycinin has a lower amino acid score and lower functional gelling properties compared to glycinin, another major soybean seed protein. In addition, the α, α', and β subunits also contain major allergens. A wild soybean (Glycine soja Sieb et Zucc.) line, 'QT2', lacks all of the β-conglycinin subunits, and the deficiency is controlled by a single dominant gene, Scg-1 (Suppressor of β-conglycinin). This gene was characterized using a soybean cultivar 'Fukuyutaka', 'QY7-25', (its near-isogenic line carrying the Scg-1 gene), and the F₂ population derived from them. The physical map of the Scg-1 region covered by lambda phage genomic clones revealed that the two α-subunit genes, a β-subunit gene, and a pseudo α-subunit gene were closely organized. The two α-subunit genes were arranged in a tail-to-tail orientation, and the genes were separated by 197 bp in Scg-1 compared to 3.3 kb in the normal allele (scg-1). In addition, small RNA was detected in immature seeds of the mutants by northern blot analysis using an RNA probe of the α subunit. These results strongly suggest that β-conglycinin deficiency in QT2 is controlled by post-transcriptional gene silencing through the inverted repeat of the α subunits.

  12. RPG: the Ribosomal Protein Gene database

    OpenAIRE

    Nakao, Akihiro; Yoshihama, Maki; Kenmochi, Naoya

    2004-01-01

    RPG (http://ribosome.miyazaki-med.ac.jp/) is a new database that provides detailed information about ribosomal protein (RP) genes. It contains data from humans and other organisms, including Drosophila melanogaster, Caenorhabditis elegans, Saccharo myces cerevisiae, Methanococcus jannaschii and Escherichia coli. Users can search the database by gene name and organism. Each record includes sequences (genomic, cDNA and amino acid sequences), intron/exon structures, genomic locations and informa...

  13. Polymorphic tandem repeats within gene promoters act as modifiers of gene expression and DNA methylation in humans.

    Science.gov (United States)

    Quilez, Javier; Guilmatre, Audrey; Garg, Paras; Highnam, Gareth; Gymrek, Melissa; Erlich, Yaniv; Joshi, Ricky S; Mittelman, David; Sharp, Andrew J

    2016-05-05

    Despite representing an important source of genetic variation, tandem repeats (TRs) remain poorly studied due to technical difficulties. We hypothesized that TRs can operate as expression (eQTLs) and methylation (mQTLs) quantitative trait loci. To test this we analyzed the effect of variation at 4849 promoter-associated TRs, genotyped in 120 individuals, on neighboring gene expression and DNA methylation. Polymorphic promoter TRs were associated with increased variance in local gene expression and DNA methylation, suggesting functional consequences related to TR variation. We identified >100 TRs associated with expression/methylation levels of adjacent genes. These potential eQTL/mQTL TRs were enriched for overlaps with transcription factor binding and DNaseI hypersensitivity sites, providing a rationale for their effects. Moreover, we showed that most TR variants are poorly tagged by nearby single nucleotide polymorphisms (SNPs) markers, indicating that many functional TR variants are not effectively assayed by SNP-based approaches. Our study assigns biological significance to TR variations in the human genome, and suggests that a significant fraction of TR variations exert functional effects via alterations of local gene expression or epigenetics. We conclude that targeted studies that focus on genotyping TR variants are required to fully ascertain functional variation in the genome. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. Designed armadillo repeat proteins as general peptide-binding scaffolds: consensus design and computational optimization of the hydrophobic core

    DEFF Research Database (Denmark)

    Parmeggiani, Fabio; Pellarin, Riccardo; Larsen, Anders Peter

    2007-01-01

    interactions with peptides or parts of proteins in extended conformation. The conserved binding mode of the peptide in extended form, observed for different targets, makes armadillo repeat proteins attractive candidates for the generation of modular peptide-binding scaffolds. Taking advantage of the large...... number of repeat sequences available, a consensus-based approach combined with a force field-based optimization of the hydrophobic core was used to derive soluble, highly expressed, stable, monomeric designed proteins with improved characteristics compared to natural armadillo proteins. These sequences...

  15. In silico reversal of repeat-induced point mutation (RIP identifies the origins of repeat families and uncovers obscured duplicated genes

    Directory of Open Access Journals (Sweden)

    Hane James K

    2010-11-01

    Full Text Available Abstract Background Repeat-induced point mutation (RIP is a fungal genome defence mechanism guarding against transposon invasion. RIP mutates the sequence of repeated DNA and over time renders the affected regions unrecognisable by similarity search tools such as BLAST. Results DeRIP is a new software tool developed to predict the original sequence of a RIP-mutated region prior to the occurrence of RIP. In this study, we apply deRIP to the genome of the wheat pathogen Stagonospora nodorum SN15 and predict the origin of several previously uncharacterised classes of repetitive DNA. Conclusions Five new classes of transposon repeats and four classes of endogenous gene repeats were identified after deRIP. The deRIP process is a new tool for fungal genomics that facilitates the identification and understanding of the role and origin of fungal repetitive DNA. DeRIP is open-source and is available as part of the RIPCAL suite at http://www.sourceforge.net/projects/ripcal.

  16. Distinct role of Arabidopsis mitochondrial P-type pentatricopeptide repeat protein-modulating editing protein, PPME, in nad1 RNA editing.

    Science.gov (United States)

    Leu, Kuan-Chieh; Hsieh, Ming-Hsiun; Wang, Huei-Jing; Hsieh, Hsu-Liang; Jauh, Guang-Yuh

    2016-06-02

    The mitochondrion is an important power generator in most eukaryotic cells. To preserve its function, many essential nuclear-encoded factors play specific roles in mitochondrial RNA metabolic processes, including RNA editing. RNA editing consists of post-transcriptional deamination, which alters specific nucleotides in transcripts to mediate gene expression. In plant cells, many pentatricopeptide repeat proteins (PPRs) participate in diverse organellar RNA metabolic processes, but only PLS-type PPRs are involved in RNA editing. Here, we report a P-type PPR protein from Arabidopsis thaliana, P-type PPR-Modulating Editing (PPME), which has a distinct role in mitochondrial nad1 RNA editing via RNA binding activity. In the homozygous ppme mutant, cytosine (C)-to-uracil (U) conversions at both the nad1-898 and 937 sites were abolished, disrupting Arg(300)-to-Trp(300) and Pro(313)-to-Ser(313) amino acid changes in the mitochondrial NAD1 protein. NAD1 is a critical component of mitochondrial respiration complex I; its activity is severely reduced in the homozygous ppme mutant, resulting in significantly altered growth and development. Both abolished RNA editing and defective complex I activity were completely rescued by CaMV 35S promoter- and PPME native promoter-driven PPME genomic fragments tagged with GFP in a homozygous ppme background. Our experimental results demonstrate a distinct role of a P-type PPR protein, PPME, in RNA editing in plant organelles.

  17. A pollen-specific novel calmodulin-binding protein with tetratricopeptide repeats

    Science.gov (United States)

    Safadi, F.; Reddy, V. S.; Reddy, A. S.

    2000-01-01

    Calcium is essential for pollen germination and pollen tube growth. A large body of information has established a link between elevation of cytosolic Ca(2+) at the pollen tube tip and its growth. Since the action of Ca(2+) is primarily mediated by Ca(2+)-binding proteins such as calmodulin (CaM), identification of CaM-binding proteins in pollen should provide insights into the mechanisms by which Ca(2+) regulates pollen germination and tube growth. In this study, a CaM-binding protein from maize pollen (maize pollen calmodulin-binding protein, MPCBP) was isolated in a protein-protein interaction-based screening using (35)S-labeled CaM as a probe. MPCBP has a molecular mass of about 72 kDa and contains three tetratricopeptide repeats (TPR) suggesting that it is a member of the TPR family of proteins. MPCBP protein shares a high sequence identity with two hypothetical TPR-containing proteins from Arabidopsis. Using gel overlay assays and CaM-Sepharose binding, we show that the bacterially expressed MPCBP binds to bovine CaM and three CaM isoforms from Arabidopsis in a Ca(2+)-dependent manner. To map the CaM-binding domain several truncated versions of the MPCBP were expressed in bacteria and tested for their ability to bind CaM. Based on these studies, the CaM-binding domain was mapped to an 18-amino acid stretch between the first and second TPR regions. Gel and fluorescence shift assays performed with CaM and a CaM-binding synthetic peptide further confirmed MPCBP binding to CaM. Western, Northern, and reverse transcriptase-polymerase chain reaction analysis have shown that MPCBP expression is specific to pollen. MPCBP was detected in both soluble and microsomal proteins. Immunoblots showed the presence of MPCBP in mature and germinating pollen. Pollen-specific expression of MPCBP, its CaM-binding properties, and the presence of TPR motifs suggest a role for this protein in Ca(2+)-regulated events during pollen germination and growth.

  18. Serine-Aspartate Repeat Protein D Increases Staphylococcus aureus Virulence and Survival in Blood

    Science.gov (United States)

    Uchiyama, Satoshi; Valderrama, J. Andrés; Ajayi, Clement; Sollid, Johanna U. E.; van Sorge, Nina M.; Nizet, Victor; van Strijp, Jos A. G.

    2016-01-01

    ABSTRACT Staphylococcus aureus expresses a panel of cell wall-anchored adhesins, including proteins belonging to the microbial surface components recognizing adhesive matrix molecule (MSCRAMM) family, exemplified by the serine-aspartate repeat protein D (SdrD), which serve key roles in colonization and infection. Deletion of sdrD from S. aureus subsp. aureus strain NCTC8325-4 attenuated bacterial survival in human whole blood ex vivo, which was associated with increased killing by human neutrophils. Remarkably, SdrD was able to inhibit innate immune-mediated bacterial killing independently of other S. aureus proteins, since addition of recombinant SdrD protein and heterologous expression of SdrD in Lactococcus lactis promoted bacterial survival in human blood. SdrD contributes to bacterial virulence in vivo, since fewer S. aureus subsp. aureus NCTC8325-4 ΔsdrD bacteria than bacteria of the parent strain were recovered from blood and several organs using a murine intravenous infection model. Collectively, our findings reveal a new property of SdrD as an important key contributor to S. aureus survival and the ability to escape the innate immune system in blood. PMID:27795358

  19. Quantification of interaction strengths between chaperones and tetratricopeptide repeat domain-containing membrane proteins.

    Science.gov (United States)

    Schweiger, Regina; Soll, Jürgen; Jung, Kirsten; Heermann, Ralf; Schwenkert, Serena

    2013-10-18

    The three tetratricopeptide repeat domain-containing docking proteins Toc64, OM64, and AtTPR7 reside in the chloroplast, mitochondrion, and endoplasmic reticulum of Arabidopsis thaliana, respectively. They are suggested to act during post-translational protein import by association with chaperone-bound preprotein complexes. Here, we performed a detailed biochemical, biophysical, and computational analysis of the interaction between Toc64, OM64, and AtTPR7 and the five cytosolic chaperones HSP70.1, HSP90.1, HSP90.2, HSP90.3, and HSP90.4. We used surface plasmon resonance spectroscopy in combination with Interaction Map® analysis to distinguish between chaperone oligomerization and docking protein-chaperone interactions and to calculate binding affinities for all tested interactions. Complementary to this, we applied pulldown assays as well as microscale thermophoresis as surface immobilization independent techniques. The data revealed that OM64 prefers HSP70 over HSP90, whereas Toc64 binds all chaperones with comparable affinities. We could further show that AtTPR7 is able to bind HSP90 in addition to HSP70. Moreover, differences between the HSP90 isoforms were detected and revealed a weaker binding for HSP90.1 to AtTPR7 and OM64, showing that slight differences in the amino acid composition or structure of the chaperones influence binding to the tetratricopeptide repeat domain. The combinatory approach of several methods provided a powerful toolkit to determine binding affinities of similar interaction partners in a highly quantitative manner.

  20. Repeat organization and epigenetic regulation of the DH-Cmu domain of the immunoglobulin heavy-chain gene locus.

    Science.gov (United States)

    Chakraborty, Tirtha; Chowdhury, Dipanjan; Keyes, Amanda; Jani, Anant; Subrahmanyam, Ramesh; Ivanova, Irina; Sen, Ranjan

    2007-09-07

    The first steps of murine immunoglobulin heavy-chain (IgH) gene recombination take place within a chromosomal domain that contains diversity (D(H)) and joining (J(H)) gene segments, but not variable (V(H)) gene segments. Here we show that the chromatin state of this domain is markedly heterogeneous. Specifically, only 5'- and 3'-most D(H) gene segments carry active chromatin modifications, whereas intervening D(H)s are associated with heterochromatic marks that are maintained by ongoing histone deacetylation. The intervening D(H)s form part of a tandemly repeated sequence that expresses tissue-specific, antisense oriented transcripts. We propose that the intervening D(H) genes are actively suppressed by repeat-induced epigenetic silencing, which is reflected in their infrequent representation in DJ(H) junctions compared to the flanking D(H) genes.

  1. Impact of CAG repeat length in the androgen receptor gene on male infertility - a meta-analysis.

    Science.gov (United States)

    Xiao, Feifan; Lan, Aihua; Lin, Zhidi; Song, Jianfei; Zhang, Yuening; Li, Jiatong; Gu, Kailong; Lv, Baihao; Zhao, Dong; Zeng, Siping; Zhang, Ruoheng; Zhao, Wei; Pan, Zhengyan; Deng, Xiaozhen; Yang, Xiaoli

    2016-07-01

    CAG repeats are polymorphic nucleotide repeats present in the androgen receptor gene. Many studies have estimated the association between CAG repeat length and male infertility, but the conclusions are controversial. Previous meta-analyses have come to different conclusions; however, new studies have been published. An updated meta-analysis was conducted. PubMed, CBM, CNKI and Web of Science databases were systematically searched for studies published from 1 January 2000 to 1 October 2015. Case-control studies on the association between CAG repeat length and male infertility using appropriate methodology were included. Forty studies were selected, including 3858 cases and 3161 controls. Results showed statistically significantly longer CAG repeat length among cases compared with controls (SMD = 0.14; 95% CI, 0.02-0.26). Shorter repeat length was associated with a lower risk of male infertility compared with a longer repeat length in the overall analysis (OR = 0.79, 95% CI: 0.66-0.95). Moreover, CAG repeat length was associated with male infertility in Caucasian populations, but not Asian or Egyptian populations. Subgroup analysis revealed no significant difference in German populations, but CAG repeat length was associated with male infertility in China and the USA. There were no significant differences between cases and controls in azoospermia and severe oligozoospermia.

  2. Ablation of the cardiac-specific gene leucine-rich repeat containing 10 (Lrrc10 results in dilated cardiomyopathy.

    Directory of Open Access Journals (Sweden)

    Matthew J Brody

    Full Text Available Leucine-rich repeat containing 10 (LRRC10 is a cardiac-specific protein exclusively expressed in embryonic and adult cardiomyocytes. However, the role of LRRC10 in mammalian cardiac physiology remains unknown. To determine if LRRC10 is critical for cardiac function, Lrrc10-null (Lrrc10(-/- mice were analyzed. Lrrc10(- (/- mice exhibit prenatal systolic dysfunction and dilated cardiomyopathy in postnatal life. Importantly, Lrrc10(-/- mice have diminished cardiac performance in utero, prior to ventricular dilation observed in young adults. We demonstrate that LRRC10 endogenously interacts with α-actinin and α-actin in the heart and all actin isoforms in vitro. Gene expression profiling of embryonic Lrrc10(-/- hearts identified pathways and transcripts involved in regulation of the actin cytoskeleton to be significantly upregulated, implicating dysregulation of the actin cytoskeleton as an early defective molecular signal in the absence of LRRC10. In contrast, microarray analyses of adult Lrrc10(-/- hearts identified upregulation of oxidative phosphorylation and cardiac muscle contraction pathways during the progression of dilated cardiomyopathy. Analyses of hypertrophic signal transduction pathways indicate increased active forms of Akt and PKCε in adult Lrrc10(-/- hearts. Taken together, our data demonstrate that LRRC10 is essential for proper mammalian cardiac function. We identify Lrrc10 as a novel dilated cardiomyopathy candidate gene and the Lrrc10(-/- mouse model as a unique system to investigate pediatric cardiomyopathy.

  3. Transcriptional enhancer from milk protein genes

    Energy Technology Data Exchange (ETDEWEB)

    Casperson, Gerald F. (Ballwin, MO); Schmidhauser, Christian T. (Berkeley, CA); Bissell, Mina J. (Berkeley, CA)

    1999-01-01

    The invention relates to novel enhancer nucleotide sequences which stimulate transcription of heterologous DNA in cells in culture. The enhancers are derived from major milk protein genes by the process of deletion mapping and functional analysis. The invention also relates to expression vectors containing the novel enhancers.

  4. Transcriptional enhancer from milk protein genes

    Energy Technology Data Exchange (ETDEWEB)

    Casperson, G.F.; Schmidhauser, C.T.; Bissell, M.J.

    1999-12-21

    The invention relates to novel enhancer nucleotide sequences which stimulate transcription of heterologous DNA in cells in culture. The enhancers are derived from major milk protein genes by the process of deletion mapping and functional analysis. The invention also relates to expression vectors containing the novel enhancers.

  5. Porcine lung surfactant protein B gene (SFTPB)

    DEFF Research Database (Denmark)

    Cirera Salicio, Susanna; Fredholm, Merete

    2008-01-01

    The porcine surfactant protein B (SFTPB) is a single copy gene on chromosome 3. Three different cDNAs for the SFTPB have been isolated and sequenced. Nucleotide sequence comparison revealed six nonsynonymous single nucleotide polymorphisms (SNPs), four synonymous SNPs and an in-frame deletion of 69...

  6. Intergenerational and striatal CAG repeat instability in Huntington's disease knock-in mice involve different DNA repair genes.

    Science.gov (United States)

    Dragileva, Ella; Hendricks, Audrey; Teed, Allison; Gillis, Tammy; Lopez, Edith T; Friedberg, Errol C; Kucherlapati, Raju; Edelmann, Winfried; Lunetta, Kathryn L; MacDonald, Marcy E; Wheeler, Vanessa C

    2009-01-01

    Modifying the length of the Huntington's disease (HD) CAG repeat, the major determinant of age of disease onset, is an attractive therapeutic approach. To explore this we are investigating mechanisms of intergenerational and somatic HD CAG repeat instability. Here, we have crossed HD CAG knock-in mice onto backgrounds deficient in mismatch repair genes, Msh3 and Msh6, to discern the effects on CAG repeat size and disease pathogenesis. We find that different mechanisms predominate in inherited and somatic instability, with Msh6 protecting against intergenerational contractions and Msh3 required both for increasing CAG length and for enhancing an early disease phenotype in striatum. Therefore, attempts to decrease inherited repeat size may entail a full understanding of Msh6 complexes, while attempts to block the age-dependent increases in CAG size in striatal neurons and to slow the disease process will require a full elucidation of Msh3 complexes and their function in CAG repeat instability.

  7. Nucleotide sequence, DNA damage location and protein stoichiometry influence base excision repair outcome at CAG/CTG repeats

    Science.gov (United States)

    Goula, Agathi-Vasiliki; Pearson, Christopher E.; Della Maria, Julie; Trottier, Yvon; Tomkinson, Alan E.; Wilson, David M.; Merienne, Karine

    2012-01-01

    Expansion of CAG/CTG repeats is the underlying cause of >fourteen genetic disorders, including Huntington’s disease (HD) and myotonic dystrophy. The mutational process is ongoing, with increases in repeat size enhancing the toxicity of the expansion in specific tissues. In many repeat diseases the repeats exhibit high instability in the striatum, whereas instability is minimal in the cerebellum. We provide molecular insights as to how base excision repair (BER) protein stoichiometry may contribute to the tissue-selective instability of CAG/CTG repeats by using specific repair assays. Oligonucleotide substrates with an abasic site were mixed with either reconstituted BER protein stoichiometries mimicking the levels present in HD mouse striatum or cerebellum, or with protein extracts prepared from HD mouse striatum or cerebellum. In both cases, repair efficiency at CAG/CTG repeats and at control DNA sequences was markedly reduced under the striatal conditions, likely due to the lower level of APE1, FEN1 and LIG1. Damage located towards the 5’ end of the repeat tract was poorly repaired accumulating incompletely processed intermediates as compared to an AP lesion in the centre or at the 3’ end of the repeats or within a control sequences. Moreover, repair of lesions at the 5’ end of CAG or CTG repeats involved multinucleotide synthesis, particularly under the cerebellar stoichiometry, suggesting that long-patch BER processes lesions at sequences susceptible to hairpin formation. Our results show that BER stoichiometry, nucleotide sequence and DNA damage position modulate repair outcome, and suggest that a suboptimal LP-BER activity promotes CAG/CTG repeat instability. PMID:22497302

  8. Creation and structure determination of an artificial protein with three complete sequence repeats

    Energy Technology Data Exchange (ETDEWEB)

    Adachi, Motoyasu, E-mail: adachi.motoyasu@jaea.go.jp; Shimizu, Rumi; Kuroki, Ryota [Japan Atomic Energy Agency, Shirakatashirane 2-4, Nakagun Tokaimura, Ibaraki 319-1195 (Japan); Blaber, Michael [Japan Atomic Energy Agency, Shirakatashirane 2-4, Nakagun Tokaimura, Ibaraki 319-1195 (Japan); Florida State University, Tallahassee, FL 32306-4300 (United States)

    2013-11-01

    An artificial protein with three complete sequence repeats was created and the structure was determined by X-ray crystallography. The structure showed threefold symmetry even though there is an amino- and carboxy-terminal. The artificial protein with threefold symmetry may be useful as a scaffold to capture small materials with C3 symmetry. Symfoil-4P is a de novo protein exhibiting the threefold symmetrical β-trefoil fold designed based on the human acidic fibroblast growth factor. First three asparagine–glycine sequences of Symfoil-4P are replaced with glutamine–glycine (Symfoil-QG) or serine–glycine (Symfoil-SG) sequences protecting from deamidation, and His-Symfoil-II was prepared by introducing a protease digestion site into Symfoil-QG so that Symfoil-II has three complete repeats after removal of the N-terminal histidine tag. The Symfoil-QG and SG and His-Symfoil-II proteins were expressed in Eschericha coli as soluble protein, and purified by nickel affinity chromatography. Symfoil-II was further purified by anion-exchange chromatography after removing the HisTag by proteolysis. Both Symfoil-QG and Symfoil-II were crystallized in 0.1 M Tris-HCl buffer (pH 7.0) containing 1.8 M ammonium sulfate as precipitant at 293 K; several crystal forms were observed for Symfoil-QG and II. The maximum diffraction of Symfoil-QG and II crystals were 1.5 and 1.1 Å resolution, respectively. The Symfoil-II without histidine tag diffracted better than Symfoil-QG with N-terminal histidine tag. Although the crystal packing of Symfoil-II is slightly different from Symfoil-QG and other crystals of Symfoil derivatives having the N-terminal histidine tag, the refined crystal structure of Symfoil-II showed pseudo-threefold symmetry as expected from other Symfoils. Since the removal of the unstructured N-terminal histidine tag did not affect the threefold structure of Symfoil, the improvement of diffraction quality of Symfoil-II may be caused by molecular characteristics of

  9. Gene deficiency and pharmacological inhibition of soluble epoxide hydrolase confers resilience to repeated social defeat stress.

    Science.gov (United States)

    Ren, Qian; Ma, Min; Ishima, Tamaki; Morisseau, Christophe; Yang, Jun; Wagner, Karen M; Zhang, Ji-Chun; Yang, Chun; Yao, Wei; Dong, Chao; Han, Mei; Hammock, Bruce D; Hashimoto, Kenji

    2016-03-29

    Depression is a severe and chronic psychiatric disease, affecting 350 million subjects worldwide. Although multiple antidepressants have been used in the treatment of depressive symptoms, their beneficial effects are limited. The soluble epoxide hydrolase (sEH) plays a key role in the inflammation that is involved in depression. Thus, we examined here the role of sEH in depression. In both inflammation and social defeat stress models of depression, a potent sEH inhibitor, TPPU, displayed rapid antidepressant effects. Expression of sEH protein in the brain from chronically stressed (susceptible) mice was higher than of control mice. Furthermore, expression of sEH protein in postmortem brain samples of patients with psychiatric diseases, including depression, bipolar disorder, and schizophrenia, was higher than controls. This finding suggests that increased sEH levels might be involved in the pathogenesis of certain psychiatric diseases. In support of this hypothesis, pretreatment with TPPU prevented the onset of depression-like behaviors after inflammation or repeated social defeat stress. Moreover, sEH KO mice did not show depression-like behavior after repeated social defeat stress, suggesting stress resilience. The sEH KO mice showed increased brain-derived neurotrophic factor (BDNF) and phosphorylation of its receptor TrkB in the prefrontal cortex, hippocampus, but not nucleus accumbens, suggesting that increased BDNF-TrkB signaling in the prefrontal cortex and hippocampus confer stress resilience. All of these findings suggest that sEH plays a key role in the pathophysiology of depression, and that epoxy fatty acids, their mimics, as well as sEH inhibitors could be potential therapeutic or prophylactic drugs for depression.

  10. Serum cartilage oligomeric matrix protein: is there a repeated bout effect?

    Directory of Open Access Journals (Sweden)

    Michael Behringer

    2014-10-01

    Full Text Available The primary aim of the present study was to investigate if there is a repeated bout effect for cartilage tissue, evident in the marker serum cartilage oligomeric matrix protein (sCOMP. Ten healthy male subjects (26.4±3.14 years performed two high impact interventions (100 drop jumps with a 30 second interval carried out at a 3 week interval. After each intervention, sCOMP and muscle soreness were assessed on 8 and 6 occasions respectively. Muscle soreness was determined via a visual analog scale with a maximum pain score of 10. sComp levels did not show a blunted response after the second bout (Bout 1: 12.2±3.3 U/L−1; Bout 2: 13.1±4.0 U/L−1; P>0.05. Remarkably, sCOMP increased from baseline levels by 16% after bout 1 and 15% after bout 2. Muscle soreness was blunted following the second intervention (Bout 1: 5.0±1.8; Bout 2: 1.6±0.8. Unlike the known repeated bout effect for muscle damage markers, sCOMP levels do not show a blunted response after two similar loading interventions. This information on biomarker behavior is essential to clinicians attempting to use this marker as an indicator of cartilage damage associated with the development or progression of osteoarthritis.

  11. RPG: the Ribosomal Protein Gene database.

    Science.gov (United States)

    Nakao, Akihiro; Yoshihama, Maki; Kenmochi, Naoya

    2004-01-01

    RPG (http://ribosome.miyazaki-med.ac.jp/) is a new database that provides detailed information about ribosomal protein (RP) genes. It contains data from humans and other organisms, including Drosophila melanogaster, Caenorhabditis elegans, Saccharo myces cerevisiae, Methanococcus jannaschii and Escherichia coli. Users can search the database by gene name and organism. Each record includes sequences (genomic, cDNA and amino acid sequences), intron/exon structures, genomic locations and information about orthologs. In addition, users can view and compare the gene structures of the above organisms and make multiple amino acid sequence alignments. RPG also provides information on small nucleolar RNAs (snoRNAs) that are encoded in the introns of RP genes.

  12. Force Spectroscopy of the Plasmodium falciparum Vaccine Candidate Circumsporozoite Protein Suggests a Mechanically Pliable Repeat Region.

    Science.gov (United States)

    Patra, Aditya Prasad; Sharma, Shobhona; Ainavarapu, Sri Rama Koti

    2017-02-10

    The most effective vaccine candidate of malaria is based on the Plasmodium falciparum circumsporozoite protein (CSP), a major surface protein implicated in the structural strength, motility, and immune evasion properties of the infective sporozoites. It is suspected that reversible conformational changes of CSP are required for infection of the mammalian host, but the detailed structure and dynamic properties of CSP remain incompletely understood, limiting our understanding of its function in the infection. Here, we report the structural and mechanical properties of the CSP studied using single-molecule force spectroscopy on several constructs, one including the central region of CSP, which is rich in NANP amino acid repeats (CSPrep), and a second consisting of a near full-length sequence without the signal and anchor hydrophobic domains (CSPΔHP). Our results show that the CSPrep is heterogeneous, with 40% of molecules requiring virtually no mechanical force to unfold (<10 piconewtons (pN)), suggesting that these molecules are mechanically compliant and perhaps act as entropic springs, whereas the remaining 60% are partially structured with low mechanical resistance (∼70 pN). CSPΔHP having multiple force peaks suggests specifically folded domains, with two major populations possibly indicating the open and collapsed forms. Our findings suggest that the overall low mechanical resistance of the repeat region, exposed on the outer surface of the sporozoites, combined with the flexible full-length conformations of CSP, may provide the sporozoites not only with immune evasion properties, but also with lubricating capacity required during its navigation through the mosquito and vertebrate host tissues. We anticipate that these findings would further assist in the design and development of future malarial vaccines. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Repeat use of human recombinant bone morphogenetic protein-2 for second level lumbar arthrodesis.

    Science.gov (United States)

    Singh, Kern; Dumonski, Mark; Stanley, Tom; Ponnappan, Ravi; Phillips, Frank M

    2011-02-01

    Prospective randomized controlled animal model. The purpose of this study is to determine whether the readministration of human recombinant bone morphogenetic protein-2 (rhBMP-2) induces an immune response and inhibits successful fusion in repeat posterolateral spinal surgery. Little research has been performed on the effectiveness or immunoreactivity of rhBMP-2 (Infuse, Medtronic, Memphis, TN) in the context of its reuse in posterolateral fusion spinal surgery at adjacent levels. A total of 34 New Zealand White rabbits underwent posterior intertransverse process fusion with the use of rhBMP-2 delivered on an absorbable collagen sponge (rhBMP-2/ACS). Two rabbits were killed early leaving 32 total rabbits. Serologic studies (Type I bovine collagen and rhBMP-2 antibodies) were obtained at 2-week intervals throughout the experiment. At 10 weeks, posteroanterior radiographs confirmed solid fusion masses in all rabbits. The 32 rabbits were randomly separated into 2 groups of 16, and each group underwent an adjacent level, bilateral intertransverse process fusion with either rhBMP-2/ACS or iliac crest. There was no statistical difference in fusion rates with repeat use of rhBMP-2 (n = 15/16, 94%) or iliac crest (n = 11/16, 69%) (P = 0.17) at the adjacent level. Four rabbits (n = 4/32, 13%) developed rhBMP-2 antibodies. Of these 4 rabbits, 1 developed anti-rhBMP antibodies after the first exposure and 3 developed antibodies after the second surgery. Eight rabbits (n = 8/32, 25%) developed collagen antibodies with 7 rabbits developing antibodies after the first exposure and 1 rabbit developing antibodies after the second exposure. The development of antibodies did not effect fusion rates. No rabbit demonstrated evidence of a systemic or anaphylactic reaction to repeat exposure to rhBMP-2. rhBMP-2 appears to be successful in promoting intertransverse fusions when used in both primary and repeat fusion environments. The infrequent development of antibodies to rhBMP-2 after

  14. Characterization of the gene encoding a fibrinogen-related protein expressed in Crassostrea gigas hemocytes.

    Science.gov (United States)

    Skazina, M A; Gorbushin, A M

    2016-07-01

    Four exons of the CgFrep1 gene (3333 bp long) encode a putative fibrinogen-related protein (324 aa) bearing a single C-terminal FBG domain. Transcripts of the gene obtained from hemocytes of different Pacific oysters show prominent individual variation based on SNP and indels of tandem repeats resulted in polymorphism of N-terminus of the putative CgFrep1 polypeptide. The polypeptide chain bears N-terminal coiled-coil region potentially acting as inter-subunit interface in the protein oligomerization. It is suggested that CgFrep1 gene encodes the oligomeric lectin composed of at least two subunits.

  15. Distribution of dipeptide repeat proteins in cellular models and C9orf72 mutation cases suggests link to transcriptional silencing.

    Science.gov (United States)

    Schludi, Martin H; May, Stephanie; Grässer, Friedrich A; Rentzsch, Kristin; Kremmer, Elisabeth; Küpper, Clemens; Klopstock, Thomas; Arzberger, Thomas; Edbauer, Dieter

    2015-10-01

    A massive expansion of a GGGGCC repeat upstream of the C9orf72 coding region is the most common known cause of amyotrophic lateral sclerosis and frontotemporal dementia. Despite its intronic localization and lack of a canonical start codon, both strands are translated into aggregating dipeptide repeat (DPR) proteins: poly-GA, poly-GP, poly-GR, poly-PR and poly-PA. To address conflicting findings on the predominant toxicity of the different DPR species in model systems, we compared the expression pattern of the DPR proteins in rat primary neurons and postmortem brain and spinal cord of C9orf72 mutation patients. Only poly-GA overexpression closely mimicked the p62-positive neuronal cytoplasmic inclusions commonly observed for all DPR proteins in patients. In contrast, overexpressed poly-GR and poly-PR formed nucleolar p62-negative inclusions. In patients, most of the less common neuronal intranuclear DPR inclusions were para-nucleolar and p62 positive. Neuronal nucleoli in C9orf72 cases showed normal size and morphology regardless of the presence of poly-GR and poly-PR inclusions arguing against widespread nucleolar stress, reported in cellular models. Colocalization of para-nucleolar DPR inclusions with heterochromatin and a marker of transcriptional repression (H3K9me2) indicates a link to gene transcription. In contrast, we detected numerous intranuclear DPR inclusions not associated with nucleolar structures in ependymal and subependymal cells. In patients, neuronal inclusions of poly-GR, poly-GP and the poly-GA interacting protein Unc119 were less abundant than poly-GA inclusions, but showed similar regional and subcellular distribution. Regardless of neurodegeneration, all inclusions were most abundant in neocortex, hippocampus and thalamus, with few inclusions in brain stem and spinal cord. In the granular cell layer of the cerebellum, poly-GA and Unc119 inclusions were significantly more abundant in cases with FTLD than in cases with MND and FTLD/MND. Poly

  16. Genetic Association Between Androgen Receptor Gene CAG Repeat Length Polymorphism and Male Infertility: A Meta-Analysis.

    Science.gov (United States)

    Pan, Bihui; Li, Rui; Chen, Yao; Tang, Qiuqin; Wu, Wei; Chen, Liping; Lu, Chuncheng; Pan, Feng; Ding, Hongjuan; Xia, Yankai; Hu, Lingqing; Chen, Daozhen; Sha, Jiahao; Wang, Xinru

    2016-03-01

    The association between polymorphism of androgen receptor gene CAG (AR-CAG) and male infertility in several studies was controversial. Based on studies on association between AR-CAG repeat length and male infertility in recent years, an updated meta-analysis is needed. We aimed to evaluate the association between AR-CAG repeat length and male infertility in advantage of the data in all published reports.We searched for reports published before August 2015 using PubMed, CNKI, VIP, and WanFang. Data on sample size, mean, and standard deviation (SD) of AR-CAG repeat length were extracted independently by 3 investigators.Forty-four reports were selected based on criteria. The overall infertile patients and azoospermic patients were found to have longer AR-CAG repeat length (standard mean difference (SMD) = 0.19, 95% confidence interval (CI): 0.10-0.28, P CAG repeat length was longer in infertile men in Asian, Caucasian, and mixed races (SMD = 0.25, 95% CI: 0.08-0.43, P CAG repeat length was associated with male infertility. The subgroup study on races shows that increased AR-CAG repeat length was associated with male infertility in Asian, Caucasian, and mixed races. Increased AR-CAG repeat length was also associated with azoospermia.This meta-analysis supports that increased androgen receptor CAG length is capable of causing male infertility susceptibility.

  17. Amelogenesis Imperfecta; Genes, Proteins, and Pathways

    Directory of Open Access Journals (Sweden)

    Claire E. L. Smith

    2017-06-01

    Full Text Available Amelogenesis imperfecta (AI is the name given to a heterogeneous group of conditions characterized by inherited developmental enamel defects. AI enamel is abnormally thin, soft, fragile, pitted and/or badly discolored, with poor function and aesthetics, causing patients problems such as early tooth loss, severe embarrassment, eating difficulties, and pain. It was first described separately from diseases of dentine nearly 80 years ago, but the underlying genetic and mechanistic basis of the condition is only now coming to light. Mutations in the gene AMELX, encoding an extracellular matrix protein secreted by ameloblasts during enamel formation, were first identified as a cause of AI in 1991. Since then, mutations in at least eighteen genes have been shown to cause AI presenting in isolation of other health problems, with many more implicated in syndromic AI. Some of the encoded proteins have well documented roles in amelogenesis, acting as enamel matrix proteins or the proteases that degrade them, cell adhesion molecules or regulators of calcium homeostasis. However, for others, function is less clear and further research is needed to understand the pathways and processes essential for the development of healthy enamel. Here, we review the genes and mutations underlying AI presenting in isolation of other health problems, the proteins they encode and knowledge of their roles in amelogenesis, combining evidence from human phenotypes, inheritance patterns, mouse models, and in vitro studies. An LOVD resource (http://dna2.leeds.ac.uk/LOVD/ containing all published gene mutations for AI presenting in isolation of other health problems is described. We use this resource to identify trends in the genes and mutations reported to cause AI in the 270 families for which molecular diagnoses have been reported by 23rd May 2017. Finally we discuss the potential value of the translation of AI genetics to clinical care with improved patient pathways and

  18. Amelogenesis Imperfecta; Genes, Proteins, and Pathways

    Science.gov (United States)

    Smith, Claire E. L.; Poulter, James A.; Antanaviciute, Agne; Kirkham, Jennifer; Brookes, Steven J.; Inglehearn, Chris F.; Mighell, Alan J.

