Molecular cloning and sequence analysis of complementary DNA encoding rat mammary gland medium-chain S-acyl fatty acid synthetase thio ester hydrolase

International Nuclear Information System (INIS)

Safford, R.; de Silva, J.; Lucas, C.

1987-01-01

Poly(A) + RNA from pregnant rat mammary glands was size-fractionated by sucrose gradient centrifugation, and fractions enriched in medium-chain S-acyl fatty acid synthetase thio ester hydrolase (MCH) were identified by in vitro translation and immunoprecipitation. A cDNA library was constructed, in pBR322, from enriched poly(A) + RNA and screened with two oligonucleotide probes deduced from rat MCH amino acid sequence data. Cross-hybridizing clones were isolated and found to contain cDNA inserts ranging from ∼ 1100 to 1550 base pairs (bp). A 1550-bp cDNA insert, from clone 43H09, was confirmed to encode MCH by hybrid-select translation/immunoprecipitation studies and by comparison of the amino acid sequence deduced from the DNA sequence of the clone to the amino acid sequence of the MCH peptides. Northern blot analysis revealed the size of the MCH mRNA to be 1500 nucleotides, and it is therefore concluded that the 1550-bp insert (including G x C tails) of clone 43H09 represents a full- or near-full-length copy of the MCH gene. The rat MCH sequence is the first reported sequence of a thioesterase from a mammalian source, but comparison of the deduced amino acid sequences of MCH and the recently published mallard duck medium-chain S-acyl fatty acid synthetase thioesterase reveals significant homology. In particular, a seven amino acid sequence containing the proposed active serine of the duck thioesterase is found to be perfectly conserved in rat MCH

Isolation and sequence analysis of a cDNA clone encoding the fifth complement component

DEFF Research Database (Denmark)

Lundwall, Åke B; Wetsel, Rick A; Kristensen, Torsten

1985-01-01

DNA clone of 1.85 kilobase pairs was isolated. Hybridization of the mixed-sequence probe to the complementary strand of the plasmid insert and sequence analysis by the dideoxy method predicted the expected protein sequence of C5a (positions 1-12), amino-terminal to the anticipated priming site. The sequence......, subcloned into M13 mp8, and sequenced at random by the dideoxy technique, thereby generating a contiguous sequence of 1703 base pairs. This clone contained coding sequence for the C-terminal 262 amino acid residues of the beta-chain, the entire C5a fragment, and the N-terminal 98 residues of the alpha......'-chain. The 3' end of the clone had a polyadenylated tail preceded by a polyadenylation recognition site, a 3'-untranslated region, and base pairs homologous to the human Alu concensus sequence. Comparison of the derived partial human C5 protein sequence with that previously determined for murine C3 and human...

Chimeric infectious DNA clones, chimeric porcine circoviruses and uses thereof

OpenAIRE

2011-01-01

The present invention relates to infectious DNA clones, infectious chimeric DNA clones of porcine circovirus (PCV), vaccines and means of protecting pigs against viral infection or postweaning multisystemic wasting syndrome (PMWS) caused by PCV2. The new chimeric infectious DNA clone and its derived, avirulent chimeric virus are constructed from the nonpathogenic PCV1 in which the immunogenic ORF gene of the pathogenic PCV2 replaces a gene of the nonpathogenic PCV1, preferably in the same pos...

Cloning of a Gene Whose Expression is Increased in Scrapie and in Senile Plaques in Human Brain

Science.gov (United States)

Wietgrefe, S.; Zupancic, M.; Haase, A.; Chesebro, B.; Race, R.; Frey, W.; Rustan, T.; Friedman, R. L.

1985-12-01

A complementary DNA library was constructed from messenger RNA's extracted from the brains of mice infected with the scrapie agent. The library was differentially screened with the objectives of finding clones that might be used as markers of infection and finding clones of genes whose increased expression might be correlated with the pathological changes common to scrapie and Alzheimer's disease. A gene was identified whose expression is increased in scrapie. The complementary DNA corresponding to this gene hybridized preferentially and focally to cells in the brains of scrapie-infected animals. The cloned DNA also hybridized to the neuritic plaques found with increased frequency in brains of patients with Alzheimer's disease.

Molecular cloning of growth hormone encoding cDNA of Indian

Indian Academy of Sciences (India)

A modified rapid amplification of cDNA ends (RACE) strategy has been developed for cloning highly conserved cDNA sequences. Using this modified method, the growth hormone (GH) encoding cDNA sequences of Labeo rohita, Cirrhina mrigala and Catla catla have been cloned, characterized and overexpressed in ...

MOLECULAR CLONING OF OVINE cDNA LEPTIN GENE

Directory of Open Access Journals (Sweden)

CLAUDIA TEREZIA SOCOL

2008-05-01

Full Text Available An efficient bacterial transformation system suitable for cloning the coding sequence of the ovine leptin gene in E. coli DH5α host cells using the pGEMT easy vector it is described in this paper. The necessity of producing leptin is based on the fact that the role of this molecule in the animal and human organism is still unknown, leptin not existing as commercial product on the Romanian market. The results obtained in the bacterial transformation, cloning, recombinant clones selection, control of the insertion experiments and DNA computational analysis represent the first steps in further genetic engineering experiments such as production of DNA libraries, DNA sequencing, protein expression, etc., for a further contribution in elucidating the role of leptin in the animal and human organism.

Infectious Maize rayado fino virus from cloned cDNA

Science.gov (United States)

Maize rayado fino virus (MRFV) is the type member of the marafiviruses within the family Tymoviridae. A cDNA clone from which infectious RNA can be transcribed was produced from a US isolate of MRFV (MRFV-US). Infectivity of transcripts derived from cDNA clones was demonstrated by infection of mai...

Molecular cloning of lupin leghemoglobin cDNA

DEFF Research Database (Denmark)

Konieczny, A; Jensen, E O; Marcker, K A

1987-01-01

Poly(A)+ RNA isolated from root nodules of yellow lupin (Lupinus luteus, var. Ventus) has been used as a template for the construction of a cDNA library. The ds cDNA was synthesized and inserted into the Hind III site of plasmid pBR 322 using synthetic Hind III linkers. Clones containing sequences...... specific for nodules were selected by differential colony hybridization using 32P-labeled cDNA synthesized either from nodule poly(A)+ RNA or from poly(A)+ RNA of uninfected root as probes. Among the recombinant plasmids, the cDNA gene for leghemoglobin was identified. The protein structure derived from...... its nucleotide sequence was consistent with known amino acid sequence of lupin Lb II. The cloned lupin Lb cDNA hybridized to poly(A)+ RNA from nodules only, which is in accordance with the general concept, that leghemoglobin is expressed exclusively in nodules. Udgivelsesdato: 1987-null...

Cloning human DNA repair genes

International Nuclear Information System (INIS)

Jeggo, P.A.; Carr, A.M.; Lehmann, A.R.

1994-01-01

Many human genes involved in the repair of UV damage have been cloned using different procedures and they have been of great value in assisting the understanding of the mechanism of nucleotide excision-repair. Genes involved in repair of ionizing radiation damage have proved more difficult to isolate. Positional cloning has localized the XRCC5 gene to a small region of chromosome 2q33-35, and a series of yeast artificial chromosomes covering this region have been isolated. Very recent work has shown that the XRCC5 gene encodes the 80 kDa subunit of the Ku DNA-binding protein. The Ku80 gene also maps to this region. Studies with fission yeast have shown that radiation sensitivity can result not only from defective DNA repair but also from abnormal cell cycle control following DNA damage. Several genes involved in this 'check-point' control in fission yeast have been isolated and characterized in detail. It is likely that a similar checkpoint control mechanism exists in human cells. (author)

Cloning of low dose radiation induced gene RIG1 by RACE based on non-cloned cDNA library

International Nuclear Information System (INIS)

Luo Ying; Sui Jianli; Tie Yi; Zhang Yuanping; Zhou Pingkun; Sun Zhixian

2001-01-01

Objective: To obtain full-length cDNA of radiation induced new gene RIG1 based on its EST fragment. Methods: Based on non-cloned cDNA library, enhanced nested RACE PCR and biotin-avidin labelled probe for magnetic bead purification was used to obtain full-length cDNA of RIG1. Results: About 1 kb of 3' end of RIG1 gene was successfully cloned by this set of methods and cloning of RIG1 5' end is proceeding well. Conclusion: The result is consistent with the design of experiment. This set of protocol is useful for cloning of full-length gene based on EST fragment

Cloning of Salmonella typhimurium DNA encoding mutagenic DNA repair

International Nuclear Information System (INIS)

Thomas, S.M.; Sedgwick, S.G.

1989-01-01

Mutagenic DNA repair in Escherichia coli is encoded by the umuDC operon. Salmonella typhimurium DNA which has homology with E. coli umuC and is able to complement E. coli umuC122::Tn5 and umuC36 mutations has been cloned. Complementation of umuD44 mutants and hybridization with E. coli umuD also occurred, but these activities were much weaker than with umuC. Restriction enzyme mapping indicated that the composition of the cloned fragment is different from the E. coli umuDC operon. Therefore, a umu-like function of S. typhimurium has been found; the phenotype of this function is weaker than that of its E. coli counterpart, which is consistent with the weak mutagenic response of S. typhimurium to UV compared with the response in E. coli

Molecular cloning of complementary DNAs encoding the heavy chain of the human 4F2 cell-surface antigen: a type II membrane glycoprotein involved in normal and neoplastic cell growth

International Nuclear Information System (INIS)

Quackenbush, E.; Clabby, M.; Gottesdiener, K.M.; Barbosa, J.; Jones, N.H.; Strominger, J.L.; Speck, S.; Leiden, J.M.

1987-01-01

Complementary DNA (cDNA) clones encoding the heavy chain of the heterodimeric human membrane glycoprotein 4F2 have been isolated by immunoscreening of a λgt11 expression library. The identity of these clones has been confirmed by hybridization to RNA and DNA prepared from mouse L-cell transfectants, which were produced by whole cell gene transfer and selected for cell-surface expression of the human 4F2 heavy chain. DNA sequence analysis suggest that the 4F2 heavy-chain cDNAs encode an approximately 526-amino acid type II membrane glycoprotein, which is composed of a large C-terminal extracellular domain, a single potential transmembrane region, and a 50-81 amino acid N-terminal intracytoplasmic domain. Southern blotting experiments have shown that the 4F2 heavy-chain cDNAs are derived from a single-copy gene that has been highly conserved during mammalian evolution

Cloning, sequencing, and expression of cDNA for human β-glucuronidase

International Nuclear Information System (INIS)

Oshima, A.; Kyle, J.W.; Miller, R.D.

1987-01-01

The authors report here the cDNA sequence for human placental β-glucuronidase (β-D-glucuronoside glucuronosohydrolase, EC 3.2.1.31) and demonstrate expression of the human enzyme in transfected COS cells. They also sequenced a partial cDNA clone from human fibroblasts that contained a 153-base-pair deletion within the coding sequence and found a second type of cDNA clone from placenta that contained the same deletion. Nuclease S1 mapping studies demonstrated two types of mRNAs in human placenta that corresponded to the two types of cDNA clones isolated. The NH 2 -terminal amino acid sequence determined for human spleen β-glucuronidase agreed with that inferred from the DNA sequence of the two placental clones, beginning at amino acid 23, suggesting a cleaved signal sequence of 22 amino acids. When transfected into COS cells, plasmids containing either placental clone expressed an immunoprecipitable protein that contained N-linked oligosaccharides as evidenced by sensitivity to endoglycosidase F. However, only transfection with the clone containing the 153-base-pair segment led to expression of human β-glucuronidase activity. These studies provide the sequence for the full-length cDNA for human β-glucuronidase, demonstrate the existence of two populations of mRNA for β-glucuronidase in human placenta, only one of which specifies a catalytically active enzyme, and illustrate the importance of expression studies in verifying that a cDNA is functionally full-length

Efficient preparation of shuffled DNA libraries through recombination (Gateway) cloning.

Science.gov (United States)

Lehtonen, Soili I; Taskinen, Barbara; Ojala, Elina; Kukkurainen, Sampo; Rahikainen, Rolle; Riihimäki, Tiina A; Laitinen, Olli H; Kulomaa, Markku S; Hytönen, Vesa P

2015-01-01

Efficient and robust subcloning is essential for the construction of high-diversity DNA libraries in the field of directed evolution. We have developed a more efficient method for the subcloning of DNA-shuffled libraries by employing recombination cloning (Gateway). The Gateway cloning procedure was performed directly after the gene reassembly reaction, without additional purification and amplification steps, thus simplifying the conventional DNA shuffling protocols. Recombination-based cloning, directly from the heterologous reassembly reaction, conserved the high quality of the library and reduced the time required for the library construction. The described method is generally compatible for the construction of DNA-shuffled gene libraries. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

An alternative method for cDNA cloning from surrogate eukaryotic cells transfected with the corresponding genomic DNA.

Science.gov (United States)

Hu, Lin-Yong; Cui, Chen-Chen; Song, Yu-Jie; Wang, Xiang-Guo; Jin, Ya-Ping; Wang, Ai-Hua; Zhang, Yong

2012-07-01

cDNA is widely used in gene function elucidation and/or transgenics research but often suitable tissues or cells from which to isolate mRNA for reverse transcription are unavailable. Here, an alternative method for cDNA cloning is described and tested by cloning the cDNA of human LALBA (human alpha-lactalbumin) from genomic DNA. First, genomic DNA containing all of the coding exons was cloned from human peripheral blood and inserted into a eukaryotic expression vector. Next, by delivering the plasmids into either 293T or fibroblast cells, surrogate cells were constructed. Finally, the total RNA was extracted from the surrogate cells and cDNA was obtained by RT-PCR. The human LALBA cDNA that was obtained was compared with the corresponding mRNA published in GenBank. The comparison showed that the two sequences were identical. The novel method for cDNA cloning from surrogate eukaryotic cells described here uses well-established techniques that are feasible and simple to use. We anticipate that this alternative method will have widespread applications.

Mitochondrial DNA heteroplasmy in ovine fetuses and sheep cloned by somatic cell nuclear transfer

Directory of Open Access Journals (Sweden)

Müller Mathias

2007-12-01

Full Text Available Abstract Background The mitochondrial DNA (mtDNA of the cloned sheep "Dolly" and nine other ovine clones produced by somatic cell nuclear transfer (SCNT was reported to consist only of recipient oocyte mtDNA without any detectable mtDNA contribution from the nucleus donor cell. In cattle, mouse and pig several or most of the clones showed transmission of nuclear donor mtDNA resulting in mitochondrial heteroplasmy. To clarify the discrepant transmission pattern of donor mtDNA in sheep clones we analysed the mtDNA composition of seven fetuses and five lambs cloned from fetal fibroblasts. Results The three fetal fibroblast donor cells used for SCNT harboured low mtDNA copy numbers per cell (A: 753 ± 54, B: 292 ± 33 and C: 561 ± 88. The ratio of donor to recipient oocyte mtDNAs was determined using a quantitative amplification refractory mutation system (ARMS PCR (i.e. ARMS-qPCR. For quantification of SNP variants with frequencies below 0.1% we developed a restriction endonuclease-mediated selective quantitative PCR (REMS-qPCR. We report the first cases (n = 4 fetuses, n = 3 lambs of recipient oocyte/nuclear donor mtDNA heteroplasmy in SCNT-derived ovine clones demonstrating that there is no species-effect hindering ovine nucleus-donor mtDNA from being transmitted to the somatic clonal offspring. Most of the heteroplasmic clones exhibited low-level heteroplasmy (0.1% to 0.9%, n = 6 indicating neutral transmission of parental mtDNAs. High-level heteroplasmy (6.8% to 46.5% was observed in one case. This clone possessed a divergent recipient oocyte-derived mtDNA genotype with three rare amino acid changes compared to the donor including one substitution at an evolutionary conserved site. Conclusion Our study using state-of-the-art techniques for mtDNA quantification, like ARMS-qPCR and the novel REMS-qPCR, documents for the first time the transmission of donor mtDNA into somatic sheep clones. MtDNA heteroplasmy was detected in seven of 12 clones

Aberrant DNA methylation in 5'regions of DNA methyltransferase genes in aborted bovine clones

Institute of Scientific and Technical Information of China (English)

2008-01-01

High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning.It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation.DNA methylation is established and maintained by DNA methyltransferases(DNMTs),therefore,it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs.Since DNA methylation can strongly inhibit gene expression,aberrant DNA methylation of DNMT genes may disturb gene expression.But presently,it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos.In our study,we analyzed methylation patterns of the 5' regions of four DNMT genes including Dnmt3a,Dnmt3b,Dnmtl and Dnmt2 in four aborted bovine clones.Using bisulfite sequencing method,we found that 3 out of 4 aborted bovine clones(AF1,AF2 and AF3)showed either hypermethylation or hypomethylation in the 5' regions of Dnmt3a and Dnmt3b.indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed.However,the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized (IVF)fetuses.Besides,we found that tle 5'regions of Dnmtl and Dnmt2 were nearly completely unmethylated in all normal adults.IVF fetuses,sperm and aborted clones.Together,our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5' regions is probably associated with the high abortion of bovine clones.

Cloning and functional expression of a human pancreatic islet glucose-transporter cDNA

International Nuclear Information System (INIS)

Permutt, M.A.; Koranyi, L.; Keller, K.; Lacy, P.E.; Scharp, D.W.; Mueckler, M.

1989-01-01

Previous studies have suggested that pancreatic islet glucose transport is mediated by a high-K m , low-affinity facilitated transporter similar to that expressed in liver. To determine the relationship between islet and liver glucose transporters, liver-type glucose-transporter cDNA clones were isolated from a human liver cDNA library. The liver-type glucose-transporter cDNA clone hybridized to mRNA transcripts of the same size in human liver and pancreatic islet RNA. A cDNA library was prepared from purified human pancreatic islet tissue and screened with human liver-type glucose-transporter cDNA. The authors isolated two overlapping cDNA clones encompassing 2600 base pairs, which encode a pancreatic islet protein identical in sequence to that of the putative liver-type glucose-transporter protein. Xenopus oocytes injected with synthetic mRNA transcribed from a full-length cDNA construct exhibited increased uptake of 2-deoxyglucose, confirming the functional identity of the clone. These cDNA clones can now be used to study regulation of expression of the gene and to assess the role of inherited defects in this gene as a candidate for inherited susceptibility to non-insulin-dependent diabetes mellitus

Brain cDNA clone for human cholinesterase

International Nuclear Information System (INIS)

McTiernan, C.; Adkins, S.; Chatonnet, A.; Vaughan, T.A.; Bartels, C.F.; Kott, M.; Rosenberry, T.L.; La Du, B.N.; Lockridge, O.

1987-01-01

A cDNA library from human basal ganglia was screened with oligonucleotide probes corresponding to portions of the amino acid sequence of human serum cholinesterase. Five overlapping clones, representing 2.4 kilobases, were isolated. The sequenced cDNA contained 207 base pairs of coding sequence 5' to the amino terminus of the mature protein in which there were four ATG translation start sites in the same reading frame as the protein. Only the ATG coding for Met-(-28) lay within a favorable consensus sequence for functional initiators. There were 1722 base pairs of coding sequence corresponding to the protein found circulating in human serum. The amino acid sequence deduced from the cDNA exactly matched the 574 amino acid sequence of human serum cholinesterase, as previously determined by Edman degradation. Therefore, our clones represented cholinesterase rather than acetylcholinesterase. It was concluded that the amino acid sequences of cholinesterase from two different tissues, human brain and human serum, were identical. Hybridization of genomic DNA blots suggested that a single gene, or very few genes coded for cholinesterase

Phenol emulsion-enhanced DNA-driven subtractive cDNA cloning: isolation of low-abundance monkey cortex-specific mRNAs

International Nuclear Information System (INIS)

Travis, G.H.; Sutcliffe, J.G.

1988-01-01

To isolate cDNA clones of low-abundance mRNAs expressed in monkey cerebral cortex but absent from cerebellum, the authors developed an improved subtractive cDNA cloning procedure that requires only modest quantities of mRNA. Plasmid DNA from a monkey cerebellum cDNA library was hybridized in large excess to radiolabeled monkey cortex cDNA in a phenol emulsion-enhanced reaction. The unhybridized cortex cDNA was isolated by chromatography on hydroxyapatite and used to probe colonies from a monkey cortex cDNA library. Of 60,000 colonies screened, 163 clones were isolated and confirmed by colony hybridization or RNA blotting to represent mRNAs, ranging from 0.001% to 0.1% abundance, specific to or highly enriched in cerebral cortex relative to cerebellum. Clones of one medium-abundance mRNA were recovered almost quantitatively. Two of the lower-abundance mRNAs were expressed at levels reduced by a factor of 10 in Alzheimer disease relative to normal human cortex. One of these was identified as the monkey preprosomatostatin I mRNA

Sequence of a cloned cDNA encoding human ribosomal protein S11

Energy Technology Data Exchange (ETDEWEB)

Lott, J B; Mackie, G A

1988-02-11

The authors have isolated a cloned cDNA that encodes human ribosomal protein (rp) S11 by screening a human fibroblast cDNA library with a labelled 204 bp DNA fragment encompassing residues 212-416 of pRS11, a rat rp Sll cDNA clone. The human rp S11 cloned cDNA consists of 15 residues of the 5' leader, the entire coding sequence and all 51 residues of the 3' untranslated region. The predicted amino acid sequence of 158 residues is identical to rat rpS11. The nucleotide sequence in the coding region differs, however, from that in rat in the first position in two codons and in the third position in 44 codons.

AFEAP cloning: a precise and efficient method for large DNA sequence assembly.

Science.gov (United States)

Zeng, Fanli; Zang, Jinping; Zhang, Suhua; Hao, Zhimin; Dong, Jingao; Lin, Yibin

2017-11-14

Recent development of DNA assembly technologies has spurred myriad advances in synthetic biology, but new tools are always required for complicated scenarios. Here, we have developed an alternative DNA assembly method named AFEAP cloning (Assembly of Fragment Ends After PCR), which allows scarless, modular, and reliable construction of biological pathways and circuits from basic genetic parts. The AFEAP method requires two-round of PCRs followed by ligation of the sticky ends of DNA fragments. The first PCR yields linear DNA fragments and is followed by a second asymmetric (one primer) PCR and subsequent annealing that inserts overlapping overhangs at both sides of each DNA fragment. The overlapping overhangs of the neighboring DNA fragments annealed and the nick was sealed by T4 DNA ligase, followed by bacterial transformation to yield the desired plasmids. We characterized the capability and limitations of new developed AFEAP cloning and demonstrated its application to assemble DNA with varying scenarios. Under the optimized conditions, AFEAP cloning allows assembly of an 8 kb plasmid from 1-13 fragments with high accuracy (between 80 and 100%), and 8.0, 11.6, 19.6, 28, and 35.6 kb plasmids from five fragments at 91.67, 91.67, 88.33, 86.33, and 81.67% fidelity, respectively. AFEAP cloning also is capable to construct bacterial artificial chromosome (BAC, 200 kb) with a fidelity of 46.7%. AFEAP cloning provides a powerful, efficient, seamless, and sequence-independent DNA assembly tool for multiple fragments up to 13 and large DNA up to 200 kb that expands synthetic biologist's toolbox.

Cloning the human lysozyme cDNA: Inverted Alu repeat in the mRNA and in situ hybridization for macrophages and Paneth cells

International Nuclear Information System (INIS)

Chung, L.P.; Keshav, S.; Gordon, S.

1988-01-01

Lysozyme is a major secretory product of human and rodent macrophages and a useful marker for myelomonocytic cells. Based on the known human lysozyme amino acid sequence, oligonucleotides were synthesized and used as probes to screen a phorbol 12-myristate 13-acetate-treated U937 cDNA library. A full-length human lysozyme cDNA clone, pHL-2, was obtained and characterized. Sequence analysis shows that human lysozyme, like chicken lysozyme, has in 18-amino-acid-long signal peptide, but unlike the chicken lysozyme cDNA, the human lysozyme cDNA has a >1-kilobase-long 3' nontranslated sequence. Interestingly, within this 3' region, an inverted repeat of the Alu family of repetitive sequences was discovered. In RNA blot analyses, DNA probes prepared from pHL-2 can be used to detect lysozyme mRNA not only from human but also from mouse and rat. Moreover, by in situ hybridization, complementary RNA transcripts have been used as probes to detect lysozyme mRNA in mouse macrophages and Paneth cells. This human lysozyme cDNA clone is therefore likely to be a useful molecular probe for studying macrophage distribution and gene expression

Accurate DNA assembly and genome engineering with optimized uracil excision cloning

DEFF Research Database (Denmark)

Cavaleiro, Mafalda; Kim, Se Hyeuk; Seppala, Susanna

2015-01-01

Simple and reliable DNA editing by uracil excision (a.k.a. USER cloning) has been described by several research groups, but the optimal design of cohesive DNA ends for multigene assembly remains elusive. Here, we use two model constructs based on expression of gfp and a four-gene pathway that pro......Simple and reliable DNA editing by uracil excision (a.k.a. USER cloning) has been described by several research groups, but the optimal design of cohesive DNA ends for multigene assembly remains elusive. Here, we use two model constructs based on expression of gfp and a four-gene pathway...... that produces β-carotene to optimize assembly junctions and the uracil excision protocol. By combining uracil excision cloning with a genomic integration technology, we demonstrate that up to six DNA fragments can be assembled in a one-tube reaction for direct genome integration with high accuracy, greatly...... facilitating the advanced engineering of robust cell factories....

Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor

International Nuclear Information System (INIS)

Antalis, T.M.; Clark, M.A.; Barnes, T.; Lehrbach, P.R.; Devine, P.L.; Schevzov, G.; Goss, N.H.; Stephens, R.W.; Tolstoshev, P.

1988-01-01

Human monocyte-derived plasminogen activator inhibitor (mPAI-2) was purified to homogeneity from the U937 cell line and partially sequenced. Oligonucleotide probes derived from this sequence were used to screen a cDNA library prepared from U937 cells. One positive clone was sequenced and contained most of the coding sequence as well as a long incomplete 3' untranslated region (1112 base pairs). This cDNA sequence was shown to encode mPAI-2 by hybrid-select translation. A cDNA clone encoding the remainder of the mPAI-2 mRNA was obtained by primer extension of U937 poly(A) + RNA using a probe complementary to the mPAI-2 coding region. The coding sequence for mPAI-2 was placed under the control of the λ P/sub L/ promoter, and the protein expressed in Escherichia coli formed a complex with urokinase that could be detected immunologically. By nucleotide sequence analysis, mPAI-2 cDNA encodes a protein containing 415 amino acids with a predicted unglycosylated M/sub r/ of 46,543. The predicted amino acid sequence of mPAI-2 is very similar to placental PAI-2 and shows extensive homology with members of the serine protease inhibitor (serpin) superfamily. mPAI-2 was found to be more homologous to ovalbumin (37%) than the endothelial plasminogen activator inhibitor, PAI-1 (26%). The 3' untranslated region of the mPAI-2 cDNA contains a putative regulatory sequence that has been associated with the inflammatory mediators

Distribution and uses of legume DNA clone resources

International Nuclear Information System (INIS)

Young, N.D.

2001-01-01

Since 1990, my lab has developed and distributed various DNA clone resources for the legumes. In the first several years, the focus was on members of the tropical genus, Vigna, including the widely cultivated species, mungbean (V. radiata) and cowpea (V. unguiculata). Both of these grain legumes play key roles in agriculture in developing countries of Asia (mungbean) and Africa (cowpea). Moreover, because there is substantial genome conservation among legumes, these genetic resources have also been utilized by a wide range of researchers in other crop species. In 1997, my lab began to focus on the development and distribution of a new generation of DNA clone resources; Bacterial Artificial Chromosomes (BAC). A library of these clones was constructed in soybean (Glycine max) the most important legume species worldwide in terms of economic value. Again, the library has become a valuable resource for the legume research community and has been widely used in studies of legume genomics. (author)

Evaluation of the efficiency and utility of recombinant enzyme-free seamless DNA cloning methods

Directory of Open Access Journals (Sweden)

Ken Motohashi

2017-03-01

Full Text Available Simple and low-cost recombinant enzyme-free seamless DNA cloning methods have recently become available. In vivo Escherichia coli cloning (iVEC can directly transform a mixture of insert and vector DNA fragments into E. coli, which are ligated by endogenous homologous recombination activity in the cells. Seamless ligation cloning extract (SLiCE cloning uses the endogenous recombination activity of E. coli cellular extracts in vitro to ligate insert and vector DNA fragments. An evaluation of the efficiency and utility of these methods is important in deciding the adoption of a seamless cloning method as a useful tool. In this study, both seamless cloning methods incorporated inserting DNA fragments into linearized DNA vectors through short (15–39 bp end homology regions. However, colony formation was 30–60-fold higher with SLiCE cloning in end homology regions between 15 and 29 bp than with the iVEC method using DH5α competent cells. E. coli AQ3625 strains, which harbor a sbcA gene mutation that activates the RecE homologous recombination pathway, can be used to efficiently ligate insert and vector DNA fragments with short-end homology regions in vivo. Using AQ3625 competent cells in the iVEC method improved the rate of colony formation, but the efficiency and accuracy of SLiCE cloning were still higher. In addition, the efficiency of seamless cloning methods depends on the intrinsic competency of E. coli cells. The competency of chemically competent AQ3625 cells was lower than that of competent DH5α cells, in all cases of chemically competent cell preparations using the three different methods. Moreover, SLiCE cloning permits the use of both homemade and commercially available competent cells because it can use general E. coli recA− strains such as DH5α as host cells for transformation. Therefore, between the two methods, SLiCE cloning provides both higher efficiency and better utility than the iVEC method for seamless DNA plasmid

Software-Supported USER Cloning Strategies for Site-Directed Mutagenesis and DNA Assembly

DEFF Research Database (Denmark)

Genee, Hans Jasper; Bonde, Mads Tvillinggaard; Bagger, Frederik Otzen

2015-01-01

USER cloning is a fast and versatile method for engineering of plasmid DNA. We have developed a user friendly Web server tool that automates the design of optimal PCR primers for several distinct USER cloning-based applications. Our Web server, named AMUSER (Automated DNA Modifications with USER...... cloning), facilitates DNA assembly and introduction of virtually any type of site-directed mutagenesis by designing optimal PCR primers for the desired genetic changes. To demonstrate the utility, we designed primers for a simultaneous two-position site-directed mutagenesis of green fluorescent protein...... (GFP) to yellow fluorescent protein (YFP), which in a single step reaction resulted in a 94% cloning efficiency. AMUSER also supports degenerate nucleotide primers, single insert combinatorial assembly, and flexible parameters for PCR amplification. AMUSER is freely available online at ....

cDNA cloning of human DNA topoisomerase I. Catalytic activity of a 67.7-kDa carboxyl-terminal fragment

International Nuclear Information System (INIS)

D'Arpa, P.; Machlin, P.S.; Ratrie, H. III; Rothfield, N.F.; Cleveland, D.W.; Earnshaw, W.C.

1988-01-01

cDNA clones encoding human topoisomerase I were isolated from an expression vector library (λgt11) screened with autoimmune anti-topoisomerase I serum. One of these clones has been expressed as a fusion protein comprised of a 32-kDa fragment of the bacterial TrpE protein linked to 67.7 kDa of protein encoded by the cDNA. Three lines of evidence indicate that the cloned cDNA encodes topoisomerase I. (i) Proteolysis maps of the fusion protein and human nuclear topoisomerase I are essentially identical. (ii) The fusion protein relaxes supercoiled DNA, an activity that can be immunoprecipitated by anti-topoisomerase I serum. (iii) Sequence analysis has revealed that the longest cDNA clone (3645 base pairs) encodes a protein of 765 amino acids that shares 42% identity with Saccharomyces cerevisiae topoisomerase I. The sequence data also show that the catalytically active 67.7-kDa fragment is comprised of the carboxyl terminus

Heterogeneity of rat tropoelastin mRNA revealed by cDNA cloning

International Nuclear Information System (INIS)

Pierce, R.A.; Deak, S.B.; Stolle, C.A.; Boyd, C.D.

1990-01-01

A λgt11 library constructed from poly(A+) RNA isolated from aortic tissue of neonatal rats was screened for rat tropoelastin cDNAs. The first, screen, utilizing a human tropoelastin cDNA clone, provided rat tropoelastin cDNAs spanning 2.3 kb of carboxy-terminal coding sequence and extended into the 3'-untranslated region. A subsequent screen using a 5' rat tropoelastin cDNA clone yielded clones extending into the amino-terminal signal sequence coding region. Sequence analysis of these clones has provided the complete derived amino acid sequence of rat tropoelastin and allowed alignment and comparison with published bovine cDNA sequence. While the overall structure of rat tropoelastin is similar to bovine sequence, numerous substitutions, deletions, and insertions demonstrated considerable heterogeneity between species. In particular, the pentapeptide repeat VPGVG, characteristic of all tropoelastins analyzed to date, is replaced in rat tropoelastin by a repeating pentapeptide, IPGVG. The hexapeptide repeat VGVAPG, the bovine elastin receptor binding peptide, is not encoded by rat tropoelastin cDNAs. Variations in coding sequence between rat tropoelastin CDNA clones were also found which may represent mRNA heterogeneity produced by alternative splicing of the rat tropoelastin pre-mRNA

[Cloning and sequencing of KIR2DL1 framework gene cDNA and identification of a novel allele].

Science.gov (United States)

Sun, Ge; Wang, Chang; Zhen, Jianxin; Zhang, Guobin; Xu, Yunping; Deng, Zhihui

2016-10-01

To develop an assay for cDNA cloning and haplotype sequencing of KIR2DL1 framework gene and determine the genotype of an ethnic Han from southern China. Total RNA was isolated from peripheral blood sample, and complementary DNA (cDNA) transcript was synthesized by RT-PCR. The entire coding sequence of the KIR2DL1 framework gene was amplified with a pair of KIR2DL1-specific PCR primers. The PCR products with a length of approximately 1.2 kb were then subjected to cloning and haplotype sequencing. A specific target fragment of the KIR2DL1 framework gene was obtained. Following allele separation, a wild-type KIR2DL1*00302 allele and a novel variant allele, KIR2DL1*031, were identified. Sequence alignment with KIR2DL1 alleles from the IPD-KIR Database showed that the novel allele KIR2DL1*031 has differed from the closest allele KIR2DL1*00302 by a non-synonymous mutation at CDS nt 188A>G (codon 42 GAG>GGG) in exon 4, which has caused an amino acid change Glu42Gly. The sequence of the novel allele KIR2DL1*031 was submitted to GenBank under the accession number KP025960 and to the IPD-KIR Database under the submission number IWS40001982. A name KIR2DL1*031 has been officially assigned by the World Health Organization (WHO) Nomenclature Committee. An assay for cDNA cloning and haplotype sequencing of KIR2DL1 has been established, which has a broad applications in KIR studies at allelic level.

Cloning of cDNA encoding steroid 11β-hydroxylase (P450c11)

International Nuclear Information System (INIS)

Chua, S.C.; Szabo, P.; Vitek, A.; Grzeschik, K.H.; John, M.; White, P.C.

1987-01-01

The authors have isolated bovine and human adrenal cDNA clones encoding the adrenal cytochrome P-450 specific for 11β-hydroxylation (P450c11). A bovine adrenal cDNA library constructed in the bacteriophage λ vector gt10 was probed with a previously isolated cDNA clone corresponding to part of the 3' untranslated region of the 4.2-kilobase (kb) mRNA encoding P450c11. Several clones with 3.2-kb cDNA inserts were isolated. Sequence analysis showed that they overlapped the original probe by 300 base pairs (bp). Combined cDNA and RNA sequence data demonstrated a continuous open reading frame of 1509 bases. P450c11 is predicted to contain 479 amino acid residues in the mature protein in addition to a 24-residue amino-terminal mitochondrial signal sequence. A bovine clone was used to isolate a homologous clone with a 3.5-kb insert from a human adrenal cDNA library. A region of 1100 bp was 81% homologous to 769 bp of the coding sequence of the bovine cDNA except for a 400-bp segment presumed to be an unprocessed intron. Hybridization of the human cDNA to DNA from a panel of human-rodent somatic cell hybrid lines and in situ hybridization to metaphase spreads of human chromosomes localized the gene to the middle of the long arm of chromosome 8. These data should be useful in developing reagents for heterozygote detection and prenatal diagnosis of 11β-hydroxylase deficiency, the second most frequent cause of congenital adrenal hyperplasia

Identification of cDNA clones expressing immunodiagnostic antigens from Trichinella spiralis

International Nuclear Information System (INIS)

Zarlenga, D.; Gamble, H.R.

1987-01-01

A cDNA expression library was built in lambda gt11 phage using poly A mRNA isolated from Trichinella spiralis muscle stage larvae. This library was screened with rabbit antibodies to parasite excretory-secretory (ES) products and greater than 180 clones were isolated. Thirteen clones producing highly immunogenic protein antigens were plaque purified and rescreened with pig antisera to T.spiralis, Trichuris suis or Ascaris suum to identify clones producing epitopes specific to T.spiralis ES products, only. Two clones, TsAc-2 and TsAc-8, which displayed strong interactions with pig antisera to T. spiralis were lysogenized in E. coli Y1089 and the protein extracted. Western blots of the crude fusion proteins revealed molecular weights of 133 kD and 129 kD, respectively. Northern blot analysis of total RNA with 32 P labelled cDNA:lambda gt11 probes indicated single RNA transcripts for each clone with molecular sizes corresponding to 800-850 nucleotides. dscDNA inserts were estimated by southern blot analysis to be 500 bp and 340 bp, respectively, with no cross-hybridization observed between the cloned sequences. Dot blots using pig sera to screen crude fusion protein preparations, total bacterial protein (negative controls) and crude worm extract or ES products from T.spiralis, T.suis and A.suum (positive controls) corroborated the specificity and sensitivity of these clones as potential diagnostic antigens for swine trichinellosis

Nuclear transfer to prevent mitochondrial DNA disorders: revisiting the debate on reproductive cloning.

Science.gov (United States)

Bredenoord, A L; Dondorp, W; Pennings, G; De Wert, G

2011-02-01

Preclinical experiments are currently performed to examine the feasibility of several types of nuclear transfer to prevent mitochondrial DNA (mtDNA) disorders. Whereas the two most promising types of nuclear transfer to prevent mtDNA disorders, spindle transfer and pronuclear transfer, do not amount to reproductive cloning, one theoretical variant, blastomere transfer does. This seems the most challenging both technically and ethically. It is prohibited by many jurisdictions and also the scientific community seems to avoid it. Nevertheless, this paper examines the moral acceptability of blastomere transfer as a method to prevent mtDNA disorders. The reason for doing so is that most objections against reproductive cloning refer to reproductive adult cloning, while blastomere transfer would amount to reproductive embryo cloning. After clarifying this conceptual difference, this paper examines whether the main non-safety objections brought forward against reproductive cloning also apply in the context of blastomere transfer. The conclusion is that if this variant were to become safe and effective, dismissing it because it would involve reproductive cloning is unjustified. Nevertheless, as it may lead to more complex ethical appraisals than the other variants, researchers should initially focus on the development of the other types of nuclear transfer to prevent mtDNA disorders. Copyright © 2010 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Isolation of full-length putative rat lysophospholipase cDNA using improved methods for mRNA isolation and cDNA cloning

International Nuclear Information System (INIS)

Han, J.H.; Stratowa, C.; Rutter, W.J.

1987-01-01

The authors have cloned a full-length putative rat pancreatic lysophospholipase cDNA by an improved mRNA isolation method and cDNA cloning strategy using [ 32 P]-labelled nucleotides. These new methods allow the construction of a cDNA library from the adult rat pancreas in which the majority of recombinant clones contained complete sequences for the corresponding mRNAs. A previously recognized but unidentified long and relatively rare cDNA clone containing the entire sequence from the cap site at the 5' end to the poly(A) tail at the 3' end of the mRNA was isolated by single-step screening of the library. The size, amino acid composition, and the activity of the protein expressed in heterologous cells strongly suggest this mRNA codes for lysophospholipase

Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor.

Science.gov (United States)

Antalis, T M; Clark, M A; Barnes, T; Lehrbach, P R; Devine, P L; Schevzov, G; Goss, N H; Stephens, R W; Tolstoshev, P

1988-02-01

Human monocyte-derived plasminogen activator inhibitor (mPAI-2) was purified to homogeneity from the U937 cell line and partially sequenced. Oligonucleotide probes derived from this sequence were used to screen a cDNA library prepared from U937 cells. One positive clone was sequenced and contained most of the coding sequence as well as a long incomplete 3' untranslated region (1112 base pairs). This cDNA sequence was shown to encode mPAI-2 by hybrid-select translation. A cDNA clone encoding the remainder of the mPAI-2 mRNA was obtained by primer extension of U937 poly(A)+ RNA using a probe complementary to the mPAI-2 coding region. The coding sequence for mPAI-2 was placed under the control of the lambda PL promoter, and the protein expressed in Escherichia coli formed a complex with urokinase that could be detected immunologically. By nucleotide sequence analysis, mPAI-2 cDNA encodes a protein containing 415 amino acids with a predicted unglycosylated Mr of 46,543. The predicted amino acid sequence of mPAI-2 is very similar to placental PAI-2 (3 amino acid differences) and shows extensive homology with members of the serine protease inhibitor (serpin) superfamily. mPAI-2 was found to be more homologous to ovalbumin (37%) than the endothelial plasminogen activator inhibitor, PAI-1 (26%). Like ovalbumin, mPAI-2 appears to have no typical amino-terminal signal sequence. The 3' untranslated region of the mPAI-2 cDNA contains a putative regulatory sequence that has been associated with the inflammatory mediators.

Construction of Infectious cDNA Clone of a Chrysanthemum stunt viroid Korean Isolate

Directory of Open Access Journals (Sweden)

Ju-Yeon Yoon

2014-03-01

Full Text Available Chrysanthemum stunt viroid (CSVd, a noncoding infectious RNA molecule, causes seriously economic losses of chrysanthemum for 3 or 4 years after its first infection. Monomeric cDNA clones of CSVd isolate SK1 (CSVd-SK1 were constructed in the plasmids pGEM-T easy vector and pUC19 vector. Linear positive-sense transcripts synthesized in vitro from the full-length monomeric cDNA clones of CSVd-SK1 could infect systemically tomato seedlings and chrysanthemum plants, suggesting that the linear CSVd RNA transcribed from the cDNA clones could be replicated as efficiently as circular CSVd in host species. However, direct inoculation of plasmid cDNA clones containing full-length monomeric cDNA of CSVd-SK1 failed to infect tomato and chrysanthemum and linear negative-sense transcripts from the plasmid DNAs were not infectious in the two plant species. The cDNA sequences of progeny viroid in systemically infected tomato and chrysanthemum showed a few substitutions at a specific nucleotide position, but there were no deletions and insertions in the sequences of the CSVd progeny from tomato and chrysanthemum plants.

DNA Methylation in Peripheral Blood Cells of Pigs Cloned by Somatic Cell Nuclear Transfer

DEFF Research Database (Denmark)

Gao, Fei; Li, Shengting; Lin, Lin

2011-01-01

To date, the genome-wide DNA methylation status of cloned pigs has not been investigated. Due to the relatively low success rate of pig cloning by somatic cell nuclear transfer, a better understanding of the epigenetic reprogramming and the global methylation patterns associated with development...... in cloned pigs is required. In this study we applied methylation-specific digital karyotyping tag sequencing by Solexa technology and investigated the genome-wide DNA methylation profiles of peripheral blood cells in cloned pigs with normal phenotypes in comparison with their naturally bred controls....... In the result, we found that globally there was no significant difference of DNA methylation patterns between the two groups. Locus-specifically, some genes involved in embryonic development presented a generally increased level of methylation. Our findings suggest that in cloned pigs with normal phenotypes...

Molecular cloning and restriction analysis of EcoRI-fragments of Vicia faba rDNA

International Nuclear Information System (INIS)

Yakura, Kimitaka; Tanifuji, Shigeyuki.

1983-01-01

EcoRI-fragments of Vicia faba rDNA were cloned in plasmid pBR325. Southern blot hybridization of BamHI-digests of these cloned plasmids and Vicia genomic DNA led to the determination of relative positions of BamHI sites in the rDNA and the physical map that had been tentatively made is corrected. (author)

Cloning and sequencing of cDNA encoding human DNA topoisomerase II and localization of the gene to chromosome region 17q21-22

International Nuclear Information System (INIS)

Tsai-Pflugfelder, M.; Liu, L.F.; Liu, A.A.; Tewey, K.M.; Whang-Peng, J.; Knutsen, T.; Huebner, K.; Croce, C.M.; Wang, J.C.

1988-01-01

Two overlapping cDNA clones encoding human DNA topoisomerase II were identified by two independent methods. In one, a human cDNA library in phage λ was screened by hybridization with a mixed oligonucleotide probe encoding a stretch of seven amino acids found in yeast and Drosophila DNA topoisomerase II; in the other, a different human cDNA library in a λgt11 expression vector was screened for the expression of antigenic determinants that are recognized by rabbit antibodies specific to human DNA topoisomerase II. The entire coding sequences of the human DNA topoisomerase II gene were determined from these and several additional clones, identified through the use of the cloned human TOP2 gene sequences as probes. Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is encoded by a single-copy gene. The location of the gene was mapped to chromosome 17q21-22 by in situ hybridization of a cloned fragment to metaphase chromosomes and by hybridization analysis with a panel of mouse-human hybrid cell lines, each retaining a subset of human chromosomes

Generation of Infectious Poliovirus with Altered Genetic Information from Cloned cDNA.

Science.gov (United States)

Bujaki, Erika

2016-01-01

The effect of specific genetic alterations on virus biology and phenotype can be studied by a great number of available assays. The following method describes the basic protocol to generate infectious poliovirus with altered genetic information from cloned cDNA in cultured cells.The example explained here involves generation of a recombinant poliovirus genome by simply replacing a portion of the 5' noncoding region with a synthetic gene by restriction cloning. The vector containing the full length poliovirus genome and the insert DNA with the known mutation(s) are cleaved for directional cloning, then ligated and transformed into competent bacteria. The recombinant plasmid DNA is then propagated in bacteria and transcribed to RNA in vitro before RNA transfection of cultured cells is performed. Finally, viral particles are recovered from the cell culture.

Horse cDNA clones encoding two MHC class I genes

Energy Technology Data Exchange (ETDEWEB)

Barbis, D.P.; Maher, J.K.; Stanek, J.; Klaunberg, B.A.; Antczak, D.F.

1994-12-31

Two full-length clones encoding MHC class I genes were isolated by screening a horse cDNA library, using a probe encoding in human HLA-A2.2Y allele. The library was made in the pcDNA1 vector (Invitrogen, San Diego, CA), using mRNA from peripheral blood lymphocytes obtained from a Thoroughbred stallion (No. 0834) homozygous for a common horse MHC haplotype (ELA-A2, -B2, -D2; Antczak et al. 1984; Donaldson et al. 1988). The clones were sequenced, using SP6 and T7 universal primers and horse-specific oligonucleotides designed to extend previously determined sequences.

Cloning and analysis of the mouse Fanconi anemia group a cDNA and an overlapping penta zinc finger cDNA

NARCIS (Netherlands)

Wong, JCY; Alon, N; Norga, K; Kruyt, FAE; Youssoufian, H; Buchwald, M

2000-01-01

Despite the cloning of four disease-associated genes for Fanconi anemia (FA), the molecular pathogenesis of FA remains largely unknown. To study FA complementation group A using the mouse as a mode I system, we cloned and characterized the mouse homolog of the human FANCA cDNA, The mouse cDNA

Cost-effective sequencing of full-length cDNA clones powered by a de novo-reference hybrid assembly.

Science.gov (United States)

Kuroshu, Reginaldo M; Watanabe, Junichi; Sugano, Sumio; Morishita, Shinichi; Suzuki, Yutaka; Kasahara, Masahiro

2010-05-07

Sequencing full-length cDNA clones is important to determine gene structures including alternative splice forms, and provides valuable resources for experimental analyses to reveal the biological functions of coded proteins. However, previous approaches for sequencing cDNA clones were expensive or time-consuming, and therefore, a fast and efficient sequencing approach was demanded. We developed a program, MuSICA 2, that assembles millions of short (36-nucleotide) reads collected from a single flow cell lane of Illumina Genome Analyzer to shotgun-sequence approximately 800 human full-length cDNA clones. MuSICA 2 performs a hybrid assembly in which an external de novo assembler is run first and the result is then improved by reference alignment of shotgun reads. We compared the MuSICA 2 assembly with 200 pooled full-length cDNA clones finished independently by the conventional primer-walking using Sanger sequencers. The exon-intron structure of the coding sequence was correct for more than 95% of the clones with coding sequence annotation when we excluded cDNA clones insufficiently represented in the shotgun library due to PCR failure (42 out of 200 clones excluded), and the nucleotide-level accuracy of coding sequences of those correct clones was over 99.99%. We also applied MuSICA 2 to full-length cDNA clones from Toxoplasma gondii, to confirm that its ability was competent even for non-human species. The entire sequencing and shotgun assembly takes less than 1 week and the consumables cost only approximately US$3 per clone, demonstrating a significant advantage over previous approaches.

Isolation and sequence of complementary DNA encoding human extracellular superoxide dismutase

International Nuclear Information System (INIS)

Hjalmarsson, K.; Marklund, S.L.; Engstroem, A.; Edlund, T.

1987-01-01

A complementary DNA (cDNA) clone from a human placenta cDNA library encoding extracellular superoxide dismutase has been isolated and the nucleotide sequence determined. The cDNA has a very high G + C content. EC-SOD is synthesized with a putative 18-amino acid signal peptide, preceding the 222 amino acids in the mature enzyme, indicating that the enzyme is a secretory protein. The first 95 amino acids of the mature enzyme show no sequence homology with other sequenced proteins and there is one possible N-glycosylation site (Asn-89). The amino acid sequence from residues 96-193 shows strong homology (∼ 50%) with the final two-thirds of the sequences of all know eukaryotic CuZn SODs, whereas the homology with the P. leiognathi CuZn SOD is clearly lower. The ligands to Cu and Zn, the cysteines forming the intrasubunit disulfide bridge in the CuZn SODs, and the arginine found in all CuZn SODs in the entrance to the active site can all be identified in EC-SOD. A comparison with bovine CuZn SOD, the three-dimensional structure of which is known, reveals that the homologies occur in the active site and the divergencies are in the part constituting the subunit contact area in CuZn SOD. Amino acid sequence 194-222 in the carboxyl-terminal end of EC-SOD is strongly hydrophilic and contains nine amino acids with a positive charge. This sequence probably confers the affinity of EC-SOD for heparin and heparan sulfate. An analysis of the amino acid sequence homologies with CuZn SODs from various species indicates that the EC-SODs may have evolved form the CuZn SODs before the evolution of fungi and plants

RFLP for Duchenne muscular dystrophy cDNA clone 44-1

Energy Technology Data Exchange (ETDEWEB)

Laing, N G; Siddique, T; Bartlett, R J; Yamaoka, L H; Chen, J C; Walker, A P; Hung, W Y; Roses, A D [Duke Univ. Medical Center, Durham, NC (USA)

1988-07-25

Clone 44-1 is one of six cDNA clones which comprise the cDNA for the Duchenne muscular dystrophy gene. It is a 0.9kb fragment in the EcoR1 site of Bluescript. Taq1 (TlCGA) identifies two alleles with bands at 6.8 and 5.7kb, as well as four constant bands at 4.8, 3.9, 3.5 and 2.5kb. Its frequency was studied in 62 unrelated individuals. Mendelian inheritance was demonstrated in one three generation and three two generation informative families, 26 individuals. There were no problems on RFLP analysis under normal stringency conditions.