    2017-01-01

    Amelogenesis imperfecta (AI) is the name given to a heterogeneous group of conditions characterized by inherited developmental enamel defects. AI enamel is abnormally thin, soft, fragile, pitted and/or badly discolored, with poor function and aesthetics, causing patients problems such as early tooth loss, severe embarrassment, eating difficulties, and pain. It was first described separately from diseases of dentine nearly 80 years ago, but the underlying genetic and mechanistic basis of the condition is only now coming to light. Mutations in the gene AMELX, encoding an extracellular matrix protein secreted by ameloblasts during enamel formation, were first identified as a cause of AI in 1991. Since then, mutations in at least eighteen genes have been shown to cause AI presenting in isolation of other health problems, with many more implicated in syndromic AI. Some of the encoded proteins have well documented roles in amelogenesis, acting as enamel matrix proteins or the proteases that degrade them, cell adhesion molecules or regulators of calcium homeostasis. However, for others, function is less clear and further research is needed to understand the pathways and processes essential for the development of healthy enamel. Here, we review the genes and mutations underlying AI presenting in isolation of other health problems, the proteins they encode and knowledge of their roles in amelogenesis, combining evidence from human phenotypes, inheritance patterns, mouse models, and in vitro studies. An LOVD resource (http://dna2.leeds.ac.uk/LOVD/) containing all published gene mutations for AI presenting in isolation of other health problems is described. We use this resource to identify trends in the genes and mutations reported to cause AI in the 270 families for which molecular diagnoses have been reported by 23rd May 2017. Finally we discuss the potential value of the translation of AI genetics to clinical care with improved patient pathways and speculate on the

  19. Androgen receptor gene CAG repeat length as modifier of the association between Persistent Organohalogen Pollutant exposure markers and semen characteristics

    DEFF Research Database (Denmark)

    Giwercman, Aleksander; Rylander, Lars; Rignell-Hydbom, Anna;

    2007-01-01

    OBJECTIVES: Exposure to persistent organohalogen pollutants was suggested to impair male reproductive function. A gene-environment interaction has been proposed. No genes modifying the effect of persistent organohalogen pollutants on reproductive organs have yet been identified. We aimed...... and morphology) and DNA fragmentation index (DFI) were determined. CAG and GGN repeat lengths were determined by direct sequencing of leukocyte DNA. RESULTS: A statistically significant interaction was found between the CB-153 group and CAG repeat category in relation to sperm concentration and total sperm count...

  20. Programmable DNA-binding proteins from Burkholderia provide a fresh perspective on the TALE-like repeat domain.

    Science.gov (United States)

    de Lange, Orlando; Wolf, Christina; Dietze, Jörn; Elsaesser, Janett; Morbitzer, Robert; Lahaye, Thomas

    2014-06-01

    The tandem repeats of transcription activator like effectors (TALEs) mediate sequence-specific DNA binding using a simple code. Naturally, TALEs are injected by Xanthomonas bacteria into plant cells to manipulate the host transcriptome. In the laboratory TALE DNA binding domains are reprogrammed and used to target a fused functional domain to a genomic locus of choice. Research into the natural diversity of TALE-like proteins may provide resources for the further improvement of current TALE technology. Here we describe TALE-like proteins from the endosymbiotic bacterium Burkholderia rhizoxinica, termed Bat proteins. Bat repeat domains mediate sequence-specific DNA binding with the same code as TALEs, despite less than 40% sequence identity. We show that Bat proteins can be adapted for use as transcription factors and nucleases and that sequence preferences can be reprogrammed. Unlike TALEs, the core repeats of each Bat protein are highly polymorphic. This feature allowed us to explore alternative strategies for the design of custom Bat repeat arrays, providing novel insights into the functional relevance of non-RVD residues. The Bat proteins offer fertile grounds for research into the creation of improved programmable DNA-binding proteins and comparative insights into TALE-like evolution.

  1. Gene expression in human skeletal muscle: alternative normalization method and effect of repeated biopsies.

    Science.gov (United States)

    Lundby, Carsten; Nordsborg, Nikolai; Kusuhara, Keiko; Kristensen, Kristina Møller; Neufer, P Darrell; Pilegaard, Henriette

    2005-10-01

    The reverse transcriptase-polymerase chain reaction (RT-PCR) method has lately become widely used to determine transcription and mRNA content in rodent and human muscle samples. However, the common use of endogenous controls for correcting for variance in cDNA between samples is not optimal. Specifically, we investigated (1) a new normalization method based on determining the cDNA content by the flourophores PicoGreen and OliGreen, (2) effect of repeated muscle biopsies on mRNA gene expression, and (3) the spatial heterogeneity in mRNA expression across the muscle. Standard curves using oligo standards revealed a high degree of sensitivity and linearity (2.5-45 ng; R2>0.99) with OliGreen reagent, as was the case for OliGreen analyses with standard curves constructed from serial dilutions of representative RT samples (R2 >0.99 for a ten times dilution range of a representative reversed transcribed (RT) sample). Likewise, PicoGreen reagent detected the RNA:DNA hybrid content in RT samples with great sensitivity. Standard curves constructed from both double-stranded lambda DNA (1-10 ng) and from serial dilutions of representative RT samples consistently resulted in linearity with R2 >0.99. The present determination of cDNA content in reversed transcribed human skeletal muscle RNA samples by both PicoGreen and OliGreen analyses suggests that these fluorophores provide a potential alternative normalization procedure for human gene expression studies. In addition, the present study shows that multiple muscle biopsies obtained from the same muscle do not influence the mRNA response induced by an acute exercise bout for any of the genes examined.

  2. [Prolonging the vase life of carnation "Mabel" through integrating repeated ACC oxidase genes into its genome].

    Science.gov (United States)

    Yu, Yi-Xun; Bao, Man-Zhu

    2004-10-01

    Carnation (Dianthus caryophyllus L.) is one of the most important cut flowers. The cultivar "Mabel" of carnation was transformed with direct repeat gene of ACC oxidase, the key enzyme in ethylene synthesis, driven by the CaMV35S promoter mediated by Agrobacterium tumefacien. Hygromycin phosphotransferase (HPT) gene was used as selection marker. Leaf explants were pre-cultured on shoot-inducing medium for 2 d, then immersed in Agrobacterium suspension for 8-12 min. Co-cultivation was carried out on the medium (MS+BA 1.0 mg/L+NAA 0.3 mg/L +Acetosyringone 100 micromol/L, pH 5.8-6.0) for 3 d. After that transformants were obtained by transferring explants to selection medium supplemented with 5 mg/L hygromycin (Hyg) and 400 mg/L cefotaxime (Cef). Southern blotting detection showed that a foreign gene was integrated into the carnation genome and 3 transgenic lines (T257, T299 and T273 line) obtained. Addition of acetosyringone and the time of co-culture were the main factors that influenced transformation frequency. After being transplanted to soil, transgenic plants were grew normally in greenhouse. Ethylene production of cut flower of transgenic T257 line was 95% lower than that of the control, and that of T299 line was reduced by 90% than that of the control, while that of transgenic T273 line has no of significantly different from control. Vase life of transgenic T257 line was 5 d longer than that of the control line at 25 degrees C.

  3. Repeated Gene Transfection Impairs the Engraftment of Transplanted Porcine Neonatal Pancreatic Cells

    Directory of Open Access Journals (Sweden)

    Min Koo Seo

    2011-02-01

    Full Text Available BackgroundPreviously, we reported that neonatal porcine pancreatic cells transfected with hepatocyte growth factor (HGF gene in an Epstein-Barr virus (EBV-based plasmid (pEBVHGF showed improved proliferation and differentiation compared to those of the control. In this study, we examined if pancreatic cells transfected repeatedly with pEBVHGF can be successfully grafted to control blood glucose in a diabetes mouse model.MethodsNeonatal porcine pancreatic cells were cultured as a monolayer and were transfected with pEBVHGF every other day for a total of three transfections. The transfected pancreatic cells were re-aggregated and transplanted into kidney capsules of diabetic nude mice or normal nude mice. Blood glucose level and body weight were measured every other day after transplantation. The engraftment of the transplanted cells and differentiation into beta cells were assessed using immunohistochemistry.ResultsRe-aggregation of the pancreatic cells before transplantation improved engraftment of the cells and facilitated neovascularization of the graft. Right before transplantation, pancreatic cells that were transfected with pEBVHGF and then re-aggregated showed ductal cell marker expression. However, ductal cells disappeared and the cells underwent fibrosis in a diabetes mouse model two to five weeks after transplantation; these mice also did not show controlled blood glucose levels. Furthermore, pancreatic cells transplanted into nude mice with normal blood glucose showed poor graft survival regardless of the type of transfected plasmid (pCEP4, pHGF, or pEBVHGF.ConclusionFor clinical application of transfected neonatal porcine pancreatic cells, further studies are required to develop methods of overcoming the damage for the cells caused by repeated transfection and to re-aggregate them into islet-like structures.

  4. A highly parallel method for synthesizing DNA repeats enables the discovery of 'smart' protein polymers.

    Science.gov (United States)

    Amiram, Miriam; Quiroz, Felipe Garcia; Callahan, Daniel J; Chilkoti, Ashutosh

    2011-02-01

    Robust high-throughput synthesis methods are needed to expand the repertoire of repetitive protein-polymers for different applications. To address this need, we developed a new method, overlap extension rolling circle amplification (OERCA), for the highly parallel synthesis of genes encoding repetitive protein-polymers. OERCA involves a single PCR-type reaction for the rolling circle amplification of a circular DNA template and simultaneous overlap extension by thermal cycling. We characterized the variables that control OERCA and demonstrated its superiority over existing methods, its robustness, high-throughput and versatility by synthesizing variants of elastin-like polypeptides (ELPs) and protease-responsive polymers of glucagon-like peptide-1 analogues. Despite the GC-rich, highly repetitive sequences of ELPs, we synthesized remarkably large genes without recursive ligation. OERCA also enabled us to discover 'smart' biopolymers that exhibit fully reversible thermally responsive behaviour. This powerful strategy generates libraries of repetitive genes over a wide and tunable range of molecular weights in a 'one-pot' parallel format.

  5. Recombinant expression of TLR5 proteins by ligand supplementation and a leucine-rich repeat hybrid technique

    OpenAIRE

    Hong, Minsun; Yoon, Sung-il; Wilson, Ian A.

    2012-01-01

    Vertebrate TLR5 directly binds bacterial flagellin proteins and activates innate immune responses against pathogenic flagellated bacteria. Structural and biochemical studies on the TLR5/flagellin interaction have been challenging due to the technical difficulty in obtaining active recombinant proteins of TLR5 ectodomain (TLR5-ECD). We recently succeeded in production of the N-terminal leucine rich repeats (LRRs) of Danio rerio (dr) TLR5-ECD in a hybrid with another LRR protein, hagfish variab...

  6. The energy landscapes of repeat-containing proteins: topology, cooperativity, and the folding funnels of one-dimensional architectures.

    Directory of Open Access Journals (Sweden)

    Diego U Ferreiro

    2008-05-01

    Full Text Available Repeat-proteins are made up of near repetitions of 20- to 40-amino acid stretches. These polypeptides usually fold up into non-globular, elongated architectures that are stabilized by the interactions within each repeat and those between adjacent repeats, but that lack contacts between residues distant in sequence. The inherent symmetries both in primary sequence and three-dimensional structure are reflected in a folding landscape that may be analyzed as a quasi-one-dimensional problem. We present a general description of repeat-protein energy landscapes based on a formal Ising-like treatment of the elementary interaction energetics in and between foldons, whose collective ensemble are treated as spin variables. The overall folding properties of a complete "domain" (the stability and cooperativity of the repeating array can be derived from this microscopic description. The one-dimensional nature of the model implies there are simple relations for the experimental observables: folding free-energy (DeltaG(water and the cooperativity of denaturation (m-value, which do not ordinarily apply for globular proteins. We show how the parameters for the "coarse-grained" description in terms of foldon spin variables can be extracted from more detailed folding simulations on perfectly funneled landscapes. To illustrate the ideas, we present a case-study of a family of tetratricopeptide (TPR repeat proteins and quantitatively relate the results to the experimentally observed folding transitions. Based on the dramatic effect that single point mutations exert on the experimentally observed folding behavior, we speculate that natural repeat proteins are "poised" at particular ratios of inter- and intra-element interaction energetics that allow them to readily undergo structural transitions in physiologically relevant conditions, which may be intrinsically related to their biological functions.

  7. Invertebrate and vertebrate class III myosins interact with MORN repeat-containing adaptor proteins.

    Directory of Open Access Journals (Sweden)

    Kirk L Mecklenburg

    Full Text Available In Drosophila photoreceptors, the NINAC-encoded myosin III is found in a complex with a small, MORN-repeat containing, protein Retinophilin (RTP. Expression of these two proteins in other cell types showed NINAC myosin III behavior is altered by RTP. NINAC deletion constructs were used to map the RTP binding site within the proximal tail domain of NINAC. In vertebrates, the RTP ortholog is MORN4. Co-precipitation experiments demonstrated that human MORN4 binds to human myosin IIIA (MYO3A. In COS7 cells, MORN4 and MYO3A, but not MORN4 and MYO3B, co-localize to actin rich filopodia extensions. Deletion analysis mapped the MORN4 binding to the proximal region of the MYO3A tail domain. MYO3A dependent MORN4 tip localization suggests that MYO3A functions as a motor that transports MORN4 to the filopodia tips and MORN4 may enhance MYO3A tip localization by tethering it to the plasma membrane at the protrusion tips. These results establish conserved features of the RTP/MORN4 family: they bind within the tail domain of myosin IIIs to control their behavior.

  8. Novel Mutations of the Tetratricopeptide Repeat Domain 7A Gene and Phenotype/Genotype Comparison

    Directory of Open Access Journals (Sweden)

    Reyin Lien

    2017-09-01

    Full Text Available The gastrointestinal tract contains the largest lymphoid organ to react with pathogenic microorganisms and suppress excess inflammation. Patients with primary immunodeficiency diseases (PIDs can suffer from refractory diarrhea. In this study, we present two siblings who began to suffer from refractory diarrhea with a poor response to aggressive antibiotic and immunosuppressive treatment after surgical release of neonatal intestinal obstruction. Their lymphocyte proliferation was low, but superoxide production and IL-10 signaling were normal. Candidate genetic approach targeted to genes involved in PIDs with inflammatory bowel disease (IBD-like manifestation was unrevealing. Whole-genome sequencing revealed novel heterozygous mutations Glu75Lys and nucleotide 520–521 CT deletion in the tetratricopeptide repeat domain 7A (TTC7A gene. A Medline search identified 49 patients with TTC7A mutations, of whom 20 survived. Their phenotypes included both multiple intestinal atresia (MIA and combined T and/or B immunodeficiency (CID in 16, both IBD and CID in 14, isolated MIA in 8, MIA, IBD, and CID complex in 8, and isolated IBD in 3. Of these 98 mutant alleles over-through the coding region clustering on exon 2 (40 alleles, exon 7 (12 alleles, and exon 20 (10 alleles, 2 common hotspot mutations were c.211 G>A (p.E71K in exon 2 in 26 alleles and AAGT deletion in exon 7 (+3 in 10 alleles. Kaplan–Meier analysis showed that those with biallelic missense mutations (p = 0.0168, unaffected tetratricopeptide repeat domains (p = 0.0311, and developing autoimmune disorders (p = 0.001 had a relatively better prognosis. Hematopoietic stem cell transplantation (HSCT restored immunity and seemed to decrease the frequency of infections; however, refractory diarrhea persisted. Clinical improvement was reported upon intestinal and liver transplantation in a child with CID and MIA of unknown genetic etiology. In conclusion, patients with TTC7A mutations

  9. Expression and its significance of stem cells marker leucine-rich repeat containing G protein coupled receptor 5 gene in human colorectal cancer%人结直肠癌干细胞标志物富含亮氨酸重复单位的G蛋白耦联受体5基因的表达及意义

    Institute of Scientific and Technical Information of China (English)

    孙艳; 盛春华; 文大成; 李玉林; 迟宝荣

    2013-01-01

    目的 探讨人结直肠癌组织及外周血中干细胞标志物富含亮氨酸重复单位的G蛋白耦联受体5(lgr5)基因的表达及其与临床病理特征间的关系.方法 采用SYBR Green实时定量PCR方法检测27份结直肠癌组织及配对的正常组织中、1 7例患者及8名健康对照者外周血中lgr5mRNA的表达.应用Wilcoxon秩和检验分析lgr5 mRNA在不同组织间、临床病理参数之间的表达差异.结果 结直肠癌组织中lgr5 mRNA表达水平为1.000(0.012,496.353),高于配对正常组织的0.147(0.004,73.002),差异有统计学意义(Z=8.029,P<0.01).结直肠癌患者外周血中lgr5mRNA表达水平为0.742(0.077,456.566),高于健康对照组的0.104(0.034,0.274),差异有统计学意义(Z=2.048,P<0.05).结直肠癌组织lgr5 mRNA在不同性别、年龄、肿瘤原发部位、肿瘤大小、组织学类型组间表达差异均无统计学意义(P均>0.05),但有淋巴结转移组lgr5 mRNA表达高于无淋巴结转移组,差异有统计学意义(Z=2.066,P<0.05).结论 lgr5在结直肠癌组织及外周血中的表达上调可能参与了结直肠癌的生长及转移.%Objective To investigate the expression of stem cell marker leucine-rich repeat containing G protein coupled receptor 5 (lgr5) gene in human colorectal cancer tissues and peripheral blood and its correlation with clinical pathological characteristics.Methods The expression of lgr5 at mRNA level was detected by SYBR Green quantitative real-time polymerase chain reaction (PCR) in 27 human colorectal cancer tissues and corresponding non-cancerous tissues as well as in peripheral blood of 17 patients and eight healthy controls.The differences of lgr5 mRNA expression in different tissues and clinical pathology parameters were analyzed by Wilcoxon test.Results The expression of lgr5 at mRNA level in colorectal cancer tissues was 1.000 (0.012,496.353),which was higher than that of corresponding non-cancerous tissues 0.147 (0.004,73.002),the

  10. A group of Giardia lamblia variant-specific surface protein (VSP) genes with nearly identical 5' regions.

    Science.gov (United States)

    Yang, Y; Adam, R D

    1995-12-01

    The surfaces of Giardia lamblia trophozoites contain one of a set of variant-specific surface proteins. The genes encoding these proteins are highly conserved at the 3' terminus, but frequently demonstrate little similarity in the remainder of the coding region. This report describes a family of vsp genes highly similar to a repeat-containing vsp gene (vspC5) at the 5' coding and flanking regions, but which diverge abruptly from vspC5 in the first repeat and do not themselves contain full copies of the repeat. This observation suggests the possibility that recombination among different vsp genes may have played a role in development of the vsp gene repertoire.

  11. Utilization of ELISA using thioredoxin peroxidase-1 and tandem repeat proteins for diagnosis of Schistosoma japonicum infection among water buffaloes.

    Directory of Open Access Journals (Sweden)

    Jose Ma M Angeles

    Full Text Available BACKGROUND: The presence of animal reservoirs in Schistosoma japonicum infection has been a major obstacle in the control of schistosomiasis. Previous studies have proven that the inclusion of control measures on animal reservoir hosts for schistosomiasis contributed to the decrease of human cases. Animal surveillance should therefore be included to strengthen and improve the capabilities of current serological tests. METHODOLOGY/PRINCIPAL FINDINGS: Thioredoxin peroxidase-1 (SjTPx-1 and four tandem repeat proteins (Sj1TR, Sj2TR, Sj4TR, Sj7TR were initially evaluated against human sera. The previous test showed high sensitivity and specificity for antibody detection against SjTPx-1 and Sj7TR. In this study, the immunodiagnostic potential of these recombinant proteins was evaluated using enzyme-linked immunoassay on 50 water buffalo serum samples collected in Cagayan, the Philippines as compared with the soluble egg antigen (SEA. For specificity, 3 goat serum samples positive with Fasciola hepatica were used and among the antigens used, only SEA showed cross-reaction. Stool PCR targeting the S. japonicum 82 bp mitochondrial NAD 1 gene was done to confirm the true positives and served as the standard test. Twenty three samples were positive for stool PCR. SjTPx-1 and Sj1TR gave the highest sensitivity among the recombinant proteins tested for water buffalo samples with 82.61% and 78.26% respectively which were higher than that of SEA (69.57%. CONCLUSIONS/SIGNIFICANCE: These results prove that SjTPx-1 works both for humans and water buffaloes making it a good candidate antigen for zoonotic diagnosis. Sj1TR showed good results for water buffaloes and therefore can also be used as a possible candidate for detecting animal schistosome infection.

  12. Widely predicting specific protein functions based on protein-protein interaction data and gene expression profile

    Institute of Scientific and Technical Information of China (English)

    GAO Lei; LI Xia; GUO Zheng; ZHU MingZhu; LI YanHui; RAO ShaoQi

    2007-01-01

    GESTs (gene expression similarity and taxonomy similarity), a gene functional prediction approach previously proposed by us, is based on gene expression similarity and concept similarity of functional classes defined in Gene Ontology (GO). In this paper, we extend this method to protein-protein interaction data by introducing several methods to filter the neighbors in protein interaction networks for a protein of unknown function(s). Unlike other conventional methods, the proposed approach automatically selects the most appropriate functional classes as specific as possible during the learning process, and calls on genes annotated to nearby classes to support the predictions to some small-sized specific classes in GO. Based on the yeast protein-protein interaction information from MIPS and a dataset of gene expression profiles, we assess the performances of our approach for predicting protein functions to "biology process" by three measures particularly designed for functional classes organized in GO. Results show that our method is powerful for widely predicting gene functions with very specific functional terms. Based on the GO database published in December 2004, we predict some proteins whose functions were unknown at that time, and some of the predictions have been confirmed by the new SGD annotation data published in April, 2006.

  13. Widely predicting specific protein functions based on protein-protein interaction data and gene expression profile

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    GESTs (gene expression similarity and taxonomy similarity), a gene functional prediction approach previously proposed by us, is based on gene expression similarity and concept similarity of functional classes defined in Gene Ontology (GO). In this paper, we extend this method to protein-protein interac-tion data by introducing several methods to filter the neighbors in protein interaction networks for a protein of unknown function(s). Unlike other conventional methods, the proposed approach automati-cally selects the most appropriate functional classes as specific as possible during the learning proc-ess, and calls on genes annotated to nearby classes to support the predictions to some small-sized specific classes in GO. Based on the yeast protein-protein interaction information from MIPS and a dataset of gene expression profiles, we assess the performances of our approach for predicting protein functions to “biology process” by three measures particularly designed for functional classes organ-ized in GO. Results show that our method is powerful for widely predicting gene functions with very specific functional terms. Based on the GO database published in December 2004, we predict some proteins whose functions were unknown at that time, and some of the predictions have been confirmed by the new SGD annotation data published in April, 2006.

  14. Analysis of the trinucleotide CAG repeat from the human mitochondrial DNA polymerase gene in healthy and diseased individuals.

    Science.gov (United States)

    Rovio, A; Tiranti, V; Bednarz, A L; Suomalainen, A; Spelbrink, J N; Lecrenier, N; Melberg, A; Zeviani, M; Poulton, J; Foury, F; Jacobs, H T

    1999-01-01

    The human nuclear gene (POLG) for the catalytic subunit of mitochondrial DNA polymerase (DNA polymerase gamma) contains a trinucleotide CAG microsatellite repeat within the coding sequence. We have investigated the frequency of different repeat-length alleles in populations of diseased and healthy individuals. The predominant allele of 10 CAG repeats was found at a very similar frequency (approximately 88%) in both Finnish and ethnically mixed population samples, with homozygosity close to the equilibrium prediction. Other alleles of between 5 and 13 repeat units were detected, but no larger, expanded alleles were found. A series of 51 British myotonic dystrophy patients showed no significant variation from controls, indicating an absence of generalised CAG repeat instability. Patients with a variety of molecular lesions in mtDNA, including sporadic, clonal deletions, maternally inherited point mutations, autosomally transmitted mtDNA depletion and autosomal dominant multiple deletions showed no differences in POLG trinucleotide repeat-length distribution from controls. These findings rule out POLG repeat expansion as a common pathogenic mechanism in disorders characterised by mitochondrial genome instability.

  15. Huntington CAG repeat size does not modify onset age in familial Parkinson’s disease: The GenePD Study

    Science.gov (United States)

    McNicoll, Christopher F.; Latourelle, Jeanne C.; MacDonald, Marcy E.; Lew, Mark F.; Suchowersky, Oksana; Klein, Christine; Golbe, Lawrence I.; Mark, Margery H.; Growdon, John H.; Wooten, G. Frederick; Watts, Ray L.; Guttman, Mark; Racette, Brad A.; Perlmutter, Joel S.; Ahmed, Anwar; Shill, Holly A.; Singer, Carlos; Saint-Hilaire, Marie H.; Massood, Tiffany; Huskey, Karen W.; DeStefano, Anita L.; Gillis, Tammy; Mysore, Jayalakshmi; Goldwurm, Stefano; Pezzoli, Gianni; Baker, Kenneth B.; Itin, Ilia; Litvan, Irene; Nicholson, Garth; Corbett, Alastair; Nance, Martha; Drasby, Edward; Isaacson, Stuart; Burn, David J.; Chinnery, Patrick F.; Pramstaller, Peter P.; Al-hinti, Jomana; Moller, Anette T.; Ostergaard, Karen; Sherman, Scott J.; Roxburgh, Richard; Snow, Barry; Slevin, John T.; Cambi, Franca; Gusella, James F.; Myers, Richard H.

    2009-01-01

    The ATP/ADP ratio reflects mitochondrial function and has been reported to be influenced by the size of the Huntington disease gene (HD) repeat. Impaired mitochondrial function has long been implicated in the pathogenesis of Parkinson’s disease (PD) and therefore, we evaluated the relationship of the HD CAG repeat size to PD onset age in a large sample of familial PD cases. PD affected siblings (n=495) with known onset ages from 248 families, were genotyped for the HD CAG repeat. Genotyping failed in 11 cases leaving 484 for analysis, including 35 LRRK2 carriers. All cases had HD CAG repeats (range 15 to 34) below the clinical range for HD, although 5.2 percent of the sample (n=25) had repeats in the intermediate range (the intermediate range lower limit=27; upper limit=35 repeats), suggesting that the prevalence of intermediate allele carriers in the general population is significant. No relation between the HD CAG repeat size and the age at onset for PD was found in this sample of familial PD. PMID:18649400

  16. Phylogenomic approaches to common problems encountered in the analysis of low copy repeats: The sulfotransferase 1A gene family example

    Directory of Open Access Journals (Sweden)

    Benner Steven A

    2005-03-01

    Full Text Available Abstract Background Blocks of duplicated genomic DNA sequence longer than 1000 base pairs are known as low copy repeats (LCRs. Identified by their sequence similarity, LCRs are abundant in the human genome, and are interesting because they may represent recent adaptive events, or potential future adaptive opportunities within the human lineage. Sequence analysis tools are needed, however, to decide whether these interpretations are likely, whether a particular set of LCRs represents nearly neutral drift creating junk DNA, or whether the appearance of LCRs reflects assembly error. Here we investigate an LCR family containing the sulfotransferase (SULT 1A genes involved in drug metabolism, cancer, hormone regulation, and neurotransmitter biology as a first step for defining the problems that those tools must manage. Results Sequence analysis here identified a fourth sulfotransferase gene, which may be transcriptionally active, located on human chromosome 16. Four regions of genomic sequence containing the four human SULT1A paralogs defined a new LCR family. The stem hominoid SULT1A progenitor locus was identified by comparative genomics involving complete human and rodent genomes, and a draft chimpanzee genome. SULT1A expansion in hominoid genomes was followed by positive selection acting on specific protein sites. This episode of adaptive evolution appears to be responsible for the dopamine sulfonation function of some SULT enzymes. Each of the conclusions that this bioinformatic analysis generated using data that has uncertain reliability (such as that from the chimpanzee genome sequencing project has been confirmed experimentally or by a "finished" chromosome 16 assembly, both of which were published after the submission of this manuscript. Conclusion SULT1A genes expanded from one to four copies in hominoids during intra-chromosomal LCR duplications, including (apparently one after the divergence of chimpanzees and humans. Thus, LCRs may

  17. Association analysis of a highly polymorphic CAG Repeat in the human potassium channel gene KCNN3 and migraine susceptibility

    Directory of Open Access Journals (Sweden)

    Ovcaric Mick

    2005-09-01

    Full Text Available Abstract Background Migraine is a polygenic multifactorial disease, possessing environmental and genetic causative factors with multiple involved genes. Mutations in various ion channel genes are responsible for a number of neurological disorders. KCNN3 is a neuronal small conductance calcium-activated potassium channel gene that contains two polyglutamine tracts, encoded by polymorphic CAG repeats in the gene. This gene plays a critical role in determining the firing pattern of neurons and acts to regulate intracellular calcium channels. Methods The present association study tested whether length variations in the second (more 3' polymorphic CAG repeat in exon 1 of the KCNN3 gene, are involved in susceptibility to migraine with and without aura (MA and MO. In total 423 DNA samples from unrelated individuals, of which 202 consisted of migraine patients and 221 non-migraine controls, were genotyped and analysed using a fluorescence labelled primer set on an ABI310 Genetic Analyzer. Allele frequencies were calculated from observed genotype counts for the KCNN3 polymorphism. Analysis was performed using standard contingency table analysis, incorporating the chi-squared test of independence and CLUMP analysis. Results Overall, there was no convincing evidence that KCNN3 CAG lengths differ between Caucasian migraineurs and controls, with no significant difference in the allelic length distribution of CAG repeats between the population groups (P = 0.090. Also the MA and MO subtypes did not differ significantly between control allelic distributions (P > 0.05. The prevalence of the long CAG repeat (>19 repeats did not reach statistical significance in migraineurs (P = 0.15, nor was there a significant difference between the MA and MO subgroups observed compared to controls (P = 0.46 and P = 0.09, respectively, or between MA vs MO (P = 0.40. Conclusion This association study provides no evidence that length variations of the second polyglutamine array in

  18. Crystal Structure of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Csn2 Protein Revealed Ca[superscript 2+]-dependent Double-stranded DNA Binding Activity

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Ki Hyun; Kurinov, Igor; Ke, Ailong (Cornell); (NWU)

    2012-05-22

    Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) are widespread in bacteria and archaea. They form a line of RNA-based immunity to eradicate invading bacteriophages and malicious plasmids. A key molecular event during this process is the acquisition of new spacers into the CRISPR loci to guide the selective degradation of the matching foreign genetic elements. Csn2 is a Nmeni subtype-specific cas gene required for new spacer acquisition. Here we characterize the Enterococcus faecalis Csn2 protein as a double-stranded (ds-) DNA-binding protein and report its 2.7 {angstrom} tetrameric ring structure. The inner circle of the Csn2 tetrameric ring is {approx}26 {angstrom} wide and populated with conserved lysine residues poised for nonspecific interactions with ds-DNA. Each Csn2 protomer contains an {alpha}/{beta} domain and an {alpha}-helical domain; significant hinge motion was observed between these two domains. Ca{sup 2+} was located at strategic positions in the oligomerization interface. We further showed that removal of Ca{sup 2+} ions altered the oligomerization state of Csn2, which in turn severely decreased its affinity for ds-DNA. In summary, our results provided the first insight into the function of the Csn2 protein in CRISPR adaptation by revealing that it is a ds-DNA-binding protein functioning at the quaternary structure level and regulated by Ca{sup 2+} ions.

  19. Crystal structure of clustered regularly interspaced short palindromic repeats (CRISPR)-associated Csn2 protein revealed Ca2+-dependent double-stranded DNA binding activity.

    Science.gov (United States)

    Nam, Ki Hyun; Kurinov, Igor; Ke, Ailong

    2011-09-02

    Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) are widespread in bacteria and archaea. They form a line of RNA-based immunity to eradicate invading bacteriophages and malicious plasmids. A key molecular event during this process is the acquisition of new spacers into the CRISPR loci to guide the selective degradation of the matching foreign genetic elements. Csn2 is a Nmeni subtype-specific cas gene required for new spacer acquisition. Here we characterize the Enterococcus faecalis Csn2 protein as a double-stranded (ds-) DNA-binding protein and report its 2.7 Å tetrameric ring structure. The inner circle of the Csn2 tetrameric ring is ∼26 Å wide and populated with conserved lysine residues poised for nonspecific interactions with ds-DNA. Each Csn2 protomer contains an α/β domain and an α-helical domain; significant hinge motion was observed between these two domains. Ca(2+) was located at strategic positions in the oligomerization interface. We further showed that removal of Ca(2+) ions altered the oligomerization state of Csn2, which in turn severely decreased its affinity for ds-DNA. In summary, our results provided the first insight into the function of the Csn2 protein in CRISPR adaptation by revealing that it is a ds-DNA-binding protein functioning at the quaternary structure level and regulated by Ca(2+) ions.

  20. Normal CAG and CCG repeats in the Huntington`s disease genes of Parkinson`s disease patients

    Energy Technology Data Exchange (ETDEWEB)

    Rubinsztein, D.C.; Leggo, J.; Barton, D.E. [Cambridge Univ. (United Kingdom)] [and others

    1995-04-24

    The clinical features of Parkinson`s disease, particularly rigidity and bradykinesia and occasionally tremor, are seen in juvenile-onset Huntington`s disease. Therefore, the CAG and CCG repeats in the Huntington`s disease gene were investigated in 45 Parkinson`s disease patients and compared to 40 control individuals. All of the Parkinson`s disease chromosomes fell within the normal size ranges. In addition, the distributions of the two repeats in the Parkinson`s disease patients did not differ significantly from those of the control population. Therefore, abnormalities of these trinucleotide repeats in the Huntington`s disease gene are not likely to contribute to the pathogenesis of Parkinson`s disease. 12 refs., 2 figs.

  1. Mining disease genes using integrated protein-protein interaction and gene-gene co-regulation information.

    Science.gov (United States)

    Li, Jin; Wang, Limei; Guo, Maozu; Zhang, Ruijie; Dai, Qiguo; Liu, Xiaoyan; Wang, Chunyu; Teng, Zhixia; Xuan, Ping; Zhang, Mingming

    2015-01-01

    In humans, despite the rapid increase in disease-associated gene discovery, a large proportion of disease-associated genes are still unknown. Many network-based approaches have been used to prioritize disease genes. Many networks, such as the protein-protein interaction (PPI), KEGG, and gene co-expression networks, have been used. Expression quantitative trait loci (eQTLs) have been successfully applied for the determination of genes associated with several diseases. In this study, we constructed an eQTL-based gene-gene co-regulation network (GGCRN) and used it to mine for disease genes. We adopted the random walk with restart (RWR) algorithm to mine for genes associated with Alzheimer disease. Compared to the Human Protein Reference Database (HPRD) PPI network alone, the integrated HPRD PPI and GGCRN networks provided faster convergence and revealed new disease-related genes. Therefore, using the RWR algorithm for integrated PPI and GGCRN is an effective method for disease-associated gene mining.

  2. An inverted repeat motif stabilizes binding of E2F and enhances transcription of the dihydrofolate reductase gene

    DEFF Research Database (Denmark)

    Wade, M; Blake, M C; Jambou, R C

    1995-01-01

    An overlapping inverted repeat sequence that binds the eukaryotic transcription factor E2F is 100% conserved near the major transcription start sites in the promoters of three mammalian genes encoding dihydrofolate reductase, and is also found in the promoters of several other important cellular ...

  3. Lactococcus garvieae carries a chromosomally encoded pentapeptide repeat protein that confers reduced susceptibility to quinolones in Escherichia coli producing a cytotoxic effect.

    Science.gov (United States)

    Gibello, Alicia; Díaz de Alba, Paula; Blanco, M Mar; Machuca, Jesus; Cutuli, M Teresa; Rodríguez-Martínez, José Manuel

    2014-09-01

    This study characterises a chromosomal gene of Lactococcus garvieae encoding a pentapeptide repeat protein designated as LgaQnr. This gene has been implicated in reduced susceptibility to quinolones in this bacterium, which is of relevance to both veterinary and human medicine. All of the L. garvieae isolates analysed were positive for the lgaqnr gene. The expression of lgaqnr in Escherichia coli reduced the susceptibility to quinolones, producing an adverse effect. The reduced susceptibility to ciprofloxacin was 16-fold in E. coli ATCC 25922 and 32-fold in E. coli DH10B, compared to the control strains. The minimum inhibitory concentration of nalidixic acid was also increased 4 or 5-fold. The effect of the expression of lgaqnr in E. coli was investigated by electron microscopy and was observed to affect the structure of the cell and the inner membrane of the recombinant cells.

  4. Genome-wide analyses and functional classification of proline repeat-rich proteins: potential role of eIF5A in eukaryotic evolution.

    Directory of Open Access Journals (Sweden)

    Ajeet Mandal

    Full Text Available The eukaryotic translation factor, eIF5A has been recently reported as a sequence-specific elongation factor that facilitates peptide bond formation at consecutive prolines in Saccharomyces cerevisiae, as its ortholog elongation factor P (EF-P does in bacteria. We have searched the genome databases of 35 representative organisms from six kingdoms of life for PPP (Pro-Pro-Pro and/or PPG (Pro-Pro-Gly-encoding genes whose expression is expected to depend on eIF5A. We have made detailed analyses of proteome data of 5 selected species, Escherichia coli, Saccharomyces cerevisiae, Drosophila melanogaster, Mus musculus and Homo sapiens. The PPP and PPG motifs are low in the prokaryotic proteomes. However, their frequencies markedly increase with the biological complexity of eukaryotic organisms, and are higher in newly derived proteins than in those orthologous proteins commonly shared in all species. Ontology classifications of S. cerevisiae and human genes encoding the highest level of polyprolines reveal their strong association with several specific biological processes, including actin/cytoskeletal associated functions, RNA splicing/turnover, DNA binding/transcription and cell signaling. Previously reported phenotypic defects in actin polarity and mRNA decay of eIF5A mutant strains are consistent with the proposed role for eIF5A in the translation of the polyproline-containing proteins. Of all the amino acid tandem repeats (≥3 amino acids, only the proline repeat frequency correlates with functional complexity of the five organisms examined. Taken together, these findings suggest the importance of proline repeat-rich proteins and a potential role for eIF5A and its hypusine modification pathway in the course of eukaryotic evolution.

  5. A prefoldin-associated WD-repeat protein (WDR92) is required for the correct architectural assembly of motile cilia

    Science.gov (United States)

    Patel-King, Ramila S.; King, Stephen M.

    2016-01-01

    WDR92 is a highly conserved WD-repeat protein that has been proposed to be involved in apoptosis and also to be part of a prefoldin-like cochaperone complex. We found that WDR92 has a phylogenetic signature that is generally compatible with it playing a role in the assembly or function of specifically motile cilia. To test this hypothesis, we performed an RNAi-based knockdown of WDR92 gene expression in the planarian Schmidtea mediterranea and were able to achieve a robust reduction in mRNA expression to levels undetectable under our standard RT-PCR conditions. We found that this treatment resulted in a dramatic reduction in the rate of organismal movement that was caused by a switch in the mode of locomotion from smooth, cilia-driven gliding to muscle-based, peristaltic contractions. Although the knockdown animals still assembled cilia of normal length and in similar numbers to controls, these structures had reduced beat frequency and did not maintain hydrodynamic coupling. By transmission electron microscopy we observed that many cilia had pleiomorphic defects in their architecture, including partial loss of dynein arms, incomplete closure of the B-tubule, and occlusion or replacement of the central pair complex by accumulated electron-dense material. These observations suggest that WDR92 is part of a previously unrecognized cytoplasmic chaperone system that is specifically required to fold key components necessary to build motile ciliary axonemes. PMID:26912790

  6. Pentatricopeptide-repeat family protein RF6 functions with hexokinase 6 to rescue rice cytoplasmic male sterility.

    Science.gov (United States)

    Huang, Wenchao; Yu, Changchun; Hu, Jun; Wang, Lili; Dan, Zhiwu; Zhou, Wei; He, Chunlan; Zeng, Yafei; Yao, Guoxin; Qi, Jianzhao; Zhang, Zhihong; Zhu, Renshan; Chen, Xuefeng; Zhu, Yingguo

    2015-12-01

    Cytoplasmic male sterility (CMS) has been extensively used for hybrid seed production in many major crops. Honglian CMS (HL-CMS) is one of the three major types of CMS in rice and has contributed greatly to food security worldwide. The HL-CMS trait is associated with an aberrant chimeric mitochondrial transcript, atp6-orfH79, which causes pollen sterility and can be rescued by two nonallelic restorer-of-fertility (Rf) genes, Rf5 or Rf6. Here, we report the identification of Rf6, which encodes a novel pentatricopeptide repeat (PPR) family protein with a characteristic duplication of PPR motifs 3-5. RF6 is targeted to mitochondria, where it physically associates with hexokinase 6 (OsHXK6) and promotes the processing of the aberrant CMS-associated transcript atp6-orfH79 at nucleotide 1238, which ensures normal pollen development and restores fertility. The duplicated motif 3 of RF6 is essential for RF6-OsHXK6 interactions, processing of the aberrant transcript, and restoration of fertility. Furthermore, reductions in the level of OsHXK6 result in atp6-orfH79 transcript accumulation and male sterility. Together these results reveal a novel mechanism for CMS restoration by which RF6 functions with OsHXK6 to restore HL-CMS fertility. The present study also provides insight into the function of hexokinase 6 in regulating mitochondrial RNA metabolism and may facilitate further exploitation of heterosis in rice.

  7. An intron in a ribosomal protein gene from Tetrahymena

    DEFF Research Database (Denmark)

    Nielsen, Henrik; Andreasen, Per Hove; Dreisig, Hanne

    1986-01-01

    We have cloned and sequenced a single copy gene encoding a ribosomal protein from the ciliate Tetrahymena thermophila. The gene product was identified as ribosomal protein S25 by comparison of the migration in two-dimensional polyacrylamide gels of the protein synthesized by translation in vitro...... of hybrid-selected mRNA and authentic ribosomal proteins. The proteins show strong homology to ribosomal protein S12 from Escherichia coli. The coding region of the gene is interrupted by a 979-bp intron 68 bp downstream of the translation start. This is the first intron in a protein encoding gene...

  8. CAG repeat polymorphism in androgen receptor gene is not directly associated with polycystic ovary syndrome but influences serum testosterone levels.

    Science.gov (United States)

    Skrgatic, L; Baldani, D Pavicic; Cerne, J Z; Ferk, P; Gersak, K

    2012-02-01

    Hyperandrogenemia has been the most consistent feature of polycystic ovary syndrome (PCOS). Androgens exert their effects through androgen receptors (ARs). The expansion of the codon CAG trinucleotide repeat polymorphism in exon 1 of the AR gene represents a type of genetic alteration associated with changes in the AR gene function. The purpose of this study was to establish a possible association of the AR gene CAG repeat length polymorphism with PCOS, and its influence on clinical and biochemical androgen traits. Two hundred and fourteen Croatian women with PCOS and 209 healthy control women of reproductive age were enrolled. Phenotypic hyperandrogenism, BMI and waist to hip ratio were recorded. Hormonal profiles, fasting insulin and glucose levels were measured on cycle days 3-5. Genotyping of the CAG repeat polymorphism in the AR gene was performed. We found no significant difference in the mean CAG repeat number between the PCOS patients and controls (22.1±3.4 vs. 21.9±3.2, P=0.286). There was a positive correlation between the CAG repeat length and total testosterone (TT) in the PCOS group (R=0.225, P=0.015). A multiple linear regression model using mean CAG repeat length, BMI, age and HOMA-IR as predictors explained 8.5% (adjusted R²) of the variability in serum TT levels. In this model the CAG repeat polymorphism was found to be a significant predictor of serum TT levels in PCOS patients (P=0.015). The logistic regression analysis revealed that the CAG repeat length is not a significant predictor of hirsutism and acne status (P=0.921 and P=0.437, respectively). The model was adjusted for serum TT, free testosterone, androstendione and DHEAS levels as independent variables, which were also not found to be significant predictors of hirsutism (P=0.687, P=0.194, P=0.675 and P=0.938, respectively) or acne status (P=0.594, P=0.095, P=0.290 and P=0.151, respectively). In conclusion, the AR CAG repeat polymorphism is not a major determinant of PCOS in the

  9. Complete mitochondrial genome of Coelomactra antiquata (Mollusca: Bivalvia): The first representative from the family Mactridae with novel gene order and unusual tandem repeats.

    Science.gov (United States)

    Meng, Xueping; Zhao, Nana; Shen, Xin; Hao, Jue; Liang, Meng; Zhu, Xiaolin; Cheng, Hanliang; Yan, Binlun; Liu, Zhaopu

    2012-06-01

    The complete mitochondrial genome plays an important role in the accurate inference of phylogenetic relationships among metazoans. Mactridae, also known as trough shells or duck clams, is an important family of marine bivalve clams in the order Veneroida. Here we present the complete mitochondrial genome sequence of the Xishishe Coelomactra antiquata (Mollusca: Bivalvia), which is the first representative from the family Mactridae. The mitochondrial genome of C. antiquata is of 17,384bp in length, and encodes 35 genes, including 12 protein-coding, 21 transfer RNA, and 2 ribosomal RNA genes. Compared with the typical gene content of animal mitochondrial genomes, atp8 and tRNAS(2) are missing. Gene order of the mitochondrial genome of C. antiquata is unique compared with others from Veneroida. In the mitochondrial genome of the C. antiquata, a total of 2189bp of non-coding nucleotides are scattered among 26 non-coding regions. The largest non-coding region contains one section of tandem repeats (99 bp×11), which is the second largest tandem repeats found in the mitochondrial genomes from Veneroida. The phylogenetic trees based on mitochondrial genomes support the monophyly of Veneridae and Lucinidae, and the relationship at the family level: ((Veneridae+Mactridae)+(Cardiidae+Solecurtidae))+Lucinidae. The phylogenetic result is consistent with the morphological classification. Meanwhile, bootstrap values are very high (BP=94-100), suggesting that the evolutionary relationship based on mitochondrial genomes is very reliable. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. Insulin regulation of a novel WD-40 repeat protein in adipocytes.

    Science.gov (United States)

    Rodgers, B D; Levine, M A; Bernier, M; Montrose-Rafizadeh, C

    2001-02-01

    A 400 bp PCR product generated with degenerate primers derived from the glucagon-like peptide-1 receptor was used to screen a rat skeletal muscle cDNA library. The predicted amino acid sequence of the 978 bp open reading frame has a predicted M(r) of 35 804, an estimated isoelectric point (pI) of 5.31 and contains seven WD-40 repeats, which are common to G-protein beta subunits (Gbeta). Although chemically and structurally similar to Gbeta subunits, the predicted amino acid sequence, when compared with the previously cloned Gbeta isoforms, was found to be only 31-41% similar and thus was named Gbeta-like (GbetaL, 'Gable'). Western blotting of whole-cell lysates and immunoprecipitates of membrane and cytosolic fractions of HEK 293 cells stably overexpressing a carboxy-terminal His-tagged GbetaL indicates that the protein is cytosolic and that it migrates at 42 kDa. A 4 kb transcript was detected in all tissues surveyed by northern blotting; however, an additional 2 kb transcript was detected in testis. Expression of GbetaL mRNA was highest in the brain and testis, followed by lung, heart, kidney, skeletal muscle, spleen and liver. In addition, reverse transcriptase/PCR showed that several other tissues and cell lines express GbetaL. The ubiquitous nature of the tissue expression pattern of GbetaL is similar to that of the insulin receptor, which suggests that insulin may influence GbetaL expression. Indeed, GbetaL protein and mRNA levels, in fully differentiated 3T3-L1 adipocytes, were upregulated by insulin in a concentration-dependent fashion. These changes were highly sensitive to insulin stimulation, being minimally affected by doses as low as 0.1 nM and maximally elevated by 1 nM doses. These data suggest that insulin regulates GbetaL production and imply that some of the actions of insulin may be mediated, in part, by this novel intracellular protein.

  11. The 1.7 Å resolution structure of At2g44920, a pentapeptide-repeat protein in the thylakoid lumen of Arabidopsis thaliana

    Energy Technology Data Exchange (ETDEWEB)

    Ni, Shuisong; McGookey, Michael E.; Tinch, Stuart L.; Jones, Alisha N.; Jayaraman, Seetharaman; Tong, Liang; Kennedy, Michael A. (Miami U); (Columbia)

    2012-01-09

    At2g44920 belongs to a diverse family (Pfam PF00805) of pentapeptide-repeat proteins (PRPs) that are present in all known organisms except yeast. PRPs contain at least eight tandem-repeating sequences of five amino acids with an approximate consensus sequence (STAV)(D/N)(L/F)(S/T/R)(X). Recent crystal structures show that PRPs adopt a highly regular four-sided right-handed {beta}-helical structure consisting mainly of type II and type IV {beta}-turns, sometimes referred to as a repeated five-residue (or Rfr) fold. Among sequenced genomes, PRP genes are most abundant in cyanobacteria, leading to speculation that PRPs play an important role in the unique lifestyle of photosynthetic cyanobacteria. Despite the recent structural characterization of several cyanobacterial PRPs, most of their functions remain unknown. Plants, whose chloroplasts are of cyanobacterial origin, have only four PRP genes in their genomes. At2g44920 is one of three PRPs located in the thylakoid lumen. Here, the crystal structure of a double methionine mutant of residues 81-224 of At2g44920, the naturally processed fragment of one of its full-length isoforms, is reported at 1.7 {angstrom} resolution. The structure of At2g44920 consists of the characteristic Rfr fold with five uninterrupted coils made up of 25 pentapeptide repeats and {alpha}-helical elements capping both termini. A disulfide bridge links the two {alpha}-helices with a conserved loop between the helical elements at its C-terminus. This structure represents the first structure of a PRP protein whose subcellular location has been experimentally confirmed to be the thylakoid lumen in a plant species.

  12. Identification of Interferon-Stimulated Gene Proteins That Inhibit Human Parainfluenza Virus Type 3.

    Science.gov (United States)

    Rabbani, M A G; Ribaudo, Michael; Guo, Ju-Tao; Barik, Sailen

    2016-12-15

    A major arm of cellular innate immunity is type I interferon (IFN), represented by IFN-α and IFN-β. Type I IFN transcriptionally induces a large number of cellular genes, collectively known as IFN-stimulated gene (ISG) proteins, which act as antivirals. The IFIT (interferon-induced proteins with tetratricopeptide repeats) family proteins constitute a major subclass of ISG proteins and are characterized by multiple tetratricopeptide repeats (TPRs). In this study, we have interrogated IFIT proteins for the ability to inhibit the growth of human parainfluenza virus type 3 (PIV3), a nonsegmented negative-strand RNA virus of the Paramyxoviridae family and a major cause of respiratory disease in children. We found that IFIT1 significantly inhibited PIV3, whereas IFIT2, IFIT3, and IFIT5 were less effective or not at all. In further screening a set of ISG proteins we discovered that several other such proteins also inhibited PIV3, including IFITM1, IDO (indoleamine 2,3-dioxygenase), PKR (protein kinase, RNA activated), and viperin (virus inhibitory protein, endoplasmic reticulum associated, interferon inducible)/Cig5. The antiviral effect of IDO, the enzyme that catalyzes the first step of tryptophan degradation, could be counteracted by tryptophan. These results advance our knowledge of diverse ISG proteins functioning as antivirals and may provide novel approaches against PIV3.

  13. A Cluster of Nucleotide-Binding Site-Leucine-Rich Repeat Genes Resides in a Barley Powdery Mildew Resistance Quantitative Trait Loci on 7HL.

    Science.gov (United States)

    Cantalapiedra, Carlos P; Contreras-Moreira, Bruno; Silvar, Cristina; Perovic, Dragan; Ordon, Frank; Gracia, María Pilar; Igartua, Ernesto; Casas, Ana M

    2016-07-01

    Powdery mildew causes severe yield losses in barley production worldwide. Although many resistance genes have been described, only a few have already been cloned. A strong QTL (quantitative trait locus) conferring resistance to a wide array of powdery mildew isolates was identified in a Spanish barley landrace on the long arm of chromosome 7H. Previous studies narrowed down the QTL position, but were unable to identify candidate genes or physically locate the resistance. In this study, the exome of three recombinant lines from a high-resolution mapping population was sequenced and analyzed, narrowing the position of the resistance down to a single physical contig. Closer inspection of the region revealed a cluster of closely related NBS-LRR (nucleotide-binding site-leucine-rich repeat containing protein) genes. Large differences were found between the resistant lines and the reference genome of cultivar Morex, in the form of PAV (presence-absence variation) in the composition of the NBS-LRR cluster. Finally, a template-guided assembly was performed and subsequent expression analysis revealed that one of the new assembled candidate genes is transcribed. In summary, the results suggest that NBS-LRR genes, absent from the reference and the susceptible genotypes, could be functional and responsible for the powdery mildew resistance. The procedure followed is an example of the use of NGS (next-generation sequencing) tools to tackle the challenges of gene cloning when the target gene is absent from the reference genome.

  14. A Cluster of Nucleotide-Binding Site–Leucine-Rich Repeat Genes Resides in a Barley Powdery Mildew Resistance Quantitative Trait Loci on 7HL

    Directory of Open Access Journals (Sweden)

    Carlos P. Cantalapiedra

    2016-07-01

    Full Text Available Powdery mildew causes severe yield losses in barley production worldwide. Although many resistance genes have been described, only a few have already been cloned. A strong QTL (quantitative trait locus conferring resistance to a wide array of powdery mildew isolates was identified in a Spanish barley landrace on the long arm of chromosome 7H. Previous studies narrowed down the QTL position, but were unable to identify candidate genes or physically locate the resistance. In this study, the exome of three recombinant lines from a high-resolution mapping population was sequenced and analyzed, narrowing the position of the resistance down to a single physical contig. Closer inspection of the region revealed a cluster of closely related NBS-LRR (nucleotide-binding site–leucine-rich repeat containing protein genes. Large differences were found between the resistant lines and the reference genome of cultivar Morex, in the form of PAV (presence-absence variation in the composition of the NBS-LRR cluster. Finally, a template-guided assembly was performed and subsequent expression analysis revealed that one of the new assembled candidate genes is transcribed. In summary, the results suggest that NBS-LRR genes, absent from the reference and the susceptible genotypes, could be functional and responsible for the powdery mildew resistance. The procedure followed is an example of the use of NGS (next-generation sequencing tools to tackle the challenges of gene cloning when the target gene is absent from the reference genome.

  15. Knowledge-based design of reagentless fluorescent biosensors from a designed ankyrin repeat protein.

    Science.gov (United States)

    Brient-Litzler, Elodie; Plückthun, Andreas; Bedouelle, Hugues

    2010-04-01

    Designed ankyrin repeat proteins (DARPins) can be selected from combinatorial libraries to bind any target antigen. They show high levels of recombinant expression, solubility and stability, and contain no cysteine residue. The possibility of obtaining, from any DARPin and at high yields, fluorescent conjugates which respond to the binding of the antigen by a variation of fluorescence, would have numerous applications in micro- and nano-analytical sciences. This possibility was explored with Off7, a DARPin directed against the maltose binding protein (MalE) from Escherichia coli, with known crystal structure of the complex. Eight residues of Off7, whose solvent accessible surface area varies on association with the antigen but which are not in direct contact with the antigen, were individually mutated into cysteine and then chemically coupled with a fluorophore. The conjugates were ranked according to their relative sensitivities. All of them showed an increase in their fluorescence intensity on antigen binding by >1.7-fold. The best conjugate retained the same affinity as the parental DARPin. Its signal increased linearly and specifically with the concentration of antigen, up to 15-fold in buffer and 3-fold in serum when fully saturated, the difference being mainly due to the absorption of light by serum. Its lower limit of detection was equal to 0.3 nM with a standard spectrofluorometer. Titrations with potassium iodide indicated that the fluorescence variation was due to a shielding of the fluorescent group from the solvent by the antigen. These results suggest rules for the design of reagentless fluorescent biosensors from any DARPin.

  16. Designed ankyrin repeat proteins: a new approach to mimic complex antigens for diagnostic purposes?

    Directory of Open Access Journals (Sweden)

    Stefanie Hausammann

    Full Text Available Inhibitory antibodies directed against coagulation factor VIII (FVIII can be found in patients with acquired and congenital hemophilia A. Such FVIII-inhibiting antibodies are routinely detected by the functional Bethesda Assay. However, this assay has a low sensitivity and shows a high inter-laboratory variability. Another method to detect antibodies recognizing FVIII is ELISA, but this test does not allow the distinction between inhibitory and non-inhibitory antibodies. Therefore, we aimed at replacing the intricate antigen FVIII by Designed Ankyrin Repeat Proteins (DARPins mimicking the epitopes of FVIII inhibitors. As a model we used the well-described inhibitory human monoclonal anti-FVIII antibody, Bo2C11, for the selection on DARPin libraries. Two DARPins were selected binding to the antigen-binding site of Bo2C11, which mimic thus a functional epitope on FVIII. These DARPins inhibited the binding of the antibody to its antigen and restored FVIII activity as determined in the Bethesda assay. Furthermore, the specific DARPins were able to recognize the target antibody in human plasma and could therefore be used to test for the presence of Bo2C11-like antibodies in a large set of hemophilia A patients. These data suggest, that our approach might be used to isolate epitopes from different sets of anti-FVIII antibodies in order to develop an ELISA-based screening assay allowing the distinction of inhibitory and non-inhibitory anti-FVIII antibodies according to their antibody signatures.

  17. The Immature Fiber Mutant Phenotype of Cotton (Gossypium hirsutum Is Linked to a 22-bp Frame-Shift Deletion in a Mitochondria Targeted Pentatricopeptide Repeat Gene

    Directory of Open Access Journals (Sweden)

    Gregory N. Thyssen

    2016-06-01

    Full Text Available Cotton seed trichomes are the most important source of natural fibers globally. The major fiber thickness properties influence the price of the raw material, and the quality of the finished product. The recessive immature fiber (im gene reduces the degree of fiber cell wall thickening by a process that was previously shown to involve mitochondrial function in allotetraploid Gossypium hirsutum. Here, we present the fine genetic mapping of the im locus, gene expression analysis of annotated proteins near the locus, and association analysis of the linked markers. Mapping-by-sequencing identified a 22-bp deletion in a pentatricopeptide repeat (PPR gene that is completely linked to the immature fiber phenotype in 2837 F2 plants, and is absent from all 163 cultivated varieties tested, although other closely linked marker polymorphisms are prevalent in the diversity panel. This frame-shift mutation results in a transcript with two long open reading frames: one containing the N-terminal transit peptide that targets mitochondria, the other containing only the RNA-binding PPR domains, suggesting that a functional PPR protein cannot be targeted to mitochondria in the im mutant. Taken together, these results suggest that PPR gene Gh_A03G0489 is involved in the cotton fiber wall thickening process, and is a promising candidate gene at the im locus. Our findings expand our understanding of the molecular mechanisms that modulate cotton fiber fineness and maturity, and may facilitate the development of cotton varieties with superior fiber attributes.

  18. Leucine-rich repeat-containing G-protein-coupled receptors as markers of adult stem cells

    NARCIS (Netherlands)

    Barker, N.; Clevers, H.

    2010-01-01

    Molecular markers are used to characterize and track adult stem cells. Colon cancer research has led to the identification of 2 related receptors, leucine-rich repeat-containing, G-protein-coupled receptors (Lgr)5 and Lgr6, that are expressed by small populations of cells in a variety of adult organ

  19. Cardiac ankyrin repeat protein and atherosclerosis%心锚重复蛋白与冠状动脉粥样硬化

    Institute of Scientific and Technical Information of China (English)

    周丹

    2010-01-01

    @@ 心锚重复蛋白(cardiac ankyrin repeat protein,CARP)也常被称为ANKRD1蛋白(cardiac ankyrin repeat domain1protein),其他别名有C-193、MCARP等,是1985年发现的一个核转录辅助因子,属于锚蛋白(ANK)重复序列蛋白家族(muscle ankyrin repeat proteins,MARP)的保守基因.

  20. Genetic Association Between Androgen Receptor Gene CAG Repeat Length Polymorphism and Male Infertility: A Meta-Analysis

    OpenAIRE

    Pan, Bihui; Li, Rui; CHEN, YAO; Tang, Qiuqin; Wu, Wei; Chen, Liping; Lu, Chuncheng; Pan, Feng; Hongjuan DING; Xia,Yankai; Hu, Lingqing; Chen, Daozhen; Sha, Jiahao; Wang, Xinru

    2016-01-01

    Abstract The association between polymorphism of androgen receptor gene CAG (AR-CAG) and male infertility in several studies was controversial. Based on studies on association between AR-CAG repeat length and male infertility in recent years, an updated meta-analysis is needed. We aimed to evaluate the association between AR-CAG repeat length and male infertility in advantage of the data in all published reports. We searched for reports published before August 2015 using PubMed, CNKI, VIP, an...

  1. The ACR11 encodes a novel type of chloroplastic ACT domain repeat protein that is coordinately expressed with GLN2 in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Hsu Chih-Ping

    2011-08-01

    Full Text Available Abstract Background The ACT domain, named after bacterial aspartate kinase, chorismate mutase and TyrA (prephenate dehydrogenase, is a regulatory domain that serves as an amino acid-binding site in feedback-regulated amino acid metabolic enzymes. We have previously identified a novel type of ACT domain-containing protein family, the ACT domain repeat (ACR protein family, in Arabidopsis. Members of the ACR family, ACR1 to ACR8, contain four copies of the ACT domain that extend throughout the entire polypeptide. Here, we describe the identification of four novel ACT domain-containing proteins, namely ACR9 to ACR12, in Arabidopsis. The ACR9 and ACR10 proteins contain three copies of the ACT domain, whereas the ACR11 and ACR12 proteins have a putative transit peptide followed by two copies of the ACT domain. The functions of these plant ACR proteins are largely unknown. Results The ACR11 and ACR12 proteins are predicted to target to chloroplasts. We used protoplast transient expression assay to demonstrate that the Arabidopsis ACR11- and ACR12-green fluorescent fusion proteins are localized to the chloroplast. Analysis of an ACR11 promoter-β-glucuronidase (GUS fusion in transgenic Arabidopsis revealed that the GUS activity was mainly detected in mature leaves and sepals. Interestingly, coexpression analysis revealed that the GLN2, which encodes a chloroplastic glutamine synthetase, has the highest mutual rank in the coexpressed gene network connected to ACR11. We used RNA gel blot analysis to confirm that the expression pattern of ACR11 is similar to that of GLN2 in various organs from 6-week-old Arabidopsis. Moreover, the expression of ACR11 and GLN2 is highly co-regulated by sucrose and light/dark treatments in 2-week-old Arabidopsis seedlings. Conclusions This study reports the identification of four novel ACT domain repeat proteins, ACR9 to ACR12, in Arabidopsis. The ACR11 and ACR12 proteins are localized to the chloroplast, and the expression

  2. Over-expression of rice leucine-rich repeat protein results in activation of defense response, thereby enhancing resistance to bacterial soft rot in Chinese cabbage.

    Science.gov (United States)

    Park, Young Ho; Choi, Changhyun; Park, Eun Mi; Kim, Hyo Sun; Park, Hong Jae; Bae, Shin Cheol; Ahn, Ilpyung; Kim, Min Gab; Park, Sang Ryeol; Hwang, Duk-Ju

    2012-10-01

    Pectobacterium carotovorum subsp. carotovorum causes soft rot disease in various plants, including Chinese cabbage. The simple extracellular leucine-rich repeat (eLRR) domain proteins have been implicated in disease resistance. Rice leucine-rich repeat protein (OsLRP), a rice simple eLRR domain protein, is induced by pathogens, phytohormones, and salt. To see whether OsLRP enhances disease resistance to bacterial soft rot, OsLRP was introduced into Chinese cabbage by Agrobacterium-mediated transformation. Two independent transgenic lines over-expressing OsLRP were generated and further analyzed. Transgenic lines over-expressing OsLRP showed enhanced disease resistance to bacterial soft rot compared to non-transgenic control. Bacterial growth was retarded in transgenic lines over-expressing OsLRP compared to non-transgenic controls. We propose that OsLRP confers enhanced resistance to bacterial soft rot. Monitoring expression of defense-associated genes in transgenic lines over-expressing OsLRP, two different glucanases and Brassica rapa polygalacturonase inhibiting protein 2, PDF1 were constitutively activated in transgenic lines compared to non-transgenic control. Taken together, heterologous expression of OsLRP results in the activation of defense response and enhanced resistance to bacterial soft rot.

  3. Engineering genes for predictable protein expression.

    Science.gov (United States)

    Gustafsson, Claes; Minshull, Jeremy; Govindarajan, Sridhar; Ness, Jon; Villalobos, Alan; Welch, Mark

    2012-05-01

    The DNA sequence used to encode a polypeptide can have dramatic effects on its expression. Lack of readily available tools has until recently inhibited meaningful experimental investigation of this phenomenon. Advances in synthetic biology and the application of modern engineering approaches now provide the tools for systematic analysis of the sequence variables affecting heterologous expression of recombinant proteins. We here discuss how these new tools are being applied and how they circumvent the constraints of previous approaches, highlighting some of the surprising and promising results emerging from the developing field of gene engineering.

  4. Multifunctional protein:cardiac ankyrin repeat protein%心锚重复蛋白的研究进展

    Institute of Scientific and Technical Information of China (English)

    Na ZHANG; Xiao-jie XIE; Jian-an WANG‡

    2016-01-01

    概心锚重复蛋白(CARP)是一个双重定位的蛋白,既可以在胞浆中作为肌节的结构组成蛋白,又定位于细胞核中作为转录共刺激因子调节其他基因的表达。研究发现CARP在多种心血管疾病及肌肉疾病中表达升高,但其在疾病中的作用尚存在争议。本文就CARP的研究进展进行综述,概述CARP的发现过程和结构,并对CARP在疾病中的作用的争议进行总结分析。%Cardiac ankyrin repeat protein (CARP) not only serves as an important component of muscle sarcomere in the cytoplasm, but also acts as a transcription co-factor in the nucleus. Previous studies have demonstrated that CARP is up-regulated in some cardiovascular disorders and muscle diseases;however, its role in these diseases remains controversial now. In this review, we wil discuss the continued progress in the research related to CARP, including its discovery, structure, and the role it plays in cardiac development and heart diseases.

  5. DNA Replication Dynamics of the GGGGCC Repeat of the C9orf72 Gene.

    Science.gov (United States)

    Thys, Ryan Griffin; Wang, Yuh-Hwa

    2015-11-27

    DNA has the ability to form a variety of secondary structures in addition to the normal B-form DNA, including hairpins and quadruplexes. These structures are implicated in a number of neurological diseases and cancer. Expansion of a GGGGCC repeat located at C9orf72 is associated with familial amyotrophic lateral sclerosis and frontotemporal dementia. This repeat expands from two to 24 copies in normal individuals to several hundreds or thousands of repeats in individuals with the disease. Biochemical studies have demonstrated that as little as four repeats have the ability to form a stable DNA secondary structure known as a G-quadruplex. Quadruplex structures have the ability to disrupt normal DNA processes such as DNA replication and transcription. Here we examine the role of GGGGCC repeat length and orientation on DNA replication using an SV40 replication system in human cells. Replication through GGGGCC repeats leads to a decrease in overall replication efficiency and an increase in instability in a length-dependent manner. Both repeat expansions and contractions are observed, and replication orientation is found to influence the propensity for expansions or contractions. The presence of replication stress, such as low-dose aphidicolin, diminishes replication efficiency but has no effect on instability. Two-dimensional gel electrophoresis analysis demonstrates a replication stall with as few as 20 GGGGCC repeats. These results suggest that replication of the GGGGCC repeat at C9orf72 is perturbed by the presence of expanded repeats, which has the potential to result in further expansion, leading to disease.

  6. Checkpoint genes and Exo1 regulate nearby inverted repeat fusions that form dicentric chromosomes in Saccharomyces cerevisiae.

    Science.gov (United States)

    Kaochar, Salma; Shanks, Lisa; Weinert, Ted

    2010-12-14

    Genomic rearrangements are common, occur by largely unknown mechanisms, and can lead to human diseases. We previously demonstrated that some genome rearrangements occur in budding yeast through the fusion of two DNA sequences that contain limited sequence homology, lie in inverted orientation, and are within 5 kb of one another. This inverted repeat fusion reaction forms dicentric chromosomes, which are well-known intermediates to additional rearrangements. We have previously provided evidence indicating that an error of stalled or disrupted DNA replication forks can cause inverted repeat fusion. Here we analyze how checkpoint protein regulatory pathways known to stabilize stalled forks affect this form of instability. We find that two checkpoint pathways suppress inverted repeat fusion, and that their activities are distinguishable by their interactions with exonuclease 1 (Exo1). The checkpoint kinase Rad53 (Chk2) and recombination protein complex MRX(MRN) inhibit Exo1 in one pathway, whereas in a second pathway the ATR-like kinases Mec1 and Tel1, adaptor protein Rad9, and effector kinases Chk1 and Dun1 act independently of Exo1 to prevent inverted repeat fusion. We provide a model that indicates how in Rad53 or MRX mutants, an inappropriately active Exo1 may facilitate faulty template switching between nearby inverted repeats to form dicentric chromosomes. We further investigate the role of Rad53, using hypomorphic alleles of Rad53 and null mutations in Rad9 and Mrc1, and provide evidence that only local, as opposed to global, activity of Rad53 is sufficient to prevent inverted repeat fusion.

  7. Prevalence of antibodies to the repeat epitope of the circumsporozoite protein of Plasmodium vivax in San Luis Potosi, Mexico.

    Science.gov (United States)

    Mota, J; Coreño, O; Cochrane, A H; Ramos, C

    1996-01-01

    The prevalence of antibodies against the repeat epitope of the circumsporozoite protein (cs) of the standard (PV210) and variant (PVK247) strain of Plasmodium vivax was determined by ELISA in 1170 sera from individual residents of seven localities of the Region Huasteca of San Luis Potosi, Mexico. The capture antigens were the synthetic peptides DDAAD and (ANGAGNQPG) that correspond to the repeats of the PV210 and PVK247 cs proteins, respectively. Of the analyzed serum samples, 34.1% (400/1170) were positive with one or both of these antigens. Of the sera, 18.2% (214/1170) reacted with the DDAAD peptide and 6.6% (78/1170) were positive with the variant synthetic peptide. Additionally, 9.2% (108/1170) of the samples reacted with both peptides. A sample of 10% of positive sera for the variant cs repeat (18/78) was tested with the cs repeat peptide of P. malariae/P. brasilianum (NAAG); almost all of them (16/18, 89%) being positive. These results confirm that the transmission of the variant strain of P. vivax is a common phenomenon in endemic regions in Latin America, as well as in other tropical regions of the world. These findings may have implications for the development of aP. vivax vaccine since that based on the standard cs repeat only would not be universally protective.

  8. Bov-B long interspersed repeated DNA (LINE) sequences are present in Vipera ammodytes phospholipase A2 genes and in genomes of Viperidae snakes.

    Science.gov (United States)

    Kordis, D; Gubensek, F

    1997-06-15

    Ammodytin L is a myotoxic Ser49 phospholipase A2 (PLA2) homologue, which is tissue-specifically expressed in the venom glands of Vipera ammodytes. The complete DNA sequence of the gene and its 5' and 3' flanking regions has been determined. The gene consists of five exons separated by four introns. Comparative analysis of the ammodytin L and ammodytoxin C genes shows that all intron and flanking sequences are considerably more conserved (93-97%) than the mature protein-coding exons. The pattern of nucleotide substitutions in protein-coding exons is not random but occurs preferentially on the first and the second positions of codons, which suggests positive Darwinian evolution for a new function. An Ruminantia specific ART-2 retroposon, recently recognised as a 5'-truncated Bov-B long interspersed repeated DNA (LINE) sequence, was identified in the fourth intron of both genes. This result suggests that ammodytin L and ammodytoxin C genes are derived by duplication of a common ancestral gene. The phylogenetic distribution of Bov-B LINE among vertebrate classes shows that, besides the Ruminantia, it is limited to Viperidae snakes (Vipera ammodytes, Vipera palaestinae, Echis coloratus, Bothrops alternatus, Trimeresurus flavoviridis and Trimeresurus gramineus). The copy number of the 3' end of Bov-B LINE in the Vipera ammodytes genome is between 62,000 and 75,000. The absence of Bov-B LINE at orthologous positions in other snake PLA2 genes indicates that its retrotransposition in the V. ammodytes PLA2 gene locus has occurred quite recently, about 5 My ago. The amplification of Bov-B LINEs in snakes may have occurred before the divergence of the Viperinae and Crotalinae subfamilies. Due to its wide distribution in Viperidae snakes it may be a valuable phylogenetic marker. The neighbor-joining phylogenetic tree shows two clusters of truncated Bov-B LINE, a Bovidae and a snake cluster, indicating an early horizontal transfer of this transposable element.