(PCR) for direct cloning of blunt-end DNA fragments

African Journals Online (AJOL)

Administrator

2011-09-19

Sep 19, 2011 ... Key words: Blunt-end cloning, phosphorylated DNA fragment, dephosphorylated blunt-end vector. INTRODUCTION ... With this method, a lot of steps are saved, which includes restriction .... pBSK-blunt (data not shown).

Cloning, sequencing and expression of cDNA encoding growth ...

Indian Academy of Sciences (India)

Unknown

of medicine, animal husbandry, fish farming and animal ..... northern pike (Esox lucius) growth hormone; Mol. Mar. Biol. ... prolactin 1-luciferase fusion gene in African catfish and ... 1988 Cloning and sequencing of cDNA that encodes goat.

Isolation and characterization of cDNA clones for carrot extensin and a proline-rich 33-kDa protein

International Nuclear Information System (INIS)

Chen, J.; Varner, J.E.

1985-01-01

Extensins are hydroxyproline-rich glycoproteins associated with most dicotyledonous plant cell walls. To isolate cDNA clones encoding extensin, the authors started by isolating poly(A) + RNA from carrot root tissue, and then translating the RNA in vitro, in the presence of tritiated leucine or proline. A 33-kDa peptide was identified in the translation products as a putative extensin precursor. From a cDNA library constructed with poly(A) + RNA from wounded carrots, one cDNA clone (pDC5) was identified that specifically hybridized to poly(A) + RNA encoding this 33-kDa peptide. They isolated three cDNA clones (pDC11, pDC12, and pDC16) from another cDNA library using pCD5 as a probe. DNA sequence data, RNA hybridization analysis, and hybrid released in vitro translation indicate that the cDNA clones pDC11 encodes extensin and that cDNA clones pDC12 and pDC16 encode the 33-kDa peptide, which as yet has an unknown identity and function. The assumption that the 33-kDa peptide was an extensin precursor was invalid. RNA hybridization analysis showed that RNA encoded by both clone types is accumulated upon wounding

Luciferase assay to study the activity of a cloned promoter DNA fragment.

Science.gov (United States)

Solberg, Nina; Krauss, Stefan

2013-01-01

Luciferase based assays have become an invaluable tool for the analysis of cloned promoter DNA fragments, both for verifying the ability of a potential promoter fragment to drive the expression of a luciferase reporter gene in various cellular contexts, and for dissecting binding elements in the promoter. Here, we describe the use of the Dual-Luciferase(®) Reporter Assay System created by Promega (Promega Corporation, Wisconsin, USA) to study the cloned 6.7 kilobases (kb) mouse (m) Tcf3 promoter DNA fragment in mouse embryonic derived neural stem cells (NSC). In this system, the expression of the firefly luciferase driven by the cloned mTcf3 promoter DNA fragment (including transcription initiation sites) is correlated with a co-transfected control reporter expressing Renilla luciferase from the herpes simplex virus (HSV) thymidine kinase promoter. Using an internal control reporter allows to normalize the activity of the experimental reporter to the internal control, which minimizes experimental variability.

Infectious Maize rayado fino virus from Cloned cDNA.

Science.gov (United States)

Edwards, Michael C; Weiland, John J; Todd, Jane; Stewart, Lucy R

2015-06-01

A full-length cDNA clone was produced from a U.S. isolate of Maize rayado fino virus (MRFV), the type member of the genus Marafivirus within the family Tymoviridae. Infectivity of transcripts derived from cDNA clones was demonstrated by infection of maize plants and protoplasts, as well as by transmission via the known leafhopper vectors Dalbulus maidis and Graminella nigrifrons that transmit the virus in a persistent-propagative manner. Infection of maize plants through vascular puncture inoculation of seed with transcript RNA resulted in the induction of fine stipple stripe symptoms typical of those produced by wild-type MRFV and a frequency of infection comparable with that of the wild type. Northern and Western blotting confirmed the production of MRFV-specific RNAs and proteins in infected plants and protoplasts. An unanticipated increase in subgenomic RNA synthesis over levels in infected plants was observed in protoplasts infected with either wild-type or cloned virus. A conserved cleavage site motif previously demonstrated to function in both Oat blue dwarf virus capsid protein and tymoviral nonstructural protein processing was identified near the amino terminus of the MRFV replicase polyprotein, suggesting that cleavage at this site also may occur.

Strong spurious transcription likely contributes to DNA insert bias in typical metagenomic clone libraries.

Science.gov (United States)

Lam, Kathy N; Charles, Trevor C

2015-01-01

Clone libraries provide researchers with a powerful resource to study nucleic acid from diverse sources. Metagenomic clone libraries in particular have aided in studies of microbial biodiversity and function, and allowed the mining of novel enzymes. Libraries are often constructed by cloning large inserts into cosmid or fosmid vectors. Recently, there have been reports of GC bias in fosmid metagenomic libraries, and it was speculated to be a result of fragmentation and loss of AT-rich sequences during cloning. However, evidence in the literature suggests that transcriptional activity or gene product toxicity may play a role. To explore possible mechanisms responsible for sequence bias in clone libraries, we constructed a cosmid library from a human microbiome sample and sequenced DNA from different steps during library construction: crude extract DNA, size-selected DNA, and cosmid library DNA. We confirmed a GC bias in the final cosmid library, and we provide evidence that the bias is not due to fragmentation and loss of AT-rich sequences but is likely occurring after DNA is introduced into Escherichia coli. To investigate the influence of strong constitutive transcription, we searched the sequence data for promoters and found that rpoD/σ(70) promoter sequences were underrepresented in the cosmid library. Furthermore, when we examined the genomes of taxa that were differentially abundant in the cosmid library relative to the original sample, we found the bias to be more correlated with the number of rpoD/σ(70) consensus sequences in the genome than with simple GC content. The GC bias of metagenomic libraries does not appear to be due to DNA fragmentation. Rather, analysis of promoter sequences provides support for the hypothesis that strong constitutive transcription from sequences recognized as rpoD/σ(70) consensus-like in E. coli may lead to instability, causing loss of the plasmid or loss of the insert DNA that gives rise to the transcription. Despite

Enhanced specificity in immunoscreening of expression cDNA clones using radiolabeled antigen overlay

International Nuclear Information System (INIS)

Chao, S.; Chao, L.; Chao, J.

1989-01-01

A highly sensitive and specific method has been developed for immunoscreening clones from an expression cDNA library. The procedures utilize a radiolabeled antigen detection method described originally for the immunoblotting of plasma proteins. Screening of rat alpha 1-antitrypsin clones was used. Comparison between Western blots of alpha 1-antitrypsin using both labeled antigen and protein A detection methods showed that the former yielded lower background and greater sensitivity than the latter. Further, this technique was shown to have a lower detection limit of less than 20 ng through Western blot analysis of varying concentrations of alpha 1-antitrypsin. The procedures are based on the expression of the protein by cDNA clones containing the DNA inserts in the correct reading frame. Following the transfer of phage proteins to nitrocellulose membranes, the bivalent antibodies bind monovalently to both nitrocellulose-bound-antigen in the phage lysates and radiolabeled antigen. The radiolabeled antigen overlay method is superior to the protein A detection method in sensitivity, specificity and reproducibility. This improved method can be applied in general for screening expression cDNA libraries, provided that the specific antiserum and radiolabeled antigen are available

Complementation of radiation-sensitive Ataxia telangiectasia cells after transfection of cDNA expression libraries and cosmid clones from wildtype cells

International Nuclear Information System (INIS)

Fritz, E.

1994-06-01

In this Ph.D.-thesis, phenotypic complementation of AT-cells (AT5BIVA) by transfection of cDNA-expression-libraries was adressed: After stable transfection of cDNA-expression-libraries G418 resistant clones were selected for enhanced radioresistance by a fractionated X-ray selection. One surviving transfectant clone (clone 514) exhibited enhanced radiation resistance in dose-response experiments and further X-ray selections. Cell cycle analysis revealed complementation of untreated and irradiated 514-cells in cell cycle progression. The rate of DNA synthesis, however, is not diminished after irradiation but shows the reverse effect. A transfected cDNA-fragment (AT500-cDNA) was isolated from the genomic DNA of 514-cells and proved to be an unknown DNA sequence. A homologous sequence could be detected in genomic DNA from human cell lines, but not in DNA from other species. The cDNA-sequence could be localized to human chromosome 11. In human cells the cDNA sequence is part of two large mRNAs. 4 different cosmid clones containing high molecular genomic DNA from normal human cells could be isolated from a library, each hybridizing to the AT500-cDNA. After stable transfection into AT-cells, one cosmid-clone was able to confer enhanced radiation resistance both in X-ray selections and dose-response experiments. The results indicate that the cloned cDNA-fragment is based on an unknown gene from human chromosome 11 which partially complements the radiosensitivity and the defective cell cycle progression in AT5BIVA cells. (orig.) [de

Exponential megapriming PCR (EMP cloning--seamless DNA insertion into any target plasmid without sequence constraints.

Directory of Open Access Journals (Sweden)

Alexander Ulrich

Full Text Available We present a fast, reliable and inexpensive restriction-free cloning method for seamless DNA insertion into any plasmid without sequence limitation. Exponential megapriming PCR (EMP cloning requires two consecutive PCR steps and can be carried out in one day. We show that EMP cloning has a higher efficiency than restriction-free (RF cloning, especially for long inserts above 2.5 kb. EMP further enables simultaneous cloning of multiple inserts.

cDNA, genomic cloning and sequence analysis of ribosomal protein ...

African Journals Online (AJOL)

enoh

2012-03-13

Mar 13, 2012 ... cDNA and the genomic sequence of RPS4X were cloned successfully from ... S4 genes plays a role in Turner syndrome; however, this ..... Project of Educational Committee of Sichuan Province ... Molecular biology of the cell.

Molecular cloning and characterization of arginine kinase gene of Toxocara canis.

Science.gov (United States)

Sahu, Shivani; Samanta, S; Harish, D R; Sudhakar, N R; Raina, O K; Shantaveer, S B; Madhu, D N; Kumar, Ashok

2015-06-01

Toxocara canis is an important gastrointestinal nematode of dogs and also a causative agent of visceral larva migrans in humans. Arginine kinase (AK) gene is one of the important biomolecule of phosphagen kinase of T. canis which is emerging as an exciting novel diagnostic target in toxocarosis. The present study was carried out to clone and characterize AK gene of T. canis for future utilization as a diagnostic molecule. Total RNA was extracted from intact adult worms and reverse transcription was done with oligo dT primers to obtain complementary DNA (cDNA). Polymerase chain reaction (PCR) was carried out using cDNA as template with specific primers which amplified a product of 1,202 bp. The amplicon was cloned into pDrive cloning vector and clone was confirmed by colony PCR and restriction endonuclease analysis. Sequence analysis of the gene showed 99.8 and 77.9 % homology with the published AK gene of T. canis (EF015466.1) and Ascaris suum respectively. Structural analysis shown that the mature AK protein consist of 400 amino acids with a molecular wt of 45360.73 Da. Further expression studies are required for producing the recombinant protein for its evaluation in the diagnosis of T. canis infection in humans as well as in adult dogs.

Anti-DNA antibodies: Sequencing, cloning, and expression

Energy Technology Data Exchange (ETDEWEB)

Barry, M.M.

1992-01-01

To gain some insight into the mechanism of systemic lupus erythematosus, and the interactions involved in proteins binding to DNA four anti-DNA antibodies have been investigated. Two of the antibodies, Hed 10 and Jel 242, have previously been prepared from female NZB/NZW mice which develop an autoimmune disease resembling human SLE. The remaining two antibodies, Jel 72 and Jel 318, have previously been produced via immunization of C57BL/6 mice. The isotypes of the four antibodies investigated in this thesis were determined by an enzyme-linked-immunosorbent assay. All four antibodies contained [kappa] light chains and [gamma]2a heavy chains except Jel 318 which contains a [gamma]2b heavy chain. The complete variable regions of the heavy and light chains of these four antibodies were sequenced from their respective mRNAs. The gene segments and variable gene families expressed in each antibody were identified. Analysis of the genes used in the autoimmune anti-DNA antibodies and those produced by immunization indicated no obvious differences to account for their different origins. Examination of the amino acid residues present in the complementary-determining regions of these four antibodies indicates a preference for aromatic amino acids. Jel 72 and Jel 242 contain three arginine residues in the third complementary-determining region. A single-chain Fv and the variable region of the heavy chain of Hed 10 were expressed in Escherichia coli. Expression resulted in the production of a 26,000 M[sub r] protein and a 15,000 M[sub r] protein. An immunoblot indicated that the 26,000 M[sub r] protein was the Fv for Hed 10, while the 15,000 M[sub r] protein was shown to bind poly (dT). The contribution of the heavy chain to DNA binding was assessed.

Isolation and characterization of full-length cDNA clones coding for cholinesterase from fetal human tissues

International Nuclear Information System (INIS)

Prody, C.A.; Zevin-Sonkin, D.; Gnatt, A.; Goldberg, O.; Soreq, H.

1987-01-01

To study the primary structure and regulation of human cholinesterases, oligodeoxynucleotide probes were prepared according to a consensus peptide sequence present in the active site of both human serum pseudocholinesterase and Torpedo electric organ true acetylcholinesterase. Using these probes, the authors isolated several cDNA clones from λgt10 libraries of fetal brain and liver origins. These include 2.4-kilobase cDNA clones that code for a polypeptide containing a putative signal peptide and the N-terminal, active site, and C-terminal peptides of human BtChoEase, suggesting that they code either for BtChoEase itself or for a very similar but distinct fetal form of cholinesterase. In RNA blots of poly(A) + RNA from the cholinesterase-producing fetal brain and liver, these cDNAs hybridized with a single 2.5-kilobase band. Blot hybridization to human genomic DNA revealed that these fetal BtChoEase cDNA clones hybridize with DNA fragments of the total length of 17.5 kilobases, and signal intensities indicated that these sequences are not present in many copies. Both the cDNA-encoded protein and its nucleotide sequence display striking homology to parallel sequences published for Torpedo AcChoEase. These finding demonstrate extensive homologies between the fetal BtChoEase encoded by these clones and other cholinesterases of various forms and species

Isolation and characterization of two cDNA clones encoding for glutamate dehydrogenase in Nicotiana plumbaginifolia.

Science.gov (United States)

Ficarelli, A; Tassi, F; Restivo, F M

1999-03-01

We have isolated two full length cDNA clones encoding Nicotiana plumbaginifolia NADH-glutamate dehydrogenase. Both clones share amino acid boxes of homology corresponding to conserved GDH catalytic domains and putative mitochondrial targeting sequence. One clone shows a putative EF-hand loop. The level of the two transcripts is affected differently by carbon source.

Construction of Agrobacterium tumefaciens-mediated tomato black ring virus infectious cDNA clones.

Science.gov (United States)

Zarzyńska-Nowak, Aleksandra; Ferriol, Inmaculada; Falk, Bryce W; Borodynko-Filas, Natasza; Hasiów-Jaroszewska, Beata

2017-02-15

Tomato black ring virus (TBRV, genus Nepovirus) infects a wide range of economically important plants such as tomato, potato, tobacco and cucumber. Here, a successful construction of infectious full-length cDNA clones of the TBRV genomic RNAs (RNA1 and RNA2) is reported for the first time. The engineered constructs consisting of PCR-amplified DNAs were cloned into binary vector pJL89 immediately downstream of a double cauliflower mosaic virus (CaMV) 35S promoter, and upstream of the hepatitis delta virus (HDV) ribozyme and nopaline synthase terminator (NOS). The symptoms induced on plants agroinoculated with both constructs were indistinguishable from those caused by the wild-type virus. The infectivity of obtained clones was verified by reinoculation to Nicotiana tabacum cv. Xanthi, Chenopodium quinoa and Cucumis sativus. The presence of viral particles and RNA was confirmed by electron microscopy and reverse transcription polymerase chain reaction, respectively. Constructed full-length infectious cDNA clones will serve as an excellent tool to study virus-host-vector interactions. Copyright © 2017 Elsevier B.V. All rights reserved.

Molecular characterization of a Leishmania donovani cDNA clone with similarity to human 20S proteasome a-type subunit

DEFF Research Database (Denmark)

Christensen, C B; Jørgensen, L; Jensen, A T

2000-01-01

Using plasma from patients infected or previously infected with Leishmania donovanii, we isolated a L. donovanii cDNA clone with similarity to the proteasome a-type subunit from humans and other eukaryotes. The cDNA clone, designated LePa, was DNA sequenced and Northern blot analysis of L....... donovanii poly(A(+))mRNA indicated the isolation of a full length cDNA clone with a transcript size of 1.9 kb. The expressed recombinant LePa fusion protein induced proliferation of peripheral blood mononuclear cells in one out of seven patients who had suffered from visceral leishmaniasis. Plasma from 16...

Cloning of the cDNA and gene for a human D2 dopamine receptor

International Nuclear Information System (INIS)

Grady, D.K.; Makam, H.; Stofko, R.E.; Bunzow, J.R.; Civelli, O.; Marchionni, M.A.; Alfano, M.; Frothingham, L.; Fischer, J.B.; Burke-Howie, K.J.; Server, A.C.

1989-01-01

A clone encoding a human D 2 dopamine receptor was isolated from a pituitary cDNA library and sequenced. The deduced protein sequence is 96% identical with that of the cloned rat receptor with one major difference: the human receptor contains an additional 29 amino acids in its putative third cytoplasmic loop. Southern blotting demonstrated the presence of only one human D 2 receptor gene. Two overlapping phage containing the gene were isolated and characterized. DNA sequence analysis of these clones showed that the coding sequence is interrupted by six introns and that the additional amino acids present in the human pituitary receptor are encoded by a single exon of 87 base pairs. The involvement of this sequence in alternative splicing and its biological significance are discussed

Human placental Na+, K+-ATPase α subunit: cDNA cloning, tissue expression, DNA polymorphism, and chromosomal localization

International Nuclear Information System (INIS)

Chehab, F.F.; Kan, Y.W.; Law, M.L.; Hartz, J.; Kao, F.T.; Blostein, R.

1987-01-01

A 2.2-kilobase clone comprising a major portion of the coding sequence of the Na + , K + -ATPase α subunit was cloned from human placenta and its sequence was identical to that encoding the α subunit of human kidney and HeLa cells. Transfer blot analysis of the mRNA products of the Na + , K + -ATPase gene from various human tissues and cell lines revealed only one band (≅ 4.7 kilobases) under low and high stringency washing conditions. The levels of expression in the tissues were intestine > placenta > liver > pancreas, and in the cell lines the levels were human erythroleukemia > butyrate-induced colon > colon > brain > HeLa cells. mRNA was undetectable in reticulocytes, consistent with the authors failure to detect positive clones in a size-selected ( > 2 kilobases) λgt11 reticulocyte cDNA library. DNA analysis revealed by a polymorphic EcoRI band and chromosome localization by flow sorting and in situ hybridization showed that the α subunit is on the short is on the short arm (band p11-p13) of chromosome 1

cDNA, genomic sequence cloning and overexpression of ribosomal ...

African Journals Online (AJOL)

RPS16 of eukaryote is a component of the 40S small ribosomal subunit encoded by RPS16 gene and is also a homolog of prokaryotic RPS9. The cDNA and genomic sequence of RPS16 was cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) using reverse transcription-polymerase chain ...

Cloning of the DNA repair gene, uvsF, by transformation of Aspergillus nidulans.

Science.gov (United States)

Oza, K; Käfer, E

1990-06-01

As a first step in the cloning of the DNA repair gene uvsF of Aspergillus nidulans, uvsF pyrG double mutant strains were transformed with a genomic library which carried the complementing Neurospora pyr-4 gene in the vector. Rare pyr+ uvs+ cotransformants were obtained on media lacking pyrimidines, overlayed with MMS (methyl-methane sulfonate) to which uvsF is hypersensitive. Among MMS-resistant transformants, Southerns revealed two types which showed single bands of different sizes when BglII-digested genomic DNA was probed with the vector. Both types produced uvsF- recombinants without vector sequences in homozygous crosses, but only those with the larger band also produced haploid uvs+ progeny. Using BglII-digested genomic DNA to transform Escherichia coli, plasmids of the corresponding two sizes could be rescued. Their inserts had a short internal region in common, giving evidence of rearrangement(s). In secondary transformation of uvsF mutants, only the plasmids with the larger insert showed complementation and these were used to screen Aspergillus libraries. Three types of genomic and two overlapping cDNA clones were identified. The cDNAs hybridized not only to each other, but also to the common region of the rescued plasmids. Therefore, cDNA subclones were used to map the putative uvsF sequences to a short segment in one genomic clone. In Northerns, the complementing large plasmid hybridized to three mRNAs, while the cDNA subclone identified one of these as the probable uvsF message.

Recombinational Cloning Using Gateway and In-Fusion Cloning Schemes

Science.gov (United States)

Throop, Andrea L.; LaBaer, Joshua

2015-01-01

The comprehensive study of protein structure and function, or proteomics, depends on the obtainability of full-length cDNAs in species-specific expression vectors and subsequent functional analysis of the expressed protein. Recombinational cloning is a universal cloning technique based on site-specific recombination that is independent of the insert DNA sequence of interest, which differentiates this method from the classical restriction enzyme-based cloning methods. Recombinational cloning enables rapid and efficient parallel transfer of DNA inserts into multiple expression systems. This unit summarizes strategies for generating expression-ready clones using the most popular recombinational cloning technologies, including the commercially available Gateway® (Life Technologies) and In-Fusion® (Clontech) cloning technologies. PMID:25827088

Molecular analysis of RAPD DNA based markers: their potential use for the detection of genetic variability in jojoba (Simmondsia chinensis L Schneider).

Science.gov (United States)

Amarger, V; Mercier, L

1995-01-01

We have applied the recently developed technique of random amplified polymorphic DNA (RAPD) for the discrimination between two jojoba clones at the genomic level. Among a set of 30 primers tested, a simple reproducible pattern with three distinct fragments for clone D and two distinct fragments for clone E was obtained with primer OPB08. Since RAPD products are the results of arbitrarily priming events and because a given primer can amplify a number of non-homologous sequences, we wondered whether or not RAPD bands, even those of similar size, were derived from different loci in the two clones. To answer this question, two complementary approaches were used: i) cloning and sequencing of the amplification products from clone E; and ii) complementary Southern analysis of RAPD gels using cloned or amplified fragments (directly recovered from agarose gels) as RFLP probes. The data reported here show that the RAPD reaction generates multiple amplified fragments. Some fragments, although resolved as a single band on agarose gels, contain different DNA species of the same size. Furthermore, it appears that the cloned RAPD products of known sequence that do not target repetitive DNA can be used as hybridization probes in RFLP to detect a polymorphism among individuals.

Cloning and sequence analysis of cDNA coding for rat nucleolar protein C23

International Nuclear Information System (INIS)

Ghaffari, S.H.; Olson, M.O.J.

1986-01-01

Using synthetic oligonucleotides as primers and probes, the authors have isolated and sequenced cDNA clones encoding protein C23, a putative nucleolus organizer protein. Poly(A + ) RNA was isolated from rat Novikoff hepatoma cells and enriched in C23 mRNA by sucrose density gradient ultracentrifugation. Two deoxyoligonuleotides, a 48- and a 27-mer, were synthesized on the basis of amino acid sequence from the C-terminal half of protein C23 and cDNA sequence data from CHO cell protein. The 48-mer was used a primer for synthesis of cDNA which was then inserted into plasmid pUC9. Transformed bacterial colonies were screened by hybridization with 32 P labeled 27-mer. Two clones among 5000 gave a strong positive signal. Plasmid DNAs from these clones were purified and characterized by blotting and nucleotide sequence analysis. The length of C23 mRNA was estimated to be 3200 bases in a northern blot analysis. The sequence of a 267 b.p. insert shows high homology with the CHO cDNA with only 9 nucleotide differences and an identical amino acid sequence. These studies indicate that this region of the protein is highly conserved

Cloning and expression of a cDNA coding for a human monocyte-derived plasminogen activator inhibitor.

OpenAIRE

Antalis, T M; Clark, M A; Barnes, T; Lehrbach, P R; Devine, P L; Schevzov, G; Goss, N H; Stephens, R W; Tolstoshev, P

1988-01-01

Human monocyte-derived plasminogen activator inhibitor (mPAI-2) was purified to homogeneity from the U937 cell line and partially sequenced. Oligonucleotide probes derived from this sequence were used to screen a cDNA library prepared from U937 cells. One positive clone was sequenced and contained most of the coding sequence as well as a long incomplete 3' untranslated region (1112 base pairs). This cDNA sequence was shown to encode mPAI-2 by hybrid-select translation. A cDNA clone encoding t...