  9. Coordination of Meristem Doming and the Floral Transition by Late Termination, a Kelch Repeat Protein.

    Science.gov (United States)

    Tal, Lior; Friedlander, Gilgi; Gilboa, Netta Segal; Unger, Tamar; Gilad, Shlomit; Eshed, Yuval

    2017-04-01

    Enlargement and doming of the shoot apical meristem (SAM) is a hallmark of the transition from vegetative growth to flowering. While this change is widespread, its role in the flowering process is unknown. The late termination (ltm) tomato (Solanum lycopersicum) mutant shows severely delayed flowering and precocious doming of the vegetative SAM LTM encodes a kelch domain-containing protein, with no link to known meristem maintenance or flowering time pathways. LTM interacts with the TOPLESS corepressor and with several transcription factors that can provide specificity for its functions. A subgroup of flowering-associated genes is precociously upregulated in vegetative stages of ltm SAMs, among them, the antiflorigen gene SELF PRUNING (SP). A mutation in SP restored the structure of vegetative SAMs in ltm sp double mutants, and late flowering was partially suppressed, suggesting that LTM functions to suppress SP in the vegetative SAM In agreement, SP-overexpressing wild-type plants exhibited precocious doming of vegetative SAMs combined with late flowering, as found in ltm plants. Strong flowering signals can result in termination of the SAM, usually by its differentiation into a flower. We propose that activation of a floral antagonist that promotes SAM growth in concert with floral transition protects it from such terminating effects. © 2017 American Society of Plant Biologists. All rights reserved.

  10. Predicting gene ontology annotations of orphan GWAS genes using protein-protein interactions.

    Science.gov (United States)

    Kuppuswamy, Usha; Ananthasubramanian, Seshan; Wang, Yanli; Balakrishnan, Narayanaswamy; Ganapathiraju, Madhavi K

    2014-04-03

    The number of genome-wide association studies (GWAS) has increased rapidly in the past couple of years, resulting in the identification of genes associated with different diseases. The next step in translating these findings into biomedically useful information is to find out the mechanism of the action of these genes. However, GWAS studies often implicate genes whose functions are currently unknown; for example, MYEOV, ANKLE1, TMEM45B and ORAOV1 are found to be associated with breast cancer, but their molecular function is unknown. We carried out Bayesian inference of Gene Ontology (GO) term annotations of genes by employing the directed acyclic graph structure of GO and the network of protein-protein interactions (PPIs). The approach is designed based on the fact that two proteins that interact biophysically would be in physical proximity of each other, would possess complementary molecular function, and play role in related biological processes. Predicted GO terms were ranked according to their relative association scores and the approach was evaluated quantitatively by plotting the precision versus recall values and F-scores (the harmonic mean of precision and recall) versus varying thresholds. Precisions of ~58% and ~ 40% for localization and functions respectively of proteins were determined at a threshold of ~30 (top 30 GO terms in the ranked list). Comparison with function prediction based on semantic similarity among nodes in an ontology and incorporation of those similarities in a k-nearest neighbor classifier confirmed that our results compared favorably. This approach was applied to predict the cellular component and molecular function GO terms of all human proteins that have interacting partners possessing at least one known GO annotation. The list of predictions is available at http://severus.dbmi.pitt.edu/engo/GOPRED.html. We present the algorithm, evaluations and the results of the computational predictions, especially for genes identified in

  11. Single sperm analysis of the trinucleotide repeat in the Huntington`s disease gene

    Energy Technology Data Exchange (ETDEWEB)

    Leeflang, E.P.; Zhang, L.; Hubert, R. [Univ. of Southern California, Los Angeles, CA (United States)] [and others

    1994-09-01

    Huntington`s disease (HD) is one of several genetic diseases caused by trinucleotide repeat expansion. The CAG repeat is very unstable, with size changes occurring in more than 80% of transmissions. The degree of instability of this repeat in the male germline can be determined by analysis of individual sperm cells. An easy and sensitive PCR assay has been developed to amplify this trinucleotide repeat region from single sperm using two rounds of PCR. As many as 90% of the single sperm show amplification for the HD repeat. The PCR product can be easily detected on an ethidium bromide-stained agarose gel. Single sperm samples from an HD patient with 18 and 49 repeats were studied. We observed size variations for the expanded alleles while the size of the normal allele in sperm is very consistent. We did not detect any significant bias in the amplification of normal alleles over the larger HD alleles. Our preliminary study supports the observation made by PCR of total sperm that instability of the HD trinucleotide repeat occurs in the germline. HD preimplantation diagnosis on single embryo blastomeres may also possible.

  12. Differential effects of the HESR/HEY transcription factor family on dopamine transporter reporter gene expression via variable number of tandem repeats.

    Science.gov (United States)

    Kanno, Kouta; Ishiura, Shoichi

    2011-04-01

    The 3'-untranslated region (UTR) of the human dopamine transporter (DAT1) gene contains a variable number of tandem repeats (VNTR) domain, which is thought to be associated with dopamine-related psychiatric disorders, personality, and behavior. However, the molecular and neuronal functions of polymorphisms within the VNTR domain are unknown. We previously identified the transcription factor HESR1 (HEY1) as a VNTR-binding protein. Hesr1 knockout mice exhibit DAT up-regulation in the brain and low levels of spontaneous activity. Other members of the HESR (HEY) family, including HESR2 (HEY2) and 3 (HEYL), have similar DNA-binding domains. In this study, we analyzed the effects of HESR1, -2, and -3 on DAT1 expression in human neuroblastoma SH-SY5Y cells using luciferase reporter assays. We found that the VNTR domain played an inhibitory role in DAT1 reporter gene expression and that HESR1 and -2 inhibited expression via both the core promoter and the VNTR. The inhibitory effects of HESR family members on DAT reporter gene expression differed depending on the number of repeats in the VNTR domain. We also found that each Hesr was expressed in the dopaminergic neurons in the mouse midbrain. These results suggest that the HESR family is involved in DAT expression via the VNTR domain.

  13. Hexanucleotide repeats in ALS/FTD form length-dependent RNA foci, sequester RNA binding proteins, and are neurotoxic.

    Science.gov (United States)

    Lee, Youn-Bok; Chen, Han-Jou; Peres, João N; Gomez-Deza, Jorge; Attig, Jan; Stalekar, Maja; Troakes, Claire; Nishimura, Agnes L; Scotter, Emma L; Vance, Caroline; Adachi, Yoshitsugu; Sardone, Valentina; Miller, Jack W; Smith, Bradley N; Gallo, Jean-Marc; Ule, Jernej; Hirth, Frank; Rogelj, Boris; Houart, Corinne; Shaw, Christopher E

    2013-12-12

    The GGGGCC (G4C2) intronic repeat expansion within C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Intranuclear neuronal RNA foci have been observed in ALS and FTD tissues, suggesting that G4C2 RNA may be toxic. Here, we demonstrate that the expression of 38× and 72× G4C2 repeats form intranuclear RNA foci that initiate apoptotic cell death in neuronal cell lines and zebrafish embryos. The foci colocalize with a subset of RNA binding proteins, including SF2, SC35, and hnRNP-H in transfected cells. Only hnRNP-H binds directly to G4C2 repeats following RNA immunoprecipitation, and only hnRNP-H colocalizes with 70% of G4C2 RNA foci detected in C9ORF72 mutant ALS and FTD brain tissues. We show that expanded G4C2 repeats are potently neurotoxic and bind hnRNP-H and other RNA binding proteins. We propose that RNA toxicity and protein sequestration may disrupt RNA processing and contribute to neurodegeneration.

  14. Downregulation of Notch-regulated Ankyrin Repeat Protein Exerts Antitumor Activities against Growth of Thyroid Cancer

    Institute of Scientific and Technical Information of China (English)

    Bing-Feng Chu; Yi-Yu Qin; Sheng-Lai Zhang; Zhi-Wei Quan; Ming-Di Zhang; Jian-Wei Bi

    2016-01-01

    Background:The Notch-regulated ankyrin repeat protein (NRARP) is recently found to promote proliferation of breast cancer cells.The role of NRARP in carcinogenesis deserves extensive investigations.This study attempted to investigate the expression of NRARP in thyroid cancer tissues and assess the influence of NRARP on cell proliferation,apoptosis,cell cycle,and invasion in thyroid cancer.Methods:Thirty-four cases with thyroid cancer were collected from the Department of General Surgery,Xinhua Hospital,Shanghai Jiao Tong University School of Medicine between 2011 and 2012.Immunohistochemistry was used to detect the level of NRARP in cancer tissues.Lentivirus carrying NRARP-shRNA (Lenti-NRARP-shRNA) was applied to down-regulate NRARP expression.Cell viability was tested after treatment with Lenti-NRARP-shRNA using 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide assay.Apoptosis and cell cycle distribution were determined by flow cytometry.Cell invasion was tested using Transwell invasion assay.In addition,expressions of several cell cycle-associated and apoptosis-associated proteins were examined using Western blotting after transfection.Student's t-test,one-way analysis of variance (ANOVA),or Kaplan-Meier were used to analyze the differences between two group or three groups.Results:NRARP was highly expressed in thyroid cancer tissues.Lenti-NRARP-shRNA showed significantly inhibitory activities against cell growth at a multiplicity of infection of 10 or higher (P < 0.05).Lenti-NRARP-shRNA-induced G1 arrest (BHT 101:72.57% ± 5.32%;8305C:75.45% ± 5.26%) by promoting p21 expression,induced apoptosis by promoting bax expression and suppressing bcl-2 expression,and inhibited cell invasion by suppressing matrix metalloproteinase-9 expression.Conclusion:Downregulation of NRARP expression exerts significant antitumor activities against cell growth and invasion of thyroid cancer,that suggests a potential role of NRARP in thyroid cancer targeted

  15. In silico structural and functional analysis of fragments of the ankyrin repeat protein p18(INK4c).

    Science.gov (United States)

    Sklenovsý, P; Otyepka, M

    2010-02-01

    Ankyrin repeat proteins (ARPs) are ubiquitous proteins that play critical regulatory roles in organisms and consist of repeating motifs (ankyrin repeats) stacked in non-globular, almost linear, "quasi one-dimensional" configurations. They also have highly unusual mechanical properties, notably ARPs can behave as nano-springs. Both their essential cellular functions and distinctive nano-mechanical properties have aroused interest in ARPs for potential applications in medicine and nanotechnology. Further, the modular architecture of ARPs, which lack the long-range contacts that typically stabilize globular proteins, provides a new paradigm for understanding protein stability and folding mechanisms of proteins. In the present study, the stability of ARP p18INK4c (p18) and fifty p18 fragments was investigated by all- atomic molecular dynamics (MD) simulations in explicit water on a ~3.3 micro- seconds timescale. The fragment simulations indicate that p18 alpha-helices are significantly stabilized by tertiary interactions, because in the absence of their native context they readily melt. All single p18 ARs and their structural elements are also unstable outside their native context. The minimal stable motifs are pairs of ARs, implying that inter-repeat contacts are essential for AR stability. Further, pairs of internal ARs are less stable than pairs that include a native capping AR. The MD simulations also provide indications of the functional roles of p18 turns and loops; the turns appear to be essential for the stability of the protein, while the loops both help to stabilize the p18 structure and are involved in recognition processes. Temperature-induced unfolding analysis shows that the p18 melts from the N-terminus to the C- terminus.

  16. (AC)n dinucleotide repeat polymorphism in 5' beta-globin gene in native and Mestizo Mexican populations.

    Science.gov (United States)

    Peñaloza, R; Delgado, P; Arenas, D; Barrientos, C; Buentello, L; Loeza, F; Salamanca, F

    2001-12-01

    Repeated sequences are dispersed along the human genome. These sequences are useful as markers in diagnosis of inherited diseases, in forensic medicine, and in tracking the origin and evolution of human populations. The (AC)n repeated element is the most frequent in the human genome. In this paper, the (AC)n repeated element located in the 5' flanking region of the beta-globin gene was studied by single-strand conformation polymorphism (SSCP). Four ethnic Mexican groups (Mixteca, Nahua, Otomí, Purépecha) and a Mestizo population were analyzed. We observed three alleles, A [(AC)16, B [(AC)14], and C [(AC)18], with a frequency of between 68.2% and 86.9%, 13.1% and 18.2%, and 6.7% and 13.7%, respectively. Allele C was present only in Purépecha and Mestizo groups. The absence of this allele in the other ethnic groups studied suggests that there is low genetic admixture of Purépecha and that this is a relatively isolated population. However, it could be that the C allele occurs in low frequencies in the other groups as a result of small sample sizes. The (AC)n repeat polymorphism in the beta-globin gene has not been previously studied in Amerindian populations.

  17. Comparative population genetic analysis of bocaccio rockfish Sebastes paucispinis using anonymous and gene-associated simple sequence repeat loci.

    Science.gov (United States)

    Buonaccorsi, Vincent P; Kimbrell, Carol A; Lynn, Eric A; Hyde, John R

    2012-01-01

    Comparative population genetic analyses of traditional and emergent molecular markers aid in determining appropriate use of new technologies. The bocaccio rockfish Sebastes paucispinis is a high gene-flow marine species off the west coast of North America that experienced strong population decline over the past 3 decades. We used 18 anonymous and 13 gene-associated simple sequence repeat (SSR) loci (expressed sequence tag [EST]-SSRs) to characterize range-wide population structure with temporal replicates. No F(ST)-outliers were detected using the LOSITAN program, suggesting that neither balancing nor divergent selection affected the loci surveyed. Consistent hierarchical structuring of populations by geography or year class was not detected regardless of marker class. The EST-SSRs were less variable than the anonymous SSRs, but no correlation between F(ST) and variation or marker class was observed. General linear model analysis showed that low EST-SSR variation was attributable to low mean repeat number. Comparative genomic analysis with Gasterosteus aculeatus, Takifugu rubripes, and Oryzias latipes showed consistently lower repeat number in EST-SSRs than SSR loci that were not in ESTs. Purifying selection likely imposed functional constraints on EST-SSRs resulting in low repeat numbers that affected diversity estimates but did not affect the observed pattern of population structure.

  18. Characterization of expressed Pgip genes in rice and wheat reveals similar extent of sequence variation to dicot PGIPs and identifies an active PGIP lacking an entire LRR repeat.

    Science.gov (United States)

    Janni, Michela; Di Giovanni, Michela; Roberti, Serena; Capodicasa, Cristina; D'Ovidio, Renato

    2006-11-01

    Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat (LRR) proteins involved in plant defence. A number of PGIPs have been characterized from dicot species, whereas only a few data are available from monocots. Database searches and genome-specific cloning strategies allowed the identification of four rice (Oryza sativa L.) and two wheat (Triticum aestivum L.) Pgip genes. The rice Pgip genes (Ospgip1, Ospgip2, Ospgip3 and Ospgip4) are distributed over a 30 kbp region of the short arm of chromosome 5, whereas the wheat Pgip genes, Tapgip1 and Tapgip2, are localized on the short arm of chromosome 7B and 7D, respectively. Deduced amino acid sequences show the typical LRR modular organization and a conserved distribution of the eight cysteines at the N- and C-terminal regions. Sequence comparison suggests that monocot and dicot PGIPs form two separate clusters sharing about 40% identity and shows that this value is close to the extent of variability observed within each cluster. Gene-specific RT-PCR and biochemical analyses demonstrate that both Ospgips and Tapgips are expressed in the whole plant or in a tissue-specific manner, and that OsPGIP1, lacking an entire LRR repeat, is an active inhibitor of fungal polygalacturonases. This last finding can contribute to define the molecular features of PG-PGIP interactions and highlights that the genetic events that can generate variability at the Pgip locus are not only limited to substitutions or small insertions/deletions, as so far reported, but can also involve variation in the number of LRRs.

  19. CTG repeats distribution and Alu insertion polymorphism at myotonic dystrophy (DM) gene in Amhara and Oromo populations of Ethiopia.

    Science.gov (United States)

    Gennarelli, M; Pavoni, M; Cruciani, F; De Stefano, G; Dallapiccola, B; Novelli, G

    1999-01-01

    Myotonic dystrophy (DM) is a dominantly inherited neuromuscular disease, highly variable and multisystemic, which is caused by the expansion of a CTG repeat located in the 3' untranslated region of the DMPK gene. Normal alleles show a copy number of 5-37 repeats on normal chromosomes, amplified to 50-3000 copies on DM chromosomes. The trinucleotide repeat shows a trimodal allele distribution in the majority of the examined population. The first class includes alleles carrying (CTG)5, the second class, alleles in the range 7-18 repeats, and the third class, alleles (CTG) > or =19. The frequency of this third class is directly related to the prevalence of DM in different populations, suggesting that normal large-sized alleles predispose toward DM. We studied CTG repeat allele distribution and Alu insertion and/or deletion polymorphism at the myotonic dystrophy locus in two major Ethiopian populations, the Amhara and Oromo. CTG allele distribution and haplotype analysis on a total of 224 normal chromosomes showed significant differences between the two ethnic groups. These differences have a bearing on the out-of-Africa hypothesis for the origin of the DM mutation. In addition, (CTG) > or =19 were exclusively detected in the Amhara population, confirming the predisposing role of these alleles compared with the DM expansion-mutation.

  20. TAA repeat variation in the GRIK2 gene does not influence age at onset in Huntington's disease

    Science.gov (United States)

    Lee, Ji-Hyun; Lee, Jong-Min; Ramos, Eliana Marisa; Gillis, Tammy; Mysore, Jayalakshmi S.; Kishikawa, Shotaro; Hadzi, Tiffany; Hendricks, Audrey E.; Hayden, Michael R.; Morrison, Patrick J.; Nance, Martha; Ross, Christopher A.; Margolis, Russell L.; Squitieri, Ferdinando; Gellera, Cinzia; Gomez-Tortosa, Estrella; Ayuso, Carmen; Suchowersky, Oksana; Trent, Ronald J.; McCusker, Elizabeth; Novelletto, Andrea; Frontali, Marina; Jones, Randi; Ashizawa, Tetsuo; Frank, Samuel; Saint-Hilaire, Marie-Helene; Hersch, Steven M.; Rosas, Herminia D.; Lucente, Diane; Harrison, Madaline B.; Zanko, Andrea; Abramson, Ruth K.; Marder, Karen; Sequeiros, Jorge; Landwehrmeyer, G. Bernhard; Network, Ira Shoulson; Myers, Richard H.; MacDonald, Marcy E.; Gusella, James F.

    2013-01-01

    Huntington's disease is a neurodegenerative disorder caused by an expanded CAG trinucleotide repeat whose length is the major determinant of age at onset but remaining variation appears to be due in part to the effect of genetic modifiers. GRIK2, which encodes GluR6, a mediator of excitatory neurotransmission in the brain, has been suggested in several studies to be a modifier gene based upon a 3′ untranslated region TAA trinucleotide repeat polymorphism. Prior to investing in detailed studies of the functional impact of this polymorphism, we sought to confirm its effect on age at onset in a much larger dataset than in previous investigations. We genotyped the HD CAG repeat and the GRIK2 TAA repeat in DNA samples from 2,911 Huntington's disease subjects with known age at onset, and tested for a potential modifier effect of GRIK2 using a variety of statistical approaches. Unlike previous reports, we detected no evidence of an influence of the GRIK2 TAA repeat polymorphism on age at motor onset. Similarly, the GRIK2 polymorphism did not show significant modifier effect on psychiatric and cognitive age at onset in HD. Comprehensive analytical methods applied to a much larger sample than in previous studies do not support a role for GRIK2 as a genetic modifier of age at onset of clinical symptoms in Huntington's disease. PMID:22771793

  1. Absence of association of a polymorphic GGC repeat at the 5' untranslated region of the reelin gene with schizophrenia.

    Science.gov (United States)

    Huang, Chia-Hsing; Chen, Chia-Hsiang

    2006-05-30

    Reelin is an extracellular matrix glycoprotein that plays an important role in guiding neuronal migration, lamination and connection during embryonic brain development. Several reports suggest that reduced reelin expression is associated with human mental illnesses such as schizophrenia, mood disorders and autism. Human reelin cDNA has been cloned and contains a polymorphic GGC repeat at the 5' untranslated region. In view of the possible regulation of reelin gene expression by this GGC polymorphism, we investigated the association of the polymorphic GGC repeat with schizophrenia in a Chinese Han population from Taiwan. We found no differences of allelic and genotypic distributions of the polymorphic GGC triplets between 162 schizophrenic patients and 176 controls in this study. Our findings do not support the involvement of the polymorphic GGC triplets of the reelin gene in the pathogenesis of schizophrenia in the population studied.

  2. A protein-tagging system for signal amplification in gene expression and fluorescence imaging.

    Science.gov (United States)

    Tanenbaum, Marvin E; Gilbert, Luke A; Qi, Lei S; Weissman, Jonathan S; Vale, Ronald D

    2014-10-23

    Signals in many biological processes can be amplified by recruiting multiple copies of regulatory proteins to a site of action. Harnessing this principle, we have developed a protein scaffold, a repeating peptide array termed SunTag, which can recruit multiple copies of an antibody-fusion protein. We show that the SunTag can recruit up to 24 copies of GFP, thereby enabling long-term imaging of single protein molecules in living cells. We also use the SunTag to create a potent synthetic transcription factor by recruiting multiple copies of a transcriptional activation domain to a nuclease-deficient CRISPR/Cas9 protein and demonstrate strong activation of endogenous gene expression and re-engineered cell behavior with this system. Thus, the SunTag provides a versatile platform for multimerizing proteins on a target protein scaffold and is likely to have many applications in imaging and controlling biological outputs.

  3. Plasmodium Cysteine Repeat Modular Proteins 3 and 4 are essential for malaria parasite transmission from the mosquito to the host

    Directory of Open Access Journals (Sweden)

    Mota Maria M

    2011-03-01

    Full Text Available Abstract Background The Plasmodium Cysteine Repeat Modular Proteins (PCRMP are a family of four conserved proteins of malaria parasites, that contain a number of motifs implicated in host-parasite interactions. Analysis of mutants of the rodent parasite Plasmodium berghei lacking expression of PCRMP1 or 2 showed that these proteins are essential for targeting of P. berghei sporozoites to the mosquito salivary gland and, hence, for transmission from the mosquito to the mouse. Methods In this work, the role of the remaining PCRMP family members, PCRMP3 and 4, has been investigated throughout the Plasmodium life cycle by generation and analysis of P. berghei gene deletion mutants, Δpcrmp3 and Δpcrmp4. The role of PCRMP members during the transmission and hepatic stages of the Plasmodium lifecycle has been evaluated by light- and electron microscopy and by analysis of liver stage development in HEPG2 cells in vitro and by infecting mice with mutant sporozoites. In addition, mice were immunized with live Δpcrmp3 and Δpcrmp4 sporozoites to evaluate their immunization potential as a genetically-attenuated parasite-based vaccine. Results Disruption of pcrmp3 and pcrmp4 in P. berghei revealed that they are also essential for transmission of the parasite through the mosquito vector, although acting in a distinct way to pbcrmp1 and 2. Mutants lacking expression of PCRMP3 or PCRMP4 show normal blood stage development and oocyst formation in the mosquito and develop into morphologically normal sporozoites, but these have a defect in egress from oocysts and do not enter the salivary glands. Sporozoites extracted from oocysts perform gliding motility and invade and infect hepatocytes but do not undergo further development and proliferation. Furthermore, the study shows that immunization with Δcrmp3 and Δcrmp4 sporozoites does not confer protective immunity upon subsequent challenge. Conclusions PCRMP3 and 4 play multiple roles during the Plasmodium life

  4. The protein network surrounding the human telomere repeat binding factors TRF1, TRF2, and POT1.

    Directory of Open Access Journals (Sweden)

    Richard J Giannone

    Full Text Available Telomere integrity (including telomere length and capping is critical in overall genomic stability. Telomere repeat binding factors and their associated proteins play vital roles in telomere length regulation and end protection. In this study, we explore the protein network surrounding telomere repeat binding factors, TRF1, TRF2, and POT1 using dual-tag affinity purification in combination with multidimensional protein identification technology liquid chromatography--tandem mass spectrometry (MudPIT LC-MS/MS. After control subtraction and data filtering, we found that TRF2 and POT1 co-purified all six members of the telomere protein complex, while TRF1 identified five of six components at frequencies that lend evidence towards the currently accepted telomere architecture. Many of the known TRF1 or TRF2 interacting proteins were also identified. Moreover, putative associating partners identified for each of the three core components fell into functional categories such as DNA damage repair, ubiquitination, chromosome cohesion, chromatin modification/remodeling, DNA replication, cell cycle and transcription regulation, nucleotide metabolism, RNA processing, and nuclear transport. These putative protein-protein associations may participate in different biological processes at telomeres or, intriguingly, outside telomeres.

  5. The protein network surrounding the human telomere repeat binding factors TRF1, TRF2, and POT1

    Energy Technology Data Exchange (ETDEWEB)

    Giannone, Richard J [ORNL; McDonald, W Hayes [ORNL; Hurst, Gregory {Greg} B [ORNL; Shen, Rong-Fong [National Institute on Aging, National Institutes of Health; Wang, Yisong [ORNL; Liu, Yie [National Institute on Aging, Baltimore

    2010-01-01

    Telomere integrity (including telomere length and capping) is critical in overall genomic stability. Telomere repeat binding factors and their associated proteins play vital roles in telomere length regulation and end protection. In this study, we explore the protein network surrounding telomere repeat binding factors, TRF1, TRF2, and POT1 using dual-tag affinity purification in combination with multidimensional protein identification technology liquid chromatography - tandem mass spectrometry (MudPIT LC-MS/MS). After control subtraction and data filtering, we found that TRF2 and POT1 co-purified all six members of the telomere protein complex, while TRF1 identified five of six components at frequencies that lend evidence towards the currently accepted telomere architecture. Many of the known TRF1 or TRF2 interacting proteins were also identified. Moreover, putative associating partners identified for each of the three core components fell into functional categories such as DNA damage repair, ubiquitination, chromosome cohesion, chromatin modification/remodeling, DNA replication, cell cycle and transcription regulation, nucleotide metabolism, RNA processing, and nuclear transport. These putative protein-protein associations may participate in different biological processes at telomeres or, intriguingly, outside telomeres.

  6. Simultaneous silencing of multiple genes in the apple scab fungus, Venturia inaequalis, by expression of RNA with chimeric inverted repeats

    NARCIS (Netherlands)

    Fitzgerald, A.; Kan, van J.A.L.; Plummer, K.M.

    2004-01-01

    RNA-mediated gene silencing has been demonstrated in plants, animals, and more recently in filamentous fungi. Here, we report high frequency, RNA-mediated gene silencing in the apple scab fungus, Venturia inaequalis. The green fluorescent protein (GFP) transgene was silenced in a GFP-expressing tran

  7. New Repeat Polymorphism in the AKT1 Gene Predicts Striatal Dopamine D2/D3 Receptor Availability and Stimulant-Induced Dopamine Release in the Healthy Human Brain.

    Science.gov (United States)

    Shumay, Elena; Wiers, Corinde E; Shokri-Kojori, Ehsan; Kim, Sung Won; Hodgkinson, Colin A; Sun, Hui; Tomasi, Dardo; Wong, Christopher T; Weinberger, Daniel R; Wang, Gene-Jack; Fowler, Joanna S; Volkow, Nora D

    2017-05-10

    The role of the protein kinase Akt1 in dopamine neurotransmission is well recognized and has been implicated in schizophrenia and psychosis. However, the extent to which variants in the AKT1 gene influence dopamine neurotransmission is not well understood. Here we investigated the effect of a newly characterized variant number tandem repeat (VNTR) polymorphism in AKT1 [major alleles: L- (eight repeats) and H- (nine repeats)] on striatal dopamine D2/D3 receptor (DRD2) availability and on dopamine release in healthy volunteers. We used PET and [(11)C]raclopride to assess baseline DRD2 availability in 91 participants. In 54 of these participants, we also measured intravenous methylphenidate-induced dopamine release to measure dopamine release. Dopamine release was quantified as the difference in specific binding of [(11)C]raclopride (nondisplaceable binding potential) between baseline values and values following methylphenidate injection. There was an effect of AKT1 genotype on DRD2 availability at baseline for the caudate (F(2,90) = 8.2, p = 0.001) and putamen (F(2,90) = 6.6, p = 0.002), but not the ventral striatum (p = 0.3). For the caudate and putamen, LL showed higher DRD2 availability than HH; HL were in between. There was also a significant effect of AKT1 genotype on dopamine increases in the ventral striatum (F(2,53) = 5.3, p = 0.009), with increases being stronger in HH > HL > LL. However, no dopamine increases were observed in the caudate (p = 0.1) or putamen (p = 0.8) following methylphenidate injection. Our results provide evidence that the AKT1 gene modulates both striatal DRD2 availability and dopamine release in the human brain, which could account for its association with schizophrenia and psychosis. The clinical relevance of the newly characterized AKT1 VNTR merits investigation.SIGNIFICANCE STATEMENT The AKT1 gene has been implicated in schizophrenia and psychosis. This association is likely to reflect modulation of dopamine signaling by Akt1 kinase

  8. A tetratricopeptide repeat domain-containing protein SSR1 located in mitochondria is involved in root development and auxin polar transport in Arabidopsis.

    Science.gov (United States)

    Zhang, Min; Wang, Cuiping; Lin, Qingfang; Liu, Aihua; Wang, Ting; Feng, Xuanjun; Liu, Jie; Han, Huiling; Ma, Yan; Bonea, Diana; Zhao, Rongmin; Hua, Xuejun

    2015-08-01

    Auxin polar transport mediated by a group of Pin-formed (PIN) transporters plays important roles in plant root development. However, the mechanism underlying the PIN expression and targeting in response to different developmental and environmental stimuli is still not fully understood. Here, we report a previously uncharacterized gene SSR1, which encodes a mitochondrial protein with tetratricopeptide repeat (TPR) domains, and show its function in root development in Arabidopsis thaliana. In ssr1-2, a SSR1 knock-out mutant, the primary root growth was dramatically inhibited due to severely impaired cell proliferation and cell elongation. Significantly lowered level of auxin was found in ssr1-2 roots by auxin measurement and was further supported by reduced expression of DR5-driven reporter gene. As a result, the maintenance of the root stem cell niche is compromised in ssr1-2. It is further revealed that the expression level of several PIN proteins, namely, PIN1, PIN2, PIN3, PIN4 and PIN7, were markedly reduced in ssr1-2 roots. In particular, we showed that the reduced protein level of PIN2 on cell membrane in ssr1-2 is due to impaired retrograde trafficking, possibly resulting from a defect in retromer sorting system, which destines PIN2 for degradation in vacuoles. In conclusion, our results indicated that SSR1 is functioning in root development in Arabidopsis, possibly by affecting PIN protein expression and subcellular targeting.

  9. Identification of a novel Leucine-rich repeat protein and candidate PP1 regulatory subunit expressed in developing spermatids

    Directory of Open Access Journals (Sweden)

    Sperry Ann O

    2008-01-01

    Full Text Available Abstract Background Spermatogenesis is comprised of a series of highly regulated developmental changes that transform the precursor germ cell into a highly specialized spermatozoon. The last phase of spermatogenesis, termed spermiogenesis, involves dramatic morphological change including formation of the acrosome, elongation and condensation of the nucleus, formation of the flagella, and disposal of unnecessary cytoplasm. A prominent cytoskeletal component of the developing spermatid is the manchette, a unique microtubular structure that surrounds the nucleus of the developing spermatid and is thought to assist in both the reshaping of the nucleus and redistribution of spermatid cytoplasm. Although the molecular motor KIFC1 has been shown to associate with the manchette, its precise role in function of the manchette and the identity of its testis specific protein partners are unknown. The purpose of this study was to identify proteins in the testis that interact with KIFC1 using a yeast 2 hybrid screen of a testis cDNA library. Results Thirty percent of the interacting clones identified in our screen contain an identical cDNA encoding a 40 kD protein. This interacting protein has 4 leucine-rich repeats in its amino terminal half and is expressed primarily in the testis; therefore we have named this protein testis leucine-rich repeat protein or TLRR. TLRR was also found to associate tightly with the KIFC1 targeting domain using affinity chromatography. In addition to the leucine-rich repeats, TLRR contains a consensus-binding site for protein phosphatase-1 (PP1. Immunocytochemistry using a TLRR specific antibody demonstrates that this protein is found near the manchette of developing spermatids. Conclusion We have identified a previously uncharacterized leucine-rich repeat protein that is expressed abundantly in the testis and associates with the manchette of developing spermatids, possibly through its interaction with the KIFC1 molecular motor

  10. Protein-Protein Interaction Network and Gene Ontology

    Science.gov (United States)

    Choi, Yunkyu; Kim, Seok; Yi, Gwan-Su; Park, Jinah

    Evolution of computer technologies makes it possible to access a large amount and various kinds of biological data via internet such as DNA sequences, proteomics data and information discovered about them. It is expected that the combination of various data could help researchers find further knowledge about them. Roles of a visualization system are to invoke human abilities to integrate information and to recognize certain patterns in the data. Thus, when the various kinds of data are examined and analyzed manually, an effective visualization system is an essential part. One instance of these integrated visualizations can be combination of protein-protein interaction (PPI) data and Gene Ontology (GO) which could help enhance the analysis of PPI network. We introduce a simple but comprehensive visualization system that integrates GO and PPI data where GO and PPI graphs are visualized side-by-side and supports quick reference functions between them. Furthermore, the proposed system provides several interactive visualization methods for efficiently analyzing the PPI network and GO directedacyclic- graph such as context-based browsing and common ancestors finding.

  11. Androgen receptor gene CAG repeat polymorphism and ovarian cancer risk: A meta-analysis.

    Science.gov (United States)

    Deng, Yang; Wang, Jue; Wang, Ling; Du, Yan

    2017-02-28

    Ovarian cancer is one of the common gynecological malignancies worldwide. It is usually diagnosed at a later stage, thus missing the best opportunity for treatment. Despite the advancement of ovarian cancer treatment, the prognosis is still poor. Androgen receptor (AR) may play a role in ovarian carcinogenesis. Previous studies regarding the association between AR CAG repeat length and ovarian cancer risk reported inconsistent results. Therefore, we conducted a meta-analysis to evaluate the association between AR CAG repeat length and ovarian cancer risk following the MOOSE guidelines. PubMed, Web of Science, EBSCO and other databases were searched up to September 15(th) 2016. Case control studies with clear definition of CAG repeat length and detailed genotype information were included. Two authors independently reviewed and extracted data. Pooled analysis and subgroup analysis stratified by ethnicity were performed for different genetic models. Begg's funnel plot and Egger's test were performed for publication bias estimation. Overall, there was no association between the AR CAG repeat polymorphism and ovarian cancer risk. However, short CAG repeat polymorphism was associated with increased ovarian cancer risk in African Americans and Chinese under the dominant model, whereas a reverse association was observed in Caucasians and Italians under the other three models. Our study results should be interpreted with caution. Further well-designed epidemiological and functional studies are needed to elucidate the role of AR in ovarian carcinogenesis.

  12. Tumoral Environment Triggers Transcript Anomalies in Established Tumors: Induction of Altered Gene Expression and of Aberrant, Truncated and B2 Repeat-Containing Gene Transcripts

    Directory of Open Access Journals (Sweden)

    Pieter Rottiers

    1999-12-01

    Full Text Available In addition to eugenetic changes, cancerous cells exhibit extensive modifications in the expression levels of a variety of genes. The phenotypic switch observed after inoculation of T lymphoma cells into syngenic mice illustrates the active participation of tumoral environment in the induction of an aberrant gene expression pattern. To further substantiate this contribution, we performed polymerase chain reaction (PCR-based subtraction suppression hybridization (SSH to identify genes that are differentially expressed in tumor-derived EL4/13.3 cells compared to the same cells isolated from cultures. Besides a number of unknown genes, the subtracted library contained several known genes that have been reported to be expressed at increased levels in tumors and/or to contribute to carcinogenesis. Apart from clones representing translated transcripts, the subtracted library also contained a high number of clones representing B2 repeat elements, viz. short interspersed repetitive elements that are transcribed by RNA polymerase III. Northern blotting confirmed the induction of B2 transcripts in tumor tissue and also revealed induction of chimeric, B2 repeat-containing mRNA. The appearance of chimeric transcripts was accompanied by aberrant, shorter-than-full-length transcripts, specifically from upregulated genes. Accordingly, in addition to altered gene expression, tumoral environmental triggers constitute a potent mechanism to create an epigenetic diversity in cancers by inducing extensive transcript anomalies.

  13. Development of the designed ankyrin repeat protein (DARPin) G3 for HER2 molecular imaging

    Energy Technology Data Exchange (ETDEWEB)

    Goldstein, Robert; Livanos, Maria; Bhavsar, Gaurav; Rashid, Mohammed; Miranda, Enrique; Tolner, Berend; Meyer, Tim; Chester, Kerry [UCL Cancer Institute, London (United Kingdom); Sosabowski, Jane; Leyton, Julius; Mather, Stephen [Queen Mary University of London, Centre for Molecular Oncology, Barts Cancer Institute, London (United Kingdom); Vigor, Kim [Clare Hall Laboratories, Biotherapeutics Development Unit, Cancer Research UK, South Mimms (United Kingdom); Nagy-Davidescu, Gabriela; Plueckthun, Andreas [Universitaet Zuerich, Biochemisches Institut, Zuerich (Switzerland); Yeung, Jenny [UCL Cancer Institute, London (United Kingdom); UCL Institute of Child Health, London (United Kingdom)

    2014-11-13

    Human epidermal growth factor receptor-2 (HER2) overexpression is a predictor of response to anti-HER2 therapy in breast and gastric cancer. Currently, HER2 status is assessed by tumour biopsy, but this may not be representative of the larger tumour mass or other metastatic sites, risking misclassification and selection of suboptimal therapy. The designed ankyrin repeat protein (DARPin) G3 binds HER2 with high affinity at an epitope that does not overlap with trastuzumab and is biologically inert. We hypothesized that radiolabelled DARPin G3 would be capable of selectively imaging HER2-positive tumours, and aimed to identify a suitable format for clinical application. G3 DARPins tagged with hexahistidine (His{sub 6}) or with histidine glutamate (HE){sub 3} and untagged G3 DARPins were manufactured using a GMP-compatible Pichia pastoris protocol and radiolabelled with {sup 125}I, or with {sup 111}In via DOTA linked to a C-terminal cysteine. BALB/c mice were injected with radiolabelled G3 and tissue biodistribution was evaluated by gamma counting. The lead construct ((HE){sub 3}-G3) was assessed in mice bearing HER2-positive human breast tumour (BT474) xenografts. For both isotopes, (HE){sub 3}-G3 had significantly lower liver uptake than His{sub 6}-G3 and untagged G3 counterparts in non-tumour-bearing mice, and there was no significantly different liver uptake between His{sub 6}-G3 and untagged G3. (HE){sub 3}-G3 was taken forward for evaluation in mice bearing HER2-positive tumour xenografts. The results demonstrated that radioactivity from {sup 111}In-(HE){sub 3}-G3 was better maintained in tumours and cleared faster from serum than radioactivity from {sup 125}I-(HE){sub 3}-G3, achieving superior tumour-to-blood ratios (343.7 ± 161.3 vs. 22.0 ± 11.3 at 24 h, respectively). On microSPECT/CT, {sup 111}In-labelled and {sup 125}I-labelled (HE){sub 3}-G3 could image HER2-positive tumours at 4 h after administration, but there was less normal tissue uptake of

  14. Crystal structure of the dimeric protein core of decorin, the archetypal small leucine-rich repeat proteoglycan.

    Science.gov (United States)

    Scott, Paul G; McEwan, Paul A; Dodd, Carole M; Bergmann, Ernst M; Bishop, Paul N; Bella, Jordi

    2004-11-02

    Decorin is a ubiquitous extracellular matrix proteoglycan with a variety of important biological functions that are mediated by its interactions with extracellular matrix proteins, cytokines, and cell surface receptors. Decorin is the prototype of the family of small leucine-rich repeat proteoglycans and proteins (SLRPs), characterized by a protein core composed of leucine-rich repeats (LRRs), flanked by two cysteine-rich regions. We report here the crystal structure of the dimeric protein core of decorin, the best characterized member of the SLRP family. Each monomer adopts the curved solenoid fold characteristic of LRR domains, with a parallel beta-sheet on the inside interwoven with loops containing short segments of beta-strands, 3(10) helices, and polyproline II helices on the outside. Two main features are unique to this structure. First, decorin dimerizes through the concave surfaces of the LRR domains, which have been implicated previously in protein-ligand interactions. The amount of surface buried in this dimer rivals the buried surfaces of some of the highest-affinity macromolecular complexes reported to date. Second, the C-terminal region adopts an unusual capping motif that involves a laterally extended LRR and a disulfide bond. This motif seems to be unique to SLRPs and has not been observed in any other LRR protein structure to date. Possible implications of these features for decorin ligand binding and SLRP function are discussed.

  15. A Low Protein Diet Alters Bone Material Level Properties and the Response to In Vitro Repeated Mechanical Loading

    Directory of Open Access Journals (Sweden)

    Victor Dubois-Ferrière

    2014-01-01

    Full Text Available Low protein intake is associated with an alteration of bone microstructure and material level properties. However, it remains unknown whether these alterations of bone tissue could influence the response to repeated mechanical loading. The authors investigated the in vitro effect of repeated loading on bone strength in humeri collected from 20 6-month-old female rats pair-fed with a control (15% casein or an isocaloric low protein (2.5% casein diet for 10 weeks. Bone specimens were cyclically loaded in three-point bending under load control for 2000 cycles. Humeri were then monotonically loaded to failure. The load-displacement curve of the in vitro cyclically loaded humerus was compared to the contralateral noncyclically loaded humerus and the influence of both protein diets. Material level properties were also evaluated through a nanoindentation test. Cyclic loading decreased postyield load and plastic deflection in rats fed a low protein diet, but not in those on a regular diet. Bone material level properties were altered in rats fed a low protein diet. This suggests that bone biomechanical alterations consequent to cyclic loading are more likely to occur in rats fed a low protein diet than in control animals subjected to the same in vitro cyclic loading regimen.

  16. Cytogenetic and molecular analysis of infertile Chinese men: karyotypic abnormalities, Y-chromosome microdeletions, and CAG and GGN repeat polymorphisms in the androgen receptor gene.

    Science.gov (United States)

    Han, T T; Ran, J; Ding, X P; Li, L J; Zhang, L Y; Zhang, Y P; Nie, S S; Chen, L

    2013-07-08

    Chromosome abnormalities, Y-chromosome microdeletions, and androgen receptor gene CAG and GGN repeat polymorphisms in infertile Chinese men featuring severe oligospermia and azoospermia were analyzed. Ninety-six fertile men and 189 non-obstructive infertile men, including 125 patients with azoospermia and 64 with severe oligozoospermia, were studied. Seventeen infertile men (9.0%) carried a chromosome abnormality. Twenty (10.6%) carried a Y-chromosome microdeletion. In the remainder of the patients and controls, GGN and CAG repeats were sequenced. Short GGN repeats (n repeats strongly correlated with sperm counts. No significant difference in CAG repeats was found between patients and controls, nor were CAG repeats correlated with sperm counts. However, for CAG repeats ranging between 24 and 25, there was a >2.5-fold risk (OR = 2.539, 95%CI = 1.206-5.344, P repeats in Chinese male infertility.

  17. LEA (Late Embryogenesis Abundant proteins and their encoding genes in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Hincha Dirk K

    2008-03-01

    Full Text Available Abstract Background LEA (late embryogenesis abundant proteins have first been described about 25 years ago as accumulating late in plant seed development. They were later found in vegetative plant tissues following environmental stress and also in desiccation tolerant bacteria and invertebrates. Although they are widely assumed to play crucial roles in cellular dehydration tolerance, their physiological and biochemical functions are largely unknown. Results We present a genome-wide analysis of LEA proteins and their encoding genes in Arabidopsis thaliana. We identified 51 LEA protein encoding genes in the Arabidopsis genome that could be classified into nine distinct groups. Expression studies were performed on all genes at different developmental stages, in different plant organs and under different stress and hormone treatments using quantitative RT-PCR. We found evidence of expression for all 51 genes. There was only little overlap between genes expressed in vegetative tissues and in seeds and expression levels were generally higher in seeds. Most genes encoding LEA proteins had abscisic acid response (ABRE and/or low temperature response (LTRE elements in their promoters and many genes containing the respective promoter elements were induced by abscisic acid, cold or drought. We also found that 33% of all Arabidopsis LEA protein encoding genes are arranged in tandem repeats and that 43% are part of homeologous pairs. The majority of LEA proteins were predicted to be highly hydrophilic and natively unstructured, but some were predicted to be folded. Conclusion The analyses indicate a wide range of sequence diversity, intracellular localizations, and expression patterns. The high fraction of retained duplicate genes and the inferred functional diversification indicate that they confer an evolutionary advantage for an organism under varying stressful environmental conditions. This comprehensive analysis will be an important starting point for

  18. Clinical features of Chinese patients with Huntington's disease carrying CAG repeats beyond 60 within HTT gene.

    Science.gov (United States)

    Liu, Z-J; Sun, Y-M; Ni, W; Dong, Y; Shi, S-S; Wu, Z-Y

    2014-02-01

    Patients with Huntington's disease (HD) carrying CAG repeats beyond 60 are less frequently seen and clinical features of them have been rarely reported. We identified four unrelated patients carrying CAG repeats beyond 60 (84.0 ± 13.76, ranging from 74 to 104) from 119 Chinese HD patients via direct sequencing. These four were all early onset with a mean age at presenting symptom of 9.8 ± 1.71 years. Paternal transmission was found in three of them and the fourth was apparently sporadic. In addition, they had atypical onset symptoms including epilepsy, intellectual decline, tics and walking instability, which might lead the clinicians to make the wrong diagnosis in the early stage of disease. Our work explores clinical features of Chinese HD patients with an expanded CAG repeat over 60 and may help the clinicians make a correct diagnosis in the early stage of disease.

  19. Entamoeba dispar: genetic diversity of Iranian isolates based on serine-rich Entamoeba dispar protein gene.

    Science.gov (United States)

    Haghighi, A; Rasti, S; Nazemalhosseini Mojarad, E; Kazemi, B; Bandehpour, M; Nochi, Z; Hooshyar, H; Rezaian, M

    2008-12-01

    The nucleotide sequences of Serine-Rich Entamoeba histolytica Protein (SREHP) gene have already exhibited stable and significant polymorphism in the gene studies. Serine-rich protein is also present and polymorphic in Entamoeba dispar which called SREDP. The polymorphism of the Serine-Rich Entamoeba dispar Protein (SREDP) gene among 8 isolates obtained from Iranian cyst carriers were analyzed by a nested PCR-RFLP followed by sequencing of the PCR products. From those isolates, six distinct DNA patterns were observed after PCR-RFLP of the nested PCR, whereas sequencing showed 8 different patterns among the isolates. The results demonstrate an extensive genetic variability among Iranian E. dispar isolates. The repeat-containing region of the SREDP was found extensively polymorphic in size, number and order of repeat units. Genetic diversity of Iranian E. dispar isolates based on the SREDP was more polymorphic in comparison of Serine-Rich Entamoeba histolytica Protein (SREHP) of the E. histolytica isolates as well as were different from a few known SREDP genes.

  20. Gene regulation in response to protein disulphide isomerase deficiency

    DEFF Research Database (Denmark)

    Nørgaard, Per; Tachibana, Christine; Bruun, Anette W

    2003-01-01

    We have examined the activities of promoters of a number of yeast genes encoding resident endoplasmic reticulum proteins, and found increased expression in a strain with severe protein disulphide isomerase deficiency. Serial deletion in the promoter of the MPD1 gene, which encodes a PDI1-homologue...... element. The sequence (GACACG) does not resemble the unfolded protein response element. It is present in the upstream regions of the MPD1, MPD2, KAR2, PDI1 and ERO1 genes....

  1. Tandem CAG repeats of the androgen receptor gene and prostate cancer risk in black and white men.

    Science.gov (United States)

    Panz, V R; Joffe, B I; Spitz, I; Lindenberg, T; Farkas, A; Haffejee, M

    2001-07-01

    The most common malignancy in men worldwide is cancer of the prostate. Androgens play a direct role in normal and malignant growth of prostate cells via the androgen receptor (AR). This study analyzed the polymorphic CAG repeat sequence in exon 1 of the AR gene to determine if the number of repeats might be an indicator of prostate cancer risk or aggressive disease. DNA was extracted from blood samples of 20 black and 20 white men with well-documented prostate cancer and 40 healthy controls (20 blacks and 20 whites). PCR amplification was followed by gel electrophoresis and DNA sequencing. This region normally contains between 9 and 29 repeats. Patients and controls both had minor variations in the number of repeats, which ranged from 13 to 27 with 21 being the most frequent allele. Black controls and patients both had a mean of 20 +/- 3 repeats; in whites the mean was significantly lower in patients than controls (21 +/- 2 versus 23 +/- 2; p = 0.004). Combined black and white patients also had a lower number than the combined group of controls (20 +/- 3 versus 22 +/- 3; p = 0.02). Similarly, black and white patients with aggressive disease had a lower number than patients whose disease was more slowly progressive (19 +/- 2 versus 22 +/- 3; p = 0.02). We conclude that the small differences in the number of CAG repeats in both black and white patients do not appear to be a strong indicator of risk or aggressive disease but that this size polymorphism may be one of many genetic and environmental risk factors involved in prostate cancer.

  2. Evolution of human alpha 1-acid glycoprotein genes and surrounding Alu repeats.

    Science.gov (United States)

    Merritt, C M; Easteal, S; Board, P G

    1990-04-01

    There is a mosaic pattern of variation between the two tandemly arranged human alpha 1-acid glycoprotein genes. Both the synonymous and the nonsynonymous sites of exons 3 and 4 are more divergent than the rest of the gene, suggesting that they have had a different evolutionary history. Comparisons of the two gene sequences with rat AGP indicate that exons 3 and 4 of AGP2 have been evolving without functional constraint since their divergence from AGP1. It is proposed that the conserved region of the gene has been homogenized recently by gene conversion with the homologous regions of AGP1. The Alu sequences surrounding the genes appear to have been involved in both the gene duplication and the gene conversion events.

  3. The sulfolobicin genes of Sulfolobus acidocaldarius encode novel antimicrobial proteins.

    Science.gov (United States)

    Ellen, Albert F; Rohulya, Olha V; Fusetti, Fabrizia; Wagner, Michaela; Albers, Sonja-Verena; Driessen, Arnold J M

    2011-09-01

    Crenarchaea, such as Sulfolobus acidocaldarius and Sulfolobus tokodaii, produce antimicrobial proteins called sulfolobicins. These antimicrobial proteins inhibit the growth of closely related species. Here we report the identification of the sulfolobicin-encoding genes in S. acidocaldarius. The active sulfolobicin comprises two proteins that are equipped with a classical signal sequence. These proteins are secreted by the cells and found to be membrane vesicle associated. Gene inactivation studies demonstrate that both proteins are required for the bacteriostatic antimicrobial activity. Sulfolobicins constitute a novel class of antimicrobial proteins without detectable homology to any other protein.

  4. The Sulfolobicin Genes of Sulfolobus acidocaldarius Encode Novel Antimicrobial Proteins

    NARCIS (Netherlands)

    Ellen, Albert F.; Rohulya, Olha V.; Fusetti, Fabrizia; Wagner, Michaela; Albers, Sonja-Verena; Driessen, Arnold J. M.

    2011-01-01

    Crenarchaea, such as Sulfolobus acidocaldarius and Sulfolobus tokodaii, produce antimicrobial proteins called sulfolobicins. These antimicrobial proteins inhibit the growth of closely related species. Here we report the identification of the sulfolobicin-encoding genes in S. acidocaldarius. The acti

  5. CAG-repeat variant in the polymerase γ gene and male infertility in the Chinese population: a meta-analysis.

    Science.gov (United States)

    Liu, Shu-Yuan; Zhang, Chang-Jun; Peng, Hai-Ying; Yao, Yu-Feng; Shi, Lei; Chen, Jin-Bao; Lin, Ke-Qin; Yu, Liang; Shi, Li; Huang, Xiao-Qin; Sun, Hao; Chu, Jia-You

    2011-03-01

    Several studies have reported a relationship between the length of the CAG-repeat in the polymerase γ (POLG) gene and male infertility. However, other studies have not reproduced this result. In our study, the POLG-CAG-repeat length was analyzed in 535 healthy individuals from six Chinese Han populations living in different provinces. The frequencies of 10-CAG alleles and genotypes were high (97.38 and 94.13%, respectively), with no significant difference among the six Chinese Han populations. Furthermore, we determined the distribution of the POLG-CAG-repeat in 150 infertile men and 126 fertile men. Our study suggested that the distributions of POLG-CAG-repeat alleles and genotypes were not significantly different between infertile (95.67 and 92.67%, respectively) and fertile men (97.22 and 94.44%, respectively). In a subsequent meta-analysis, combining our data with data from previous studies, a comparison of the CAG-repeat alleles in fertile versus infertile men showed no obvious risk for male infertility associated with any particular allele (pooled odds ratio (OR)=0.94; 95% confidence interval (CI): 0.60-1.48). The significance level was not attained with any of the following genetic models: homozygote comparison (not 10/not 10 versus 10/10: OR=1.34; 95% CI: 0.66-2.72), heterozygote comparison (10/not 10 versus 10/10: OR=1.04; 95% CI: 0.78-1.38), dominant model comparison (not 10/not 10+10/not 10 versus 10/10: OR=1.08; 95% CI: 0.79-1.47) and recessive genetic comparison (not 10/not 10 versus 10/not 10+10/10: OR=1.31; 95% CI: 0.68-2.55). In conclusion, there is no significant difference of the frequencies of POLG-CAG-repeat variants among six Chinese Han populations, and this polymorphism may not be associated with Chinese male infertility. On the basis of a meta-analysis, there is no obvious association between CAG-repeat variants of the POLG gene and male infertility.

  6. Factors influencing the clinical expression of intermediate CAG repeat length mutations of the Huntington's disease gene.

    Science.gov (United States)

    Panegyres, Peter K; Shu, Chen-Chun; Chen, Huei-Yang; Paulsen, Jane S

    2015-02-01

    Our aim is to elucidate the clinical variables associated with the development of manifest HD in patients with intermediate CAG repeat lengths. 2,167 participants were seen throughout 44 research sites in the United States, Canada or Australia over a five-year natural history observational study (2006-2011) (Trial # NCT00313495). The Chi-square test and a generalised linear model were used to examine the differences in demographics and cognitive tests among three groups of CAG repeat length. The mixed model was then used to examine the time effect on cognitive assessments by CAG groups. No patient with CAG repeat length 27-35 developed manifest HD, whereas three patients with 36-39 did. Total motor score, maximal chorea score and maximal dystonia score were significantly different at baseline (p CAG 36-39; those with an associated university degree or higher education were less frequently diagnosed as manifest HD (OR 0.10, 95 % CI 0.02-0.54, p = 0.007). Age, smoking and lower education achievement were found to be significantly associated with higher odds of manifest HD in patients with intermediate CAG repeat length mutations.

  7. Androgen receptor gene CAG repeat polymorphism and risk of isolated hypospadias: results from a meta-analysis.

    Science.gov (United States)

    Huang, G; Shan, W; Zeng, L; Huang, L

    2015-03-06

    Studies investigating the association between the CAG repeat polymorphism and the risk of isolated hypospadias have reported conflicting results. The aim of this study was to quantitatively summarize the evidence for such a relationship. Two investigators independently searched the Medline, Embase, CNKI, and Wanfang databases. Weighted mean difference and 95% confidence intervals for the CAG repeat polymorphism and isolated hypospadias were calculated using a random-effects model. Subgroup analyses were performed by race, study design, sample for DNA extraction, and hypospadias classifications. This meta-analysis included 6 case-control studies, including 444 isolated hypospadias cases and 727 controls. The results showed that patients with isolated hypospadias had longer CAG repeats in their androgen receptor gene sequence (weighted mean difference = 1.36, 95% confidence interval = 0.60-2.13; P = 0.0005). Similarly, stratified analyses also detected significant associations in all subgroups, excluding the group with severe hypospadias (weighted mean difference = 0.35, 95% confidence interval = -0.42-1.12; P = 0.38). This meta-analysis indicated that longer CAG repeats were associated with the risk of isolated hypospadias, and that longer CAG polymorphisms may be related to the etiology of isolated hypospadias. Future studies based on Asian and African-American patients should be performed to re-evaluate this association.

  8. Ankyrin repeat domain-encoding genes in the wPip strain of Wolbachia from the Culex pipiens group

    Directory of Open Access Journals (Sweden)

    Parkhill Julian

    2007-09-01

    Full Text Available Abstract Background Wolbachia are obligate endosymbiotic bacteria maternally transmitted through the egg cytoplasm that are responsible for several reproductive disorders in their insect hosts, such as cytoplasmic incompatibility (CI in infected mosquitoes. Species in the Culex pipiens complex display an unusually high number of Wolbachia-induced crossing types, and based on present data, only the wPip strain is present. Results The sequencing of the wPip strain of Wolbachia revealed the presence of 60 ankyrin repeat domain (ANK encoding genes and expression studies of these genes were carried out in adult mosquitoes. One of these ANK genes, pk2, is shown to be part of an operon of three prophage-associated genes with sex-specific expression, and is present in two identical copies in the genome. Another homolog of pk2 is also present that is differentially expressed in different Cx. pipiens group strains. A further two ANK genes showed sex-specific regulation in wPip-infected Cx. pipiens group adults. Conclusion The high number, variability and differential expression of ANK genes in wPip suggest an important role in Wolbachia biology, and the gene family provides both markers and promising candidates for the study of reproductive manipulation.

  9. Extensive gene amplification and concerted evolution within the CPR family of cuticular proteins in mosquitoes.

    Science.gov (United States)

    Cornman, R Scott; Willis, Judith H

    2008-06-01

    Annotation of the Anopheles gambiae genome has revealed a large increase in the number of genes encoding cuticular proteins with the Rebers and Riddiford Consensus (the CPR gene family) relative to Drosophila melanogaster. This increase reflects an expansion of the RR-2 group of CPR genes, particularly the amplification of sets of highly similar paralogs. Patterns of nucleotide variation indicate that extensive concerted evolution is occurring within these clusters. The pattern of concerted evolution is complex, however, as sequence similarity within clusters is uncorrelated with gene order and orientation, and no comparable clusters occur within similarly compact arrays of the RR-1 group in mosquitoes or in either group in D. melanogaster. The dearth of pseudogenes suggests that sequence clusters are maintained by selection for high gene-copy number, perhaps due to selection for high expression rates. This hypothesis is consistent with the apparently parallel evolution of compact gene architectures within sequence clusters relative to single-copy genes. We show that RR-2 proteins from sequence-cluster genes have complex repeats and extreme amino-acid compositions relative to single-copy CPR proteins in An. gambiae, and that the amino-acid composition of the N-terminal and C-terminal sequence flanking the chitin-binding consensus region evolves in a correlated fashion.

  10. Regulation of human protein S gene (PROS1) transcription

    NARCIS (Netherlands)

    Wolf, Cornelia de

    2006-01-01

    This thesis describes the investigation of the transcriptional regulation of the gene for anticoagulant plasma Protein S, PROS1. Protein S is a cofactor for Protein C in the Protein C anticoagulant pathway. The coagulation cascade is negatively regulated by this pathway through inactivation of activ

  11. Regulation of human protein S gene (PROS1) transcription

    NARCIS (Netherlands)

    Wolf, Cornelia de

    2006-01-01

    This thesis describes the investigation of the transcriptional regulation of the gene for anticoagulant plasma Protein S, PROS1. Protein S is a cofactor for Protein C in the Protein C anticoagulant pathway. The coagulation cascade is negatively regulated by this pathway through inactivation of

  12. Molecular-intelligence correlations in young fragile X males with a mild CGG repeat expansion in the FMR1 gene

    Energy Technology Data Exchange (ETDEWEB)

    Steyaert, J. [Central of Clinical Genetics, Maastricht (Netherlands); Borghgraef, M.; Legius, E. [University Hospital Gasthuisberg, Leuven (Belgium)] [and others

    1996-08-09

    Several mechanisms can explain the occurrence of full-mutation fragile X males with an IQ level above -2 SD below mean, also called {open_quotes}high-functioning fragile X males.{close_quotes} Incomplete methylation of the CpG island at the 5{prime} end of the FMR1 gene is one of these mechanisms. The present study describes the physical and behavior phenotypes in 7 fragile X boys with CGG repeat insertions in the FMR1 gene between 600-2,400 base pairs. The degree of methylation at the FMR1-associated CpG island ranges in peripheral blood lymphocytes from 0-95%. Subjects with a low degree of methylation at this site have mild or absent physical characteristics of the fragile X syndrome, while subjects with a high degree of methylation at this site have more severe physical characteristics. In this range of CGG repeat insertion (600-2,400 base pairs), the degree of methylation at the FMR1-associated CpG island is a good predictor of intelligence, while CGG repeat insertion length is not. 13 refs., 1 fig., 1 tab.

  13. Analysis of the AHR gene proximal promoter GGGGC-repeat polymorphism in lung, breast, and colon cancer

    Energy Technology Data Exchange (ETDEWEB)

    Spink, Barbara C. [Wadsworth Center, New York State Department of Health, Albany, NY 12201 (United States); Bloom, Michael S. [Department of Environmental Health Sciences, School of Public Health, University at Albany, State University of New York, Albany, NY 12201 (United States); Wu, Susan [Wadsworth Center, New York State Department of Health, Albany, NY 12201 (United States); Sell, Stewart; Schneider, Erasmus [Wadsworth Center, New York State Department of Health, Albany, NY 12201 (United States); Department of Biomedical Sciences, School of Public Health, University at Albany, State University of New York, Albany, NY 12201 (United States); Ding, Xinxin [Wadsworth Center, New York State Department of Health, Albany, NY 12201 (United States); Department of Environmental Health Sciences, School of Public Health, University at Albany, State University of New York, Albany, NY 12201 (United States); Department of Biomedical Sciences, School of Public Health, University at Albany, State University of New York, Albany, NY 12201 (United States); Spink, David C., E-mail: spink@wadsworth.org [Wadsworth Center, New York State Department of Health, Albany, NY 12201 (United States); Department of Environmental Health Sciences, School of Public Health, University at Albany, State University of New York, Albany, NY 12201 (United States)

    2015-01-01

    The aryl hydrocarbon receptor (AhR) regulates expression of numerous genes, including those of the CYP1 gene family. With the goal of determining factors that control AHR gene expression, our studies are focused on the role of the short tandem repeat polymorphism, (GGGGC){sub n}, located in the proximal promoter of the human AHR gene. When luciferase constructs containing varying GGGGC repeats were transfected into cancer cell lines derived from the lung, colon, and breast, the number of GGGGC repeats affected AHR promoter activity. The number of GGGGC repeats was determined in DNA from 327 humans and from 38 samples representing 5 species of non-human primates. In chimpanzees and 3 species of macaques, only (GGGGC){sub 2} alleles were observed; however, in western gorilla, (GGGGC){sub n} alleles with n = 2, 4, 5, 6, 7, and 8 were identified. In all human populations examined, the frequency of (GGGGC){sub n} was n = 4 > 5 ≫ 2, 6. When frequencies of the (GGGGC){sub n} alleles in DNA from patients with lung, colon, or breast cancer were evaluated, the occurrence of (GGGGC){sub 2} was found to be 8-fold more frequent among lung cancer patients in comparison with its incidence in the general population, as represented by New York State neonates. Analysis of matched tumor and non-tumor DNA samples from the same individuals provided no evidence of microsatellite instability. These studies indicate that the (GGGGC){sub n} short tandem repeats are inherited, and that the (GGGGC){sub 2} allele in the AHR proximal promoter region should be further investigated with regard to its potential association with lung cancer susceptibility. - Highlights: • The AHR proximal promoter contains a polymorphism, (GGGGC){sub n}, where n = 4 > 5 ≫ 2, 6 • Matched tumor and non-tumor DNA did not show (GGGGC){sub n} microsatellite instability • AHR promoter activity of a construct with (GGGGC){sub 2} was lower than that of (GGGGC){sub 4} • The frequency of (GGGGC){sub 2} in lung

  14. Taxonomic distribution, repeats, and functions of the S1 domain-containing proteins as members of the OB-fold family.

    Science.gov (United States)

    Deryusheva, Evgeniia I; Machulin, Andrey V; Selivanova, Olga M; Galzitskaya, Oxana V

    2017-04-01

    Proteins of the nucleic acid-binding proteins superfamily perform such functions as processing, transport, storage, stretching, translation, and degradation of RNA. It is one of the 16 superfamilies containing the OB-fold in protein structures. Here, we have analyzed the superfamily of nucleic acid-binding proteins (the number of sequences exceeds 200,000) and obtained that this superfamily prevalently consists of proteins containing the cold shock DNA-binding domain (ca. 131,000 protein sequences). Proteins containing the S1 domain compose 57% from the cold shock DNA-binding domain family. Furthermore, we have found that the S1 domain was identified mainly in the bacterial proteins (ca. 83%) compared to the eukaryotic and archaeal proteins, which are available in the UniProt database. We have found that the number of multiple repeats of S1 domain in the S1 domain-containing proteins depends on the taxonomic affiliation. All archaeal proteins contain one copy of the S1 domain, while the number of repeats in the eukaryotic proteins varies between 1 and 15 and correlates with the protein size. In the bacterial proteins, the number of repeats is no more than 6, regardless of the protein size. The large variation of the repeat number of S1 domain as one of the structural variants of the OB-fold is a distinctive feature of S1 domain-containing proteins. Proteins from the other families and superfamilies have either one OB-fold or change slightly the repeat numbers. On the whole, it can be supposed that the repeat number is a vital for multifunctional activity of the S1 domain-containing proteins. Proteins 2017; 85:602-613. © 2016 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  15. Specific Binding of Tetratricopeptide Repeat Proteins to Heat Shock Protein 70 (Hsp70) and Heat Shock Protein 90 (Hsp90) Is Regulated by Affinity and Phosphorylation.

    Science.gov (United States)

    Assimon, Victoria A; Southworth, Daniel R; Gestwicki, Jason E

    2015-12-01

    Heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) require the help of tetratricopeptide repeat (TPR) domain-containing cochaperones for many of their functions. Each monomer of Hsp70 or Hsp90 can interact with only a single TPR cochaperone at a time, and each member of the TPR cochaperone family brings distinct functions to the complex. Thus, competition for TPR binding sites on Hsp70 and Hsp90 appears to shape chaperone activity. Recent structural and biophysical efforts have improved our understanding of chaperone-TPR contacts, focusing on the C-terminal EEVD motif that is present in both chaperones. To better understand these important protein-protein interactions on a wider scale, we measured the affinity of five TPR cochaperones, CHIP, Hop, DnaJC7, FKBP51, and FKBP52, for the C-termini of four members of the chaperone family, Hsc70, Hsp72, Hsp90α, and Hsp90β, in vitro. These studies identified some surprising selectivity among the chaperone-TPR pairs, including the selective binding of FKBP51/52 to Hsp90α/β. These results also revealed that other TPR cochaperones are only able to weakly discriminate between the chaperones or between their paralogs. We also explored whether mimicking phosphorylation of serine and threonine residues near the EEVD motif might impact affinity and found that pseudophosphorylation had selective effects on binding to CHIP but not other cochaperones. Together, these findings suggest that both intrinsic affinity and post-translational modifications tune the interactions between the Hsp70 and Hsp90 proteins and the TPR cochaperones.

  16. Recombinant myxoma virus lacking all poxvirus ankyrin-repeat proteins stimulates multiple cellular anti-viral pathways and exhibits a severe decrease in virulence.

    Science.gov (United States)

    Lamb, Stephanie A; Rahman, Masmudur M; McFadden, Grant

    2014-09-01

    Although the production of single gene knockout viruses is a useful strategy to study viral gene functions, the redundancy of many host interactive genes within a complex viral genome can obscure their collective functions. In this study, a rabbit-specific poxvirus, myxoma virus (MYXV), was genetically altered to disrupt multiple members of the poxviral ankyrin-repeat (ANK-R) protein superfamily, M-T5, M148, M149 and M150. A particularly robust activation of the NF-κB pathway was observed in A549 cells following infection with the complete ANK-R knockout (vMyx-ANKsKO). Also, an increased release of IL-6 was only observed upon infection with vMyx-ANKsKO. In virus-infected rabbit studies, vMyx-ANKsKO was the most extensively attenuated and produced the smallest primary lesion of all ANK-R mutant constructs. This study provides the first insights into the shared functions of the poxviral ANK-R protein superfamily in vitro and in vivo. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. The knockout of aquaporin 2 gene of renal inner medullary collecting duct epithelial cell line in mice by clustered regularly interspaced short palindromic repeats and associated protein 9 system%利用常间回文重复序列丛集及相关蛋白9系统敲除小鼠肾集合管上皮细胞系的水通道蛋白2基因

    Institute of Scientific and Technical Information of China (English)

    陈晓芳; 练桂丽; 黄邦邦; 谢良地

    2016-01-01

    Objective To knock out aquaporin 2 (Aqp2) gene in mouse renal inner medullary collecting duct 3 (m-IMCD3) epithelial cells by clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9) system.Methods Four small guide RNAs (sgRNA) targeting exon 1 and 4 (sgRNA1-1,sgRNA1-2,sgRNA4-1,sgRNA4-2) of Aqp2 gene were designed respectively and successfully ligated to LentiCRISPR V2 vector.The plasmid containing a corresponding sgRNA was transfected into m-IMCD3 cells.The genomic DNA of new monoclonal cell lines was extracted and the target DNA fragment was amplified by polymerase chain reaction (PCR).Then the product of PCR was sequenced.The expression of Aqp2 was determined by reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence.Results The lentiviral CRISPR vector 2 containing a corresponding sgRNA were successfully constructed.The four sgRNAs could cut the Aqp2 gene.The combination of sgRNA1-1 and sgRNA4-2 could successfully knock out the DNA fragment of 5500 bp between them.The expressions of Aqp2 mRNA and protein in the Aqp2 knockout monoclonal cell line were not detected by RT-PCR and immunofluorescence.Conclusion Aqp2 knockout m-IMCD3 epithelial cell line was established via CRISPR/Cas9 system.%目的 利用常间回文重复序列丛集(CRISPR)/CRISPR相关蛋白9(Cas9)系统敲除小鼠肾内髓集合管3(m-I MCD3)上皮细胞的水通道蛋白2(Aqp2)基因.方法 在小鼠Aqp2基因的第1、4个外显子上各设计两个小向导RNA(sgRNA),分别为sgRNA1-1、sgRNA1-2、sgRNA4-1、sgRNA4-2,并将其成功克隆进常间回文重复序列丛集慢病毒载体2上.将测序正确的质粒转染到m-IMCD3细胞中,提取各单克隆细胞系的基因组DNA,通过聚合酶链反应扩增出sgRNA作用靶点的DNA片段并测序.利用逆转录聚合酶链反应(RT-PCR)及免疫荧光检测Aqp2的表达.结果 成功构建出含有相应sgRNA的载体;4个sgRNA对Aqp2基因的

  18. HOT1 is a mammalian direct telomere repeat-binding protein contributing to telomerase recruitment

    NARCIS (Netherlands)

    Kappei, D.; Butter, F.; Benda, C.; Scheibe, M.; Draskovic, Irena; Stevense, M.; Novo, C.L.; Basquin, C.; Araki, M.; Araki, K.; Krastev, D.B.; Kittler, R.; Jessberger, R.; Londono-Vallejo, J.A.; Mann, M.; Buchholz, F.

    2013-01-01

    Telomeres are repetitive DNA structures that, together with the shelterin and the CST complex, protect the ends of chromosomes. Telomere shortening is mitigated in stem and cancer cells through the de novo addition of telomeric repeats by telomerase. Telomere elongation requires the delivery of the

  19. Leucine-rich repeat C4 protein is Involved in Nervous Tissue Development and Neurite Outgrowth, and Induction of Glioma Cell Differentiation

    Institute of Scientific and Technical Information of China (English)

    Minghua WU; Jianhong LU; Shourong SHEN; Guiyuan LI; He HUANG; Qiong CHEN; Dan LI; Zhaoyang ZENG; Wei XIONG; Yanhong ZHOU; Xiaoling LI; Ming ZHOU

    2007-01-01

    LRRC4, leucine-rich repeat C4 protein, has been identified in human (GenBank accession No.AF196976), mouse (GenBank accession No. DQ177325), rat (GenBank accession No. DQ119102) and bovine (GenBank accession No. DQ164537) with identical domains. In terms of their similarity, the genes encoding LRRC4 in these four mammalian species are orthogs and therefore correspond to the same gene entity. Based on previous research, and using in situ hybridization, we found that LRRC4 had the strongest expression in hippocampal CA1 and CA2, the granule cells of the dentate gyrus region, the mediodoral thalamic nucleus, and cerebella Purkinje cell layers. Using a P19 cell model, we also found that LRRC4 participates in the differentiation of neuron and glia cells. In addition, extracellular proteins containing both an LRR cassette and immunoglobulin domains have been shown to participate in axon guidance. Our data from neurite outgrowth assays indicated that LRRC4 promoted neurite extension of hippocampal neurons, and induced differentiation of glioblastoma U251 cells into astrocyte-like cells, confirmed by morphology observation and glial fibrillary acidic protein expression.