Cloning and cDNA sequence of the dihydrolipoamide dehydrogenase component of human α-ketoacid dehydrogenase complexes

International Nuclear Information System (INIS)

Pons, G.; Raefsky-Estrin, C.; Carothers, D.J.; Pepin, R.A.; Javed, A.A.; Jesse, B.W.; Ganapathi, M.K.; Samols, D.; Patel, M.S.

1988-01-01

cDNA clones comprising the entire coding region for human dihydrolipoamide dehydrogenase have been isolated from a human liver cDNA library. The cDNA sequence of the largest clone consisted of 2082 base pairs and contained a 1527-base open reading frame that encodes a precursor dihydrolipoamide dehydrogenase of 509 amino acid residues. The first 35-amino acid residues of the open reading frame probably correspond to a typical mitochondrial import leader sequence. The predicted amino acid sequence of the mature protein, starting at the residue number 36 of the open reading frame, is almost identical (>98% homology) with the known partial amino acid sequence of the pig heart dihydrolipoamide dehydrogenase. The cDNA clone also contains a 3' untranslated region of 505 bases with an unusual polyadenylylation signal (TATAAA) and a short poly(A) track. By blot-hybridization analysis with the cDNA as probe, two mRNAs, 2.2 and 2.4 kilobases in size, have been detected in human tissues and fibroblasts, whereas only one mRNA (2.4 kilobases) was detected in rat tissues

Cloning of a cDNA encoding the human cation-dependent mannose 6-phosphate-specific receptor

International Nuclear Information System (INIS)

Pohlmann, R.; Nagel, G.; Schmidt, B.

1987-01-01

Complementary DNA clones for the human cation-dependent mannose 6-phosphate-specific receptor have been isolated from a human placenta library in λgt11. The nucleotide sequence of the 2463-base-pair cDNA insert includes a 145-base-pair 5' untranslated region, an open reading frame of 831 base pairs corresponding to 277 amino acids, and a 1487-base-pair 3' untranslated region. The deduced amino acid sequence is colinear with that determined by amino acid sequencing of the N-terminus peptide (41 residues) and nine tryptic peptides (93 additional residues). The receptor is synthesized as a precursor with a signal peptide of 20 amino acids. The hydrophobicity profile of the receptor indicates a single membrane-spanning domain, which separates an N-terminal region containing five potential N-glycosylation sites from a C-terminal region lacking N-glycosylation sites. Thus the N-terminal (M/sub r/ = 18,299) and C-terminal (M/sub r/ ≤ 7648) segments of the mature receptor are assumed to be exposed to the extracytosolic and cytosolic sides of the membrane, respectively. Analysis of a panel of somatic cell (mouse-human) hybrids shows that the gene for the receptor is located on human chromosome 12

cDNA cloning of rat and human medium chain acyl-CoA dehydrogenase (MCAD)

International Nuclear Information System (INIS)

Matsubara, Y.; Kraus, J.P.; Rosenberg, L.E.; Tanaka, K.

1986-01-01

MCAD is one of three mitochondrial flavoenzymes which catalyze the first step in the β-oxidation of straight chain fatty acids. It is a tetramer with a subunit Mr of 45 kDa. MCAD is synthesized in the cytosol as a 49 kDa precursor polypeptide (pMCAD), imported into mitochondria, and cleaved to the mature form. Genetic deficiency of MCAD causes recurrent episodes of hypoglycemic coma accompanied by medium chain dicarboxylic aciduria. Employing a novel approach, the authors now report isolation of partial rat and human cDNA clones encoding pMCAD. mRNA encoding pMCAD was purified to near homogeneity by polysome immunoadsorption using polyclonal monospecific antibody. Single-stranded [ 32 P]labeled cDNA probe was synthesized using the enriched mRNA as template, and was used to screen directly 16,000 colonies from a total rat liver cDNA library constructed in pBR322. One clone (600 bp) was detected by in situ hybridization. Hybrid-selected translation with this cDNA yielded a 49 kDa polypeptide indistinguishable in size from rat pMCAD and immunoprecipitable with anti-MCAD antibody. Using the rat cDNA as probe, 43,000 colonies from a human liver cDNA library were screened. Four identical positive clones (400 bp) were isolated and positively identified by hybrid-selected translation and immunoprecipitation. The sizes of rat and human mRNAs encoding pMCAD were 2.2 kb and 2.4 kb, respectively, as determined by Northern blotting

Determination of radioinduced delay in DNA synthesis in two-garlic-clones cells (Allium Sativum L.)

International Nuclear Information System (INIS)

Perez Lezcano, A.; Perez Talavera, S.

1989-01-01

To contribute to tech improvement of the use of ionizing radiations as an auxiliary tool in the fitoimprovement, dose-effect curves for the 'Martinez' and 'Sancti Spiritus-3' clones were stablished by using as effect the delay induced by radiations in DNA synthesis determined by the 'Martinez' clone which induces a delay of 50% in reference to the control is approximately 11 Gy, while the dose value for the 'Sancti Spiritus-3' clone is 18 Gy, thus the 'Martinez' clones has a higher sensitivity to radiations than the other clone, therefore it coincides with what we found for these clones other indexes are used as radiosensitivity criteria

Revisiting the identification and cDNA cloning of T cell-replacing factor/interleukin-5

Directory of Open Access Journals (Sweden)

Kiyoshi eTakatsu

2014-12-01

Full Text Available This is a perspective based on the paper Cloning of complementary DNA encoding T cell replacing factor and identity with B cell growth factor II, by Kinashi T, Harada N, Severinson E, Tanabe T, Sideras P, Konishi M, Azuma C, Tominaga A, Bergstedt-Lindqvist S, Takahashi M, Matsuda F, Yaoita Y, Takatsu K, and Honjo, T. Nature (1986 32(6092: 70-3. We have been interested in understanding the molecular basis of T-B cell cooperation for antibody formation. Although many investigators had described a number of different soluble factors that appeared to have biological relevance to T-B cell interactions, molecular basis of such active substances remained unknown for a long period of time. In this perspective, I will briefly summarize the history of the initial discovery of T cell-replacing factor/B cell growth factor II that appeared to be involved in B-cell growth and differentiation, and outline the discovery and characterization of interleukin-5. Studies of interleukin-5 have provided strong evidence that a single cytokine exerts a variety of activities on diverse target cells.

cDNA cloning and transcriptional controlling of a novel low dose radiation-induced gene and its function analysis

International Nuclear Information System (INIS)

Zhou Pingkun; Sui Jianli

2002-01-01

Objective: To clone a novel low dose radiation-induced gene (LRIGx) and study its function as well as its transcriptional changes after irradiation. Methods: Its cDNA was obtained by DDRT-PCR and RACE techniques. Northern blot hybridization was used to investigate the gene transcription. Bioinformatics was employed to analysis structure and function of this gene. Results: LRIGx cDNA was cloned. The sequence of LRIGx was identical to a DNA clone located in human chromosome 20 q 11.2-12 Bioinformatics analysis predicted an encoded protein with a conserved helicase domain. Northern analysis revealed a ∼8.5 kb transcript which was induced after 0.2 Gy as well as 0.02 Gy irradiation, and the transcript level was increased 5 times at 4 h after 0.2 Gy irradiation. The induced level of LRIGx transcript by 2.0 Gy high dose was lower than by 0.2 Gy. Conclusion: A novel low dose radiation-induced gene has been cloned. It encodes a protein with a conserved helicase domain that could involve in DNA metabolism in the cellular process of radiation response

cDNA cloning and immunological characterization of the rye grass allergen Lol p I.

Science.gov (United States)

Perez, M; Ishioka, G Y; Walker, L E; Chesnut, R W

1990-09-25

The complete amino acid sequence of two "isoallergenic" forms of Lol p I, the major rye grass (Lolium perenne) pollen allergen, was deduced from cDNA sequence analysis. cDNA clones isolated from a Lolium perenne pollen library contained an open reading frame coding for a 240-amino acid protein. Comparison of the nucleotide and deduced amino acid sequence of two of these clones revealed four changes at the amino acid level and numerous nucleotide differences. Both clones contained one possible asparagine-linked glycosylation site. Northern blot analysis shows one RNA species of 1.2 kilobases. Based on the complete amino acid sequence of Lol p I, overlapping peptides covering the entire molecule were synthesized. Utilizing these peptides we have identified a determinant within the Lol p I molecule that is recognized by human leukocyte antigen class II-restricted T cells obtained from persons allergic to rye grass pollen.

Molecular cloning and nucleotide sequence of cDNA for human liver arginase

International Nuclear Information System (INIS)

Haraguchi, Y.; Takiguchi, M.; Amaya, Y.; Kawamoto, S.; Matsuda, I.; Mori, M.

1987-01-01

Arginase (EC3.5.3.1) catalyzes the last step of the urea cycle in the liver of ureotelic animals. Inherited deficiency of the enzyme results in argininemia, an autosomal recessive disorder characterized by hyperammonemia. To facilitate investigation of the enzyme and gene structures and to elucidate the nature of the mutation in argininemia, the authors isolated cDNA clones for human liver arginase. Oligo(dT)-primed and random primer human liver cDNA libraries in λ gt11 were screened using isolated rat arginase cDNA as a probe. Two of the positive clones, designated λ hARG6 and λ hARG109, contained an overlapping cDNA sequence with an open reading frame encoding a polypeptide of 322 amino acid residues (predicted M/sub r/, 34,732), a 5'-untranslated sequence of 56 base pairs, a 3'-untranslated sequence of 423 base pairs, and a poly(A) segment. Arginase activity was detected in Escherichia coli cells transformed with the plasmid carrying λ hARG6 cDNA insert. RNA gel blot analysis of human liver RNA showed a single mRNA of 1.6 kilobases. The predicted amino acid sequence of human liver arginase is 87% and 41% identical with those of the rat liver and yeast enzymes, respectively. There are several highly conserved segments among the human, rat, and yeast enzymes

Bacterial community composition in different sediments from the Eastern Mediterranean Sea: a comparison of four 16S ribosomal DNA clone libraries.

Science.gov (United States)

Polymenakou, Paraskevi N; Bertilsson, Stefan; Tselepides, Anastasios; Stephanou, Euripides G

2005-10-01

The regional variability of sediment bacterial community composition and diversity was studied by comparative analysis of four large 16S ribosomal DNA (rDNA) clone libraries from sediments in different regions of the Eastern Mediterranean Sea (Thermaikos Gulf, Cretan Sea, and South lonian Sea). Amplified rDNA restriction analysis of 664 clones from the libraries indicate that the rDNA richness and evenness was high: for example, a near-1:1 relationship among screened clones and number of unique restriction patterns when up to 190 clones were screened for each library. Phylogenetic analysis of 207 bacterial 16S rDNA sequences from the sediment libraries demonstrated that Gamma-, Delta-, and Alphaproteobacteria, Holophaga/Acidobacteria, Planctomycetales, Actinobacteria, Bacteroidetes, and Verrucomicrobia were represented in all four libraries. A few clones also grouped with the Betaproteobacteria, Nitrospirae, Spirochaetales, Chlamydiae, Firmicutes, and candidate division OPl 1. The abundance of sequences affiliated with Gammaproteobacteria was higher in libraries from shallow sediments in the Thermaikos Gulf (30 m) and the Cretan Sea (100 m) compared to the deeper South Ionian station (2790 m). Most sequences in the four sediment libraries clustered with uncultured 16S rDNA phylotypes from marine habitats, and many of the closest matches were clones from hydrocarbon seeps, benzene-mineralizing consortia, sulfate reducers, sulk oxidizers, and ammonia oxidizers. LIBSHUFF statistics of 16S rDNA gene sequences from the four libraries revealed major differences, indicating either a very high richness in the sediment bacterial communities or considerable variability in bacterial community composition among regions, or both.

Molecular cloning and characterization of human papilloma virus DNA derived from a laryngeal papilloma.

OpenAIRE

Gissmann, L; Diehl, V; Schultz-Coulon, H J; zur Hausen, H

1982-01-01

Papilloma virus DNA from a laryngeal papilloma was cloned in phage lambda L 47 and characterized after cleavage with different restriction enzymes. Hybridization with the DNAs of human papilloma virus types 1, 2, 3, 4, 5, and 8 showed no homology under stringent hybridization conditions. Human papilloma virus type 6 DNA, however, was partially identical to laryngeal papilloma virus DNA; different restriction enzyme fragments hybridizing with the other DNA were identified on each genome. The d...

FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method

Directory of Open Access Journals (Sweden)

Lu Jia

2011-10-01

Full Text Available Abstract Background Although a variety of methods and expensive kits are available, molecular cloning can be a time-consuming and frustrating process. Results Here we report a highly simplified, reliable, and efficient PCR-based cloning technique to insert any DNA fragment into a plasmid vector or into a gene (cDNA in a vector at any desired position. With this method, the vector and insert are PCR amplified separately, with only 18 cycles, using a high fidelity DNA polymerase. The amplified insert has the ends with ~16-base overlapping with the ends of the amplified vector. After DpnI digestion of the mixture of the amplified vector and insert to eliminate the DNA templates used in PCR reactions, the mixture is directly transformed into competent E. coli cells to obtain the desired clones. This technique has many advantages over other cloning methods. First, it does not need gel purification of the PCR product or linearized vector. Second, there is no need of any cloning kit or specialized enzyme for cloning. Furthermore, with reduced number of PCR cycles, it also decreases the chance of random mutations. In addition, this method is highly effective and reproducible. Finally, since this cloning method is also sequence independent, we demonstrated that it can be used for chimera construction, insertion, and multiple mutations spanning a stretch of DNA up to 120 bp. Conclusion Our FastCloning technique provides a very simple, effective, reliable, and versatile tool for molecular cloning, chimera construction, insertion of any DNA sequences of interest and also for multiple mutations in a short stretch of a cDNA.

cDNA cloning and mRNA expression of heat shock protein 70 gene ...

African Journals Online (AJOL)

In this study, the full-length heat shock protein 70 of Tegillarca granosa was cloned from cDNA library by rapid amplification of cDNA end (RACE). The open reading frame (ORF) of heat shock protein 70 was 1968 bp, and it encoded a protein of 655 amino acids with a predicted molecular weight of 71.48 kDa and an ...

Cloning and expression of cDNA coding for bouganin.

Science.gov (United States)

den Hartog, Marcel T; Lubelli, Chiara; Boon, Louis; Heerkens, Sijmie; Ortiz Buijsse, Antonio P; de Boer, Mark; Stirpe, Fiorenzo

2002-03-01

Bouganin is a ribosome-inactivating protein that recently was isolated from Bougainvillea spectabilis Willd. In this work, the cloning and expression of the cDNA encoding for bouganin is described. From the cDNA, the amino-acid sequence was deduced, which correlated with the primary sequence data obtained by amino-acid sequencing on the native protein. Bouganin is synthesized as a pro-peptide consisting of 305 amino acids, the first 26 of which act as a leader signal while the 29 C-terminal amino acids are cleaved during processing of the molecule. The mature protein consists of 250 amino acids. Using the cDNA sequence encoding the mature protein of 250 amino acids, a recombinant protein was expressed, purified and characterized. The recombinant molecule had similar activity in a cell-free protein synthesis assay and had comparable toxicity on living cells as compared to the isolated native bouganin.

Cloning, sequencing and expression of a novel xylanase cDNA from ...

African Journals Online (AJOL)

A strain SH 2016, capable of producing xylanase, was isolated and identified as Aspergillus awamori, based on its physiological and biochemical characteristics as well as its ITS rDNA gene sequence analysis. A xylanase gene of 591 bp was cloned from this newly isolated A. awamori and the ORF sequence predicted a ...

Characterization of a pollen-specific cDNA clone from Nicotiana tabacum expressed during microgametogenesis and germination.

Science.gov (United States)

Weterings, K; Reijnen, W; van Aarssen, R; Kortstee, A; Spijkers, J; van Herpen, M; Schrauwen, J; Wullems, G

1992-04-01

This report describes the isolation and characterization of a cDNA clone representing a gene specifically expressed in pollen. A cDNA library was constructed against mRNA from mature pollen of Nicotiana tabacum. It was screened differentially against cDNA from mRNA of leaf and of pollen. One clone, NTPc303, was further characterized. On northern blot this clone hybridizes to a transcript 2100 nucleotides in length. NTPc303 is abundant in pollen. Expression of the corresponding gene is restricted to pollen, because no other generative or vegetative tissue contains transcripts hybridizing to NTPc303. Expression of NTP303 is evolutionarily conserved: homologous transcripts are present in pollen from various plant species. The first NTP303 transcripts are detectable on northern blot at the early bi-nucleate stage and accumulate until the pollen has reached maturity. During germination and pollen tube growth in vitro new NTP303 transcripts appear. This transcription has been proved by northern blots as well as by pulse labelling experiments. Nucleotide sequence analysis revealed that NTPc303 has an open reading frame coding for a predicted protein of 62 kDa. This protein shares homology to ascorbate oxidase and other members of the blue copper oxidase family. A possible function for this clone during pollen germination is discussed.

Construction and characterization of the alpha form of a cardiac myosin heavy chain cDNA clone and its developmental expression in the Syrian hamster.

OpenAIRE

Liew, C C; Jandreski, M A

1986-01-01

A cDNA clone, pVHC1, was isolated from a Syrian hamster heart cDNA library and was compared to the rat alpha (pCMHC21) and beta (pCMHC5) ventricular myosin heavy chain cDNA clones. The DNA sequence and amino acid sequence deducted from the DNA show more homology with pCMHC21 than pCMHC5. This indicates that pVHC1 is an alpha ventricular myosin heavy chain cDNA clone. However, even though pVHC1 shows a high degree of nucleotide and amino acid conservation with the rat myosin heavy chain sequen...

cDNA cloning and primary structure analysis of invariant chain in ...

African Journals Online (AJOL)

cDNA cloning and primary structure analysis of invariant chain in Chinese Pengze crucian carp. X Liu, W Yu, J Li, F Chen, S Liu, C Wu, J Xu. Abstract. Invariant chain (Ii) plays an important role in MHC class II molecules assembly and exogenous peptide presentation in vertebrates. Although mammalian Ii has been ...

16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

Science.gov (United States)

The bacterial composition of chlorinated drinking water was analyzed using 16S rRNA gene clone libraries derived from DNA extracts of 12 samples and compared to clone libraries previously generated using RNA extracts from the same samples. Phylogenetic analysis of 761 DNA-based ...

Characterization and immunological identification of cDNA clones encoding two human DNA topoisomerase II isozymes

International Nuclear Information System (INIS)

Chung, T.D.Y.; Drake, F.H.; Tan, K.B.; Per, S.R.; Crooke, S.T.; Mirabelli, C.K.

1989-01-01

Several DNA topoisomerase II partial cDNA clones obtained from a human Raji-HN2 cDNA library were sequenced and two classes of nucleotide sequences were found. One member of the first class, SP1, was identical to an internal fragment of human HeLa cell Topo II cDNA described earlier. A member of the second class, SP11, shared extensive nucleotide (75%) and predicted peptide (92%) sequence similarities with the first two-thirds of HeLa Topo II. Each class of cDNAs hybridized to unique, nonoverlapping restriction enzyme fragments of genomic DNA from several human cell lines. Synthetic 24-mer oligonucleotide probes specific for each cDNA class hybridized to 6.5-kilobase mRNAs; furthermore, hybridization of probe specific for one class was not blocked by probe specific for the other. Antibodies raised against a synthetic SP1-encoded dodecapeptide specifically recognized the 170-kDa form of Topo II, while antibodies raised against the corresponding SP11-encoded dodecapeptide, or a second unique SP11-encoded tridecapeptide, selectively recognized the 180-kDa form of Topo II. These data provide genetic and immunochemical evidence for two Topo II isozymes

Toward a molecular cytogenetic map for cultivated sunflower (Helianthus annuus L.) by landed BAC/BIBAC clones.

Science.gov (United States)

Feng, Jiuhuan; Liu, Zhao; Cai, Xiwen; Jan, Chao-Chien

2013-01-01

Conventional karyotypes and various genetic linkage maps have been established in sunflower (Helianthus annuus L., 2n = 34). However, the relationship between linkage groups and individual chromosomes of sunflower remains unknown and has considerable relevance for the sunflower research community. Recently, a set of linkage group-specific bacterial /binary bacterial artificial chromosome (BAC/BIBAC) clones was identified from two complementary BAC and BIBAC libraries constructed for cultivated sunflower cv. HA89. In the present study, we used these linkage group-specific clones (~100 kb in size) as probes to in situ hybridize to HA89 mitotic chromosomes at metaphase using the BAC-fluorescence in situ hybridization (FISH) technique. Because a characteristic of the sunflower genome is the abundance of repetitive DNA sequences, a high ratio of blocking DNA to probe DNA was applied to hybridization reactions to minimize the background noise. As a result, all sunflower chromosomes were anchored by one or two BAC/BIBAC clones with specific FISH signals. FISH analysis based on tandem repetitive sequences, such as rRNA genes, has been previously reported; however, the BAC-FISH technique developed here using restriction fragment length polymorphism (RFLP)-derived BAC/BIBAC clones as probes to apply genome-wide analysis is new for sunflower. As chromosome-specific cytogenetic markers, the selected BAC/BIBAC clones that encompass the 17 linkage groups provide a valuable tool for identifying sunflower cytogenetic stocks (such as trisomics) and tracking alien chromosomes in interspecific crosses. This work also demonstrates the potential of using a large-insert DNA library for the development of molecular cytogenetic resources.

Cloning and characterization of BKV(MM) DNA and its use for detection of BKV DNA in human urine

International Nuclear Information System (INIS)

Harley, E.H.; Olliver, C.L.; Rhodes-Harrison, L.; Mew, R.T.; Lecatsas, G.; Naude, W. du T.

1982-01-01

The two fragments produced by restriction of BKV(MM) DNA with the endonucleases Pst I and Eco RI have been cloned separately into the vector pBR322 and amplified in E. coli HB101. Eight recombinant plasmids were characterized by gel electrophoresis of Pst I/Eco RI double digestions or Hind III digestions of the DNA and by hybridization of Southern gel blots to a nick-translated BKV(MM) DNA probe. Four of the recombinant plasmids contained the large Pst I/Eco RI BKV(MM) DNA fragment and four contained the small fragment. Two of these recombinant plasmids were then used to make a probe for the identification of BK DNA in a urine specimen from a patient known to be exreting particles with the morphological features of papovavirus [af

Cloning and chromosomal assignment of a human cDNA encoding a T cell- and natural killer cell-specific trypsin-like serine protease

International Nuclear Information System (INIS)

Gershenfeld, H.K.; Hershberger, R.J.; Shows, T.B.; Weissman, I.L.

1988-01-01

A cDNA clone encoding a human T cell- and natural killer cell-specific serine protease was obtained by screening a phage λgt10 cDNA library from phytohemagglutinin-stimulated human peripheral blood lymphocytes with the mouse Hanukah factor cDNA clone. In an RNA blot-hybridization analysis, this human Hanukah factor cDNA hybridized with a 1.3-kilobase band in allogeneic-stimulated cytotoxic T cells and the Jurkat cell line, but this transcript was not detectable in normal muscle, liver, tonsil, or thymus. By dot-blot hybridization, this cDNA hybridized with RNA from three cytolytic T-cell clones and three noncytolytic T-cell clones grown in vitro as well as with purified CD16 + natural killer cells and CD3 + , CD16 - T-cell large granular lymphocytes from peripheral blood lymphocytes (CD = cluster designation). The nucleotide sequence of this cDNA clone encodes a predicted serine protease of 262 amino acids. The active enzyme is 71% and 77% similar to the mouse sequence at the amino acid and DNA level, respectively. The human and mouse sequences conserve the active site residues of serine proteases--the trypsin-specific Asp-189 and all 10 cysteine residues. The gene for the human Hanukah factor serine protease is located on human chromosome 5. The authors propose that this trypsin-like serine protease may function as a common component necessary for lysis of target cells by cytotoxic T lymphocytes and natural killer cells

Molecular cloning of a mouse DNA repair gene that complements the defect of group-A xeroderma pigmentosum

International Nuclear Information System (INIS)

Tanaka, K.; Satokata, I.; Ogita, Z.; Uchida, T.; Okada, Y.

1989-01-01

For isolation of the gene responsible for xeroderma pigmentosum (XP) complementation group A, plasmid pSV2gpt and genomic DNA from a mouse embryo were cotransfected into XP2OSSV cells, a group-A XP cell line. Two primary UV-resistant XP transfectants were isolated from about 1.6 X 10(5) pSV2gpt-transformed XP colonies. pSV2gpt and genomic DNA from the primary transfectants were again cotransfected into XP2OSSV cells and a secondary UV-resistant XP transfectant was obtained by screening about 4.8 X 10(5) pSV2gpt-transformed XP colonies. The secondary transfectant retained fewer mouse repetitive sequences. A mouse gene that complements the defect of XP2OSSV cells was cloned into an EMBL3 vector from the genome of a secondary transfectant. Transfections of the cloned DNA also conferred UV resistance on another group-A XP cell line but not on XP cell lines of group C, D, F, or G. Northern blot analysis of poly(A)+ RNA with a subfragment of cloned mouse DNA repair gene as the probe revealed that an approximately 1.0 kilobase mRNA was transcribed in the donor mouse embryo and secondary transfectant, and approximately 1.0- and approximately 1.3-kilobase mRNAs were transcribed in normal human cells, but none of these mRNAs was detected in three strains of group-A XP cells. These results suggest that the cloned DNA repair gene is specific for group-A XP and may be the mouse homologue of the group-A XP human gene

ISOLASI cDNA SUCROSE TRANSPORTER (SUT DARI BATANG TANAMAN TEBU (Saccharum officinarum L.