  20. Molecular cloning, genomic organization, developmental regulation, and a knock-out mutant of a novel leu-rich repeats-containing G protein-coupled receptor (DLGR-2) from Drosophila melanogaster

    DEFF Research Database (Denmark)

    Eriksen, Kathrine Krageskov; Hauser, Frank; Schiøtt, Morten

    2000-01-01

    After screening the Berkeley Drosophila Genome Project database with sequences from a recently characterized Leu-rich repeats-containing G protein-coupled receptor (LGR) fromDrosophila (DLGR-1), we identified a second gene for a different LGR (DLGR-2) and cloned its cDNA. DLGR-2 is 1360 amino acid...... LGRs (LGR-4 and LGR-5). This homology includes the seven transmembrane region (e.g., 49% amino acid identity with the human TSH receptor) and the very large extracellular amino terminus. This amino terminus contains 18 Leu-rich repeats-in contrast with the 3 mammalian glycoprotein hormone receptors...

  1. No relationship exists between itai-itai disease and TA repeat polymorphisms of the estrogen receptor alpha gene.

    Science.gov (United States)

    Sadewa, Hamim Ahmad; Miyabe, Yuri; Nishio, Hisahide; Hayashi, Chiyo; Sutomo, Retno; Lee, Myeong Jin; Ayaki, Hitoshi; Koizumi, Naoko; Sumino, Kimiaki

    2002-08-01

    Itai-itai (ouch-ouch) disease is a syndrome accompanied by bone mineral disorders that may be related to oral cadmium exposure. Itai-itai predominantly affects postmenopausal women with a history of multiple childbirth. In a previous study we have examined the genotype distributions of PvuII and XbaI restriction fragment length polymorphisms of the estrogen receptor alpha (ER alpha) gene in patients with itai-itai disease and compared them with those of controls. However, no significant differences were shown between the genotype distributions of the patients and controls. In the present study, we determined the TA repeat polymorphisms of the patients and controls. The distributions of the patients were: HH 25.0%, HL 50.0%, and LL 25.0%; where HH includes two alleles with a high number of TA repeats (TA> or =16), HL includes one high number allele and one low number allele (TAitai-itai disease.

  2. The phenome analysis of mutant alleles in Leucine-Rich Repeat Receptor-Like Kinase genes in rice reveals new potential targets for stress tolerant cereals.

    Science.gov (United States)

    Dievart, Anne; Perin, Christophe; Hirsch, Judith; Bettembourg, Mathilde; Lanau, Nadège; Artus, Florence; Bureau, Charlotte; Noel, Nicolas; Droc, Gaétan; Peyramard, Matthieu; Pereira, Serge; Courtois, Brigitte; Morel, Jean-Benoit; Guiderdoni, Emmanuel

    2016-01-01

    Plants are constantly exposed to a variety of biotic and abiotic stresses that reduce their fitness and performance. At the molecular level, the perception of extracellular stimuli and the subsequent activation of defense responses require a complex interplay of signaling cascades, in which protein phosphorylation plays a central role. Several studies have shown that some members of the Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) family are involved in stress and developmental pathways. We report here a systematic analysis of the role of the members of this gene family by mutant phenotyping in the monocotyledon model plant rice, Oryza sativa. We have then targeted 176 of the ∼320 LRR-RLK genes (55.7%) and genotyped 288 mutant lines. Position of the insertion was confirmed in 128 lines corresponding to 100 LRR-RLK genes (31.6% of the entire family). All mutant lines harboring homozygous insertions have been screened for phenotypes under normal conditions and under various abiotic stresses. Mutant plants have been observed at several stages of growth, from seedlings in Petri dishes to flowering and grain filling under greenhouse conditions. Our results show that 37 of the LRR-RLK rice genes are potential targets for improvement especially in the generation of abiotic stress tolerant cereals.

  3. Gene cloning and characterization of the protein encoded by the Neospora caninum bradyzoite-specific antigen gene BAG1.

    Science.gov (United States)

    Kobayashi, T; Narabu, S; Yanai, Y; Hatano, Y; Ito, A; Imai, S; Ike, K

    2013-06-01

    Neospora caninum is an Apicomplexan parasite that causes repeated abortion and stillbirth in cattle. The aim of this study was to clone the gene encoding the N. caninum orthologue (NcBAG1) of the Toxoplasma gondii bradyzoite-specific protein TgBAG1 and characterize its expression pattern in the parasite. Isolation of the full-length 684-bp gene revealed that it shared 78.3% sequence similarity with TgBAG1. NcBAG1 encodes a predicted protein of 227 amino acids with 80.3% similarity to TgBAG1. A putative signal peptide sequence and an invariant GVL motif characteristic of small heat-shock proteins were identified in the predicted N. caninum amino acid sequence. We expressed the NcBAG1 gene as a recombinant glutathione S-transferase fusion protein (rNcBAG1) in Escherichia coli and used the purified 60 kDa protein to obtain a monoclonal antibody (Mab). rNcBAG1 reacted to Mabs specific for NcBAG1 and TgBAG1. No reaction between the NcBAG1 Mab and N. caninum tachyzoites was observed. Although the predicted molecular mass of NcBAG1 is 25 kDa, Western blot analysis of parasite lysates using the NcBAG1 Mab revealed a cross-reactive protein of approximately 30 kDa. Additionally, immunofluorescence assays using the tachyzoite-specific Mab for NcSAG1 and the bradyzoite-specific Mab for TgBAG1 or NcSAG4 revealed NcBAG1-specific expression in bradyzoites in cultures exposed to sodium nitroprusside, a reagent that increases the frequency of bradyzoites. Interestingly, the NcBAG1 protein was identified in the cytoplasm of the bradyzoite-stage parasites. This preliminary analysis of the NcBAG1 gene will assist investigations into the role of this protein in N. caninum .

  4. Novel histone biotinylation marks are enriched in repeat regions and participate in repression of transcriptionally competent genes.

    Science.gov (United States)

    Pestinger, Valerie; Wijeratne, Subhashinee S K; Rodriguez-Melendez, Rocio; Zempleni, Janos

    2011-04-01

    Covalent histone modifications play crucial roles in chromatin structure and genome stability. We previously reported biotinylation of lysine (K) residues in histones H2A, H3 and H4 by holocarboxylase synthetase and demonstrated that K12-biotinylated histone H4 (H4K12bio) is enriched in repeat regions and participates in gene repression. The biological functions of biotinylation marks other than H4K12bio are poorly understood. Here, novel biotinylation site-specific antibodies against H3K9bio, H3K18bio and H4K8bio were used in chromatin immunoprecipitation studies to obtain first insights into possible biological functions of these marks. Chromatin immunoprecipitation assays were conducted in human primary fibroblasts and Jurkat lymphoblastoma cells, and revealed that H3K9bio, H3K18bio and H4K8bio are enriched in repeat regions such as pericentromeric alpha satellite repeats and long-terminal repeats while being depleted in transcriptionally active promoters in euchromatin. Transcriptional stimulation of the repressed interleukin-2 promoter triggered a rapid depletion of histone biotinylation marks at this locus in Jurkat cells, which was paralleled by an increase in interleukin-2 mRNA. Importantly, the enrichment of H3K9bio, H3K18bio and H4K8bio at genomic loci depended on the concentration of biotin in culture media at nutritionally relevant levels, suggesting a novel mechanism of gene regulation by biotin. Copyright © 2011 Elsevier Inc. All rights reserved.

  5. Simple tandem repeat (TTTAn polymorphism in CYP19 (aromatase gene and breast cancer risk in Nigerian women

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    Okobia Michael N

    2006-05-01

    Full Text Available Abstract Background Breast cancer is the most common cancer and the leading cause of cancer related deaths in women worldwide. The incidence of the disease is increasing globally and this increase is occurring at a faster rate in population groups that hirtherto enjoyed low incidence. This study was designed to evaluate the role of a simple tandem repeat polymorphism (STRP in the aromatase (CYP19 gene in breast cancer susceptibility in Nigerian women, a population of indigenous sub-Saharan African ancestry. Methods A case-control study recruiting 250 women with breast cancer and 250 women without the disease from four University Teaching Hospitals in Southern Nigeria was carried out between September 2002 and April 2004. Participants were recruited from the surgical outpatient clinics and surgical wards of the Nigerian institutions. A polymerase chain reaction (PCR-based assay was employed for genotyping and product sizes were detected with an ABI 3730 DNA Analyzer. Results Conditional logistic regression analysis revealed that harboring the putative high risk genotypes conferred a 29% increased risk of breast cancer when all women in the study were considered (Odds ratio [OR] = 1.29, 95% confidence interval [CI] 0.83–2.00, although this association was not statistically significant. Subgroup analysis based on menopausal status showed similar results among premenopausal women (OR = 1.35, 95% CI 0.76–2.41 and postmenopausal women (OR = 1.27, 95% CI 0.64–2.49. The data also demonstrated marked differences in the distribution of (TTTAn repeats in Nigerian women compared with other populations. Conclusion This study has shown that harboring 10 or more repeats of the microsatellite (TTTAn repeats of the CYY19 gene is associated with a modest increased risk of breast cancer in Nigerian women.

  6. The origin and evolution of variable number tandem repeat of CLEC4M gene in the global human population.

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    Hui Li

    Full Text Available CLEC4M is a C-type lectin gene serving as cell adhesion receptor and pathogen recognition receptor. It recognizes several pathogens of important public health concern. In particular, a highly polymorphic variable number tandem repeat (VNTR at the neck-region of CLEC4M had been associated with genetic predisposition to some infectious diseases. To gain insight into the origin and evolution of this VNTR in CLEC4M, we studied 21 Africans, 20 Middle Easterns, 35 Europeans, 38 Asians, 13 Oceania, and 18 Americans (a total of 290 chromosomes from the (Human Genome Diversity Panel HGDP-CEPH panel; these samples covered most of alleles of this VNTR locus present in human populations. We identified a limited number of haplotypes among the basic repeat subunits that is 69 base pairs in length. Only 8 haplotypes were found. Their sequence identities were determined in the 290 chromosomes. VNTR alleles of different repeat length (from 4 to 9 repeats were analyzed for composition and orientation of these subunits. Our results showed that the subunit configuration of the same repeat number of VNTR locus from different populations were, in fact, virtually identical. It implies that most of the VNTR alleles existed before dispersion of modern humans outside Africa. Further analyses indicate that the present diversity profile of this locus in worldwide populations is generated from the effect of migration of different tribes and neutral evolution. Our findings do not support the hypothesis that the origin of the VNTR alleles were arisen by independent (separate mutation events and caused by differential allele advantage and natural selection as suggested by previous report based on SNP data.

  7. Interrogation of the protein-protein interactions between human BRCA2 BRC repeats and RAD51 reveals atomistic determinants of affinity.

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    Daniel J Cole

    2011-07-01

    Full Text Available The breast cancer suppressor BRCA2 controls the recombinase RAD51 in the reactions that mediate homologous DNA recombination, an essential cellular process required for the error-free repair of DNA double-stranded breaks. The primary mode of interaction between BRCA2 and RAD51 is through the BRC repeats, which are ∼35 residue peptide motifs that interact directly with RAD51 in vitro. Human BRCA2, like its mammalian orthologues, contains 8 BRC repeats whose sequence and spacing are evolutionarily conserved. Despite their sequence conservation, there is evidence that the different human BRC repeats have distinct capacities to bind RAD51. A previously published crystal structure reports the structural basis of the interaction between human BRC4 and the catalytic core domain of RAD51. However, no structural information is available regarding the binding of the remaining seven BRC repeats to RAD51, nor is it known why the BRC repeats show marked variation in binding affinity to RAD51 despite only subtle sequence variation. To address these issues, we have performed fluorescence polarisation assays to indirectly measure relative binding affinity, and applied computational simulations to interrogate the behaviour of the eight human BRC-RAD51 complexes, as well as a suite of BRC cancer-associated mutations. Our computational approaches encompass a range of techniques designed to link sequence variation with binding free energy. They include MM-PBSA and thermodynamic integration, which are based on classical force fields, and a recently developed approach to computing binding free energies from large-scale quantum mechanical first principles calculations with the linear-scaling density functional code onetep. Our findings not only reveal how sequence variation in the BRC repeats directly affects affinity with RAD51 and provide significant new insights into the control of RAD51 by human BRCA2, but also exemplify a palette of computational and

  8. Cell-to-Cell Transmission of Dipeptide Repeat Proteins Linked to C9orf72-ALS/FTD

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    Thomas Westergard

    2016-10-01

    Full Text Available Aberrant hexanucleotide repeat expansions in C9orf72 are the most common genetic change underlying amyotrophic lateral sclerosis (ALS and frontotemporal dementia (FTD. RNA transcripts containing these expansions undergo repeat-associated non-ATG translation (RAN-T to form five dipeptide repeat proteins (DPRs. DPRs are found as aggregates throughout the CNS of C9orf72-ALS/FTD patients, and some cause degeneration when expressed in vitro in neuronal cultures and in vivo in animal models. The spread of characteristic disease-related proteins drives the progression of pathology in many neurodegenerative diseases. While DPR toxic mechanisms continue to be investigated, the potential for DPRs to spread has yet to be determined. Using different experimental cell culture platforms, including spinal motor neurons derived from induced pluripotent stem cells from C9orf72-ALS patients, we found evidence for cell-to-cell spreading of DPRs via exosome-dependent and exosome-independent pathways, which may be relevant to disease.

  9. Molecular characteristics of three thymosin-repeat proteins fromMarsupenaeus japonicus and their responses to WSSV infection

    Institute of Scientific and Technical Information of China (English)

    MA Jinyou; RUAN Lingwei; XU Xun; GAO Zhaoming

    2016-01-01

    β-thymosins, a family of highly conserved peptides, play a vital role in wound-healing, angiogenesis, antimicrobial process and antiviral immunity. Three novelβ-thymosin-repeat proteins, namedmjthm4,mjthm3 andmjthm2, were cloned fromMarsupenaeus japonicus using expressed sequence tags (EST) from suppression subtractive hybridization. Themjthm4,mjthm3 andmjthm2 cDNAs possessed open reading frames that encoded 166, 128 and 90 amino acid residue polypeptides and contained four, three and twoβ-thymosin actin binding modules, respectively. Blast analysis demonstrated thatmjthm4,mjthm3 andmjthm2 shared high homology with known invertebrate multi-repeatβ-thymosins. These proteins are ubiquitously expressed in all of the examined tissues, and the transcriptional levels were highest in the intestine. Further investigation revealed thatmjthm4, mjthm3 andmjthm2 were remarkably up-regulated 6 h after WSSV infection. Moreover, whilemjthm4 transcriptional levels displayed no changes,mjthm3 andmjthm2 levels decreased in the virus-resistant shrimps. The results indicate thatmjthm4,mjthm3 andmjthm2 are novel multi-repeatβ-thymosin homologues, have a close relationship with WSSV infection, and might contribute to a better understanding of host defense and/or virus invasion interactions in shrimps.

  10. Binary gene induction and protein expression in individual cells

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    Conolly Rory B

    2006-04-01

    Full Text Available Abstract Background Eukaryotic gene transcription is believed to occur in either a binary or a graded fashion. With binary induction, a transcription activator (TA regulates the probability with which a gene template is switched from the inactive to the active state without affecting the rate at which RNA molecules are produced from the template. With graded, also called rheostat-like, induction the gene template has continuously varying levels of transcriptional activity, and the TA regulates the rate of RNA production. Support for each of these two mechanisms arises primarily from experimental studies measuring reporter proteins in individual cells, rather than from direct measurement of induction events at the gene template. Methods and results In this paper, using a computational model of stochastic gene expression, we have studied the biological and experimental conditions under which a binary induction mode operating at the gene template can give rise to differentially expressed "phenotypes" (i.e., binary, hybrid or graded at the protein level. We have also investigated whether the choice of reporter genes plays a significant role in determining the observed protein expression patterns in individual cells, given the diverse properties of commonly-used reporter genes. Our simulation confirmed early findings that the lifetimes of active/inactive promoters and half-lives of downstream mRNA/protein products are important determinants of various protein expression patterns, but showed that the induction time and the sensitivity with which the expressed genes are detected are also important experimental variables. Using parameter conditions representative of reporter genes including green fluorescence protein (GFP and β-galactosidase, we also demonstrated that graded gene expression is more likely to be observed with GFP, a longer-lived protein with low detection sensitivity. Conclusion The choice of reporter genes may determine whether protein

  11. Down-regulation of Leucine-rich Repeats and Immunoglobulin-like Domain Proteins (LRIG1-3) in HP75 Pituitary Adenoma Cell Line

    Institute of Scientific and Technical Information of China (English)

    GUO Dongsheng; HAN Lin; SHU Kai; CHEN Jian; LEI Ting

    2007-01-01

    Three human leucine-rich repeats and immunoglobulin-like domains (LRIG) genes and proteins, named LRIG1-3, has been previously characterized and it was proposed that they may act as suppressors of tumor growth. The LRIG1 protein can inhibit the growth of tumors of glial cells and the down-regulation of the LRIG1 gene may be involved in the development and progression of the tumor. Real-time reverse transcription-polymerase chain reaction (RT-PCR) is a recently developed technique for quantitative assessment of specific RNA levels. In the current study, it was demonstrated that LRIG1-3 and EGFR mRNA was detected in human pituitary adenoma cell lines and a normal pituitary sample, with differences in the expression levels. Compared to the normal pituitary samples, the expression of LRIG1-3 in HP75 cell line was lower, but the expression of EGFR in HP75 cell line was higher. The results are consistent with LRIG1-3 being tumour suppressor genes, and LRIG genes decreasing the expression of EGFR. The ratio of EGFR/LRIG1 was increased at least 13-fold in HP75 cells compared with the normal pituitary cells, which was also the case for the ratio of EGFR/LRIG2 (14-fold increase in HP75) and EGFR/LRIG3 (11-fold increase in HP75). Further studies were needed to elucidate the explicit role of LRIG genes as negative regulators of oncogenesis in human pituitary adenoma.

  12. Identification and characterization of a tandem repeat in exon III of the dopamine receptor D4 (DRD4) gene in cetaceans

    DEFF Research Database (Denmark)

    Mogensen, Line; Kinze, Carl Christian; Werge, Thomas

    2006-01-01

    in exon III of their DRD4 gene. Consequently, the 18-bp tandem repeat appears to have originated prior to the differentiation of hoofed mammals into odd-toed and even-toed ungulates. The composition of the tandem repeat in cetaceans differed markedly from that in primates, which is composed of 48-bp...

  13. Endothelial Nitric Oxide Synthase Gene Intron 4, 27 bp Repeat Polymorphism and Essential Hypertension in the Kazakh Chinese Population

    Institute of Scientific and Technical Information of China (English)

    Fengmei DENG; Huimin ZHANG; Juan ZHAO; Hua ZHONG; Ling HE; Jun LI; Le ZHANG; Shuren WANG; Qinghua HU; Bin TANG; Fang HE; Shuxia GUO; Jiang CHEN; Feng LI; Xuehua WU; Jun ZHANG

    2007-01-01

    To investigate the relationship between 27 bp repeat polymorphism in intron 4 in the endothelial nitric oxide synthase (eNOS4) gene and essential hypertension in the Kazakh Chinese population, 151 patients with essential hypertension and 138 healthy people were selected from the Boertonggu countryside of Shawan region in the Xinjiang Uygur Autonomous Region of China in 2006. The polymorphism of eNOS in the two groups was detected with polymerase chain reaction assays and the genotype frequencies in each group were calculated following the Hardy-Weinberg law. Four and five tandem 27 bp repeats were designated as "a" and "b", respectively. It was found that the frequencies of b/b, b/a and a/a genotypes of the eNOS4 gene were 84.06%, 15.22% and 0.72% in the control group, and 81.46%, 15.89% and 2.65% in the hypertension group, respectively. The frequencies of gene "b" and "a" were 91.67% and 8.33% in the control group and 89.40% and 10.60% in the hypertension group, respectively. It was found that plasma eNOS activity was not associated with genotypes and alleles of eNOS gene. Plasma eNOS activity in the hypertension group was significantly decreased compared with the control group (P<0.01). The results suggest that eNOS4 gene polymorphisms are unlikely to be the major genetic susceptibility factors for essential hypertension in the Xinjiang Kazakh population. However, a positive association between plasma eNOS activity and essential hypertension has been revealed.

  14. The repeat domain of the type III effector protein PthA shows a TPR-like structure and undergoes conformational changes upon DNA interaction.

    Science.gov (United States)

    Murakami, Mário Tyago; Sforça, Mauricio Luis; Neves, Jorge Luiz; Paiva, Joice Helena; Domingues, Mariane Noronha; Pereira, André Luiz Araujo; Zeri, Ana Carolina de Mattos; Benedetti, Celso Eduardo

    2010-12-01

    Many plant pathogenic bacteria rely on effector proteins to suppress defense and manipulate host cell mechanisms to cause disease. The effector protein PthA modulates the host transcriptome to promote citrus canker. PthA possesses unusual protein architecture with an internal region encompassing variable numbers of near-identical tandem repeats of 34 amino acids termed the repeat domain. This domain mediates protein-protein and protein-DNA interactions, and two polymorphic residues in each repeat unit determine DNA specificity. To gain insights into how the repeat domain promotes protein-protein and protein-DNA contacts, we have solved the structure of a peptide corresponding to 1.5 units of the PthA repeat domain by nuclear magnetic resonance (NMR) and carried out small-angle X-ray scattering (SAXS) and spectroscopic studies on the entire 15.5-repeat domain of PthA2 (RD2). Consistent with secondary structure predictions and circular dichroism data, the NMR structure of the 1.5-repeat peptide reveals three α-helices connected by two turns that fold into a tetratricopeptide repeat (TPR)-like domain. The NMR structure corroborates the theoretical TPR superhelix predicted for RD2, which is also in agreement with the elongated shape of RD2 determined by SAXS. Furthermore, RD2 undergoes conformational changes in a pH-dependent manner and upon DNA interaction, and shows sequence similarities to pentatricopeptide repeat (PPR), a nucleic acid-binding motif structurally related to TPR. The results point to a model in which the RD2 structure changes its compactness as it embraces the DNA with the polymorphic diresidues facing the interior of the superhelix oriented toward the nucleotide bases.

  15. Mosaic tetracycline resistance genes encoding ribosomal protection proteins.

    Science.gov (United States)

    Warburton, Philip J; Amodeo, Nina; Roberts, Adam P

    2016-12-01

    First reported in 2003, mosaic tetracycline resistance genes are a subgroup of the genes encoding ribosomal protection proteins (RPPs). They are formed when two or more RPP-encoding genes recombine resulting in a functional chimera. To date, the majority of mosaic genes are derived from sections of three RPP genes, tet(O), tet(W) and tet(32), with others comprising tet(M) and tet(S). In this first review of mosaic genes, we report on their structure, diversity and prevalence, and suggest that these genes may be responsible for an under-reported contribution to tetracycline resistance in bacteria.

  16. An Ehrlichia chaffeensis tandem repeat protein interacts with multiple host targets involved in cell signaling, transcriptional regulation, and vesicle trafficking.

    Science.gov (United States)

    Wakeel, Abdul; Kuriakose, Jeeba A; McBride, Jere W

    2009-05-01

    Ehrlichia chaffeensis is an obligately intracellular bacterium that exhibits tropism for mononuclear phagocytes forming cytoplasmic membrane-bound microcolonies called morulae. To survive and replicate within phagocytes, E. chaffeensis exploits the host cell by modulating a number of host cell processes, but the ehrlichial effector proteins involved are unknown. In this study, we determined that p47, a secreted, differentially expressed, tandem repeat (TR) protein, interacts with multiple host proteins associated with cell signaling, transcriptional regulation, and vesicle trafficking. Yeast two-hybrid analysis revealed that p47 interacts with polycomb group ring finger 5 (PCGF5) protein, Src protein tyrosine kinase FYN (FYN), protein tyrosine phosphatase non-receptor type 2 (PTPN2), and adenylate cyclase-associated protein 1 (CAP1). p47 interaction with these proteins was further confirmed by coimmunoprecipitation assays and colocalization in HeLa cells transfected with p47-green fluorescent fusion protein (AcGFP1-p47). Moreover, confocal microscopy demonstrated p47-expressing dense-cored (DC) ehrlichiae colocalized with PCGF5, FYN, PTPN2, and CAP1. An amino-terminally truncated form of p47 containing TRs interacted only with PCGF5 and not with FYN, PTPN2, and CAP1, indicating differences in p47 domains that are involved in these interactions. These results demonstrate that p47 is involved in a complex network of interactions involving numerous host cell proteins. Furthermore, this study provides a new insight into the molecular and functional distinction of DC ehrlichiae, as well as the effector proteins involved in facilitating ehrlichial survival in mononuclear phagocytes.

  17. Effects of concentric and repeated eccentric exercise on muscle damage and calpain-calpastatin gene expression in human skeletal muscle

    DEFF Research Database (Denmark)

    Vissing, K.; Overgaard, K.; Nedergaard, A.

    2008-01-01

    The purpose of this study was to compare the responsiveness of changes in Ca2+-content and calpain-calpastatin gene expression to concentric and eccentric single-bout and repeated exercise. An exercise group (n = 14) performed two bouts of bench-stepping exercise with 8 weeks between exercise bouts...... for muscle Ca2+-content and mRNA levels for calpain isoforms and calpastatin. Exercise reduced muscle strength and increased muscle soreness predominantly in the eccentric leg (P ... eccentric exercise bout (P eccentric exercise 24 h post-exercise (P

  18. A proline/arginine-rich end leucine-rich repeat protein (PRELP variant is uniquely expressed in chronic lymphocytic leukemia cells.

    Directory of Open Access Journals (Sweden)

    Eva Mikaelsson

    Full Text Available Proline/arginine-rich end leucine-rich repeat protein (PRELP belongs to the small leucine-rich proteoglycan (SLRP family, normally expressed in extracellular matrix of collagen-rich tissues. We have previously reported on another SLRP, fibromodulin (FMOD in patients with chronic lymphocytic leukemia (CLL. PRELP is structurally similar to FMOD with adjacent localization on chromosome 1 (1q32.1. As cluster-upregulation of genes may occur in malignancies, the aim of our study was to analyze PRELP expression in CLL. PRELP was expressed (RT-PCR in all CLL patients (30/30, as well as in some patients with mantle cell lymphoma (3/5, but not in healthy donor leukocytes (0/20 or tumor samples from other hematological malignancies (0/35. PRELP was also detected in CLL cell-lines (4/4 but not in cell-lines from other hematological tumors (0/9. PRELP protein was detected in all CLL samples but not in normal leukocytes. Deglycosylation experiments revealed a CLL-unique 38 kDa core protein, with an intact signal peptide. This 38 kDa protein was, in contrast to the normal 55 kDa size, not detected in serum which, in combination with the uncleaved signal peptide, suggests cellular retention. The unique expression of a 38 kDa PRELP in CLL cells may suggest involvement in the pathobiology of CLL and merits further studies.

  19. CSL encodes a leucine-rich-repeat protein implicated in red/violet light signaling to the circadian clock in Chlamydomonas.

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    Ayumi Kinoshita

    2017-03-01

    Full Text Available The green alga Chlamydomonas reinhardtii shows various light responses in behavior and physiology. One such photoresponse is the circadian clock, which can be reset by external light signals to entrain its oscillation to daily environmental cycles. In a previous report, we suggested that a light-induced degradation of the clock protein ROC15 is a trigger to reset the circadian clock in Chlamydomonas. However, light signaling pathways of this process remained unclear. Here, we screened for mutants that show abnormal ROC15 diurnal rhythms, including the light-induced protein degradation at dawn, using a luciferase fusion reporter. In one mutant, ROC15 degradation and phase resetting of the circadian clock by light were impaired. Interestingly, the impairments were observed in response to red and violet light, but not to blue light. We revealed that an uncharacterized gene encoding a protein similar to RAS-signaling-related leucine-rich repeat (LRR proteins is responsible for the mutant phenotypes. Our results indicate that a previously uncharacterized red/violet light signaling pathway is involved in the phase resetting of circadian clock in Chlamydomonas.

  20. CSL encodes a leucine-rich-repeat protein implicated in red/violet light signaling to the circadian clock in Chlamydomonas

    Science.gov (United States)

    Kinoshita, Ayumi; Niwa, Yoshimi; Onai, Kiyoshi; Fukuzawa, Hideya; Ishiura, Masahiro

    2017-01-01

    The green alga Chlamydomonas reinhardtii shows various light responses in behavior and physiology. One such photoresponse is the circadian clock, which can be reset by external light signals to entrain its oscillation to daily environmental cycles. In a previous report, we suggested that a light-induced degradation of the clock protein ROC15 is a trigger to reset the circadian clock in Chlamydomonas. However, light signaling pathways of this process remained unclear. Here, we screened for mutants that show abnormal ROC15 diurnal rhythms, including the light-induced protein degradation at dawn, using a luciferase fusion reporter. In one mutant, ROC15 degradation and phase resetting of the circadian clock by light were impaired. Interestingly, the impairments were observed in response to red and violet light, but not to blue light. We revealed that an uncharacterized gene encoding a protein similar to RAS-signaling-related leucine-rich repeat (LRR) proteins is responsible for the mutant phenotypes. Our results indicate that a previously uncharacterized red/violet light signaling pathway is involved in the phase resetting of circadian clock in Chlamydomonas. PMID:28333924

  1. Operon Gene Order Is Optimized for Ordered Protein Complex Assembly.

    Science.gov (United States)

    Wells, Jonathan N; Bergendahl, L Therese; Marsh, Joseph A

    2016-02-02

    The assembly of heteromeric protein complexes is an inherently stochastic process in which multiple genes are expressed separately into proteins, which must then somehow find each other within the cell. Here, we considered one of the ways by which prokaryotic organisms have attempted to maximize the efficiency of protein complex assembly: the organization of subunit-encoding genes into operons. Using structure-based assembly predictions, we show that operon gene order has been optimized to match the order in which protein subunits assemble. Exceptions to this are almost entirely highly expressed proteins for which assembly is less stochastic and for which precisely ordered translation offers less benefit. Overall, these results show that ordered protein complex assembly pathways are of significant biological importance and represent a major evolutionary constraint on operon gene organization.

  2. Expression of protein-coding genes embedded in ribosomal DNA

    DEFF Research Database (Denmark)

    Johansen, Steinar D; Haugen, Peik; Nielsen, Henrik

    2007-01-01

    Ribosomal DNA (rDNA) is a specialised chromosomal location that is dedicated to high-level transcription of ribosomal RNA genes. Interestingly, rDNAs are frequently interrupted by parasitic elements, some of which carry protein genes. These are non-LTR retrotransposons and group II introns...... that encode reverse transcriptase-like genes, and group I introns and archaeal introns that encode homing endonuclease genes (HEGs). Although rDNA-embedded protein genes are widespread in nuclei, organelles and bacteria, there is surprisingly little information available on how these genes are expressed....... Exceptions include a handful of HEGs from group I introns. Recent studies have revealed unusual and essential roles of group I and group I-like ribozymes in the endogenous expression of HEGs. Here we discuss general aspects of rDNA-embedded protein genes and focus on HEG expression from group I introns...

  3. Repeated Evolution of Testis-Specific New Genes: The Case of Telomere-Capping Genes in Drosophila

    Directory of Open Access Journals (Sweden)

    Raphaëlle Dubruille

    2012-01-01

    Full Text Available Comparative genome analysis has allowed the identification of various mechanisms involved in gene birth. However, understanding the evolutionary forces driving new gene origination still represents a major challenge. In particular, an intriguing and not yet fully understood trend has emerged from the study of new genes: many of them show a testis-specific expression pattern, which has remained poorly understood. Here we review the case of such a new gene, which involves a telomere-capping gene family in Drosophila. hiphop and its testis-specific paralog K81 are critical for the protection of chromosome ends in somatic cells and male gametes, respectively. Two independent functional studies recently proposed that these genes evolved under a reproductive-subfunctionalization regime. The 2011 release of new Drosophila genome sequences from the melanogaster group of species allowed us to deepen our phylogenetic analysis of the hiphop/K81 family. This work reveals an unsuspected dynamic of gene birth and death within the group, with recurrent duplication events through retroposition mechanisms. Finally, we discuss the plausibility of different evolutionary scenarios that could explain the diversification of this gene family.

  4. Androgen receptor gene CAG and GGN repeat polymorphisms in Chilean men with primary severe spermatogenic failure.

    Science.gov (United States)

    Castro-Nallar, Eduardo; Bacallao, Ketty; Parada-Bustamante, Alexis; Lardone, María C; López, Patricia V; Madariaga, Marcia; Valdevenito, Raúl; Piottante, Antonio; Ebensperger, Mauricio; Castro, Andrea

    2010-01-01

    There is ample documentation supporting the fact that androgens are required for normal spermatogenesis. A minority of infertile men have abnormal testosterone blood levels or mild androgen receptor mutations. We investigated the androgen receptor CAG and GGN repeat lengths in Chilean men with spermatogenic impairment. We studied 117 secretory azoospermic/oligozoospermic men (93 idiopathic and 24 excryptorchidic), without Y-chromosome microdeletions, and 121 controls with normal spermatogenesis (42 obstructive and 79 normozoospermic men). Peripheral blood was drawn to obtain genomic DNA for polymerase chain reaction and automated sequencing of CAG and GGN repeats. Testicular characterization included hormonal studies, physical evaluation, and seminal and biopsy analysis. The CAG and GGN polymorphism distributions were similar among idiopathic men, excryptorchidic men, and controls and among the different types of spermatogenic impairment. However, the proportion of the CAG 21 allele was significantly increased in idiopathic cases compared to controls (P = .012 by Bonferroni test, odds ratio = 2.99, 95% confidence interval, 1.27-7.0) and the CAG 32 allele only was observed in excryptorchidic patients (P CAG 21 allele (P = .024, χ(2) test). On the other hand, in idiopathic cases and controls the most common GGN allele was 23, followed by 24, but an inverse relation was found among excryptorchidic cases. The joint distribution of CAG and GGN in control, idiopathic, and excryptorchidic groups did not show an association between the 2 allele repeat polymorphisms (P > 0.05, χ(2) test). Our results suggest that the CAG 21 allele seems to increase the risk of idiopathic Sertoli cell-only syndrome. Moreover, the GGN 24 allele could be contributing to deranged androgen receptor function, associated with cryptorchidism and spermatogenic failure.

  5. Trinucleotide Repeat Expansion in the Transcription Factor 4 (TCF4) Gene Leads to Widespread mRNA Splicing Changes in Fuchs' Endothelial Corneal Dystrophy

    Science.gov (United States)

    Wieben, Eric D.; Aleff, Ross A.; Tang, Xiaojia; Butz, Malinda L.; Kalari, Krishna R.; Highsmith, Edward W.; Jen, Jin; Vasmatzis, George; Patel, Sanjay V.; Maguire, Leo J.; Baratz, Keith H.; Fautsch, Michael P.

    2017-01-01

    Purpose To identify RNA missplicing events in human corneal endothelial tissue isolated from Fuchs' endothelial corneal dystrophy (FECD). Methods Total RNA was isolated and sequenced from corneal endothelial tissue obtained during keratoplasty from 12 patients with FECD and 4 patients undergoing keratoplasty or enucleation for other indications. The length of the trinucleotide repeat (TNR) CTG in the transcription factor 4 (TCF4) gene was determined using leukocyte-derived DNA analyzed by a combination of Southern blotting and Genescan analysis. Commercial statistical software was used to quantify expression of alternatively spliced genes. Validation of selected alternative splicing events was performed by using RT-PCR. Gene sets identified were analyzed for overrepresentation using Web-based analysis system. Results Corneal endothelial tissue from FECD patients containing a CTG TNR expansion sequence in the TCF4 gene revealed widespread changes in mRNA splicing, including a novel splicing event involving FGFR2. Differential splicing of NUMA1, PPFIBP1, MBNL1, and MBNL2 transcripts were identified in all FECD samples containing a TNR expansion. The differentially spliced genes were enriched for products that localize to the cell cortex and bind cytoskeletal and cell adhesion proteins. Conclusions Corneal endothelium from FECD patients harbors a unique signature of mis-splicing events due to CTG TNR expansion in the TCF4 gene, consistent with the hypothesis that RNA toxicity contributes to the pathogenesis of FECD. Changes to the endothelial barrier function, a known event in the development of FECD, was identified as a key biological process influenced by the missplicing events. PMID:28118661

  6. The relationship among gene expression, the evolution of gene dosage, and the rate of protein evolution.

    Directory of Open Access Journals (Sweden)

    Jean-François Gout

    2010-05-01

    Full Text Available The understanding of selective constraints affecting genes is a major issue in biology. It is well established that gene expression level is a major determinant of the rate of protein evolution, but the reasons for this relationship remain highly debated. Here we demonstrate that gene expression is also a major determinant of the evolution of gene dosage: the rate of gene losses after whole genome duplications in the Paramecium lineage is negatively correlated to the level of gene expression, and this relationship is not a byproduct of other factors known to affect the fate of gene duplicates. This indicates that changes in gene dosage are generally more deleterious for highly expressed genes. This rule also holds for other taxa: in yeast, we find a clear relationship between gene expression level and the fitness impact of reduction in gene dosage. To explain these observations, we propose a model based on the fact that the optimal expression level of a gene corresponds to a trade-off between the benefit and cost of its expression. This COSTEX model predicts that selective pressure against mutations changing gene expression level or affecting the encoded protein should on average be stronger in highly expressed genes and hence that both the frequency of gene loss and the rate of protein evolution should correlate negatively with gene expression. Thus, the COSTEX model provides a simple and common explanation for the general relationship observed between the level of gene expression and the different facets of gene evolution.