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- Slameto

2010-09-01

Full Text Available Sucrose Transporter (SUT is kind of protein transporter that control in sucrose translocation. Sucrose Transporter is intermediate in translocation of sucrose from apoplasmic to simplasmic. SUT facilitates sucrose transportation from vascular tissues to parenchyma cells toward in node sugarcane stem. This research was purposed to isolate cDNA SUT from sugarcane stem, and cloned in Escherichia coli strain DH5α. Total RNA of sugarcane stem was isolated by single step method, then add with oligo dT in order to obtain the first strand of SUT cDNA then used as template for PCR. The primer used for PCR is 5’ –ggg ctg att gtg gcc atg tc- ‘3 (SUT-F and 5’ –tgc cct ttg tct ccg gaa cc- ‘3 (SUT-R. PCR was programmed as follow denaturation at 94°C for 2 minutes and 30 second, annealing at 54°C for 30 s, extension at 72°C 2 min and 7 min, and storage at 4°C for unlimited, It was for 30 cycles. Complementary DNA SUT from PCR ligalized to pTOPO bunt-end, then it cloned in to E. coli strain DH5α. The cloning resulted then be sequenced in order to observe the homologues with other nucleotides sequences of some plant using BLASTn program in GENE BANK NCBI and the level of homology determined by Genetyx program. The concentrated of total RNA isolated was 5,024 μg/μl, with purity of 1,85. Complementary DNA SUT fragment from PCR with size 2037 bp appropriated to the both of primer was used. Complementary DNA SUT fragment showed by analyzed some of restriction enzyme e.g. EcoRI, PstI and BamHI. Homologues of this cDNA SUT fragment was 100% to SoSUT 2A of sugarcane stem and 84% to OsSUT of rice plant (Casu et al ., 2003.

Human placental Na/sup +/, K/sup +/-ATPase. cap alpha. subunit: cDNA cloning, tissue expression, DNA polymorphism, and chromosomal localization

Energy Technology Data Exchange (ETDEWEB)

Chehab, F.F.; Kan, Y.W.; Law, M.L.; Hartz, J.; Kao, F.T.; Blostein, R.

1987-11-01

A 2.2-kilobase clone comprising a major portion of the coding sequence of the Na/sup +/, K/sup +/-ATPase ..cap alpha.. subunit was cloned from human placenta and its sequence was identical to that encoding the ..cap alpha.. subunit of human kidney and HeLa cells. Transfer blot analysis of the mRNA products of the Na/sup +/, K/sup +/-ATPase gene from various human tissues and cell lines revealed only one band (approx. = 4.7 kilobases) under low and high stringency washing conditions. The levels of expression in the tissues were intestine > placenta > liver > pancreas, and in the cell lines the levels were human erythroleukemia > butyrate-induced colon > colon > brain > HeLa cells. mRNA was undetectable in reticulocytes, consistent with the authors failure to detect positive clones in a size-selected ( > 2 kilobases) lambdagt11 reticulocyte cDNA library. DNA analysis revealed by a polymorphic EcoRI band and chromosome localization by flow sorting and in situ hybridization showed that the ..cap alpha.. subunit is on the short is on the short arm (band p11-p13) of chromosome 1.

Osteoponin Promoter Controlled by DNA Methylation: Aberrant Methylation in Cloned Porcine Genome

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Chih-Jie Shen

2014-01-01

Full Text Available Cloned animals usually exhibited many defects in physical characteristics or aberrant epigenetic reprogramming, especially in some important organ development. Osteoponin (OPN is an extracellular-matrix protein involved in heart and bone development and diseases. In this study, we investigated the correlation between OPN mRNA and its promoter methylation changes by the 5-aza-dc treatment in fibroblast cell and promoter assay. Aberrant methylation of porcine OPN was frequently found in different tissues of somatic nuclear transferred cloning pigs, and bisulfite sequence data suggested that the OPN promoter region −2615 to −2239 nucleotides (nt may be a crucial regulation DNA element. In pig ear fibroblast cell culture study, the demethylation of OPN promoter was found in dose-dependent response of 5-aza-dc treatment and followed the OPN mRNA reexpression. In cloned pig study, discrepant expression pattern was identified in several cloned pig tissues, especially in brain, heart, and ear. Promoter assay data revealed that four methylated CpG sites presenting in the −2615 to −2239 nt region cause significant downregulation of OPN promoter activity. These data suggested that methylation in the OPN promoter plays a crucial role in the regulation of OPN expression that we found in cloned pigs genome.

Genomic organization and developmental fate of adjacent repeated sequences in a foldback DNA clone of Tetrahymena thermophila

International Nuclear Information System (INIS)

Tschunko, A.H.; Loechel, R.H.; McLaren, N.C.; Allen, S.L.

1987-01-01

DNA sequence elimination and rearrangement occurs during the development of somatic cell lineages of eukaryotes and was first discovered over a century ago. However, the significance and mechanism of chromatin elimination are not understood. DNA elimination also occurs during the development of the somatic macronucleus from the germinal micronucleus in unicellular ciliated protozoa such as Tetrahymena thermophila. In this study foldback DNA from the micronucleus was used as a probe to isolate ten clones. All of those tested (4/4) contained sequences that were repetitive in the micronucleus and rearranged in the macronucleus. Inverted repeated sequences were present in one clone. This clone, pTtFBl, was subjected to a detailed analysis of its developmental fate. Subregions were subcloned and used as probes against Southern blots of micronuclear and macronuclear DNA. DNA was labeled with [ 33 P]-labeled dATP. The authors found that all subregions defined repeated sequence families in the micronuclear genome. A minimum of four different families was defined, two of which are retained in the macronucleus and two of which are completely eliminated. The inverted repeat family is retained with little rearrangement. Two of the families, defined by subregions that do not contain parts of the inverted repeat are totally eliminated during macronuclear development-and contain open reading frames. The significance of retained inverted repeats to the process of elimination is discussed

Survival of Skin Graft between Transgenic Cloned Dogs and Non-Transgenic Cloned Dogs

Science.gov (United States)

Kim, Geon A; Oh, Hyun Ju; Kim, Min Jung; Jo, Young Kwang; Choi, Jin; Park, Jung Eun; Park, Eun Jung; Lim, Sang Hyun; Yoon, Byung Il; Kang, Sung Keun; Jang, Goo; Lee, Byeong Chun

2014-01-01

Whereas it has been assumed that genetically modified tissues or cells derived from somatic cell nuclear transfer (SCNT) should be accepted by a host of the same species, their immune compatibility has not been extensively explored. To identify acceptance of SCNT-derived cells or tissues, skin grafts were performed between cloned dogs that were identical except for their mitochondrial DNA (mtDNA) haplotypes and foreign gene. We showed here that differences in mtDNA haplotypes and genetic modification did not elicit immune responses in these dogs: 1) skin tissues from genetically-modified cloned dogs were successfully transplanted into genetically-modified cloned dogs with different mtDNA haplotype under three successive grafts over 63 days; and 2) non-transgenic cloned tissues were accepted into transgenic cloned syngeneic recipients with different mtDNA haplotypes and vice versa under two successive grafts over 63 days. In addition, expression of the inserted gene was maintained, being functional without eliciting graft rejection. In conclusion, these results show that transplanting genetically-modified tissues into normal, syngeneic or genetically-modified recipient dogs with different mtDNA haplotypes do not elicit skin graft rejection or affect expression of the inserted gene. Therefore, therapeutically valuable tissue derived from SCNT with genetic modification might be used safely in clinical applications for patients with diseased tissues. PMID:25372489

Construction and Cloning of Reporter-Tagged Replicon cDNA for an In Vitro Replication Study of Murine Norovirus-1 (MNV-1).

Science.gov (United States)

Ahmad, Muhammad Khairi; Tabana, Yasser M; Ahmed, Mowaffaq Adam; Sandai, Doblin Anak; Mohamed, Rafeezul; Ismail, Ida Shazrina; Zulkiflie, Nurulisa; Yunus, Muhammad Amir

2017-12-01

A norovirus maintains its viability, infectivity and virulence by its ability to replicate. However, the biological mechanisms of the process remain to be explored. In this work, the NanoLuc™ Luciferase gene was used to develop a reporter-tagged replicon system to study norovirus replication. The NanoLuc™ Luciferase reporter protein was engineered to be expressed as a fusion protein for MNV-1 minor capsid protein, VP2. The foot-and-mouth disease virus 2A (FMDV2A) sequence was inserted between the 3'end of the reporter gene and the VP2 start sequence to allow co-translational 'cleavage' of fusion proteins during intracellular transcript expression. Amplification of the fusion gene was performed using a series of standard and overlapping polymerase chain reactions. The resulting amplicon was then cloned into three readily available backbones of MNV-1 cDNA clones. Restriction enzyme analysis indicated that the NanoLucTM Luciferase gene was successfully inserted into the parental MNV-1 cDNA clone. The insertion was further confirmed by using DNA sequencing. NanoLuc™ Luciferase-tagged MNV-1 cDNA clones were successfully engineered. Such clones can be exploited to develop robust experimental assays for in vitro assessments of viral RNA replication.

Construction and biological activities of the first infectious cDNA clones of the genus Foveavirus

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Meng, Baozhong, E-mail: bmeng@uoguelph.ca [Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road, Guelph, Ontario, Canada N1G2W1 (Canada); Venkataraman, Srividhya; Li, Caihong; Wang, Weizhou [Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road, Guelph, Ontario, Canada N1G2W1 (Canada); Dayan-Glick, Cathy; Mawassi, Munir [The Plant Pathology Department-The Virology Unit, Plant Protection Institute, Agricultural Research Organization, The Volcani Center, Bet-Dagan 50250 (Israel)

2013-01-20

Grapevine rupestris stem pitting-associated virus (GRSPaV, genus Foveavirus, family Betaflexiviridae) is one of the most prevalent viruses in grapevines and is associated with three distinct diseases: rupestris stem pitting, vein necrosis and Syrah decline. Little is known about the biology and pathological properties of GRSPaV. In this work, we engineered a full-length infectious cDNA clone for GRSPaV and a GFP-tagged variant, both under the transcriptional control of Cauliflower mosaic virus 35 S promoter. We demonstrated that these cDNA clones were infectious in grapevines and Nicotiana benthamiana through fluorescence microscopy, RT-PCR, Western blotting and immuno electron microscopy. Interestingly, GRSPaV does not cause systemic infection in four of the most commonly used herbaceous plants, even in the presence of the movement proteins of two other viruses which are known to complement numerous movement-defective viruses. These infectious clones are the first of members of Foveavirus which would allow further investigations into mechanisms governing different aspects of replication for GRSPaV and perhaps related viruses.

Development of three full-length infectious cDNA clones of distinct brassica yellows virus genotypes for agrobacterium-mediated inoculation.

Science.gov (United States)

Zhang, Xiao-Yan; Dong, Shu-Wei; Xiang, Hai-Ying; Chen, Xiang-Ru; Li, Da-Wei; Yu, Jia-Lin; Han, Cheng-Gui

2015-02-02

Brassica yellows virus is a newly identified species in the genus of Polerovirus within the family Luteoviridae. Brassica yellows virus (BrYV) is prevalently distributed throughout Mainland China and South Korea, is an important virus infecting cruciferous crops. Based on six BrYV genomic sequences of isolates from oilseed rape, rutabaga, radish, and cabbage, three genotypes, BrYV-A, BrYV-B, and BrYV-C, exist, which mainly differ in the 5' terminal half of the genome. BrYV is an aphid-transmitted and phloem-limited virus. The use of infectious cDNA clones is an alternative means of infecting plants that allows reverse genetic studies to be performed. In this study, full-length cDNA clones of BrYV-A, recombinant BrYV5B3A, and BrYV-C were constructed under control of the cauliflower mosaic virus 35S promoter. An agrobacterium-mediated inoculation system of Nicotiana benthamiana was developed using these cDNA clones. Three days after infiltration with full-length BrYV cDNA clones, necrotic symptoms were observed in the inoculated leaves of N. benthamiana; however, no obvious symptoms appeared in the upper leaves. Reverse transcription-PCR (RT-PCR) and western blot detection of samples from the upper leaves showed that the maximum infection efficiency of BrYVs could reach 100%. The infectivity of the BrYV-A, BrYV-5B3A, and BrYV-C cDNA clones was further confirmed by northern hybridization. The system developed here will be useful for further studies of BrYV, such as host range, pathogenicity, viral gene functions, and plant-virus-vector interactions, and especially for discerning the differences among the three genotypes. Copyright © 2014 Elsevier B.V. All rights reserved.

DNA-decorated carbon-nanotube-based chemical sensors on complementary metal oxide semiconductor circuitry

International Nuclear Information System (INIS)

Chen, Chia-Ling; Yang, Chih-Feng; Dokmeci, Mehmet R; Agarwal, Vinay; Sonkusale, Sameer; Kim, Taehoon; Busnaina, Ahmed; Chen, Michelle

2010-01-01

We present integration of single-stranded DNA (ss-DNA)-decorated single-walled carbon nanotubes (SWNTs) onto complementary metal oxide semiconductor (CMOS) circuitry as nanoscale chemical sensors. SWNTs were assembled onto CMOS circuitry via a low voltage dielectrophoretic (DEP) process. Besides, bare SWNTs are reported to be sensitive to various chemicals, and functionalization of SWNTs with biomolecular complexes further enhances the sensing specificity and sensitivity. After decorating ss-DNA on SWNTs, we have found that the sensing response of the gas sensor was enhanced (up to ∼ 300% and ∼ 250% for methanol vapor and isopropanol alcohol vapor, respectively) compared with bare SWNTs. The SWNTs coupled with ss-DNA and their integration on CMOS circuitry demonstrates a step towards realizing ultra-sensitive electronic nose applications.

A Polymerase Chain Reaction-Based Method for Isolating Clones from a Complimentary DNA Library in Sheep

Science.gov (United States)

Friis, Thor Einar; Stephenson, Sally; Xiao, Yin; Whitehead, Jon

2014-01-01

The sheep (Ovis aries) is favored by many musculoskeletal tissue engineering groups as a large animal model because of its docile temperament and ease of husbandry. The size and weight of sheep are comparable to humans, which allows for the use of implants and fixation devices used in human clinical practice. The construction of a complimentary DNA (cDNA) library can capture the expression of genes in both a tissue- and time-specific manner. cDNA libraries have been a consistent source of gene discovery ever since the technology became commonplace more than three decades ago. Here, we describe the construction of a cDNA library using cells derived from sheep bones based on the pBluescript cDNA kit. Thirty clones were picked at random and sequenced. This led to the identification of a novel gene, C12orf29, which our initial experiments indicate is involved in skeletal biology. We also describe a polymerase chain reaction-based cDNA clone isolation method that allows the isolation of genes of interest from a cDNA library pool. The techniques outlined here can be applied in-house by smaller tissue engineering groups to generate tools for biomolecular research for large preclinical animal studies and highlights the power of standard cDNA library protocols to uncover novel genes. PMID:24447069

Microsatellite DNA fingerprinting, differentiation, and genetic relationships of clones, cultivars, and varieties of six poplar species from three sections of the genus Populus.

Science.gov (United States)

Rahman, Muhammad H; Rajora, Om P

2002-12-01

Accurate identification of Populus clones and cultivars is essential for effective selection, breeding, and genetic resource management programs. The unit of cultivation and breeding in poplars is a clone, and individual cultivars are normally represented by a single clone. Microsatellite DNA markers of 10 simple sequence repeat loci were used for genetic fingerprinting and differentiation of 96 clones/cultivars and varieties belonging to six Populus species (P. deltoides, P. nigra, P. balsamifera, P. trichocarpa, P. grandidentata, and P maximowiczii) from three sections of the genus. All 96 clones/cultivars could be uniquely fingerprinted based on their single- or multilocus microsatellite genotypes. The five P. grandidentata clones could be differentiated based on their single-locus genotypes, while six clones of P. trichocarpa and 11 clones of P. maximowiczii could be identified by their two-locus genotypes. Twenty clones of P. deltoides and 25 clones of P. nigra could be differentiated by their multilocus genotypes employing three loci, and 29 clones of P. balsamifera required the use of multilocus genotypes at five loci for their genetic fingerprinting and differentiation. The loci PTR3, PTR5, and PTR7 were found to be the most informative for genetic fingerprinting and differentiation of the clones. The mean number of alleles per locus ranged from 2.9 in P. trichocarpa or P. grandidentata to 6.0 in P. balsamifera and 11.2 in 96 clones of the six species. The mean number of observed genotypes per locus ranged from 2.4 in P. grandidentata to 7.4 in P. balsamifera and 19.6 in 96 clones of the six species. The mean number of unique genotypes per locus ranged from 1.3 in P. grandidentata to 3.9 in P. deltoides and 8.8 in 96 clones of the six species. The power of discrimination of the microsatellite DNA markers in the 96 clones ranged from 0.726 for PTR4 to 0.939 for PTR7, with a mean of 0.832 over the 10 simple sequence repeat loci. Clones/cultivars from the same

RFLP for Duchenne muscular dystrophy cDNA clone 30-2

Energy Technology Data Exchange (ETDEWEB)

Walker, A P; Bartlett, R J; Laing, N G; Siddique, T; Yamaoka, L H; Chen, J C; Hung, W Y; Roses, A D [Duke Univ. Medical Center, Durham, NC (USA)

1988-09-26

30-2 is one of 6 cDNA clones which comprise the cDNA for the Duchenne muscular dystrophy gene. It is a 1.15 kb fragment in the EcoRI site of Bluescribe. TaqI (T{down arrow}CGA) identifies two bands with alleles at 3.7 and 3.5 kb, as well as eight constant bands at 9.0, 7.5, 4.6, 3.6, 3.4, 2.5, 1.7 and 1.4 kb. The allele frequency was studied in 47 unrelated DMD males: 3.7 kb allele 0.45; and 3.5 kb allele 0.55. Co-dominant X-linked segregation was demonstrated in two 2-generation families. 1.1% agarose gels required to resolve the bands. The polymorphism is also recognized by PERT 87-15.

Cloning and analysis of the mouse Fanconi anemia group A cDNA and an overlapping penta zinc finger cDNA.

Science.gov (United States)

Wong, J C; Alon, N; Norga, K; Kruyt, F A; Youssoufian, H; Buchwald, M

2000-08-01

Despite the cloning of four disease-associated genes for Fanconi anemia (FA), the molecular pathogenesis of FA remains largely unknown. To study FA complementation group A using the mouse as a model system, we cloned and characterized the mouse homolog of the human FANCA cDNA. The mouse cDNA (Fanca) encodes a 161-kDa protein that shares 65% amino acid sequence identity with human FANCA. Fanca is located at the distal region of mouse chromosome 8 and has a ubiquitous pattern of expression in embryonic and adult tissues. Expression of the mouse cDNA in human FA-A cells restores the cellular drug sensitivity to normal levels. Thus, the expression pattern, protein structure, chromosomal location, and function of FANCA are conserved in the mouse. We also isolated a novel zinc finger protein, Zfp276, which has five C(2)H(2) domains. Interestingly, Zfp276 is situated in the Fanca locus, and the 3'UTR of its cDNA overlaps with the last four exons of Fanca in a tail-to-tail manner. Zfp276 is expressed in the same tissues as Fanca, but does not complement the mitomycin C (MMC)-sensitive phenotype of FA-A cells. The overlapping genomic organization between Zfp276 and Fanca may have relevance to the disease phenotype of FA. Copyright 2000 Academic Press.

The DNA-instability test as a specific marker of malignancy and its application to detect cancer clones in borderline malignancy

Directory of Open Access Journals (Sweden)

M Fukuda

2009-06-01

Full Text Available Recent progress in cytogenetic and biochemical mutator assay technologies has enabled us to detect single gene alterations and gross chromosomal rearrangements, and it became clear that all cancer cells are genetically unstable. In order to detect the genome-wide instability of cancer cells, a new simple method, the DNA-instability test, was developed. The methods to detect genomic instability so far reported have only demonstrated the presence of qualitative and quantitative alterations in certain specific genomic loci. In contrast to these commonly used methods to reveal the genomic instability at certain specific DNA regions, the newly introduced DNA-instability test revealed the presence of physical DNA-instability in the entire DNA molecule of a cancer cell nucleus as revealed by increased liability to denature upon HCl hydrolysis or formamide exposure. When this test was applied to borderline malignancies, cancer clones were detected in all cases at an early-stage of cancer progression. We proposed a new concept of “procancer” clones to define those cancer clones with “functional atypia” showing positivities for various cancer markers, as well as DNA-instability testing, but showing no remarkable ordinary “morphological atypia” which is commonly used as the basis of histopathological diagnosis of malignancy.

Molecular cloning and sequence analysis of growth hormone cDNA of Neotropical freshwater fish Pacu (Piaractus mesopotamicus

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Janeth Silva Pinheiro

2008-01-01

Full Text Available RT-PCR was used for amplifying Piaractus mesopotamicus growth hormone (GH cDNA obtained from mRNA extracted from pituitary cells. The amplified fragment was cloned and the complete cDNA sequence was determined. The cloned cDNA encompassed a sequence of 543 nucleotides that encoded a polypeptide of 178 amino acids corresponding to mature P. mesopotamicus GH. Comparison with other GH sequences showed a gap of 10 amino acids localized in the N terminus of the putative polypeptide of P. mesopotamicus. This same gap was also observed in other members of the family. Neighbor-joining tree analysis with GH sequences from fishes belonging to different taxonomic groups placed the P. mesopotamicus GH within the Otophysi group. To our knowledge, this is the first GH sequence of a Neotropical characiform fish deposited in GenBank.

Purification, characterization and molecular cloning of TGP1, a novel G-DNA binding protein from Tetrahymena thermophila.

OpenAIRE

Lu, Q; Schierer, T; Kang, S G; Henderson, E

1998-01-01

G-DNA, a polymorphic family of four-stranded DNA structures, has been proposed to play roles in a variety of biological processes including telomere function, meiotic recombination and gene regulation. Here we report the purification and cloning of TGP1, a G-DNA specific binding protein from Tetrahymena thermophila. TGP1 was purified by three-column chromatographies, including a G-DNA affinity column. Two major proteins (approximately 80 and approximately 40 kDa) were present in the most high...

Cloning and expression of a human kidney cDNA for an α2-adrenergic receptor subtype

International Nuclear Information System (INIS)

Regan, J.W.; Kobilka, T.S.; Yang-Feng, T.L.; Caron, M.G.; Lefkowitz, R.J.; Kobilka, B.K.

1988-01-01

An α 2 -adrenergic receptor subtype has been cloned from a human kidney cDNA library using the gene for the human platelet α 2 -adrenergic receptor as a probe. The deduced amino acid sequence resembles the human platelet α 2 -adrenergic receptor and is consistent with the structure of other members of he family of guanine nucleotide-binding protein-coupled receptors. The cDNA was expressed in a mammalian cell line (COS-7), and the α 2 -adrenergic ligand [ 3 H]rauwolscine was bound. Competition curve analysis with a variety of adrenergic ligands suggests that this cDNA clone represents the α 2 B-adrenergic receptor. The gene for this receptor is on human chromosome 4, whereas the gene for the human platelet α 2 -adrenergic receptor (α 2 A) lies on chromosome 10. This ability to express the receptor in mammalian cells, free of other adrenergic receptor subtypes, should help in developing more selective α-adrenergic ligands

Cloning of the PYR3 gene of Ustilago maydis and its use in DNA transformation

Energy Technology Data Exchange (ETDEWEB)

Banks, G.R.; Taylor, S.Y. (National Institute for Medical Research, London (England))

1988-12-01

The Ustilago maydis PYR3 gene encoding dihydroorotase activity was cloned by direct complementation of Escherichia coli pyrC mutations. PYR3 transformants of E. coli pyrC mutants expressed homologous transcripts of a variety of sizes and regained dihydroorotase activity. PYR3 also complemented Saccharomyces cerevisiae ura4 mutations, and again multiple transcripts were expressed in transformants, and enzyme activity was regained. A 1.25-kilobase poly(rA)+ PYR3 transcript was detected in U. maydis itself. Linear DNA carrying the PYR3 gene transformed a U. maydis pyr3-1 pyrimidine auxotroph to prototrophy. Hybridization analysis revealed that three different types of transformants could be generated, depending on the structure of the transforming DNA used. The first type involved exchange of chromosomal mutant gene sequences with the cloned wild-type plasmid sequences. A second type had integrated linear transforming DNA at the chromosomal PYR3 locus, probably via a single crossover event. The third type had integrated transforming DNA sequences at multiple sites in the U. maydis genome. In the last two types, tandemly reiterated copies of the transforming DNA were found to have been integrated. All three types had lost the sensitivity of the parental pyr3-1 mutant to UV irradiation. They had also regained dihydroorotase activity, although its level did not correlate with the PYR3 gene copy number.