  7. Gene Repression in Haloarchaea Using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas I-B System*

    Science.gov (United States)

    Stachler, Aris-Edda; Marchfelder, Anita

    2016-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system is used by bacteria and archaea to fend off foreign genetic elements. Since its discovery it has been developed into numerous applications like genome editing and regulation of transcription in eukaryotes and bacteria. For archaea currently no tools for transcriptional repression exist. Because molecular biology analyses in archaea become more and more widespread such a tool is vital for investigating the biological function of essential genes in archaea. Here we use the model archaeon Haloferax volcanii to demonstrate that its endogenous CRISPR-Cas system I-B can be harnessed to repress gene expression in archaea. Deletion of cas3 and cas6b genes results in efficient repression of transcription. crRNAs targeting the promoter region reduced transcript levels down to 8%. crRNAs targeting the reading frame have only slight impact on transcription. crRNAs that target the coding strand repress expression only down to 88%, whereas crRNAs targeting the template strand repress expression down to 8%. Repression of an essential gene results in reduction of transcription levels down to 22%. Targeting efficiencies can be enhanced by expressing a catalytically inactive Cas3 mutant. Genes can be targeted on plasmids or on the chromosome, they can be monocistronic or part of a polycistronic operon. PMID:27226589

  8. Gene Repression in Haloarchaea Using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas I-B System.

    Science.gov (United States)

    Stachler, Aris-Edda; Marchfelder, Anita

    2016-07-15

    The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system is used by bacteria and archaea to fend off foreign genetic elements. Since its discovery it has been developed into numerous applications like genome editing and regulation of transcription in eukaryotes and bacteria. For archaea currently no tools for transcriptional repression exist. Because molecular biology analyses in archaea become more and more widespread such a tool is vital for investigating the biological function of essential genes in archaea. Here we use the model archaeon Haloferax volcanii to demonstrate that its endogenous CRISPR-Cas system I-B can be harnessed to repress gene expression in archaea. Deletion of cas3 and cas6b genes results in efficient repression of transcription. crRNAs targeting the promoter region reduced transcript levels down to 8%. crRNAs targeting the reading frame have only slight impact on transcription. crRNAs that target the coding strand repress expression only down to 88%, whereas crRNAs targeting the template strand repress expression down to 8%. Repression of an essential gene results in reduction of transcription levels down to 22%. Targeting efficiencies can be enhanced by expressing a catalytically inactive Cas3 mutant. Genes can be targeted on plasmids or on the chromosome, they can be monocistronic or part of a polycistronic operon. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Extended gene diversity at the FMR1 locus and neighbouring CA repeats in a sub-Saharan population

    Energy Technology Data Exchange (ETDEWEB)

    Chiurazzi, Genuardi, M.; Neri, G. [Instituto di Genetica Medica, Roma (Italy)] [and others

    1996-07-12

    We report on the allele distributions in a normal black African population at two microsatellite loci neighbouring the FRAXA locus and at the CGG repeat in the 5{prime} end of the FMR1 gene, which causes the fragile X syndrome. The CGG repeat distribution was found to be similar to that of other ethnic groups, as well as to that of other non-human primates, possibly predicting a comparable prevalence of fragile X in Africa. Significant linkage disequilibrium has been observed between fragile X mutations and alleles of the DXS548 and FRAXAC1 loci in European and Asian populations, and some founder chromosomes may be extremely old. Those associated with FRAXAC1-A and DXS548-2 alleles are not present in the Asian fragile X samples. We searched for these alleles and their frequency in the well defined Bamileke population of Cameroon. All previously described alleles and some new ones were found in this sample, supporting the hypothesis of their pre-existence and subsequent loss in Asian populations. Finally, the heterozygosity of the Bamileke sample was significantly higher at both marker loci and comparable to that of Europeans at the CGG repeat, confirming the notion that genetic diversity is greater in Africans than in other groups and supporting the view that evolution of modern man started in Africa. 31 refs., 1 fig., 1 tab.

  10. Gene composer: database software for protein construct design, codon engineering, and gene synthesis.

    Science.gov (United States)

    Lorimer, Don; Raymond, Amy; Walchli, John; Mixon, Mark; Barrow, Adrienne; Wallace, Ellen; Grice, Rena; Burgin, Alex; Stewart, Lance

    2009-04-21

    To improve efficiency in high throughput protein structure determination, we have developed a database software package, Gene Composer, which facilitates the information-rich design of protein constructs and their codon engineered synthetic gene sequences. With its modular workflow design and numerous graphical user interfaces, Gene Composer enables researchers to perform all common bio-informatics steps used in modern structure guided protein engineering and synthetic gene engineering. An interactive Alignment Viewer allows the researcher to simultaneously visualize sequence conservation in the context of known protein secondary structure, ligand contacts, water contacts, crystal contacts, B-factors, solvent accessible area, residue property type and several other useful property views. The Construct Design Module enables the facile design of novel protein constructs with altered N- and C-termini, internal insertions or deletions, point mutations, and desired affinity tags. The modifications can be combined and permuted into multiple protein constructs, and then virtually cloned in silico into defined expression vectors. The Gene Design Module uses a protein-to-gene algorithm that automates the back-translation of a protein amino acid sequence into a codon engineered nucleic acid gene sequence according to a selected codon usage table with minimal codon usage threshold, defined G:C% content, and desired sequence features achieved through synonymous codon selection that is optimized for the intended expression system. The gene-to-oligo algorithm of the Gene Design Module plans out all of the required overlapping oligonucleotides and mutagenic primers needed to synthesize the desired gene constructs by PCR, and for physically cloning them into selected vectors by the most popular subcloning strategies. We present a complete description of Gene Composer functionality, and an efficient PCR-based synthetic gene assembly procedure with mis-match specific endonuclease

  11. Gene Composer: database software for protein construct design, codon engineering, and gene synthesis

    Directory of Open Access Journals (Sweden)

    Mixon Mark

    2009-04-01

    Full Text Available Abstract Background To improve efficiency in high throughput protein structure determination, we have developed a database software package, Gene Composer, which facilitates the information-rich design of protein constructs and their codon engineered synthetic gene sequences. With its modular workflow design and numerous graphical user interfaces, Gene Composer enables researchers to perform all common bio-informatics steps used in modern structure guided protein engineering and synthetic gene engineering. Results An interactive Alignment Viewer allows the researcher to simultaneously visualize sequence conservation in the context of known protein secondary structure, ligand contacts, water contacts, crystal contacts, B-factors, solvent accessible area, residue property type and several other useful property views. The Construct Design Module enables the facile design of novel protein constructs with altered N- and C-termini, internal insertions or deletions, point mutations, and desired affinity tags. The modifications can be combined and permuted into multiple protein constructs, and then virtually cloned in silico into defined expression vectors. The Gene Design Module uses a protein-to-gene algorithm that automates the back-translation of a protein amino acid sequence into a codon engineered nucleic acid gene sequence according to a selected codon usage table with minimal codon usage threshold, defined G:C% content, and desired sequence features achieved through synonymous codon selection that is optimized for the intended expression system. The gene-to-oligo algorithm of the Gene Design Module plans out all of the required overlapping oligonucleotides and mutagenic primers needed to synthesize the desired gene constructs by PCR, and for physically cloning them into selected vectors by the most popular subcloning strategies. Conclusion We present a complete description of Gene Composer functionality, and an efficient PCR-based synthetic gene

  12. Identification and characterization of multiple Spidroin 1 genes encoding major ampullate silk proteins in Nephila clavipes.

    Science.gov (United States)

    Gaines, W A; Marcotte, W R

    2008-09-01

    Spider dragline silk is primarily composed of proteins called major ampullate spidroins (MaSps) that consist of a large repeat array flanked by nonrepetitive N- and C-terminal domains. Until recently, there has been little evidence for more than one gene encoding each of the two major spidroin silk proteins, MaSp1 and MaSp2. Here, we report the deduced N-terminal domain sequences for two distinct MaSp1 genes from Nephila clavipes (MaSp1A and MaSp1B) and for MaSp2. All three MaSp genes are co-expressed in the major ampullate gland. A search of the GenBank database also revealed two distinct MaSp1 C-terminal domain sequences. Sequencing confirmed that both MaSp1 genes are present in all seven Nephila clavipes spiders examined. The presence of nucleotide polymorphisms in these genes confirmed that MaSp1A and MaSp1B are distinct genetic loci and not merely alleles of the same gene. We experimentally determined the transcription start sites for all three MaSp genes and established preliminary pairing between the two MaSp1 N- and C-terminal domains. Phylogenetic analysis of these new sequences and other published MaSp N- and C-terminal domain sequences illustrated that duplications of MaSp genes may be widespread among spider species.

  13. Survey of clustered regularly interspaced short palindromic repeats and their associated Cas proteins (CRISPR/Cas) systems in multiple sequenced strains of Klebsiella pneumoniae.

    Science.gov (United States)

    Ostria-Hernández, Martha Lorena; Sánchez-Vallejo, Carlos Javier; Ibarra, J Antonio; Castro-Escarpulli, Graciela

    2015-08-04

    In recent years the emergence of multidrug resistant Klebsiella pneumoniae strains has been an increasingly common event. This opportunistic species is one of the five main bacterial pathogens that cause hospital infections worldwide and multidrug resistance has been associated with the presence of high molecular weight plasmids. Plasmids are generally acquired through horizontal transfer and therefore is possible that systems that prevent the entry of foreign genetic material are inactive or absent. One of these systems is CRISPR/Cas. However, little is known regarding the clustered regularly interspaced short palindromic repeats and their associated Cas proteins (CRISPR/Cas) system in K. pneumoniae. The adaptive immune system CRISPR/Cas has been shown to limit the entry of foreign genetic elements into bacterial organisms and in some bacteria it has been shown to be involved in regulation of virulence genes. Thus in this work we used bioinformatics tools to determine the presence or absence of CRISPR/Cas systems in available K. pneumoniae genomes. The complete CRISPR/Cas system was identified in two out of the eight complete K. pneumoniae genomes sequences and in four out of the 44 available draft genomes sequences. The cas genes in these strains comprises eight cas genes similar to those found in Escherichia coli, suggesting they belong to the type I-E group, although their arrangement is slightly different. As for the CRISPR sequences, the average lengths of the direct repeats and spacers were 29 and 33 bp, respectively. BLAST searches demonstrated that 38 of the 116 spacer sequences (33%) are significantly similar to either plasmid, phage or genome sequences, while the remaining 78 sequences (67%) showed no significant similarity to other sequences. The region where the CRISPR/Cas systems were located is the same in all the Klebsiella genomes containing it, it has a syntenic architecture, and is located among genes encoding for proteins likely involved in

  14. Intergenerational Instability of the CAG Repeat of the Gene for Machado-Joseph Disease (MJD1) is Affected by the Genotype of the Normal Chromosome

    OpenAIRE

    五十嵐, 修一; Igarashi, Shuichi

    1997-01-01

    Machado-Joseph disease (MJD) is an autosomal dominant neurodegenerative disorder caused by unstable expansion of a CAG repeat in the MJD1 gene at 14q32.1. To identify elements affecting the intergenerational instability of the CAG repeat, we investigated whether the CGG/GGG polymorphism at the 3' end of the CAG repeat affects the intergenerational instability of the CAG repeat. The [expanded (CAG) n-CGG]/[normal (CAG) n-GGG] haplotypes were found to result in significantly greater instability...

  15. A systematic evaluation of short tandem repeats in lipid candidate genes: riding on the SNP-wave.

    Directory of Open Access Journals (Sweden)

    Claudia Lamina

    Full Text Available Structural genetic variants as short tandem repeats (STRs are not targeted in SNP-based association studies and thus, their possible association signals are missed. We systematically searched for STRs in gene regions known to contribute to total cholesterol, HDL cholesterol, LDL cholesterol and triglyceride levels in two independent studies (KORA F4, n = 2553 and SAPHIR, n = 1648, resulting in 16 STRs that were finally evaluated. In a combined dataset of both studies, the sum of STR alleles was regressed on each phenotype, adjusted for age and sex. The association analyses were repeated for SNPs in a 200 kb region surrounding the respective STRs in the KORA F4 Study. Three STRs were significantly associated with total cholesterol (within LDLR, the APOA1/C3/A4/A5/BUD13 gene region and ABCG5/8, five with HDL cholesterol (3 within CETP, one in LPL and one inAPOA1/C3/A4/A5/BUD13, three with LDL cholesterol (LDLR, ABCG5/8 and CETP and two with triglycerides (APOA1/C3/A4/A5/BUD13 and LPL. None of the investigated STRs, however, showed a significant association after adjusting for the lead or adjacent SNPs within that gene region. The evaluated STRs were found to be well tagged by the lead SNP within the respective gene regions. Therefore, the STRs reflect the association signals based on surrounding SNPs. In conclusion, none of the STRs contributed additionally to the SNP-based association signals identified in GWAS on lipid traits.

  16. A Systematic Evaluation of Short Tandem Repeats in Lipid Candidate Genes: Riding on the SNP-Wave

    Science.gov (United States)

    Lamina, Claudia; Haun, Margot; Coassin, Stefan; Kloss-Brandstätter, Anita; Gieger, Christian; Peters, Annette; Grallert, Harald; Strauch, Konstantin; Meitinger, Thomas; Kedenko, Lyudmyla; Paulweber, Bernhard; Kronenberg, Florian

    2014-01-01

    Structural genetic variants as short tandem repeats (STRs) are not targeted in SNP-based association studies and thus, their possible association signals are missed. We systematically searched for STRs in gene regions known to contribute to total cholesterol, HDL cholesterol, LDL cholesterol and triglyceride levels in two independent studies (KORA F4, n = 2553 and SAPHIR, n = 1648), resulting in 16 STRs that were finally evaluated. In a combined dataset of both studies, the sum of STR alleles was regressed on each phenotype, adjusted for age and sex. The association analyses were repeated for SNPs in a 200 kb region surrounding the respective STRs in the KORA F4 Study. Three STRs were significantly associated with total cholesterol (within LDLR, the APOA1/C3/A4/A5/BUD13 gene region and ABCG5/8), five with HDL cholesterol (3 within CETP, one in LPL and one inAPOA1/C3/A4/A5/BUD13), three with LDL cholesterol (LDLR, ABCG5/8 and CETP) and two with triglycerides (APOA1/C3/A4/A5/BUD13 and LPL). None of the investigated STRs, however, showed a significant association after adjusting for the lead or adjacent SNPs within that gene region. The evaluated STRs were found to be well tagged by the lead SNP within the respective gene regions. Therefore, the STRs reflect the association signals based on surrounding SNPs. In conclusion, none of the STRs contributed additionally to the SNP-based association signals identified in GWAS on lipid traits. PMID:25050552

  17. Atomic model of human Rcd-1 reveals an armadillo-like-repeat protein with in vitro nucleic acid binding properties.

    Science.gov (United States)

    Garces, Robert G; Gillon, Wanda; Pai, Emil F

    2007-02-01

    Rcd-1, a protein highly conserved across eukaryotes, was initially identified as a factor essential for nitrogen starvation-invoked differentiation in fission yeast, and its Saccharomyces cerevisiae homolog, CAF40, has been identified as part of the CCR4-NOT transcription complex, where it interacts with the NOT1 protein. Mammalian homologs are involved in various cellular differentiation processes including retinoic acid-induced differentiation and hematopoetic cell development. Here, we present the 2.2 A X-ray structure of the highly conserved region of human Rcd-1 and investigate possible functional abilities of this and the full-length protein. The monomer is made up of six armadillo repeats forming a solvent-accessible, positively-charged cleft 21-22 A wide that, in contrast to other armadillo proteins, stays fully exposed in the dimer. Prompted by this finding, we established that Rcd-1 can bind to single- and double-stranded oligonucleotides in vitro with the affinity of G/C/T > A. Mutation of an arginine residue within the cleft strongly reduced or abolished oligonucleotide binding. Rcd-1's ability to bind to nucleic acids, in addition to the previously reported protein-protein interaction with NOT1, suggests a new feature in Rcd-1's role in regulation of overall cellular differentiation processes.

  18. Design and analysis of post-fusion 6-helix bundle of heptad repeat regions from Newcastle disease virus F protein.

    Science.gov (United States)

    Zhu, Jieqing; Li, Pengyun; Wu, Tinghe; Gao, Feng; Ding, Yi; Zhang, Catherine W-H; Rao, Zihe; Gao, George F; Tien, Po

    2003-05-01

    Fusion of paramyxovirus to the cell involves receptor binding of the HN glycoprotein and a number of conformational changes of F glycoprotein. The F protein is expressed as a homotrimer on the virus surface. In the present model, there are at least three conformations of F protein, i.e. native form, pre-hairpin intermediate and the post-fusion state. In the post-fusion state, the two highly conserved heptad repeat (HR) regions of F protein form a stable 6-helix coiled-coil bundle. However, no crystal structure is known for this state for the Newcastle disease virus, although the crystal structure of the F protein native form has been solved recently. Here we deployed an Escherichia coli in vitro expression system to engineer this 6-helix bundle by fusion of either the two HR regions (HR1, linker and HR2) or linking the 6-helix [3 x (HR1, linker and HR2)] together as a single chain. Subsequently, both of them form a stable 6-helix bundle in vitro judging by gel filtration and chemical cross-linking and the proteins show salient features of an alpha-helix structure. Crystals diffracting X-rays have been obtained from both protein preparations and the structure determination is under way. This method could be used for crystallization of the post-fusion state HR structures of other viruses.

  19. Molecular identification and characterization of clustered regularly interspaced short palindromic repeat (CRISPR) gene cluster in Taylorella equigenitalis.

    Science.gov (United States)

    Hara, Yasushi; Hayashi, Kyohei; Nakajima, Takuya; Kagawa, Shizuko; Tazumi, Akihiro; Moore, John E; Matsuda, Motoo

    2013-09-01

    Clustered regularly interspaced short palindromic repeats (CRISPRs), of approximately 10,000 base pairs (bp) in length, were shown to occur in the Japanese Taylorella equigenitalis strain, EQ59. The locus was composed of the putative CRISPRs-associated with 5 (cas5), RAMP csd1, csd2, recB, cas1, a leader region, 13 CRISPR consensus sequence repeats (each 32 bp; 5'-TCAGCCACGTTCGCGTGGCTGTGTGTTTAAAG-3'). These were in turn separated by 12 non repetitive unique spacer regions of similar length. In addition, a leader region, a transposase/IS protein, a leader region, and cas3 were also seen. All seven putative open reading frames carry their ribosome binding sites. Promoter consensus sequences at the -35 and -10 regions and putative intrinsic ρ-independent transcription terminator regions also occurred. A possible long overlap of 170 bp in length occurred between the recB and cas1 loci. Positive reverse transcription PCR signals of cas5, RAMP csd1, csd2-recB/cas1, and cas3 were generated. A putative secondary structure of the CRISPR consensus repeats was constructed. Following this, CRISPR results of the T. equigenitalis EQ59 isolate were subsequently compared with those from the Taylorella asinigenitalis MCE3 isolate.

  20. A repeat sequence domain of the ring-exported protein-1 of Plasmodium falciparum controls export machinery architecture and virulence protein trafficking.

    Science.gov (United States)

    McHugh, Emma; Batinovic, Steven; Hanssen, Eric; McMillan, Paul J; Kenny, Shannon; Griffin, Michael D W; Crawford, Simon; Trenholme, Katharine R; Gardiner, Donald L; Dixon, Matthew W A; Tilley, Leann

    2015-12-01

    The malaria parasite Plasmodium falciparum dramatically remodels its host red blood cell to enhance its own survival, using a secretory membrane system that it establishes outside its own cell. Cisternal organelles, called Maurer's clefts, act as a staging point for the forward trafficking of virulence proteins to the red blood cell (RBC) membrane. The Ring-EXported Protein-1 (REX1) is a Maurer's cleft resident protein. We show that inducible knockdown of REX1 causes stacking of Maurer's cleft cisternae without disrupting the organization of the knob-associated histidine-rich protein at the RBC membrane. Genetic dissection of the REX1 sequence shows that loss of a repeat sequence domain results in the formation of giant Maurer's cleft stacks. The stacked Maurer's clefts are decorated with tether-like structures and retain the ability to dock onto the RBC membrane skeleton. The REX1 mutant parasites show deficient export of the major virulence protein, PfEMP1, to the red blood cell surface and markedly reduced binding to the endothelial cell receptor, CD36. REX1 is predicted to form a largely α-helical structure, with a repetitive charge pattern in the repeat sequence domain, providing potential insights into the role of REX1 in Maurer's cleft sculpting.

  1. Short repeats in the spa gene of Staphylococcus aureus are prone to nonsense mutations: stop codons can be found in strains isolated from patients with generalized infection.

    Science.gov (United States)

    Khrustalev, Vladislav Victorovich; Ghaznavi-Rad, Ehsanollah; Neela, Vasanthakumari; Shamsudin, Mariana-Nor; Amouzandeh-Nobaveh, Alireza; Barkovsky, Eugene Victorovich

    2013-11-01

    Fifteen sequences with stop codons have been obtained in the course of standard methicillin-resistant Staphylococcus aureus (MRSA) spa typing. In nine of those sequences, stop codons occurred due to nonsense G-T and A-T transversions. G-T transversions would appear to be frequent in the spa gene, mostly due to symmetric mutational AT-pressure in the whole S. aureus genome and due to replication-associated mutational pressure characteristic of lagging strands of the "chromosome". A-T transversions would appear to be frequent in the spa gene mostly due to transcription-associated mutational pressure. Relative to other S. aureus genes, short repeats in spa are enriched by nonsense sites for G-T and A-T transversions; the probability of being nonsense for A-T transversion is high in that part of spa coding region. 13 out of 15 (87%) of the sequences with stop codons were obtained from strains isolated from patients with generalized S. aureus infection. Truncation of spa at its C-terminus is predicted to result in a protein that possesses functional IgG binding domains unable to be linked to the cell wall. This is discussed in light of the known fact that extracellular spa is a strong virulence factor involved in immune evasion. Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  2. Double hairpin elements and tandem repeats in the non-coding region of Adenoides eludens chloroplast gene minicircles.

    Science.gov (United States)

    Nelson, Martha J; Green, Beverley R

    2005-09-26

    Dinoflagellate plastid genomes are unique in having a reduced number of genes, most of which are found on unigenic minicircles of 2-3 kb. Although the dinoflagellate Adenoides eludens has larger minicircles of about 5 kb, they still carry only one gene. In addition, digenic circles of about 10 kb were detected and mapped by PCR. The non-coding regions of both unigenic and digenic circles share a number of common features including a pair of conserved cores in opposite orientation, four large families of tandem repeats and a number of double hairpin elements (DHEs). They most closely resemble the non-coding regions of the Symbiodinium psbA minicircles, but are much longer, less conserved and have an even greater variety of DHEs and tandem repeats. The presence of so many recombinogenic elements suggests models for the origin of minicircles from a multigenic ancestral chloroplast genome, and raises the possibility of recombination-directed replication rather than defined replication origins in the minicircles.

  3. Polyalanine repeat expansion mutation of the HOXD13 gene in a Chinese family with unusual clinical manifestations of synpolydactyly.

    Science.gov (United States)

    Gong, Licheng; Wang, Binbin; Wang, Jing; Yu, Haibo; Ma, Xu; Yang, Jun

    2011-01-01

    Synpolydactyly (SPD) is an autosomal dominant limb malformation caused by mutations in the gene HOXD13. We investigated a Chinese family in which three individuals across three generations were affected with distinctive limb malformations. We extracted genomic DNA from the affected and three unaffected individuals from this family as well as 100 unrelated controls, for mutation detection by DNA sequencing. The family was characterized by camptodactyly and symphalangism of fingers two to five, transverse phalanx and osseous fusion of the third metacarpal with the proximal phalanx, as well as the coexistence of mild and more severe bilateral phenotypes. We identified a duplication mutation, c. 186-212dup, in exon 1 of the HOXD13 gene in the affected individuals from this family; it was not present in the unaffected individuals or the 100 unrelated individuals. And we also did not find polymorphism among the controls. This study has expanded the phenotypic spectrum of known HOXD13 polyalanine repeat mutations and provided more information about the polymorphic nature of the polyalanine repeat. In addition, new clinical manifestations have been added to the spectrum of possible synpolydactyly phenotypes.

  4. A human-specific de novo protein-coding gene associated with human brain functions.

    Directory of Open Access Journals (Sweden)

    Chuan-Yun Li

    2010-03-01

    Full Text Available To understand whether any human-specific new genes may be associated with human brain functions, we computationally screened the genetic vulnerable factors identified through Genome-Wide Association Studies and linkage analyses of nicotine addiction and found one human-specific de novo protein-coding gene, FLJ33706 (alternative gene symbol C20orf203. Cross-species analysis revealed interesting evolutionary paths of how this gene had originated from noncoding DNA sequences: insertion of repeat elements especially Alu contributed to the formation of the first coding exon and six standard splice junctions on the branch leading to humans and chimpanzees, and two subsequent substitutions in the human lineage escaped two stop codons and created an open reading frame of 194 amino acids. We experimentally verified FLJ33706's mRNA and protein expression in the brain. Real-Time PCR in multiple tissues demonstrated that FLJ33706 was most abundantly expressed in brain. Human polymorphism data suggested that FLJ33706 encodes a protein under purifying selection. A specifically designed antibody detected its protein expression across human cortex, cerebellum and midbrain. Immunohistochemistry study in normal human brain cortex revealed the localization of FLJ33706 protein in neurons. Elevated expressions of FLJ33706 were detected in Alzheimer's brain samples, suggesting the role of this novel gene in human-specific pathogenesis of Alzheimer's disease. FLJ33706 provided the strongest evidence so far that human-specific de novo genes can have protein-coding potential and differential protein expression, and be involved in human brain functions.

  5. A human-specific de novo protein-coding gene associated with human brain functions.

    Directory of Open Access Journals (Sweden)

    Chuan-Yun Li

    2010-03-01

    Full Text Available To understand whether any human-specific new genes may be associated with human brain functions, we computationally screened the genetic vulnerable factors identified through Genome-Wide Association Studies and linkage analyses of nicotine addiction and found one human-specific de novo protein-coding gene, FLJ33706 (alternative gene symbol C20orf203. Cross-species analysis revealed interesting evolutionary paths of how this gene had originated from noncoding DNA sequences: insertion of repeat elements especially Alu contributed to the formation of the first coding exon and six standard splice junctions on the branch leading to humans and chimpanzees, and two subsequent substitutions in the human lineage escaped two stop codons and created an open reading frame of 194 amino acids. We experimentally verified FLJ33706's mRNA and protein expression in the brain. Real-Time PCR in multiple tissues demonstrated that FLJ33706 was most abundantly expressed in brain. Human polymorphism data suggested that FLJ33706 encodes a protein under purifying selection. A specifically designed antibody detected its protein expression across human cortex, cerebellum and midbrain. Immunohistochemistry study in normal human brain cortex revealed the localization of FLJ33706 protein in neurons. Elevated expressions of FLJ33706 were detected in Alzheimer's brain samples, suggesting the role of this novel gene in human-specific pathogenesis of Alzheimer's disease. FLJ33706 provided the strongest evidence so far that human-specific de novo genes can have protein-coding potential and differential protein expression, and be involved in human brain functions.

  6. RNA quality and gene expression analysis of ovarian tumor tissue undergoing repeated thaw-freezing

    DEFF Research Database (Denmark)

    Jochumsen, Kirsten Marie; Tan, Qihua; Dahlgaard, Jesper

    2007-01-01

    Gene expression profiles evaluated by microarray-based quantization of RNA are used in studies of differential diagnosis and prognosis in cancer. RNA of good quality is mandatory for this evaluation. The RNA most often comes from tumor banks with limited amount of tissue, and the tissue often......, and three thaw-freeze cycles. RNA from each aliquot was extracted on the day of division, and quantity and quality were evaluated. RNA from all three aliquots of four tumor samples underwent microarray analysis on Affymetrix Human Genome U133A 2.0 arrays. Microarray data were evaluated using both...... unsupervised, and supervised multivariate statistical methods, reliability analysis, as well as verification using published gene lists in ovarian cancer studies. RNA quality and quantity did not change during the division procedure and microarray data showed insignificant difference in gene expression. Tumor...

  7. Coevolution of gene expression among interacting proteins

    OpenAIRE

    2004-01-01

    Physically interacting proteins or parts of proteins are expected to evolve in a coordinated manner that preserves proper interactions. Such coevolution at the amino acid-sequence level is well documented and has been used to predict interacting proteins, domains, and amino acids. Interacting proteins are also often precisely coexpressed with one another, presumably to maintain proper stoichiometry among interacting components. Here, we show that the expression levels of physically inter...

  8. Conserved leucines in N-terminal heptad repeat HR1 of envelope fusion protein F of group II nucleopolyhedroviruses are important for correct processing and essential for fusogenicity

    NARCIS (Netherlands)

    Long, G.; Pan, X.; Vlak, J.M.

    2008-01-01

    The heptad repeat (HR), a conserved structural motif of class I viral fusion proteins, is responsible for the formation of a six-helix bundle structure during the envelope fusion process. The insect baculovirus F protein is a newly found budded virus envelope fusion protein which possesses common fe

  9. Is Asp-His-Ser/Thr-Trp tetrad hydrogen-bond network important to WD40-repeat proteins: a statistical and theoretical study.

    Science.gov (United States)

    Wu, Xian-Hui; Zhang, Hui; Wu, Yun-Dong

    2010-04-01

    WD40-repeat proteins are abundant and play important roles in forming protein complexes. The domain usually has seven WD40 repeats, which folds into a seven beta-sheet propeller with each beta-sheet in a four-strand structure. An analysis of 20 available WD40-repeat proteins in Protein Data Bank reveals that each protein has at least one Asp-His-Ser/Thr-Trp (D-H-S/T-W) hydrogen-bonded tetrad, and some proteins have up to six or seven such tetrads. The relative positions of the four residues in the tetrads are also found to be conserved. A sequence alignment analysis of 560 WD40-repeat protein sequences in human reveals very similar features, indicating that such tetrad may be a general feature of WD40-repeat proteins. We carried out density functional theory and found that these tetrads can lead to significant stabilization including hydrogen-bonding cooperativity. The hydrogen bond involving Trp is significant. These results lead us to propose that the tetrads may be critical to the stability and the mechanism of folding of these proteins.

  10. Expression of a new chimeric protein with a highly repeated sequence in tobacco cells.

    Science.gov (United States)

    Saumonneau, Amélie; Rottier, Karine; Conrad, Udo; Popineau, Yves; Guéguen, Jacques; Francin-Allami, Mathilde

    2011-07-01

    In wheat, the high-molecular weight (HMW) glutenin subunits are known to contribute to gluten viscoelasticity, and show some similarities to elastomeric animal proteins as elastin. When combining the sequence of a glutenin with that of elastin is a way to create new chimeric functional proteins, which could be expressed in plants. The sequence of a glutenin subunit was modified by the insertion of several hydrophobic and elastic motifs derived from elastin (elastin-like peptide, ELP) into the hydrophilic repetitive domain of the glutenin subunit to create a triblock protein, the objective being to improve the mechanical (elastomeric) properties of this wheat storage protein. In this study, we investigated an expression model system to analyze the expression and trafficking of the wild-type HMW glutenin subunit (GS(W)) and an HMW glutenin subunit mutated by the insertion of elastin motifs (GS(M)-ELP). For this purpose, a series of constructs was made to express wild-type subunits and subunits mutated by insertion of elastin motifs in fusion with green fluorescent protein (GFP) in tobacco BY-2 cells. Our results showed for the first time the expression of HMW glutenin fused with GFP in tobacco protoplasts. We also expressed and localized the chimeric protein composed of plant glutenin and animal elastin-like peptides (ELP) in BY-2 protoplasts, and demonstrated its presence in protein body-like structures in the endoplasmic reticulum. This work, therefore, provides a basis for heterologous production of the glutenin-ELP triblock protein to characterize its mechanical properties.

  11. Huntington's disease and mitochondrial DNA deletions: event or regular mechanism for mutant huntingtin protein and CAG repeats expansion?!

    Science.gov (United States)

    Banoei, Mohammad Mehdi; Houshmand, Massoud; Panahi, Mehdi Shafa Shariat; Shariati, Parvin; Rostami, Maryam; Manshadi, Masoumeh Dehghan; Majidizadeh, Tayebeh

    2007-11-01

    The mitochondrial DNA (mtDNA) may play an essential role in the pathogenesis of the respiratory chain complex activities in neurodegenerative disorders such as Huntington's disease (HD). Research studies were conducted to determine the possible levels of mitochondrial defect (deletion) in HD patients and consideration of interaction between the expanded Huntingtin gene as a nuclear gene and mitochondria as a cytoplasmic organelle. To determine mtDNA damage, we investigated deletions based in four areas of mitochondrial DNA, in a group of 60 Iranian patients clinically diagnosed with HD and 70 healthy controls. A total of 41 patients out of 60 had CAG expansion (group A). About 19 patients did not show expansion but had the clinical symptoms of HD (group B). MtDNA deletions were classified into four groups according to size; 9 kb, 7.5 kb, 7 kb, and 5 kb. We found one of the four-mtDNA deletions in at least 90% of samples. Multiple deletions have also been observed in 63% of HD patients. None of the normal control (group C) showed mtDNA deletions. The sizes or locations of the deletions did not show a clear correlation with expanded CAG repeat and age in our samples. The study presented evidence that HD patients had higher frequencies of mtDNA deletions in lymphocytes in comparison to the controls. It is thus proposed that CAG repeats instability and mutant Htt are causative factor in mtDNA damage.

  12. Disruption of human papillomavirus 16 E6 gene by clustered regularly interspaced short palindromic repeat/Cas system in human cervical cancer cells

    Directory of Open Access Journals (Sweden)

    Yu L

    2014-12-01

    Full Text Available Lan Yu, Xiaoli Wang, Da Zhu, Wencheng Ding, Liming Wang, Changlin Zhang, Xiaohui Jiang, Hui Shen, Shujie Liao, Ding Ma, Zheng Hu, Hui Wang Cancer Biology Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People's Republic of China Abstract: High-risk human papillomavirus (HPV, especially HPV16, is considered a main causative agent of cervical cancer. Upon HPV infection, the viral oncoprotein E6 disrupts the host tumor-suppressor protein p53, thus promoting malignant transformation of normal cervical cells. Here, we used the newly developed programmable ribonucleic acid-guided clustered regularly interspaced short palindromic repeat (CRISPR/Cas system to disrupt the HPV16 E6 gene. We showed that HPV16 E6 deoxyribonucleic acid was cleaved at specific sites, leading to apoptosis and growth inhibition of HPV16-positive SiHa and CaSki cells, but not HPV-negative C33A or human embryonic kidney 293 cells. We also observed downregulation of the E6 protein and restoration of the p53 protein. These data proved that the HPV16 E6 ribonucleic acid-guided CRISPR/Cas system might be an effective therapeutic agent in treating HPV infection-related cervical malignancy. Keywords: CRISPR/Cas system, E6, p53, SiHa, CaSki, cervical cancer

  13. 6-alkynyl fucose is a bioorthogonal analog for O-fucosylation of epidermal growth factor-like repeats and thrombospondin type-1 repeats by protein O-fucosyltransferases 1 and 2.

    Science.gov (United States)

    Al-Shareffi, Esam; Chaubard, Jean-Luc; Leonhard-Melief, Christina; Wang, Sheng-Kai; Wong, Chi-Huey; Haltiwanger, Robert S

    2013-02-01

    Protein O-fucosyltransferase 1 (Pofut1) and protein O-fucosyltransferase 2 (Pofut2) add O-linked fucose at distinct consensus sequences in properly folded epidermal growth factor (EGF)-like repeats and thrombospondin type-1 (TSR) repeats, respectively. Glycan chain elongation past O-fucose can occur to yield a tetrasaccharide on EGF repeats and a disaccharide on TSRs. Elimination of Pofut1 in mice causes embryonic lethality with Notch-like phenotypes demonstrating that O-fucosylation of Notch is essential for its function. Similarly, elimination of Pofut2 results in an early embryonic lethal phenotype in mice, although the molecular mechanism for the lethality is unknown. The recent development of sugar analogs has revolutionized the study of glycans by providing a convenient method for labeling and tracking glycosylation. In order to study O-fucosylation, we took advantage of the recently developed reporter, 6-alkynyl fucose. Using the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), or "click" reaction, azido-biotin allows tagging and detection of 6AF-modified proteins. Here we examine whether proteins containing EGF repeats or TSRs with O-fucose consensus sequences are specifically modified with 6AF in cell culture. Using mass spectrometry (MS), we demonstrate that 6AF is efficiently incorporated onto the appropriate consensus sequences on EGF repeats and TSRs. Furthermore, the elongation of the O-fucose monosaccharide on EGF repeats and TSRs is not hampered when 6AF is used. These results show that 6AF is efficiently utilized in a truly bioorthogonal manner by Pofut1, Pofut2 and the enzymes that elongate O-fucose, providing evidence that 6AF is a significant new tool in the study of protein O-fucosylation.

  14. Exons I and VII of the gene (Ker10) encoding human keratin 10 undergo structural rearrangements within repeats.

    Science.gov (United States)

    Tkachenko, A V; Buchman, V L; Bliskovsky, V V; Shvets YuP; Kisselev, L L

    1992-07-15

    A genomic fragment containing the K51 gene previously isolated from a rat genomic library by hybridization with the v-mos probe in nonstringent conditions [Chumakov et al., Dokl. Akad. Nauk SSSR 290 (1986) 1252-1254], resembles a human keratin type-I-encoding gene [Shvets et al., Mol. Biol. 24 (1990) 663-677]. This genomic clone, K51, has been used as a probe to search for related human genes. A recombinant clone, HK51, with a 1.5-kb insert, was isolated from a human embryonic skin cDNA library, and its nucleotide (nt) sequence was determined. Analysis has shown that the cloned cDNA encodes human keratin 10 (Ker10). All presently known nt sequences of the human Ker10-encoding gene (Ker10) are not identical. Differences are concentrated in the 5'-end of the first exon and in the middle of the seventh exon within repeats. In spite of structural rearrangements in two of eight exons, the reading frame and position of the stop codon are preserved. The genetic rearrangements cause changes in hydrophobicity profiles of the N and C termini of Ker10. It was also noticed that insertion of one nt leads to the formation of an unusual 3'-end of the transcript.

  15. Analysis of polyglutamine-coding repeats in the TATA-binding protein in different human populations and in patients with schizophrenia an bipolar affective disorder

    Energy Technology Data Exchange (ETDEWEB)

    Rubinsztein, D.C.; Leggo, J. [Addenbrooke`s National Health Service Trust, Cambridge (United Kingdom); Crow, T.J. [Cambridge Univ. (United Kingdom)] [and others

    1996-09-20

    A new class of disease (including Huntington disease, Kennedy disease, and spinocerebellar ataxias types 1 and 3) results from abnormal expansions of CAG trinucleotides in the coding regions of genes. In all of these diseases the CAG repeats are thought to be translated into polyglutamine tracts. There is accumulating evidence arguing for CAG trinucleotide expansions as one of the causative disease mutations in schizophrenia and bipolar affective disorder. We and others believe that the TATA-binding protein (TBP) is an important candidate to investigate in these diseases as it contains a highly polymorphic stretch of glutamine codons, which are close to the threshold length where the polyglutamine tracts start to be associated with disease. Thus, we examined the lengths of this polyglutamine repeat in normal unrelated East Anglians, South African Blacks, sub-Saharan Africans mainly from Nigeria, and Asian Indians. We also examined 43 bipolar affective disorder patients and 65 schizophrenic patients. The range of polyglutamine tract-lengths that we found in humans was from 26-42 codons. No patients with bipolar affective disorder and schizophrenia had abnormal expansions at this locus. 22 refs., 1 tab.

  16. Evidence that C9ORF72 Dipeptide Repeat Proteins Associate with U2 snRNP to Cause Mis-splicing in ALS/FTD Patients

    Directory of Open Access Journals (Sweden)

    Shanye Yin

    2017-06-01

    Full Text Available Hexanucleotide repeat expansion in the C9ORF72 gene results in production of dipeptide repeat (DPR proteins that may disrupt pre-mRNA splicing in amyotrophic lateral sclerosis (ALS and frontotemporal dementia (FTD patients. At present, the mechanisms underlying this mis-splicing are not understood. Here, we show that addition of proline-arginine (PR and glycine-arginine (GR toxic DPR peptides to nuclear extracts blocks spliceosome assembly and splicing, but not other types of RNA processing. Proteomic and biochemical analyses identified the U2 small nuclear ribonucleoprotein particle (snRNP as a major interactor of PR and GR peptides. In addition, U2 snRNP, but not other splicing factors, mislocalizes from the nucleus to the cytoplasm both in C9ORF72 patient induced pluripotent stem cell (iPSC-derived motor neurons and in HeLa cells treated with the toxic peptides. Bioinformatic studies support a specific role for U2-snRNP-dependent mis-splicing in C9ORF72 patient brains. Together, our data indicate that DPR-mediated dysfunction of U2 snRNP could account for as much as ∼44% of the mis-spliced cassette exons in C9ORF72 patient brains.

  17. Evidence that C9ORF72 Dipeptide Repeat Proteins Associate with U2 snRNP to Cause Mis-splicing in ALS/FTD Patients.

    Science.gov (United States)

    Yin, Shanye; Lopez-Gonzalez, Rodrigo; Kunz, Ryan C; Gangopadhyay, Jaya; Borufka, Carl; Gygi, Steven P; Gao, Fen-Biao; Reed, Robin

    2017-06-13

    Hexanucleotide repeat expansion in the C9ORF72 gene results in production of dipeptide repeat (DPR) proteins that may disrupt pre-mRNA splicing in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients. At present, the mechanisms underlying this mis-splicing are not understood. Here, we show that addition of proline-arginine (PR) and glycine-arginine (GR) toxic DPR peptides to nuclear extracts blocks spliceosome assembly and splicing, but not other types of RNA processing. Proteomic and biochemical analyses identified the U2 small nuclear ribonucleoprotein particle (snRNP) as a major interactor of PR and GR peptides. In addition, U2 snRNP, but not other splicing factors, mislocalizes from the nucleus to the cytoplasm both in C9ORF72 patient induced pluripotent stem cell (iPSC)-derived motor neurons and in HeLa cells treated with the toxic peptides. Bioinformatic studies support a specific role for U2-snRNP-dependent mis-splicing in C9ORF72 patient brains. Together, our data indicate that DPR-mediated dysfunction of U2 snRNP could account for as much as ∼44% of the mis-spliced cassette exons in C9ORF72 patient brains. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  18. Matrix Gla protein and osteocalcin: from gene duplication to neofunctionalization.

    Science.gov (United States)

    Cancela, M Leonor; Laizé, Vincent; Conceição, Natércia

    2014-11-01

    Osteocalcin (OC or bone Gla protein, BGP) and matrix Gla protein (MGP) are two members of the growing family of vitamin K-dependent (VKD) proteins. They were the first VKD proteins found not to be involved in coagulation and synthesized outside the liver. Both proteins were isolated from bone although it is now known that only OC is synthesized by bone cells under normal physiological conditions, but since both proteins can bind calcium and hydroxyapatite, they can also accumulate in bone. Both OC and MGP share similar structural features, both in terms of protein domains and gene organization. OC gene is likely to have appeared from MGP through a tandem gene duplication that occurred concomitantly with the appearance of the bony vertebrates. Despite their relatively close relationship and the fact that both can bind calcium and affect mineralization, their functions are not redundant and they also have other unrelated functions. Interestingly, these two proteins appear to have followed quite different evolutionary strategies in order to acquire novel functionalities, with OC following a gene duplication strategy while MGP variability was obtained mostly by the use of multiple promoters and alternative splicing, leading to proteins with additional functional characteristics and alternative gene regulatory pathways. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Identification of genes involved in radioresistance of nasopharyngeal carcinoma by integrating gene ontology and protein-protein interaction networks.

    Science.gov (United States)

    Guo, Ya; Zhu, Xiao-Dong; Qu, Song; Li, Ling; Su, Fang; Li, Ye; Huang, Shi-Ting; Li, Dan-Rong

    2012-01-01

    Radioresistance remains one of the important factors in relapse and metastasis of nasopharyngeal carcinoma. Thus, it is imperative to identify genes involved in radioresistance and explore the underlying biological processes in the development of radioresistance. In this study, we used cDNA microarrays to select differential genes between radioresistant CNE-2R and parental CNE-2 cell lines. One hundred and eighty-three significantly differentially expressed genes (pgenes were upregulated and 45 genes were downregulated in CNE-2R. We further employed publicly available bioinformatics related software, such as GOEAST and STRING to examine the relationship among differentially expressed genes. The results show that these genes were involved in type I interferon-mediated signaling pathway biological processes; the nodes tended to have high connectivity with the EGFR pathway, IFN-related pathways, NF-κB. The node STAT1 has high connectivity with other nodes in the protein-protein interaction (PPI) networks. Finally, the reliability of microarray data was validated for selected genes by semi-quantitative RT-PCR and Western blotting. The results were consistent with the microarray data. Our study suggests that microarrays combined with gene ontology and protein interaction networks have great value in the identification of genes of radioresistance in nasopharyngeal carcinoma; genes involved in several biological processes and protein interaction networks may be relevant to NPC radioresistance; in particular, the verified genes CCL5, STAT1-α, STAT2 and GSTP1 may become potential biomarkers for predicting NPC response to radiotherapy.

  20. Structural model for the interaction of a designed Ankyrin Repeat Protein with the human epidermal growth factor receptor 2.

    Directory of Open Access Journals (Sweden)

    V Chandana Epa

    Full Text Available Designed Ankyrin Repeat Proteins are a class of novel binding proteins that can be selected and evolved to bind to targets with high affinity and specificity. We are interested in the DARPin H10-2-G3, which has been evolved to bind with very high affinity to the human epidermal growth factor receptor 2 (HER2. HER2 is found to be over-expressed in 30% of breast cancers, and is the target for the FDA-approved therapeutic monoclonal antibodies trastuzumab and pertuzumab and small molecule tyrosine kinase inhibitors. Here, we use computational macromolecular docking, coupled with several interface metrics such as shape complementarity, interaction energy, and electrostatic complementarity, to model the structure of the complex between the DARPin H10-2-G3 and HER2. We analyzed the interface between the two proteins and then validated the structural model by showing that selected HER2 point mutations at the putative interface with H10-2-G3 reduce the affinity of binding up to 100-fold without affecting the binding of trastuzumab. Comparisons made with a subsequently solved X-ray crystal structure of the complex yielded a backbone atom root mean square deviation of 0.84-1.14 Ångstroms. The study presented here demonstrates the capability of the computational techniques of structural bioinformatics in generating useful structural models of protein-protein interactions.

  1. Alu Sx repeat-induced homozygous deletion of the StAR gene causes lipoid congenital adrenal hyperplasia.

    Science.gov (United States)

    Eiden-Plach, Antje; Nguyen, Huy-Hoang; Schneider, Ursula; Hartmann, Michaela F; Bernhardt, Rita; Hannemann, Frank; Wudy, Stefan A

    2012-05-01

    Lipoid congenital adrenal hyperplasia (Lipoid CAH) is the most severe form of the autosomal recessive disorder CAH. A general loss of the steroid biosynthetic activity caused by defects in the StAR gene manifests as life-threatening primary adrenal insufficiency. We report a case of Lipoid CAH caused by a so far not described homozygous deletion of the complete StAR gene and provide diagnostic results based on a GC-MS steroid metabolomics and molecular genetic analysis. The patient presented with postnatal hypoglycemia, vomiting, adynamia, increasing pigmentation and hyponatremia. The constellation of urinary steroid metabolites suggested Lipoid CAH and ruled out all other forms of CAH or defects of aldosterone biosynthesis. After treatment with sodium supplementation, hydrocortisone and fludrocortisone the child fully recovered. Molecular genetic analysis demonstrated a homozygous 12.1 kb deletion in the StAR gene locus. The breakpoints of the deletion are embedded into two typical genomic repetitive Alu Sx elements upstream and downstream of the gene leading to the loss of all exons and regulatory elements. We established deletion-specific and intact allele-specific PCR methods and determined the StAR gene status of all available family members over three generations. This analysis revealed that one of the siblings, who died a few weeks after birth, carried the same genetic defect. Since several Alu repeats at the StAR gene locus increase the probability of deletions, patients with typical symptoms of lipoid CAH lacking evidence for the presence of both StAR alleles should be analyzed carefully for this kind of disorder.

  2. De Novo Origin of Human Protein-Coding Genes

    Science.gov (United States)

    Wu, Dong-Dong; Irwin, David M.; Zhang, Ya-Ping

    2011-01-01

    The de novo origin of a new protein-coding gene from non-coding DNA is considered to be a very rare occurrence in genomes. Here we identify 60 new protein-coding genes that originated de novo on the human lineage since divergence from the chimpanzee. The functionality of these genes is supported by both transcriptional and proteomic evidence. RNA–seq data indicate that these genes have their highest expression levels in the cerebral cortex and testes, which might suggest that these genes contribute to phenotypic traits that are unique to humans, such as improved cognitive ability. Our results are inconsistent with the traditional view that the de novo origin of new genes is very rare, thus there should be greater appreciation of the importance of the de novo origination of genes. PMID:22102831

  3. The four-transmembrane protein IP39 of Euglena forms strands by a trimeric unit repeat.

    Science.gov (United States)

    Suzuki, Hiroshi; Ito, Yasuyuki; Yamazaki, Yuji; Mineta, Katsuhiko; Uji, Masami; Abe, Kazuhiro; Tani, Kazutoshi; Fujiyoshi, Yoshinori; Tsukita, Sachiko

    2013-01-01

    Euglenoid flagellates have striped surface structures comprising pellicles, which allow the cell shape to vary from rigid to flexible during the characteristic movement of the flagellates. In Euglena gracilis, the pellicular strip membranes are covered with paracrystalline arrays of a major integral membrane protein, IP39, a putative four-membrane-spanning protein with the conserved sequence motif of the PMP-22/EMP/MP20/Claudin superfamily. Here we report the three-dimensional structure of Euglena IP39 determined by electron crystallography. Two-dimensional crystals of IP39 appear to form a striated pattern of antiparallel double-rows in which trimeric IP39 units are longitudinally polymerised, resulting in continuously extending zigzag-shaped lines. Structural analysis revealed an asymmetric molecular arrangement in the trimer, and suggested that at least four different interactions between neighbouring protomers are involved. A combination of such multiple interactions would be important for linear strand formation of membrane proteins in a lipid bilayer.

  4. Murine protein H is comprised of 20 repeating units, 61 amino acids in length

    DEFF Research Database (Denmark)

    Kristensen, Torsten; Tack, B F

    1986-01-01

    A cDNA library constructed from size-selected (greater than 28 S) poly(A)+ RNA isolated from the livers of C57B10. WR mice was screened by using a 249-base-pair (bp) cDNA fragment encoding 83 amino acid residues of human protein H as a probe. Of 120,000 transformants screened, 30 hybridized......, 448 bp of 3'-untranslated sequence, and a polyadenylylated tail of undetermined length. Murine pre-protein H was deduced to consist of an 18-amino acid signal peptide and 1216 residues of H-protein sequence. Murine H was composed of 20 repetitive units, each about 61 amino acid residues in length...

  5. Conservation of ribosomal protein gene ordering in 16 complete genomes

    Institute of Scientific and Technical Information of China (English)

    王宁; 陈润生; 王永雄

    2000-01-01

    The organization of ribosomal proteins in 16 prokaryotic genomes was studied as an example of comparative genome analyses of gene systems. Hypothetical ribosomal protein-containing operons were constructed. These operons also contained putative genes and other non-ribosomal genes. The correspondences among these genes across different organisms were clarified by sequence homology computations. In this way a cross tabulation of 70 ribosomal proteins genes was constructed. On average, these were organized into 9-14 operons in each genome. There were also 25 non-ribosomal or putative genes in these mainly ribosomal protein operons. Hence the table contains 95 genes in total. It was found that: (i) the conservation of the block of about 20 r-proteins in the L3 and L4 operons across almost the entire eubacteria and ar-chaebacteria is remarkable; (ii) some operons only belong to eubacteria or archaebacte-ria; (iii) although the ribosomal protein operons are highly conserved within domain, there are fine variat

  6. Conservation of ribosomal protein gene ordering in 16 complete genomes

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The organization of ribosomal proteins in 16 prokaryotic genomes was studied as an example of comparative genome analyses of gene systems. Hypothetical ribosomal protein-containing operons were constructed. These operons also contained putative genes and other non-ribosomal genes. The correspondences among these genes across different organisms were clarified by sequence homology computations. In this way a cross tabulation of 70 ribosomal proteins genes was constructed. On average, these were organized into 9-14 operons in each genome. There were also 25 non-ribosomal or putative genes in these mainly ribosomal protein operons. Hence the table contains 95 genes in total. It was found that: (i) the conservation of the block of about 20 r-proteins in the L3 and L4 operons across almost the entire eubacteria and archaebacteria is remarkable; (ii) some operons only belong to eubacteria or archaebacteria; (iii) although the ribosomal protein operons are highly conserved within domain, there are fine variations in some operons across different organisms within each domain, and these variations are informative on the evolutionary relations among the organisms. This method provides a new potential for studying the origin and evolution of old species.

  7. Regulator of complement activation (RCA) locus in chicken: identification of chicken RCA gene cluster and functional RCA proteins.

    Science.gov (United States)

    Oshiumi, Hiroyuki; Shida, Kyoko; Goitsuka, Ryo; Kimura, Yuko; Katoh, Jun; Ohba, Shinya; Tamaki, Yuichiroh; Hattori, Takashi; Yamada, Nozomi; Inoue, Norimitsu; Matsumoto, Misako; Mizuno, Shigeki; Seya, Tsukasa

    2005-08-01

    A 150-kb DNA fragment, which contains the gene of the chicken complement regulatory protein CREM (formerly named Cremp), was isolated from a microchromosome by screening bacterial artificial chromosome library. Within 100 kb of the cloned region, three complete genes encoding short consensus repeats (SCRs, motifs with tandemly arranged 60 aa) were identified by exon-trap method and 3'- or 5'-RACE. A chicken orthologue of the human gene 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2, which exists in close proximity to the regulator of complement activation genes in humans and mice, was located near this chicken SCR gene cluster. Moreover, additional genes encoding SCR proteins appeared to be present in this region. Three distinct transcripts were detected in RNA samples from a variety of chicken organs and cell lines. Two novel genes named complement regulatory secretory protein of chicken (CRES) and complement regulatory GPI-anchored protein of chicken (CREG) besides CREM were identified by cloning corresponding cDNA. Based on the predicted primary structures and properties of the expressed molecules, CRES is a secretory protein, whereas CREG is a GPI-anchored membrane protein. CREG and CREM were protected host cells from chicken complement-mediated cytolysis. Likewise, a membrane-bound form of CRES, which was artificially generated, also protected host cells from chicken complement. Taken together, the chicken possesses an regulator of complement activation locus similar to those of the mammals, and the gene products function as complement regulators.

  8. Identification of oral cancer related candidate genes by integrating protein-protein interactions, gene ontology, pathway analysis and immunohistochemistry.

    Science.gov (United States)

    Kumar, Ravindra; Samal, Sabindra K; Routray, Samapika; Dash, Rupesh; Dixit, Anshuman

    2017-05-30

    In the recent years, bioinformatics methods have been reported with a high degree of success for candidate gene identification. In this milieu, we have used an integrated bioinformatics approach assimilating information from gene ontologies (GO), protein-protein interaction (PPI) and network analysis to predict candidate genes related to oral squamous cell carcinoma (OSCC). A total of 40973 PPIs were considered for 4704 cancer-related genes to construct human cancer gene network (HCGN). The importance of each node was measured in HCGN by ten different centrality measures. We have shown that the top ranking genes are related to a significantly higher number of diseases as compared to other genes in HCGN. A total of 39 candidate oral cancer target genes were predicted by combining top ranked genes and the genes corresponding to significantly enriched oral cancer related GO terms. Initial verification using literature and available experimental data indicated that 29 genes were related with OSCC. A detailed pathway analysis led us to propose a role for the selected candidate genes in the invasion and metastasis in OSCC. We further validated our predictions using immunohistochemistry (IHC) and found that the gene FLNA was upregulated while the genes ARRB1 and HTT were downregulated in the OSCC tissue samples.

  9. An intron in a ribosomal protein gene from Tetrahymena

    DEFF Research Database (Denmark)

    Nielsen, Henrik; Andreasen, Per Hove; Dreisig, Hanne

    1986-01-01

    of hybrid-selected mRNA and authentic ribosomal proteins. The proteins show strong homology to ribosomal protein S12 from Escherichia coli. The coding region of the gene is interrupted by a 979-bp intron 68 bp downstream of the translation start. This is the first intron in a protein encoding gene...... of a ciliate to be described at the nucleotide sequence level. The intron obeys the GT/AG rule for splice junctions of nuclear mRNA introns from higher eukaryotes but lacks the pyrimidine stretch usually found in the immediate vicinity of the 3' splice junction. The structure of the intron and the fact...

  10. Characterization of membrane occupation and recognition nexus repeat containing 3, meiosis expressed gene 1 binding partner, in mouse male germ cells

    Institute of Scientific and Technical Information of China (English)

    Ling Zhang; ZhiBing Zhang; XueJun Shang; HongFei Li; YuQin Shi; Wei Li; Maria E Teves; ZhiQiong Wang; GaoFeng Jiang; ShiZhen Song

    2015-01-01

    Mammalian spermatogenesis is a well‑organized process of cell development and differentiation. Meiosis expressed gene 1 (MEIG1) plays an essential role in the regulation of spermiogenesis. To explore potential mechanisms of MEIG1’s action, a yeast two‑hybrid screen was conducted, and several potential binding partners were identified; one of them was membrane occupation and recognition nexus repeat containing 3 (MORN3). MORN3 mRNA is only abundant in mouse testis. In the testis, Morn3 mRNA is highly expressed in the spermiogenesis stage. Specific anti‑MORN3 polyclonal antibody was generated against N‑terminus of the full‑length MORN3 protein, and MORN3 expression and localization was examined in vitro and in vivo. In transfected Chinese hamster ovary cells, the antibody specifically crossed‑reacted the full‑length MORN3 protein, and immunofluorescence staining revealed that MORN3 was localized throughout the cytoplasm. Among multiple mouse tissues, about 25 kDa protein, was identified only in the testis. The protein was highly expressed after day 20 of birth. Immunofluorescence staining on mixed testicular cells isolated from adult wild‑type mice demonstrated that MORN3 was expressed in the acrosome in germ cells throughout spermiogenesis. The protein was also present in the manchette of elongating spermatids. The total MORN3 expression and acrosome localization were not changed in the Meig 1‑deficient mice. However, its expression in manchette was dramatically reduced in the mutant mice. Our studies suggest that MORN3 is another regulator for spermatogenesis, probably together with MEIG1.

  11. Murine protein H is comprised of 20 repeating units, 61 amino acids in length

    DEFF Research Database (Denmark)

    Kristensen, Torsten; Tack, B F

    1986-01-01

    A cDNA library constructed from size-selected (greater than 28 S) poly(A)+ RNA isolated from the livers of C57B10. WR mice was screened by using a 249-base-pair (bp) cDNA fragment encoding 83 amino acid residues of human protein H as a probe. Of 120,000 transformants screened, 30 hybridized...... with this cDNA probe. Ten positives were colony-purified, and the largest plasmid cDNA insert, MH8 (4.4 kb), was sequenced by the dideoxy chain termination method. MH8 contained the complete coding sequence for the precursor of murine complement protein factor H (3702 bp), 100 bp of 5'-untranslated sequence......, 448 bp of 3'-untranslated sequence, and a polyadenylylated tail of undetermined length. Murine pre-protein H was deduced to consist of an 18-amino acid signal peptide and 1216 residues of H-protein sequence. Murine H was composed of 20 repetitive units, each about 61 amino acid residues in length...

  12. Variation in CAG and GGN repeat lengths and CAG/GGN haplotype in androgen receptor gene polymorphism and prostate carcinoma in Nigerians.

    Science.gov (United States)

    Akinloye, O; Gromoll, J; Simoni, M

    2011-01-01

    Prostate cancer has become the most common cancer in Nigerian men. The growth of the prostate gland depends on circulating androgens and intracellular steroid signalling pathways. The effects of androgens are mediated through the androgen receptor (AR), a nuclear transcription factor encoded by the AR gene. The common polymorphisms, CAG and GGN repeats, in exon 1 of this gene have been implicated as possible risk factors. Thus far, existing supporting data are scanty and none are from sub-Saharan African populations. Therefore, this study investigates the possible association between AR polymorphism repeat length (CAG and GGN) and prostate cancer in Nigerians. A total of 261 subjects (70 with prostate cancer, 68 with benign prostate hyperplasia [BPH], 123 age-matched apparently normal subjects as controls) were studied. CAG and GGN repeats length were determined by fragment length analysis using GeneScan. The CAG repeat length in prostate cancer and in BPH compared to the controls was significantly different (P CAG repeats showing a significant odds ratio (OR) in both cases. However, this was not observed in GGN repeat length, which showed no significant difference between cases and controls (P > 0.05). CAG and GGN haplotype variation showed no significant difference between cases and controls (P > 0.05), except that the haplotypes CAG > or =21 and GGN CAG repeat length is a risk factor for prostate cancer, and also suggests an association with BPH.

  13. The characterization of cytoplasmic ribosomal protein genes in ...

    African Journals Online (AJOL)

    USER

    2012-04-17

    Apr 17, 2012 ... 2Experimental Teaching Center, Chongqing Medical University, Chongqing 400016, ... ribosomal protein genes of N. bombycis were located in syntenic blocks, .... genome distribution of all RPGs have been displayed and.

  14. Selection for Genes Encoding Secreted Proteins and Receptors

    Science.gov (United States)

    Klein, Robert D.; Gu, Qimin; Goddard, Audrey; Rosenthal, Arnon

    1996-07-01

    Extracellular proteins play an essential role in the formation, differentiation, and maintenance of multicellular organisms. Despite that, the systematic identification of genes encoding these proteins has not been possible. We describe here a highly efficient method to isolate genes encoding secreted and membrane-bound proteins by using a single-step selection in yeast. Application of this method, termed signal peptide selection, to various tissues yielded 559 clones that appear to encode known or novel extracellular proteins. These include members of the transforming growth factor and epidermal growth factor protein families, endocrine hormones, tyrosine kinase receptors, serine/threonine kinase receptors, seven transmembrane receptors, cell adhesion molecules, extracellular matrix proteins, plasma proteins, and ion channels. The eventual identification of most, or all, extracellular signaling molecules will advance our understanding of fundamental biological processes and our ability to intervene in disease states.

  15. The KP4 killer protein gene family

    Science.gov (United States)

    Killer protein 4 (KP4) is a well studied toxin secreted by the maize smut fungus Ustilago maydis that kills sensitive Ustilago strains as well as inhibits Fusarium and plant root growth. This small, cysteine rich protein is encoded by a virus that depends on host survival for replication. KP4 functi...

  16. Epstein-Barr virus immediate-early gene product trans-activates gene expression from the human immunodeficiency virus long terminal repeat

    Energy Technology Data Exchange (ETDEWEB)

    Kenney, S.; Kamine, J.; Markovitz, D.; Fenrick, R.; Pagano, J.

    1988-03-01

    Acquired immunodeficiency syndrome patients are frequently coinfected with Epstein-Barr virus (EBV). In this report, the authors demonstrate that an EBV immediate-early gene product, BamHI MLF1, stimulates expression of the bacterial chloramphenicol acetyltransferase (CAT) gene linked to the human immunodeficiency virus (HIV) promoter. The HIV promoter sequences necessary for trans-activation by EBV do not include the tat-responsive sequences. In addition, in contrast to the other herpesvirus trans-activators previously studied, the EBV BamHI MLF1 gene product appears to function in part by a posttranscriptional mechanism, since it increases pHIV-CAT protein activity more than it increases HIV-CAT mRNA. This ability of an EBV gene product to activate HIV gene expression may have biologic consequences in persons coinfected with both viruses.

  17. Short (GT)n microsatellite repeats in the heme oxygenase-1 gene promoter are associated with antioxidant and anti-inflammatory status in Mexican pediatric patients with sepsis.

    Science.gov (United States)

    Vázquez-Armenta, Gabriela; González-Leal, Natalia; J Vázquez-de la Torre, Mayra; Muñoz-Valle, José Francisco; Ramos-Márquez, Martha E; Hernández-Cañaveral, Iván; Plascencia-Hernández, Arturo; Siller-López, Fernando

    2013-01-01

    An adequate immune and antioxidant response is a key to the resolution of sepsis. Heme oxygenase-1 (HMOX1) is a stress protein with a polymorphic (GT)n repeat in its gene promoter that regulates its expression in response to oxidative injury, such as that present in sepsis. HMOX1 is the rate-limiting enzyme of heme degradation, and the heme breakdown products, CO, Fe, and bilirubin, are considered to be biologically active metabolites with direct or indirect antioxidant and anti-inflammatory properties. In this study, we investigated the inflammatory and antioxidant response and the relationship with the HMOX1 levels and HMOX1 polymorphism in Mexican septic pediatric patients. In a case-control pilot study, we enrolled 64 septic patients and 72 hospitalized control patients without a diagnosis of sepsis. DNA extracted from buffy coat was genotyped for HMOX1 (GT)n polymorphism by PCR and markers of antioxidant and inflammatory status were quantified in plasma by analysis of the oxygen radical absorbance capacity (ORAC), protein carbonyl (PC), interleukin (IL) 6, IL10, and HMOX1 levels. In septic children, oxidative and inflammatory markers were elevated, and HMOX1 levels were positively correlated with IL10 levels. Genotypic and allelic distribution of HMOX1 polymorphism showed no difference between groups. HMOX1 short-allele septic carriers (< 25 GT repeats) presented favorable ORAC, PC and IL10 levels. This study confirms that an active response against pediatric sepsis involves the expression of HMOX1 and IL10, suggesting that the high antioxidant status associated with HMOX1 short-allele septic carriers might provide a beneficial environment for sepsis resolution.

  18. Associating genes and protein complexes with disease via network propagation.

    Directory of Open Access Journals (Sweden)

    Oron Vanunu

    2010-01-01

    Full Text Available A fundamental challenge in human health is the identification of disease-causing genes. Recently, several studies have tackled this challenge via a network-based approach, motivated by the observation that genes causing the same or similar diseases tend to lie close to one another in a network of protein-protein or functional interactions. However, most of these approaches use only local network information in the inference process and are restricted to inferring single gene associations. Here, we provide a global, network-based method for prioritizing disease genes and inferring protein complex associations, which we call PRINCE. The method is based on formulating constraints on the prioritization function that relate to its smoothness over the network and usage of prior information. We exploit this function to predict not only genes but also protein complex associations with a disease of interest. We test our method on gene-disease association data, evaluating both the prioritization achieved and the protein complexes inferred. We show that our method outperforms extant approaches in both tasks. Using data on 1,369 diseases from the OMIM knowledgebase, our method is able (in a cross validation setting to rank the true causal gene first for 34% of the diseases, and infer 139 disease-related complexes that are highly coherent in terms of the function, expression and conservation of their member proteins. Importantly, we apply our method to study three multi-factorial diseases for which some causal genes have been found already: prostate cancer, alzheimer and type 2 diabetes mellitus. PRINCE's predictions for these diseases highly match the known literature, suggesting several novel causal genes and protein complexes for further investigation.

  19. Androgen receptor gene methylation and exon one CAG repeat length in ovarian cancer: differences from breast cancer.

    Science.gov (United States)

    Kassim, Samar; Zoheiry, Nivan M; Hamed, Wael M; Going, James J; Craft, John A

    2004-07-01

    More than one neoplastic founder clone can exist in benign epithelial tumours. Although theories of clonal selection make pluriclonality appear unlikely in carcinomas, published data do not exclude this possibility. This study looked for evidence of multiclonal X inactivation in ovarian carcinoma using AR methylation as a marker. Fifteen unifocal ovarian carcinomas and 14 multifocal carcinomas all in Scottish patients were studied. One representative formalin-fixed paraffin-embedded tumour block was chosen for each of the former and two for the latter. From each of these 43 tumour blocks three samples each of approximately 10(4) carcinoma cells were obtained by microdissection (129 in all). DNA released by proteinase K digestion was subjected to PCR amplification of the androgen receptor gene AR exon I CAG repeat polymorphism with and without prior digestion with methylation-sensitive restriction enzymes HpaII and HhaI. Complex amplification patterns were consistent with mosaic X inactivation in some ovarian carcinomas but acquired anomalies of AR methylation cannot be excluded. Parallel analysis of other X-linked polymorphic loci would strengthen the inference of clonality status from DNA methylation data in tumour X studies. Strikingly, the number of CAG repeats in the 29 ovarian tumour patients (median 16, range 11 - 20) was substantially fewer than in 34 previously studied breast cancer patients from the same scottish population (median 21, range 14 - 26; P CAG repeat were over-represented in the ovarian cancer patients but not in the breast cancer series. These findings reinforce recent suggestions that AR may have a role in ovarian carcinogenesis.

  20. Intermediate CAG repeat expansion in the ATXN2 gene is a unique genetic risk factor for ALS--a systematic review and meta-analysis of observational studies.

    Directory of Open Access Journals (Sweden)

    Ming-Dong Wang

    Full Text Available Amyotrophic lateral sclerosis (ALS is a rare degenerative condition of the motor neurons. Over 10% of ALS cases are linked to monogenic mutations, with the remainder thought to be due to other risk factors, including environmental factors, genetic polymorphisms, and possibly gene-environmental interactions. We examined the association between ALS and an intermediate CAG repeat expansion in the ATXN2 gene using a meta-analytic approach. Observational studies were searched with relevant disease and gene terms from MEDLINE, EMBASE, and PsycINFO from January 2010 through to January 2014. All identified articles were screened using disease terms, gene terms, population information, and CAG repeat information according to PRISMA guidelines. The final list of 17 articles was further evaluated based on the study location, time period, and authors to exclude multiple usage of the same study populations: 13 relevant articles were retained for this study. The range 30-33 CAG repeats in the ATXN2 gene was most strongly associated with ALS. The meta-analysis revealed that the presence of an intermediate CAG repeat (30-33 in the ATXN2 gene was associated with an increased risk of ALS [odds ratio (OR = 4.44, 95%CI: 2.91-6.76] in Caucasian ALS patients. There was no significant difference in the association of this CAG intermediate repeat expansion in the ATXN2 gene between familial ALS cases (OR = 3.59, 1.58-8.17 and sporadic ALS cases (OR = 3.16, 1.88-5.32. These results indicate that the presence of intermediate CAG repeat expansion in the ATXN2 gene is a specific genetic risk factor for ALS, unlike monogenic mutations with an autosomal dominant transmission mode, which cause a more severe phenotype of ALS, with a higher prevalence in familial ALS.

  1. Intermediate CAG repeat expansion in the ATXN2 gene is a unique genetic risk factor for ALS--a systematic review and meta-analysis of observational studies.

    Science.gov (United States)

    Wang, Ming-Dong; Gomes, James; Cashman, Neil R; Little, Julian; Krewski, Daniel

    2014-01-01

    Amyotrophic lateral sclerosis (ALS) is a rare degenerative condition of the motor neurons. Over 10% of ALS cases are linked to monogenic mutations, with the remainder thought to be due to other risk factors, including environmental factors, genetic polymorphisms, and possibly gene-environmental interactions. We examined the association between ALS and an intermediate CAG repeat expansion in the ATXN2 gene using a meta-analytic approach. Observational studies were searched with relevant disease and gene terms from MEDLINE, EMBASE, and PsycINFO from January 2010 through to January 2014. All identified articles were screened using disease terms, gene terms, population information, and CAG repeat information according to PRISMA guidelines. The final list of 17 articles was further evaluated based on the study location, time period, and authors to exclude multiple usage of the same study populations: 13 relevant articles were retained for this study. The range 30-33 CAG repeats in the ATXN2 gene was most strongly associated with ALS. The meta-analysis revealed that the presence of an intermediate CAG repeat (30-33) in the ATXN2 gene was associated with an increased risk of ALS [odds ratio (OR) = 4.44, 95%CI: 2.91-6.76)] in Caucasian ALS patients. There was no significant difference in the association of this CAG intermediate repeat expansion in the ATXN2 gene between familial ALS cases (OR = 3