Cloning Should Be Simple: Escherichia coli DH5α-Mediated Assembly of Multiple DNA Fragments with Short End Homologies

Science.gov (United States)

Richardson, Ruth E.; Suzuki, Yo

2015-01-01

Numerous DNA assembly technologies exist for generating plasmids for biological studies. Many procedures require complex in vitro or in vivo assembly reactions followed by plasmid propagation in recombination-impaired Escherichia coli strains such as DH5α, which are optimal for stable amplification of the DNA materials. Here we show that despite its utility as a cloning strain, DH5α retains sufficient recombinase activity to assemble up to six double-stranded DNA fragments ranging in size from 150 bp to at least 7 kb into plasmids in vivo. This process also requires surprisingly small amounts of DNA, potentially obviating the need for upstream assembly processes associated with most common applications of DNA assembly. We demonstrate the application of this process in cloning of various DNA fragments including synthetic genes, preparation of knockout constructs, and incorporation of guide RNA sequences in constructs for clustered regularly interspaced short palindromic repeats (CRISPR) genome editing. This consolidated process for assembly and amplification in a widely available strain of E. coli may enable productivity gain across disciplines involving recombinant DNA work. PMID:26348330

[Fingerprints identification of Gynostemma pentaphyllum by RAPD and cloning and analysis of its specific DNA fragment].

Science.gov (United States)

Jiang, Jun-fu; Li, Xiong-ying; Wu, Yao-sheng; Luo, Yu; Zhao, Rui-qiang; Lan, Xiu-wan

2009-02-01

To identify the resources of Gynostemma pentaphyllum and its spurious breed plant Cayratia japonica at level of DNA. Two random primers ( WGS001, WGS004) screened were applied to do random amplification with genomic DNA extracted from Gynostemma pentaphyllum and Cayratia japonica which were collected from different habitats. After amplificated with WGS004, one characteristic fragment about 500 bp which was common to all Gynostemma pentaphyllum samples studied but not to Cayratia japonica was cloned and sequenced. Then these sequences obtained were analyzed for identity and compared by Blastn program in GenBank. There were obvious different bands amplified by above two primers in their fingerprints of genomic DNA. On the basis of these different bands of DNA fingerprints, they could distinguish Gynostemma pentaphyllum and Cayratia japonica obviously. Sequence alignment of seven cloned bands showed that their identities ranged from 45.7% - 94.5%. There was no similar genome sequences searched in GenBank. This indicated that these seven DNA fragments had not been reported before and they should be new sequences. RAPD technique can be used for the accurate identification of Gynostemma pentaphyllum and its counterfeit goods Cayratia japonica. Besides, these specific DNA sequences for Gynostemmna pentaphyllum in this study are useful for the further research on identification of species and assisted selection breeding in Gynostemma pentaphyllum.

The identification of specific cDNA clones from tall and dwarf rice plants

International Nuclear Information System (INIS)

Youssefian, S.; Kamada, I.; Sano, H.

1990-01-01

Full text: The use of dwarfing genes in rice breeding has proceeded for several years without a clear understanding of the genetic, hormonal and physiological mechanisms involved. This issue was addressed by focussing on the isolation of specific clones from tall- and dwarf-derived cDNA libraries. The materials used include near-isogenic lines of the tall rice cultivar 'Shiokari', differing at the DGWG or 'Tanginbozu' dwarfing gene loci. Also used were tall and dwarf 'Ginbozu' rice, the latter having been induced by treatment with 5-azacytidine, a potent demethylating agent. Subtractive and differential hybridisation have, to date, identified several candidate tall- and dwarf-specific clones. Their further characterisation is currently underway. (author)

Purification, cDNA Cloning, and Developmental Expression of the Nodule-Specific Uricase from Phaseolus vulgaris L. 1

Science.gov (United States)

Sánchez, Federico; Campos, Francisco; Padilla, Jaime; Bonneville, Jean-Marc; Enríquez, Consuelo; Caput, Daniel

1987-01-01

Nodule-specific uricase (uricase II) from Phaseolus vulgaris L. was purified to homogeneity by chromatographic methods. Purification data indicated that uricase II is approximately 2% of the total soluble protein from mature nodules. Specific antiserum was raised and used to determine the developmental expression and for immunoselection of polysomes. Uricase II was antigenically detected early in nodule development, 2 to 3 days before nitrogen fixation. Uricase-encoding cDNA clones were isolated by hybridizing a nodule-specific pUC9 cDNA library with labeled mRNA from immunoselected polysomes and a 35,000 molecular weight uricase II-encoding cDNA from soybean. An homologous clone (pNF-UR07) was used to assess the expression pattern of the specific transcript during development. Northern-blot analysis indicated that uricase II mRNA is exclusively expressed in nodule tissue. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:16665575

Complementation of the UV-sensitive phenotype of a xeroderma pigmentosum human cell line by transfection with a cDNA clone library

International Nuclear Information System (INIS)

Teitz, T.; Naiman, T.; Avissar, S.S.; Bar, S.; Okayama, H.; Canaani, D.

1987-01-01

In previous work, a xeroderma pigmentosum cell line belonging to complementation group C was established by transformation with origin-defective simian virus 40. We now report the complementation of the UV sensitivity of this cell line by gene transfer. A human cDNA clone library constructed in a mammalian expression vector, and itself incorporated in a lambda phage vector, was introduced into the cells as a calcium phosphate precipitate. Following selection to G418 resistance, provided by the neo gene of the vector, transformants were selected for UV resistance. Twenty-one cell clones were obtained with UV-resistance levels typical of normal human fibroblasts. All transformants contained vector DNA sequences in their nuclei. Upon further propagation in the absence of selection for G418 resistance, about half of the primary transformants remained UV-resistant. Secondary transformants were generated by transfection with a partial digest of total chromosomal DNA from one of these stable transformants. This resulted in 15 G418-resistant clones, 2 of which exhibited a UV-resistant phenotype. The other primary clones lost UV resistance rapidly when subcultured in the absence of G418. Importantly, several retained UV resistance under G418 selection pressure. The acquisition of UV resistance by secondary transformants derived by transfection of DNA from a stable primary transformant, and the linkage between G418 and UV resistances in the unstable primary transformants, strongly suggests that the transformants acquired UV resistance through DNA-mediated gene transfer and not by reversion

Cloning of oleosin, a putative new hazelnut allergen, using a hazelnut cDNA library

NARCIS (Netherlands)

Akkerdaas, Jaap H.; Schocker, Frauke; Vieths, Stefan; Versteeg, Serge; Zuidmeer, Laurian; Hefle, Sue L.; Aalberse, Rob C.; Richter, Klaus; Ferreira, Fatima; van Ree, Ronald

2006-01-01

The clinical presentation of non-pollen related allergy to hazelnut can be severe and systemic. So far, only a limited number of non-pollen related hazelnut allergens have been identified and characterized. The aim of this study was to identify and clone new hazelnut allergens. A lambda ZAP cDNA

Construction of a cDNA library from human retinal pigment epithelial cells challenged with rod outer segments.

Science.gov (United States)

Cavaney, D M; Rakoczy, P E; Constable, I J

1995-05-01

To study genes expressed by retinal pigment epithelial (RPE) cells during phagocytosis and digestion of rod outer segments (ROS), a complementary (c)DNA library was produced using an in-vitro model. The cDNA library can be used to study molecular changes which contribute to the development of diseases due to a failure in outer segment phagocytosis and digestion by RPE cells. Here we demonstrate a way to study genes and their functions using a molecular biological approach and describing the first step involved in this process, the construction of a cDNA library. Human RPE cells obtained from the eyes of a seven-year-old donor were cultured and challenged with bovine ROS. The culture was harvested and total RNA was extracted. Complementary DNA was transcribed from the messenger (m)RNA and was directionally cloned into the LambdaGEM-4 bacteriophage vector successfully. Some clones were picked and the DNA extracted, to determine the size of the inserts as a measure of the quality of the library. Molecular biology and cell culture are important tools to be used in eye research, especially in areas where tissue is limiting and animal models are not available. We now have a ROS challenged RPE cDNA library which will be used to identify genes responsible for degrading phagocytosed debris within the retinal pigment epithelium.

Molecular cloning and sequence analysis of hamster CENP-A cDNA

Directory of Open Access Journals (Sweden)

Valdivia Manuel M

2002-05-01

Full Text Available Abstract Background The centromere is a specialized locus that mediates chromosome movement during mitosis and meiosis. This chromosomal domain comprises a uniquely packaged form of heterochromatin that acts as a nucleus for the assembly of the kinetochore a trilaminar proteinaceous structure on the surface of each chromatid at the primary constriction. Kinetochores mediate interactions with the spindle fibers of the mitotic apparatus. Centromere protein A (CENP-A is a histone H3-like protein specifically located to the inner plate of kinetochore at active centromeres. CENP-A works as a component of specialized nucleosomes at centromeres bound to arrays of repeat satellite DNA. Results We have cloned the hamster homologue of human and mouse CENP-A. The cDNA isolated was found to contain an open reading frame encoding a polypeptide consisting of 129 amino acid residues with a C-terminal histone fold domain highly homologous to those of CENP-A and H3 sequences previously released. However, significant sequence divergence was found at the N-terminal region of hamster CENP-A that is five and eleven residues shorter than those of mouse and human respectively. Further, a human serine 7 residue, a target site for Aurora B kinase phosphorylation involved in the mechanism of cytokinesis, was not found in the hamster protein. A human autoepitope at the N-terminal region of CENP-A described in autoinmune diseases is not conserved in the hamster protein. Conclusions We have cloned the hamster cDNA for the centromeric protein CENP-A. Significant differences on protein sequence were found at the N-terminal tail of hamster CENP-A in comparison with that of human and mouse. Our results show a high degree of evolutionary divergence of kinetochore CENP-A proteins in mammals. This is related to the high diverse nucleotide repeat sequences found at the centromere DNA among species and support a current centromere model for kinetochore function and structural

cDNA cloning and nucleotide sequence comparison of Chinese hamster metallothionein I and II mRNAs

Energy Technology Data Exchange (ETDEWEB)

Griffith, B B; Walters, R A; Enger, M D; Hildebrand, C E; Griffith, J K

1983-01-01

Polyadenylated RNA was extracted from a cadmium resistant Chinese hamster (CHO) cell line, enriched for metal-induced, abundant RNA sequences and cloned as double-stranded cDNA in the plasmid pBR322. Two cDNA clones, pCHMT1 and pCHMT2, encoding two Chinese hamster isometallothioneins were identified, and the nucleotide sequence of each insert was determined. The two Chinese hamster metallothioneins show nucleotide sequence homologies of 80% in the protein coding region and approximately 35% in both the 5' and 3' untranslated regions. Interestingly, an 8 nucleotide sequence (TGTAAATA) has been conserved in sequence and position in the 3' untranslated regions of each metallothionein mRNA sequenced thus far. Estimated nucleotide substitution rates derived from interspecies comparisons were used to calculate a metallothionein gene duplication time of 45 to 120 million years ago. 39 references, 1 figure, 1 table.

Isolation and characterization of cDNA clones for human erythrocyte β-spectrin

International Nuclear Information System (INIS)

Prchal, J.T.; Morley, B.J.; Yoon, S.H.; Coetzer, T.L.; Palek, J.; Conboy, J.G.; Kan, Y.W.

1987-01-01

Spectrin is an important structural component of the membrane skeleton that underlies and supports the erythrocyte plasma membrane. It is composed of nonidentical α (M/sub r/ 240,000) and β (M/sub r/ 220,000) subunits, each of which contains multiple homologous 106-amino acid segments. The authors report here the isolation and characterization of a human erythroid-specific β-spectrin cDNA clone that encodes parts of the β-9 through β-12 repeat segments. This cDNA was used as a hybridization probe to assign the β-spectrin gene to human chromosome 14 and to begin molecular analysis of the gene and its mRNA transcripts. RNA transfer blot analysis showed that the reticulocyte β-spectrin mRNA is 7.8 kilobases in length. Southern blot analysis of genomic DNA revealed the presence of restriction fragment length polymorphisms (RFLPs) within the β-spectrin gene locus. The isolation of human spectrin cDNA probes and the identification of closely linked RFLPs will facilitate analysis of mutant spectrin genes causing congenital hemolytic anemias associated with quantitative and qualitative spectrin abnormalities

Complete amino acid sequence of human intestinal aminopeptidase N as deduced from cloned cDNA

DEFF Research Database (Denmark)

Cowell, G M; Kønigshøfer, E; Danielsen, E M

1988-01-01

The complete primary structure (967 amino acids) of an intestinal human aminopeptidase N (EC 3.4.11.2) was deduced from the sequence of a cDNA clone. Aminopeptidase N is anchored to the microvillar membrane via an uncleaved signal for membrane insertion. A domain constituting amino acid 250...

Cloning of the DNA Repair Gene, Uvsf, by Transformation of Aspergillus Nidulans

OpenAIRE

Oza, K.; Kafer, E.

1990-01-01

As a first step in the cloning of the DNA repair gene uvsF of Aspergillus nidulans, uvsF pyrG double mutant strains were transformed with a genomic library which carried the complementing Neurospora pyr-4 gene in the vector. Rare pyr(+) uvs(+) cotransformants were obtained on media lacking pyrimidines, overlayed with MMS (methyl-methane sulfonate) to which uvsF is hypersensitive. Among MMS-resistant transformants, Southerns revealed two types which showed single bands of different sizes when ...

3G vector-primer plasmid for constructing full-length-enriched cDNA libraries.

Science.gov (United States)

Zheng, Dong; Zhou, Yanna; Zhang, Zidong; Li, Zaiyu; Liu, Xuedong

2008-09-01

We designed a 3G vector-primer plasmid for the generation of full-length-enriched complementary DNA (cDNA) libraries. By employing the terminal transferase activity of reverse transcriptase and the modified strand replacement method, this plasmid (assembled with a polydT end and a deoxyguanosine [dG] end) combines priming full-length cDNA strand synthesis and directional cDNA cloning. As a result, the number of steps involved in cDNA library preparation is decreased while simplifying downstream gene manipulation, sequencing, and subcloning. The 3G vector-primer plasmid method yields fully represented plasmid primed libraries that are equivalent to those made by the SMART (switching mechanism at 5' end of RNA transcript) approach.

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

Directory of Open Access Journals (Sweden)

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.

New Approaches to Attenuated Hepatitis a Vaccine Development: Cloning and Sequencing of Cell-Culture Adapted Viral cDNA.

Science.gov (United States)

1987-10-13

after multiple passages in vivo and in vitro. J. Gen. Virol. 67, 1741- 1744. Sabin , A.B. (1985). Oral poliovirus vaccine : history of its development...IN (N NEW APPROACHES TO ATTENUATED HEPATITIS A VACCINE DEVELOPMENT: Q) CLONING AND SEQUENCING OF CELL-CULTURE ADAPTED VIRAL cDNA I ANNUAL REPORT...6ll02Bsl0 A 055 11. TITLE (Include Security Classification) New Approaches to Attenuated Hepatitis A Vaccine Development: Cloning and Sequencing of Cell

Parallel Sequencing of Expressed Sequence Tags from Two Complementary DNA Libraries for High and Low Phosphorus Adaptation in Common Beans

Directory of Open Access Journals (Sweden)

Matthew W. Blair

2011-11-01

Full Text Available Expressed sequence tags (ESTs have proven useful for gene discovery in many crops. In this work, our objective was to construct complementary DNA (cDNA libraries from root tissues of common beans ( L. grown under low and high P hydroponic conditions and to conduct EST sequencing and comparative analyses of the libraries. Expressed sequence tag analysis of 3648 clones identified 2372 unigenes, of which 1591 were annotated as known genes while a total of 465 unigenes were not associated with any known gene. Unigenes with hits were categorized according to biological processes, molecular function, and cellular compartmentalization. Given the young tissue used to make the root libraries, genes for catalytic activity and binding were highly expressed. Comparisons with previous root EST sequencing and between the two libraries made here resulted in a set of genes to study further for differential gene expression and adaptation to low P, such as a 14 kDa praline-rich protein, a metallopeptidase, tonoplast intrinsic protein, adenosine triphosphate (ATP citrate synthase, and cell proliferation genes expressed in the low P treated plants. Given that common beans are often grown on acid soils of the tropics and subtropics that are usually low in P these genes and the two parallel libraries will be useful for selection for better uptake of this essential macronutrient. The importance of EST generation for common bean root tissues under low P and other abiotic soil stresses is also discussed.

Molecular cloning and expression of the gene encoding the kinetoplast-associated type II DNA topoisomerase of Crithidia fasciculata.

Science.gov (United States)

Pasion, S G; Hines, J C; Aebersold, R; Ray, D S

1992-01-01

A type II DNA topoisomerase, topoIImt, was shown previously to be associated with the kinetoplast DNA of the trypanosomatid Crithidia fasciculata. The gene encoding this kinetoplast-associated topoisomerase has been cloned by immunological screening of a Crithidia genomic expression library with monoclonal antibodies raised against the purified enzyme. The gene CfaTOP2 is a single copy gene and is expressed as a 4.8-kb polyadenylated transcript. The nucleotide sequence of CfaTOP2 has been determined and encodes a predicted polypeptide of 1239 amino acids with a molecular mass of 138,445. The identification of the cloned gene is supported by immunoblot analysis of the beta-galactosidase-CfaTOP2 fusion protein expressed in Escherichia coli and by analysis of tryptic peptide sequences derived from purified topoIImt. CfaTOP2 shares significant homology with nuclear type II DNA topoisomerases of other eukaryotes suggesting that in Crithidia both nuclear and mitochondrial forms of topoisomerase II are encoded by the same gene.

Isolation and expression of human cytokine synthesis inhibitory factor cDNA clones: Homology to Epstein-Barr virus open reading frame BCRFI

Energy Technology Data Exchange (ETDEWEB)

Vieira, P.; De Waal-Malefyt, R.; Dang, M.N.; Johnson, K.E.; Kastelein, R.; Fiorentino, D.F.; DeVries, J.E.; Roncarolo, M.G.; Mosmann, T.R.; Moore, K.W. (DNAX Research Inst. of Molecular and Cellular Biology, Palo Alto, CA (United States))

1991-02-15

The authors demonstrated the existence of human cytokine synthesis inhibitory factor (DSIF) (interleukin 10 (IL-10)). cDNA clones encoding human IL-10 (hIL-10) were isolated from a tetanus toxin-specific human T-cell clone. Like mouse IL-10, hIL-10 exhibits strong DNA and amino acid sequence homology to an open reading frame in the Epstein-Barr virus, BDRFL. hIL-10 and the BCRFI product inhibit cytokine synthesis by activated human peripheral blood mononuclear cells and by a mouse Th1 clone. Both hIL-10 and mouse IL-10 sustain the viability of a mouse mast cell line in culture, but BCRFI lacks comparable activity in this way, suggesting that BCRFI may have conserved only a subset of hIL-10 activities.

Isolation and expression of human cytokine synthesis inhibitory factor cDNA clones: Homology to Epstein-Barr virus open reading frame BCRFI

International Nuclear Information System (INIS)

Vieira, P.; De Waal-Malefyt, R.; Dang, M.N.; Johnson, K.E.; Kastelein, R.; Fiorentino, D.F.; DeVries, J.E.; Roncarolo, M.G.; Mosmann, T.R.; Moore, K.W.

1991-01-01

The authors demonstrated the existence of human cytokine synthesis inhibitory factor (DSIF) [interleukin 10 (IL-10)]. cDNA clones encoding human IL-10 (hIL-10) were isolated from a tetanus toxin-specific human T-cell clone. Like mouse IL-10, hIL-10 exhibits strong DNA and amino acid sequence homology to an open reading frame in the Epstein-Barr virus, BDRFL. hIL-10 and the BCRFI product inhibit cytokine synthesis by activated human peripheral blood mononuclear cells and by a mouse Th1 clone. Both hIL-10 and mouse IL-10 sustain the viability of a mouse mast cell line in culture, but BCRFI lacks comparable activity in this way, suggesting that BCRFI may have conserved only a subset of hIL-10 activities

Molecular cloning and expression of the calmodulin gene from guinea pig hearts.

Science.gov (United States)

Feng, Rui; Liu, Yan; Sun, Xuefei; Wang, Yan; Hu, Huiyuan; Guo, Feng; Zhao, Jinsheng; Hao, Liying

2015-06-01

The aim of the present study was to isolate and characterize a complementary DNA (cDNA) clone encoding the calmodulin (CaM; GenBank accession no. FJ012165) gene from guinea pig hearts. The CaM gene was amplified from cDNA collected from guinea pig hearts and inserted into a pGEM®-T Easy vector. Subsequently, CaM nucleotide and protein sequence similarity analysis was conducted between guinea pigs and other species. In addition, reverse transcription-polymerase chain reaction (RT-PCR) was performed to investigate the CaM 3 expression patterns in different guinea pig tissues. Sequence analysis revealed that the CaM gene isolated from the guinea pig heart had ∼90% sequence identity with the CaM 3 genes in humans, mice and rats. Furthermore, the deduced peptide sequences of CaM 3 in the guinea pig showed 100% homology to the CaM proteins from other species. In addition, the RT-PCR results indicated that CaM 3 was widely and differentially expressed in guinea pigs. In conclusion, the current study provided valuable information with regard to the cloning and expression of CaM 3 in guinea pig hearts. These findings may be helpful for understanding the function of CaM3 and the possible role of CaM3 in cardiovascular diseases.

Phylogenetic characterization of a biogas plant microbial community integrating clone library 16S-rDNA sequences and metagenome sequence data obtained by 454-pyrosequencing.

Science.gov (United States)

Kröber, Magdalena; Bekel, Thomas; Diaz, Naryttza N; Goesmann, Alexander; Jaenicke, Sebastian; Krause, Lutz; Miller, Dimitri; Runte, Kai J; Viehöver, Prisca; Pühler, Alfred; Schlüter, Andreas

2009-06-01

The phylogenetic structure of the microbial community residing in a fermentation sample from a production-scale biogas plant fed with maize silage, green rye and liquid manure was analysed by an integrated approach using clone library sequences and metagenome sequence data obtained by 454-pyrosequencing. Sequencing of 109 clones from a bacterial and an archaeal 16S-rDNA amplicon library revealed that the obtained nucleotide sequences are similar but not identical to 16S-rDNA database sequences derived from different anaerobic environments including digestors and bioreactors. Most of the bacterial 16S-rDNA sequences could be assigned to the phylum Firmicutes with the most abundant class Clostridia and to the class Bacteroidetes, whereas most archaeal 16S-rDNA sequences cluster close to the methanogen Methanoculleus bourgensis. Further sequences of the archaeal library most probably represent so far non-characterised species within the genus Methanoculleus. A similar result derived from phylogenetic analysis of mcrA clone sequences. The mcrA gene product encodes the alpha-subunit of methyl-coenzyme-M reductase involved in the final step of methanogenesis. BLASTn analysis applying stringent settings resulted in assignment of 16S-rDNA metagenome sequence reads to 62 16S-rDNA amplicon sequences thus enabling frequency of abundance estimations for 16S-rDNA clone library sequences. Ribosomal Database Project (RDP) Classifier processing of metagenome 16S-rDNA reads revealed abundance of the phyla Firmicutes, Bacteroidetes and Euryarchaeota and the orders Clostridiales, Bacteroidales and Methanomicrobiales. Moreover, a large fraction of 16S-rDNA metagenome reads could not be assigned to lower taxonomic ranks, demonstrating that numerous microorganisms in the analysed fermentation sample of the biogas plant are still unclassified or unknown.

Hibiscus latent Fort Pierce virus in Brazil and synthesis of its biologically active full-length cDNA clone.

Science.gov (United States)

Gao, Ruimin; Niu, Shengniao; Dai, Weifang; Kitajima, Elliot; Wong, Sek-Man

2016-10-01

A Brazilian isolate of Hibiscus latent Fort Pierce virus (HLFPV-BR) was firstly found in a hibiscus plant in Limeira, SP, Brazil. RACE PCR was carried out to obtain the full-length sequences of HLFPV-BR which is 6453 nucleotides and has more than 99.15 % of complete genomic RNA nucleotide sequence identity with that of HLFPV Japanese isolate. The genomic structure of HLFPV-BR is similar to other tobamoviruses. It includes a 5' untranslated region (UTR), followed by open reading frames encoding for a 128-kDa protein and a 188-kDa readthrough protein, a 38-kDa movement protein, 18-kDa coat protein, and a 3' UTR. Interestingly, the unique feature of poly(A) tract is also found within its 3'-UTR. Furthermore, from the total RNA extracted from the local lesions of HLFPV-BR-infected Chenopodium quinoa leaves, a biologically active, full-length cDNA clone encompassing the genome of HLFPV-BR was amplified and placed adjacent to a T7 RNA polymerase promoter. The capped in vitro transcripts from the cloned cDNA were infectious when mechanically inoculated into C. quinoa and Nicotiana benthamiana plants. This is the first report of the presence of an isolate of HLFPV in Brazil and the successful synthesis of a biologically active HLFPV-BR full-length cDNA clone.

Molecular techniques as complementary tools in orchid mutagenesis

Energy Technology Data Exchange (ETDEWEB)

Mohd Nazir Basiran; Sakinah Ariffin [Malaysian Institute for Nuclear Technology Research (MINT), Bangi (Malaysia)

2002-02-01

Orchid breeders have always been dependent on hybridization technology to produce new orchid hybrids and varieties. The technology has proven very reliable and easy to use and has produced wide range of successful cultivars with attractive combinations of spray length, bud number, flower colour and form, vase life, fragrance, seasonality, and compactness. By introducing mutagenesis however, wide variations of flower colours, form and size can still be obtained in addition to overcoming the problem of sexual incompatibility and sterility. In addition, complementary use of molecular techniques will allow breeders to target more specific characteristic changes and cut short breeding time. PCR-based techniques used to analyse the DNA of mutagenic clones found polymorphic fragments that can be developed as molecular markers. This paper describes how mutagenesis and molecular techniques can be used to enhance orchid breeding efforts. (author)

Cloning and molecular characterization of the salt-regulated jojoba ScRab cDNA encoding a small GTP-binding protein.

Science.gov (United States)

Mizrahi-Aviv, Ela; Mills, David; Benzioni, Aliza; Bar-Zvi, Dudy

2002-10-01

Salt stress results in a massive change in gene expression. An 837 bp cDNA designated ScRab was cloned from shoot cultures of the salt tolerant jojoba (Simmondsia chinesis). The cloned cDNA encodes a full length 200 amino acid long polypeptide that bears high homology to the Rab subfamily of small GTP binding proteins, particularly, the Rab5 subfamily. ScRab expression is reduced in shoots grown in the presence of salt compared to shoots from non-stressed cultures. His6-tagged ScRAB protein was expressed in E. coli, and purified to homogeneity. The purified protein bound radiolabelled GTP. The unlabelled guanine nucleotides GTP, GTP gamma S and GDP but not ATP, CTP or UTP competed with GTP binding.

Human uroporphyrinogen III synthase: Molecular cloning, nucleotide sequence, and expression of a full-length cDNA

International Nuclear Information System (INIS)

Tsai, Shihfeng; Bishop, D.F.; Desnick, R.J.

1988-01-01

Uroporphyrinogen III synthase, the fourth enzyme in the heme biosynthetic pathway, is responsible for conversion of the linear tetrapyrrole, hydroxymethylbilane, to the cyclic tetrapyrrole, uroporphyrinogen III. The deficient activity of URO-synthase is the enzymatic defect in the autosomal recessive disorder congenital erythropoietic porphyria. To facilitate the isolation of a full-length cDNA for human URO-synthase, the human erythrocyte enzyme was purified to homogeneity and 81 nonoverlapping amino acids were determined by microsequencing the N terminus and four tryptic peptides. Two synthetic oligonucleotide mixtures were used to screen 1.2 x 10 6 recombinants from a human adult liver cDNA library. Eight clones were positive with both oligonucleotide mixtures. Of these, dideoxy sequencing of the 1.3 kilobase insert from clone pUROS-2 revealed 5' and 3' untranslated sequences of 196 and 284 base pairs, respectively, and an open reading frame of 798 base pairs encoding a protein of 265 amino acids with a predicted molecular mass of 28,607 Da. The isolation and expression of this full-length cDNA for human URO-synthase should facilitate studies of the structure, organization, and chromosomal localization of this heme biosynthetic gene as well as the characterization of the molecular lesions causing congenital erythropoietic porphyria

cDNA cloning and expression of a human platelet-derived growth factor (PDGF) receptor specific for B-chain-containing PDGF molecules

International Nuclear Information System (INIS)

Claesson-Welsh, L.; Eriksson, A.; Moren, A.; Severinsson, L.; Ek, B.; Ostman, A.; Betsholtz, C.; Heldin, C.H.

1988-01-01

The structure of the human receptor for platelet-derived growth factor (PDGF) has been deduced through cDNA cloning. A 5.45-kilobase-pair cDNA clone predicts a 1,106-amino-acid polypeptide, including the cleavable signal sequence. The overall amino acid sequence similarity with the murine PDGFR receptor is 85%. After transcription of the cDNA and translation in vitro, a PDGR receptor antiserum was used to immunoprecipitate a product of predicted size, which also could be phosphorylated in vitro. Stable introduction of the cDNA into Chinese hamster ovary (CHO) cells led to the expression of a 190-kilodalton component, which was immunoprecipitated by the PDGF receptor antiserum; this most probably represents the mature PDGF receptor. Binding assays with different /sup 125/I-labeled dimeric forms of PDGF A and B chains showed that the PDGFR receptor expressed in CHO cells bound PDGF-BB and, to a lesser extent, PDGF-AB, but not PDGF-AA

Cloning and molecular analyses of a gibberellin 20-oxidase gene expressed specifically in developing seeds of watermelon.

Science.gov (United States)

Kang, H G; Jun, S H; Kim, J; Kawaide, H; Kamiya, Y; An, G

1999-10-01

To understand the biosynthesis and functional role of gibberellins (GAs) in developing seeds, we isolated Cv20ox, a cDNA clone from watermelon (Citrullus lanatus) that shows significant amino acid homology with GA 20-oxidases. The complementary DNA clone was expressed in Escherichia coli as a fusion protein, which oxidized GA(12) at C-20 to the C(19) compound GA(9), a precursor of bioactive GAs. RNA-blot analysis showed that the Cv20ox gene was expressed specifically in developing seeds. The gene was strongly expressed in the integument tissues, and it was also expressed weakly in inner seed tissues. In parthenocarpic fruits induced by 1-(2-chloro-4-pyridyl)-3-phenylurea treatment, the expression pattern of Cv20ox did not change, indicating that the GA 20-oxidase gene is expressed primarily in the maternal cells of developing seeds. The promoter of Cv20ox was isolated and fused to the beta-glucuronidase (GUS) gene. In a transient expression system, beta-glucuronidase staining was detectable only in the integument tissues of developing watermelon seeds.

Analysis of a cDNA clone expressing a human autoimmune antigen: full-length sequence of the U2 small nuclear RNA-associated B antigen

International Nuclear Information System (INIS)

Habets, W.J.; Sillekens, P.T.G.; Hoet, M.H.; Schalken, J.A.; Roebroek, A.J.M.; Leunissen, J.A.M.; Van de Ven, W.J.M.; Van Venrooij, W.J.

1987-01-01

A U2 small nuclear RNA-associated protein, designated B'', was recently identified as the target antigen for autoimmune sera from certain patients with systemic lupus erythematosus and other rheumatic diseases. Such antibodies enabled them to isolate cDNA clone λHB''-1 from a phage λgt11 expression library. This clone appeared to code for the B'' protein as established by in vitro translation of hybrid-selected mRNA. The identity of clone λHB''-1 was further confirmed by partial peptide mapping and analysis of the reactivity of the recombinant antigen with monospecific and monoclonal antibodies. Analysis of the nucleotide sequence of the 1015-base-pair cDNA insert of clone λHB''-1 revealed a large open reading frame of 800 nucleotides containing the coding sequence for a polypeptide of 25,457 daltons. In vitro transcription of the λHB''-1 cDNA insert and subsequent translation resulted in a protein product with the molecular size of the B'' protein. These data demonstrate that clone λHB''-1 contains the complete coding sequence of this antigen. The deduced polypeptide sequence contains three very hydrophilic regions that might constitute RNA binding sites and/or antigenic determinants. These findings might have implications both for the understanding of the pathogenesis of rheumatic diseases as well as for the elucidation of the biological function of autoimmune antigens

Utilization of a cloned alphoid repeating sequence of human DNA in the study of polymorphism of chromosomal heterochromatin regions

International Nuclear Information System (INIS)

Kruminya, A.R.; Kroshkina, V.G.; Yurov, Yu.B.; Aleksandrov, I.A.; Mitkevich, S.P.; Gindilis, V.M.

1988-01-01

The chromosomal distribution of the cloned PHS05 fragment of human alphoid DNA was studied by in situ hybridization in 38 individuals. It was shown that this DNA fraction is primarily localized in the pericentric regions of practically all chromosomes of the set. Significant interchromosomal differences and a weakly expressed interindividual polymorphism were discovered in the copying ability of this class of repeating DNA sequences; associations were not found between the results of hybridization and the pattern of Q-polymorphism

Cloning and comparative mapping of a human chromosome 4-specific alpha satellite DNA sequence

Energy Technology Data Exchange (ETDEWEB)

D' Aiuto, L.; Marzella, R.; Archidiacono, N.; Rocchi, M. (Universita di Bari (Italy)); Antonacci, R. (Instituto Anatomia Umana Normale, Modena (Italy))

1993-11-01

The authors have isolated and characterized two human alphoid DNA clones: p4n1/4 and pZ4.1. Clone p4n1/4 identifies specifically the centromeric region of chromosome 4; pZ4.1 recognizes a subset of alphoid DNA shared by chromosomes 4 and 9. The specificity was determined using fluorescence in situ hybridization experiments on metaphase spreads and Southern blotting analysis of human-hamster somatic cell hybrids. The genomic organization of both subsets was also investigated. Comparative mapping on chimpanzee and gorilla chromosomes was performed. p4n1/4 hybridizes to chimpanzee chromosomes 11 and 13, homologs of human chromosomes 9 and 2q, respectively. On gorilla metaphase spreads, p4n1/4 hybridizes exclusively to the centromeric region of chromosome 19, partially homologous to human chromosome 17. No hybridization signal was detected on chromosome 3 of both chimpanzee and gorilla, in both species homolog of human chromosome 4. Identical comparative mapping results were obtained using pZ4.1 probe, although the latter recognizes an alphoid subset distinct from the one recognized by p4n1/4. The implications of these results in the evolution of centromeric regions of primate chromosomes are discussed. 33 refs., 4 figs.

MPO cDNA clone identifies an RFLP with PstI

Energy Technology Data Exchange (ETDEWEB)

Miki, T; Weil, S C; Rosner, G L; Reid, M S; Kidd, K K

1988-02-25

A myeloperoxidase (MPO) cDNA clone (pHMP7: 270 base pair insert in the vector pGEM-1reverse arrow was isolated from a library created from human promyelocytic (HL-60) cell mRNA. PstI (CTGCA/G) (New England Biolabs) identifies a simple two-allele polymorphism with bands at either 2.2 kb (Al) or 2.0 kb (A2). There are three constant bands at 2.8 kb, 0.95 kb and 0.6 kb. Preliminary family data show evidence of linkage to several markers in proximal 17q, with MPO closest to the Growth Hormone cluster at 17q22-q24. Autosomal condominant segregation was observed in four large reference pedigrees with several informative matings.

Quantitative discrimination of Aggregatibacter actinomycetemcomitans highly leukotoxic JP2 clone from non-JP2 clones in diagnosis of aggressive periodontitis.

Science.gov (United States)

Yoshida, Akihiro; Ennibi, Oum-Keltoum; Miyazaki, Hideo; Hoshino, Tomonori; Hayashida, Hideaki; Nishihara, Tatsuji; Awano, Shuji; Ansai, Toshihiro

2012-10-11

Aggregatibacter actinomycetemcomitans is the etiological agent of periodontitis, and there is a strong association between clone JP2 and aggressive periodontitis in adolescents of African descent. The JP2 clone has an approximately 530-bp deletion (∆530) in the promoter region of the lkt/ltx gene, which encodes leukotoxin, and this clone has high leukotoxic activity. Therefore, this clone is very important in aggressive periodontitis. To diagnose this disease, culture methods and conventional PCR techniques are used. However, quantitative detection based on qPCR for the JP2 clone has not been developed due to genetic difficulties. In this study, we developed a qPCR-based quantification method specific to the JP2 clone. Based on our analysis of the DNA sequence of the lkt/ltx gene and its flanking region, we designed a reverse primer specific for the ∆530 deletion border sequence and developed a JP2-specific PCR-based quantification method using this primer. We also analyzed the DNA sequence of the ∆530 locus and found it to be highly conserved (97-100%) among 17 non-JP2 strains. Using the ∆530 locus, we designed a qPCR primer-probe set specific to non-JP2 clones. Next, we determined the numbers of JP2 and non-JP2 clone cells in the periodontal pockets of patients with aggressive periodontitis. The JP2-specific primers specifically amplified the genomic DNA of the A. actinomycetemcomitans JP2 clone and did not react with other bacterial DNA, whereas the non-JP2 specific primers reacted only with A. actinomycetemcomitans non-JP2 clones. Samples from the 88 periodontal sites in the 11 patients with aggressive periodontitis were analyzed. The bacterial cell numbers in 88 periodontal sites ranged from 0 to 4.8 × 10(8) (mean 1.28 × 10(7)) for JP2 clones and from 0 to 1.6 × 10(6) for non-JP2 clones (mean 1.84 × 10(5)). There were significant differences in the JP2 cell number between a clinical attachment level (CAL) ≤6 mm and a level ≥7 mm (p clones. This

Reproductive cloning combined with genetic modification.

Science.gov (United States)

Strong, C

2005-11-01

Although there is widespread opposition to reproductive cloning, some have argued that its use by infertile couples to have genetically related children would be ethically justifiable. Others have suggested that lesbian or gay couples might wish to use cloning to have genetically related children. Most of the main objections to human reproductive cloning are based on the child's lack of unique nuclear DNA. In the future, it may be possible safely to create children using cloning combined with genetic modifications, so that they have unique nuclear DNA. The genetic modifications could be aimed at giving such children genetic characteristics of both members of the couple concerned. Thus, cloning combined with genetic modification could be appealing to infertile, lesbian, or gay couples who seek genetically related children who have genetic characteristics of both members. In such scenarios, the various objections to human reproductive cloning that are based on the lack of genetic uniqueness would no longer be applicable. The author argues that it would be ethically justifiable for such couples to create children in this manner, assuming these techniques could be used safely.

Coding sequence of human rho cDNAs clone 6 and clone 9

Energy Technology Data Exchange (ETDEWEB)

Chardin, P; Madaule, P; Tavitian, A

1988-03-25

The authors have isolated human cDNAs including the complete coding sequence for two rho proteins corresponding to the incomplete isolates previously described as clone 6 and clone 9. The deduced a.a. sequences, when compared to the a.a. sequence deduced from clone 12 cDNA, show that there are in human at least three highly homologous rho genes. They suggest that clone 12 be named rhoA, clone 6 : rhoB and clone 9 : rhoC. RhoA, B and C proteins display approx. 30% a.a. identity with ras proteins,. mainly clustered in four highly homologous internal regions corresponding to the GTP binding site; however at least one significant difference is found; the 3 rho proteins have an Alanine in position corresponding to ras Glycine 13, suggesting that rho and ras proteins might have slightly different biochemical properties.

Cloning and Molecular Analyses of a Gibberellin 20-Oxidase Gene Expressed Specifically in Developing Seeds of Watermelon1

Science.gov (United States)

Kang, Hong-Gyu; Jun, Sung-Hoon; Kim, Junyul; Kawaide, Hiroshi; Kamiya, Yuji; An, Gynheung

1999-01-01

To understand the biosynthesis and functional role of gibberellins (GAs) in developing seeds, we isolated Cv20ox, a cDNA clone from watermelon (Citrullus lanatus) that shows significant amino acid homology with GA 20-oxidases. The complementary DNA clone was expressed in Escherichia coli as a fusion protein, which oxidized GA12 at C-20 to the C19 compound GA9, a precursor of bioactive GAs. RNA-blot analysis showed that the Cv20ox gene was expressed specifically in developing seeds. The gene was strongly expressed in the integument tissues, and it was also expressed weakly in inner seed tissues. In parthenocarpic fruits induced by 1-(2-chloro-4-pyridyl)-3-phenylurea treatment, the expression pattern of Cv20ox did not change, indicating that the GA 20-oxidase gene is expressed primarily in the maternal cells of developing seeds. The promoter of Cv20ox was isolated and fused to the β-glucuronidase (GUS) gene. In a transient expression system, β-glucuronidase staining was detectable only in the integument tissues of developing watermelon seeds. PMID:10517828

B-G cDNA clones have multiple small repeats and hybridize to both chicken MHC regions

DEFF Research Database (Denmark)

Kaufman, J; Salomonsen, J; Skjødt, K

1989-01-01

We used rabbit antisera to the chicken MHC erythrocyte molecule B-G and to the class I alpha chain (B-F) to screen lambda gt11 cDNA expression libraries made with RNA selected by oligo-dT from bone marrow cells of anemic B19 homozygous chickens. Eight clones were found to encode B-G molecules which...

Genetic alterations of hepatocellular carcinoma by random amplified polymorphic DNA analysis and cloning sequencing of tumor differential DNA fragment

Science.gov (United States)

Xian, Zhi-Hong; Cong, Wen-Ming; Zhang, Shu-Hui; Wu, Meng-Chao

2005-01-01

AIM: To study the genetic alterations and their association with clinicopathological characteristics of hepatocellular carcinoma (HCC), and to find the tumor related DNA fragments. METHODS: DNA isolated from tumors and corresponding noncancerous liver tissues of 56 HCC patients was amplified by random amplified polymorphic DNA (RAPD) with 10 random 10-mer arbitrary primers. The RAPD bands showing obvious differences in tumor tissue DNA corresponding to that of normal tissue were separated, purified, cloned and sequenced. DNA sequences were analyzed and compared with GenBank data. RESULTS: A total of 56 cases of HCC were demonstrated to have genetic alterations, which were detected by at least one primer. The detestability of genetic alterations ranged from 20% to 70% in each case, and 17.9% to 50% in each primer. Serum HBV infection, tumor size, histological grade, tumor capsule, as well as tumor intrahepatic metastasis, might be correlated with genetic alterations on certain primers. A band with a higher intensity of 480 bp or so amplified fragments in tumor DNA relative to normal DNA could be seen in 27 of 56 tumor samples using primer 4. Sequence analysis of these fragments showed 91% homology with Homo sapiens double homeobox protein DUX10 gene. CONCLUSION: Genetic alterations are a frequent event in HCC, and tumor related DNA fragments have been found in this study, which may be associated with hepatocarcin-ogenesis. RAPD is an effective method for the identification and analysis of genetic alterations in HCC, and may provide new information for further evaluating the molecular mechanism of hepatocarcinogenesis. PMID:15996039

A Gateway MultiSite recombination cloning toolkit.

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Lena K Petersen

Full Text Available The generation of DNA constructs is often a rate-limiting step in conducting biological experiments. Recombination cloning of single DNA fragments using the Gateway system provided an advance over traditional restriction enzyme cloning due to increases in efficiency and reliability. Here we introduce a series of entry clones and a destination vector for use in two, three, and four fragment Gateway MultiSite recombination cloning whose advantages include increased flexibility and versatility. In contrast to Gateway single-fragment cloning approaches where variations are typically incorporated into model system-specific destination vectors, our Gateway MultiSite cloning strategy incorporates variations in easily generated entry clones that are model system-independent. In particular, we present entry clones containing insertions of GAL4, QF, UAS, QUAS, eGFP, and mCherry, among others, and demonstrate their in vivo functionality in Drosophila by using them to generate expression clones including GAL4 and QF drivers for various trp ion channel family members, UAS and QUAS excitatory and inhibitory light-gated ion channels, and QUAS red and green fluorescent synaptic vesicle markers. We thus establish a starter toolkit of modular Gateway MultiSite entry clones potentially adaptable to any model system. An inventory of entry clones and destination vectors for Gateway MultiSite cloning has also been established (www.gatewaymultisite.org.

Cloning, sequencing, and expression of dnaK-operon proteins from the thermophilic bacterium Thermus thermophilus.

Science.gov (United States)

Osipiuk, J; Joachimiak, A

1997-09-12

We propose that the dnaK operon of Thermus thermophilus HB8 is composed of three functionally linked genes: dnaK, grpE, and dnaJ. The dnaK and dnaJ gene products are most closely related to their cyanobacterial homologs. The DnaK protein sequence places T. thermophilus in the plastid Hsp70 subfamily. In contrast, the grpE translated sequence is most similar to GrpE from Clostridium acetobutylicum, a Gram-positive anaerobic bacterium. A single promoter region, with homology to the Escherichia coli consensus promoter sequences recognized by the sigma70 and sigma32 transcription factors, precedes the postulated operon. This promoter is heat-shock inducible. The dnaK mRNA level increased more than 30 times upon 10 min of heat shock (from 70 degrees C to 85 degrees C). A strong transcription terminating sequence was found between the dnaK and grpE genes. The individual genes were cloned into pET expression vectors and the thermophilic proteins were overproduced at high levels in E. coli and purified to homogeneity. The recombinant T. thermophilus DnaK protein was shown to have a weak ATP-hydrolytic activity, with an optimum at 90 degrees C. The ATPase was stimulated by the presence of GrpE and DnaJ. Another open reading frame, coding for ClpB heat-shock protein, was found downstream of the dnaK operon.

Molecular cloning of a catalase cDNA from Nicotiana glutinosa L. and its repression by tobacco mosaic virus infection.

Science.gov (United States)

Yi, S Y; Yu, S H; Choi, D

1999-06-30

Recent reports revealed that catalase has a role in the plant defense mechanism against a broad range of pathogens through being inhibited by salicylic acid (SA). During an effort to clone disease resistance-responsive genes, a cDNA encoding catalase (Ngcat1; Nicotiana glutinosa cat1) was isolated from a tobacco cDNA library. In N. glutinosa, catalase is encoded by a small gene family. The deduced amino acid sequence of the Ngcat1 cDNA has 98% homology with the cat1 gene of N. plumbaginifolia. The Ngcat1 expression is controlled by the circadian clock, and its mRNA level is the most abundant in leaves. Both the expression of Ngcat1 mRNA and its enzyme activity in the tobacco plant undergoing a hypersensitive response (HR) to TMV infection were repressed. The repression of the mRNA level was also observed following treatment with SA. These results imply that SA may act as an inhibitor of catalase transcription during the HR of tobacco. Cloning and expression of the Ngcat1 in tobacco following pathogen infection and SA treatment are presented.

Mutation Processes in 293-Based Clones Overexpressing the DNA Cytosine Deaminase APOBEC3B.

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Monica K Akre

Full Text Available Molecular, cellular, and clinical studies have combined to demonstrate a contribution from the DNA cytosine deaminase APOBEC3B (A3B to the overall mutation load in breast, head/neck, lung, bladder, cervical, ovarian, and other cancer types. However, the complete landscape of mutations attributable to this enzyme has yet to be determined in a controlled human cell system. We report a conditional and isogenic system for A3B induction, genomic DNA deamination, and mutagenesis. Human 293-derived cells were engineered to express doxycycline-inducible A3B-eGFP or eGFP constructs. Cells were subjected to 10 rounds of A3B-eGFP exposure that each caused 80-90% cell death. Control pools were subjected to parallel rounds of non-toxic eGFP exposure, and dilutions were done each round to mimic A3B-eGFP induced population fluctuations. Targeted sequencing of portions of TP53 and MYC demonstrated greater mutation accumulation in the A3B-eGFP exposed pools. Clones were generated and microarray analyses were used to identify those with the greatest number of SNP alterations for whole genome sequencing. A3B-eGFP exposed clones showed global increases in C-to-T transition mutations, enrichments for cytosine mutations within A3B-preferred trinucleotide motifs, and more copy number aberrations. Surprisingly, both control and A3B-eGFP clones also elicited strong mutator phenotypes characteristic of defective mismatch repair. Despite this additional mutational process, the 293-based system characterized here still yielded a genome-wide view of A3B-catalyzed mutagenesis in human cells and a system for additional studies on the compounded effects of simultaneous mutation mechanisms in cancer cells.

Molecular Cloning, Expression and Characterization of Plasmid Encoding Rhomboid 4 (ROM4 of Tachyzoite of Toxoplasma gondii RH Strain

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Mohammad Taghi RAHIMI

2017-12-01

Full Text Available AbstractBackground: The objective of this study was to clone, express and characterize the gene encoding rhomboid 4 (ROM4 proteins, a vital gene in surface adhesion and host cell invasion process of tachyzoite of T. gondii in an appropriate expression vector and eukaryotic cell for production of recombinant protein.Methods: Toxoplasma RNA was isolated from tachyzoites (RH strain and complementary DNA was synthesized. Oligonucleotide primer pair was designed based on Toxoplasma ROM4 gene sequence with XhoI and EcoRI restriction sites at 5´ end of forward and reverse primers, respectively. ROM4 gene was amplified by PCR, cloned into pTG19-T vector and the recombinant plasmid was sequenced. The gene was subcloned into pcDNA3 plasmid and expressed in CHO cells as eukaryotic cell. SDS-PAGE and western blotting were performed for protein determination and verification.Results: Cloning of ROM4 gene in pTG19-T vector was confirmed by colony-PCR and enzymatic digestion. The results of enzymatic digestion and gene sequencing confirmed successful cloning and subcloning procedures. The nucleotide sequence of the cloned ROM4 gene showed 99% homology compared to the corresponding sequences of original gene. SDS-PAGE and western blotting analyses of the purified protein revealed a single band having expected size of 65 kDa.Conclusion: This eukaryotic expression system is an appropriate system for high-level recombinant protein production of ROM4 gene from T. gondii tachyzoites used as antigenic component for serological assay and vaccine development.

Cloning of the human carnitine-acylcarnitine carrier cDNA and identification of the molecular defect in a patient

NARCIS (Netherlands)

Huizing, M.; Iacobazzi, V.; IJlst, L.; Savelkoul, P.; Ruitenbeek, W.; van den Heuvel, L.; Indiveri, C.; Smeitink, J.; Trijbels, F.; Wanders, R.; Palmieri, F.

1997-01-01

The carnitine-acylcarnitine carrier (CAC) catalyzes the translocation of long-chain fatty acids across the inner mitochondrial membrane. We cloned and sequenced the human CAC cDNA, which has an open reading frame of 903 nucleotides. Northern blot studies revealed different expression levels of CAC

Cloning and characterisation of the sagA gene of Aspergillus nidulans: a gene which affects sensitivity to DNA-damaging agents.

Science.gov (United States)

Jones, G W; Hooley, P; Farrington, S M; Shawcross, S G; Iwanejko, L A; Strike, P

1999-03-01

Mutations within the sagA gene of Aspergillus nidulans cause sensitisation to DNA-damaging chemicals but have no effect upon spontaneous or damage-induced mutation frequency. The sagA gene was cloned on a 19-kb cosmid-derived fragment by functional complementation of a sagA1 sagC3 double mutant; subsequently, a fragment of the gene was also isolated on a 3.9-kb genomic subclone. Initial sequencing of a small section of the 19-kb fragment allowed the design of primers that were subsequently used in RTPCR experiments to show that this DNA is transcribed. A 277-bp fragment derived from the transcribed region was used to screen an A. nidulans cDNA library, resulting in the isolation of a 1.4-kb partial cDNA clone which had sequence overlap with the genomic sagA fragment. This partial cDNA was incomplete but appeared to contain the whole coding region of sagA. The sagA1 mutant was shown to possess two mutations; a G-T transversion and a+ 1 frameshift due to insertion of a T. causing disruption to the C-terminal region of the SagA protein. Translation of the sagA cDNA predicts a protein of 378 amino acids, which has homology to the Saccharomyces cerevisiae End3 protein and also to certain mammalian proteins capable of causing cell transformation.

Molecular cloning of cDNA for the human tumor-associated antigen CO-029 and identification of related transmembrane antigens

International Nuclear Information System (INIS)

Szala, S.; Kasai, Yasushi; Steplewski, Z.; Rodeck, U.; Koprowski, H.; Linnenbach, A.J.

1990-01-01

The human tumor-associated antigen CO-029 is a monoclonal antibody-defined cell surface glycoprotein of 27-34 kDa. By using the high-efficiency COS cell expression system, a full-length cDNA clone for CO-029 was isolated. When transiently expressed in COS cells, the cDNA clone directed the synthesis of an antigen reactive to monoclonal antibody CO-029 in mixed hemadsorption and immunoblot assays. Sequence analysis revealed that CO-029 belongs to a family of cell surface antigens that includes the melanoma-associated antigen ME491, the leukocyte cell surface antigen CD37, and the Sm23 antigen of the parasitic helminth Schistosoma mansoni. CO-029 and ME491 antigen expression and the effect of their corresponding monoclonal antibodies on cell growth were compared in human tumor cell lines of various histologic origins

Cloning of a cDNA encoding a novel human nuclear phosphoprotein belonging to the WD-40 family

DEFF Research Database (Denmark)

Honoré, B; Leffers, H; Madsen, Peder

1994-01-01

We have cloned and expressed in vaccinia virus a cDNA encoding an ubiquitous 501-amino-acid (aa) phosphoprotein that corresponds to protein IEF SSP 9502 (79,400 Da, pI 4.5) in the master 2-D-gel keratinocyte protein database [Celis et al., Electrophoresis 14 (1993) 1091-1198]. The deduced aa...

A new set of ESTs and cDNA clones from full-length and normalized libraries for gene discovery and functional characterization in citrus

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Alamar Santiago

2009-09-01

Full Text Available Abstract Background Interpretation of ever-increasing raw sequence information generated by modern genome sequencing technologies faces multiple challenges, such as gene function analysis and genome annotation. Indeed, nearly 40% of genes in plants encode proteins of unknown function. Functional characterization of these genes is one of the main challenges in modern biology. In this regard, the availability of full-length cDNA clones may fill in the gap created between sequence information and biological knowledge. Full-length cDNA clones facilitate functional analysis of the corresponding genes enabling manipulation of their expression in heterologous systems and the generation of a variety of tagged versions of the native protein. In addition, the development of full-length cDNA sequences has the power to improve the quality of genome annotation. Results We developed an integrated method to generate a new normalized EST collection enriched in full-length and rare transcripts of different citrus species from multiple tissues and developmental stages. We constructed a total of 15 cDNA libraries, from which we isolated 10,898 high-quality ESTs representing 6142 different genes. Percentages of redundancy and proportion of full-length clones range from 8 to 33, and 67 to 85, respectively, indicating good efficiency of the approach employed. The new EST collection adds 2113 new citrus ESTs, representing 1831 unigenes, to the collection of citrus genes available in the public databases. To facilitate functional analysis, cDNAs were introduced in a Gateway-based cloning vector for high-throughput functional analysis of genes in planta. Herein, we describe the technical methods used in the library construction, sequence analysis of clones and the overexpression of CitrSEP, a citrus homolog to the Arabidopsis SEP3 gene, in Arabidopsis as an example of a practical application of the engineered Gateway vector for functional analysis. Conclusion The new

A new set of ESTs and cDNA clones from full-length and normalized libraries for gene discovery and functional characterization in citrus

Science.gov (United States)

Marques, M Carmen; Alonso-Cantabrana, Hugo; Forment, Javier; Arribas, Raquel; Alamar, Santiago; Conejero, Vicente; Perez-Amador, Miguel A

2009-01-01

Background Interpretation of ever-increasing raw sequence information generated by modern genome sequencing technologies faces multiple challenges, such as gene function analysis and genome annotation. Indeed, nearly 40% of genes in plants encode proteins of unknown function. Functional characterization of these genes is one of the main challenges in modern biology. In this regard, the availability of full-length cDNA clones may fill in the gap created between sequence information and biological knowledge. Full-length cDNA clones facilitate functional analysis of the corresponding genes enabling manipulation of their expression in heterologous systems and the generation of a variety of tagged versions of the native protein. In addition, the development of full-length cDNA sequences has the power to improve the quality of genome annotation. Results We developed an integrated method to generate a new normalized EST collection enriched in full-length and rare transcripts of different citrus species from multiple tissues and developmental stages. We constructed a total of 15 cDNA libraries, from which we isolated 10,898 high-quality ESTs representing 6142 different genes. Percentages of redundancy and proportion of full-length clones range from 8 to 33, and 67 to 85, respectively, indicating good efficiency of the approach employed. The new EST collection adds 2113 new citrus ESTs, representing 1831 unigenes, to the collection of citrus genes available in the public databases. To facilitate functional analysis, cDNAs were introduced in a Gateway-based cloning vector for high-throughput functional analysis of genes in planta. Herein, we describe the technical methods used in the library construction, sequence analysis of clones and the overexpression of CitrSEP, a citrus homolog to the Arabidopsis SEP3 gene, in Arabidopsis as an example of a practical application of the engineered Gateway vector for functional analysis. Conclusion The new EST collection denotes an

Cloning and characterization of human DNA repair genes

International Nuclear Information System (INIS)

Thompson, L.H.; Brookman, K.W.; Weber, C.A.; Salazar, E.P.; Stewart, S.A.; Carrano, A.V.

1987-01-01

The isolation of two addition human genes that give efficient restoration of the repair defects in other CHO mutant lines is reported. The gene designated ERCC2 (Excision Repair Complementing Chinese hamster) corrects mutant UV5 from complementation group 1. They recently cloned this gene by first constructing a secondary transformant in which the human gene was shown to have become physically linked to the bacterial gpt dominant-marker gene by cotransfer in calcium phosphate precipitates in the primary transfection. Transformants expressing both genes were recovered by selecting for resistance to both UV radiation and mycophenolic acid. Using similar methods, the human gene that corrects CHO mutant EM9 was isolated in cosmids and named XRCC1 (X-ray Repair Complementing Chinese hamster). In this case, transformants were recovered by selecting for resistance to CldUrd, which kills EM9 very efficiently. In both genomic and cosmid transformants, the XRCC1 gene restored resistance to the normal range. DNA repair was studied using the kinetics of strand-break rejoining, which was measured after exposure to 137 Cs γ-rays

Gene organization in rice revealed by full-length cDNA mapping and gene expression analysis through microarray.

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Kouji Satoh

Full Text Available Rice (Oryza sativa L. is a model organism for the functional genomics of monocotyledonous plants since the genome size is considerably smaller than those of other monocotyledonous plants. Although highly accurate genome sequences of indica and japonica rice are available, additional resources such as full-length complementary DNA (FL-cDNA sequences are also indispensable for comprehensive analyses of gene structure and function. We cross-referenced 28.5K individual loci in the rice genome defined by mapping of 578K FL-cDNA clones with the 56K loci predicted in the TIGR genome assembly. Based on the annotation status and the presence of corresponding cDNA clones, genes were classified into 23K annotated expressed (AE genes, 33K annotated non-expressed (ANE genes, and 5.5K non-annotated expressed (NAE genes. We developed a 60mer oligo-array for analysis of gene expression from each locus. Analysis of gene structures and expression levels revealed that the general features of gene structure and expression of NAE and ANE genes were considerably different from those of AE genes. The results also suggested that the cloning efficiency of rice FL-cDNA is associated with the transcription activity of the corresponding genetic locus, although other factors may also have an effect. Comparison of the coverage of FL-cDNA among gene families suggested that FL-cDNA from genes encoding rice- or eukaryote-specific domains, and those involved in regulatory functions were difficult to produce in bacterial cells. Collectively, these results indicate that rice genes can be divided into distinct groups based on transcription activity and gene structure, and that the coverage bias of FL-cDNA clones exists due to the incompatibility of certain eukaryotic genes in bacteria.

Cloning the interleukin 1 receptor from human T cells

International Nuclear Information System (INIS)

Sims, J.E.; Acres, R.B.; Grubin, C.E.; McMahan, C.J.; Wignall, J.M.; March, C.J.; Dower, S.K.

1989-01-01

cDNA clones of the interleukin 1 (IL-1) receptor expressed in a human T-cell clone have been isolated by using a murine IL-1 receptor cDNA as a probe. The human and mouse receptors show a high degree of sequence conservation. Both are integral membrane proteins possessing a single membrane-spanning segment. Similar to the mouse receptor, the human IL-1 receptor contains a large cytoplasmic region and an extracellular, IL-1 binding portion composed of three immunoglobulin-like domains. When transfected into COS cells, the human IL-1 receptor cDNA clone leads to expression of two different affinity classes of receptors, with K a values indistinguishable from those determined for IL-1 receptors in the original T-cell clone. An IL-1 receptor expressed in human dermal fibroblasts has also been cloned and sequenced and found to be identical to the IL-1 receptor expressed in T cells

Molecular cloning and characterization of an acetylcholinesterase cDNA in the brown planthopper, Nilaparvata lugens.

Science.gov (United States)

Yang, Zhifan; Chen, Jun; Chen, Yongqin; Jiang, Sijing

2010-01-01

A full cDNA encoding an acetylcholinesterase (AChE, EC 3.1.1.7) was cloned and characterized from the brown planthopper, Nilaparvata lugens Stål (Hemiptera: Delphacidae). The complete cDNA (2467 bp) contains a 1938-bp open reading frame encoding 646 amino acid residues. The amino acid sequence of the AChE deduced from the cDNA consists of 30 residues for a putative signal peptide and 616 residues for the mature protein with a predicted molecular weight of 69,418. The three residues (Ser242, Glu371, and His485) that putatively form the catalytic triad and the six Cys that form intra-subunit disulfide bonds are completely conserved, and 10 out of the 14 aromatic residues lining the active site gorge of the AChE are also conserved. Northern blot analysis of poly(A)+ RNA showed an approximately 2.6-kb transcript, and Southern blot analysis revealed there likely was just a single copy of this gene in N. lugens. The deduced protein sequence is most similar to AChE of Nephotettix cincticeps with 83% amino acid identity. Phylogenetic analysis constructed with 45 AChEs from 30 species showed that the deduced N. lugens AChE formed a cluster with the other 8 insect AChE2s. Additionally, the hypervariable region and amino acids specific to insect AChE2 also existed in the AChE of N. lugens. The results revealed that the AChE cDNA cloned in this work belongs to insect AChE2 subgroup, which is orthologous to Drosophila AChE. Comparison of the AChEs between the susceptible and resistant strains revealed a point mutation, Gly185Ser, is likely responsible for the insensitivity of the AChE to methamidopho in the resistant strain.

Inverse fusion PCR cloning.

Directory of Open Access Journals (Sweden)

Markus Spiliotis

Full Text Available Inverse fusion PCR cloning (IFPC is an easy, PCR based three-step cloning method that allows the seamless and directional insertion of PCR products into virtually all plasmids, this with a free choice of the insertion site. The PCR-derived inserts contain a vector-complementary 5'-end that allows a fusion with the vector by an overlap extension PCR, and the resulting amplified insert-vector fusions are then circularized by ligation prior transformation. A minimal amount of starting material is needed and experimental steps are reduced. Untreated circular plasmid, or alternatively bacteria containing the plasmid, can be used as templates for the insertion, and clean-up of the insert fragment is not urgently required. The whole cloning procedure can be performed within a minimal hands-on time and results in the generation of hundreds to ten-thousands of positive colonies, with a minimal background.

Quantitative discrimination of Aggregatibacter actinomycetemcomitans highly leukotoxic JP2 clone from non-JP2 clones in diagnosis of aggressive periodontitis

Directory of Open Access Journals (Sweden)

Yoshida Akihiro

2012-10-01

Full Text Available Abstract Background Aggregatibacter actinomycetemcomitans is the etiological agent of periodontitis, and there is a strong association between clone JP2 and aggressive periodontitis in adolescents of African descent. The JP2 clone has an approximately 530-bp deletion (∆530 in the promoter region of the lkt/ltx gene, which encodes leukotoxin, and this clone has high leukotoxic activity. Therefore, this clone is very important in aggressive periodontitis. To diagnose this disease, culture methods and conventional PCR techniques are used. However, quantitative detection based on qPCR for the JP2 clone has not been developed due to genetic difficulties. In this study, we developed a qPCR-based quantification method specific to the JP2 clone. Methods Based on our analysis of the DNA sequence of the lkt/ltx gene and its flanking region, we designed a reverse primer specific for the ∆530 deletion border sequence and developed a JP2-specific PCR-based quantification method using this primer. We also analyzed the DNA sequence of the ∆530 locus and found it to be highly conserved (97–100% among 17 non-JP2 strains. Using the ∆530 locus, we designed a qPCR primer–probe set specific to non-JP2 clones. Next, we determined the numbers of JP2 and non-JP2 clone cells in the periodontal pockets of patients with aggressive periodontitis. Results The JP2-specific primers specifically amplified the genomic DNA of the A. actinomycetemcomitans JP2 clone and did not react with other bacterial DNA, whereas the non-JP2 specific primers reacted only with A. actinomycetemcomitans non-JP2 clones. Samples from the 88 periodontal sites in the 11 patients with aggressive periodontitis were analyzed. The bacterial cell numbers in 88 periodontal sites ranged from 0 to 4.8 × 108 (mean 1.28 × 107 for JP2 clones and from 0 to 1.6 × 106 for non-JP2 clones (mean 1.84 × 105. There were significant differences in the JP2 cell number between a clinical attachment level

Frameshift mutations in infectious cDNA clones of Citrus tristeza virus: a strategy to minimize the toxicity of viral sequences to Escherichia coli

International Nuclear Information System (INIS)

Satyanarayana, Tatineni; Gowda, Siddarame; Ayllon, Maria A.; Dawson, William O.

2003-01-01

The advent of reverse genetics revolutionized the study of positive-stranded RNA viruses that were amenable for cloning as cDNAs into high-copy-number plasmids of Escherichia coli. However, some viruses are inherently refractory to cloning in high-copy-number plasmids due to toxicity of viral sequences to E. coli. We report a strategy that is a compromise between infectivity of the RNA transcripts and toxicity to E. coli effected by introducing frameshift mutations into 'slippery sequences' near the viral 'toxicity sequences' in the viral cDNA. Citrus tristeza virus (CTV) has cDNA sequences that are toxic to E. coli. The original full-length infectious cDNA of CTV and a derivative replicon, CTV-ΔCla, cloned into pUC119, resulted in unusually limited E. coli growth. However, upon sequencing of these cDNAs, an additional uridinylate (U) was found in a stretch of U's between nts 3726 and 3731 that resulted in a change to a reading frame with a stop codon at nt 3734. Yet, in vitro produced RNA transcripts from these clones infected protoplasts, and the resulting progeny virus was repaired. Correction of the frameshift mutation in the CTV cDNA constructs resulted in increased infectivity of in vitro produced RNA transcripts, but also caused a substantial increase of toxicity to E. coli, now requiring 3 days to develop visible colonies. Frameshift mutations created in sequences not suspected to facilitate reading frame shifting and silent mutations introduced into oligo(U) regions resulted in complete loss of infectivity, suggesting that the oligo(U) region facilitated the repair of the frameshift mutation. Additional frameshift mutations introduced into other oligo(U) regions also resulted in transcripts with reduced infectivity similarly to the original clones with the +1 insertion. However, only the frameshift mutations introduced into oligo(U) regions that were near and before the toxicity region improved growth and stability in E. coli. These data demonstrate that

Isolation and Cloning of cDNA Fragment of Gene Encoding for Multidrug Resistance Associated Protein from M. affine.

Directory of Open Access Journals (Sweden)

Utut Widyastuti Suharsono

2008-11-01

Full Text Available Isolation and Cloning of cDNA Fragment of Gene Encoding for Multidrug Resistance Associated Protein from M. affine. M. affine can grow well in acid soil with high level of soluble aluminum. One of the important proteins in the detoxifying xenobiotic stress including acid and Al stresses is a multidrug resistance associated protein (MRP encoded by mrp gene. The objective of this research is to isolate and clone the cDNA fragment of MaMrp encoding MRP from M. affine. By reverse transcription, total cDNA had been synthesized from the total RNA as template. The fragment of cDNA MaMrp had been successfully isolated by PCR by using total cDNA as template and mrp primer designed from A. thaliana, yeast, and human. This fragment was successfully inserted into pGEM-T Easy and the recombinant plasmid was successfully introduced into E. coli DH5α. Nucleotide sequence analysis showed that the lenght of MaMrp fragment is 633 bp encoding 208 amino acids. Local alignment analysis based on nucleotide of mRNA showed that MaMrp fragment is 69% identical to AtMrp1 and 63% to AtMrp from A. thaliana. Based on deduced amino acid sequence, MaMRP is 84% identical to part of AtMRP13, 77% to AtMRP12, and 73% to AtMRP1 from A. thaliana respectively. Alignment analysis with AtMRP1 showed that MaMRP fragment is located in TM1 and NBF1 domains and has a specific amino acid sequence QCKAQLQNMEEE.

Cloning of cDNA sequences of a progestin-regulated mRNA from MCF7 human breast cancer cells

Energy Technology Data Exchange (ETDEWEB)

Chalbos, D; Westley, B; Alibert, C; Rochefort, H

1986-01-24

A cDNA clone corresponding to an mRNA regulated by the progestin R5020, has been isolated by differential screening of a cDNA library from the MCF7 breast cancer cell line, which contains estrogen and progesterone receptors. This probe hybridized with a single species of poly A + RNA of 8-kb molecular weight as shown by Northern blot analysis and could also be used to total RNA preparation. This recombinant cone hybridized specifically to an mRNA coding for a 250,000 daltons protein when translated in vitro. This protein was identical to the 250 kDa progestin-regulated protein that the authors previously described as shown by immunoprecipitation with specific rabbit polyclonal antibodies. Dose-response curve and specificity studies show that the accumulation of the Pg8 mRNA and that of the 250-kDa protein was increased by 5 to 30-fold following progestin treatment and that this effect was mediated by the progesterone receptor. Time course of induction indicated that the accumulation of mRNA was rapid and preceded that of the protein. This is the first report on a cloned cDNA probe of progestin-regulated mRNA in human cell lines.

Successful application of FTA Classic Card technology and use of bacteriophage phi29 DNA polymerase for large-scale field sampling and cloning of complete maize streak virus genomes.

Science.gov (United States)

Owor, Betty E; Shepherd, Dionne N; Taylor, Nigel J; Edema, Richard; Monjane, Adérito L; Thomson, Jennifer A; Martin, Darren P; Varsani, Arvind

2007-03-01

Leaf samples from 155 maize streak virus (MSV)-infected maize plants were collected from 155 farmers' fields in 23 districts in Uganda in May/June 2005 by leaf-pressing infected samples onto FTA Classic Cards. Viral DNA was successfully extracted from cards stored at room temperature for 9 months. The diversity of 127 MSV isolates was analysed by PCR-generated RFLPs. Six representative isolates having different RFLP patterns and causing either severe, moderate or mild disease symptoms, were chosen for amplification from FTA cards by bacteriophage phi29 DNA polymerase using the TempliPhi system. Full-length genomes were inserted into a cloning vector using a unique restriction enzyme site, and sequenced. The 1.3-kb PCR product amplified directly from FTA-eluted DNA and used for RFLP analysis was also cloned and sequenced. Comparison of cloned whole genome sequences with those of the original PCR products indicated that the correct virus genome had been cloned and that no errors were introduced by the phi29 polymerase. This is the first successful large-scale application of FTA card technology to the field, and illustrates the ease with which large numbers of infected samples can be collected and stored for downstream molecular applications such as diversity analysis and cloning of potentially new virus genomes.

A secreted aspartic proteinase from Glomerella cingulata: purification of the enzyme and molecular cloning of the cDNA.

Science.gov (United States)

Clark, S J; Templeton, M D; Sullivan, P A

1997-04-01

A secreted aspartic proteinase from Glomerella cingulata (GcSAP) was purified to homogeneity by ion exchange chromatography. The enzyme has an M, of 36000 as estimated by SDS-PAGE, optimal activity from pH 3.5 to pH 4.0 and is inhibited by pepstatin. The N-terminal sequence, 23 residues long, was used to design a gene-specific primer. This was used in 3' RACE (rapid amplification of cDNA ends) PCR to amplify a 1.2 kb fragment of the gcsap cDNA. A second gene-specific primer was designed and used in 5' RACE PCR to clone the 5' region. This yielded a 600 bp DNA fragment and completed the open reading frame. The gcsap open reading frame encodes a protein with a 78 residue prepro-sequence typical of other fungal secreted aspartic proteinases. Based on the deduced sequence, the mature enzyme contains 329 amino acids and shows approximately 40% identity to other fungal aspartic proteinases. Subsequent cloning and sequencing of gcsap fragments obtained from PCR with genomic DNA revealed a 73 bp intron beginning at nt 728. Southern analyses at medium and high stringency indicated that G. cingulata possesses one gene for the secreted aspartic proteinase, and Northern blots indicated that gene expression was induced by exogenous protein and repressed by ammonium salts. GcSAP is a putative pathogenicity factor of G. cingulata, and it will now be possible to create SAP-mutants and assess the role GcSAP plays in pathogenicity.

Cloning and expression of a cDNA encoding human sterol carrier protein 2

International Nuclear Information System (INIS)

Yamamoto, Ritsu; Kallen, C.B.; Babalola, G.O.; Rennert, H.; Strauss, J.F. III; Billheimer, J.T.

1991-01-01

The authors report the cloning and expression of a cDNA encoding human sterol carrier protein 2 (SCP 2 ). The 1.3-kilobase (kb) cDNA contains an open reading frame which encompasses a 143-amino acid sequence which is 89% identical to the rat SCP 2 amino acid sequence. The deduced amino acid sequence of the polypeptide reveals a 20-residue amino-terminal leader sequence in front of the mature polypeptide, which contains a carboxyl-terminal tripeptide (Ala-Lys-Leu) related to the peroxisome targeting sequence. The expressed cDNA in COS-7 cells yields a 15.3-kDa polypeptide and increased amounts of a 13.2-kDa polypeptide, both reacting with a specific rabbit antiserum to rat liver SCP 2 . The cDNA insert hybridizes with 3.2- and 1.8-kb mRNA species in human liver poly(A) + RNA. In human fibroblasts and placenta the 1.8-kb mRNA was most abundant. Southern blot analysis suggests either that there are multiple copies of the SCP 2 gene in the human genome or that the SCP 2 gene is very large. Coexpression of the SCP 2 cDNA with expression vectors for cholesterol side-chain cleavage enzyme and adrenodoxin resulted in a 2.5-fold enhancement of progestin synthesis over that obtained with expression of the steroidogenic enzyme system alone. These findings are concordant with the notion that SCP 2 plays a role in regulating steroidogenesis, among other possible functions

Generating West Nile Virus from an Infectious Clone.

Science.gov (United States)

Vandergaast, Rianna; Fredericksen, Brenda L

2016-01-01

WNV infectious clones are valuable tools for elucidating WNV biology. Nevertheless, relatively few infectious WNV clones have been generated because their construction is hampered by the instability of flaviviral genomes. More recently, advances in cloning techniques as well as the development of several two-plasmid WNV infectious clone systems have facilitated the generation of WNV infectious clones. Here we described a protocol for recovering WNV from a two-plasmid system. In this approach, large quantities of these constructs are digested with restriction enzymes to produce complementary restriction sites at the 3' end of the upstream fragment and the 5' end of the downstream fragment. These fragments are then annealed to produce linear template for in vitro transcription to synthesize infectious RNA. The resulting RNA is transfected into cells and after several days WNV is recovered in the culture supernatant. This method can be used to generate virus from infectious clones encoding high- and low-pathogenicity strains of WNV, as well as chimeric virues.

Molecular cloning of the cDNA encoding follicle-stimulating hormone beta subunit of the Chinese soft-shell turtle Pelodiscus sinensis, and its gene expression.

Science.gov (United States)

Chien, Jung-Tsun; Shen, San-Tai; Lin, Yao-Sung; Yu, John Yuh-Lin

2005-04-01

Follicle-stimulating hormone (FSH) is a member of the pituitary glycoprotein hormone family. These hormones are composed of two dissimilar subunits, alpha and beta. Very little information is available regarding the nucleotide and amino acid sequence of FSHbeta in reptilian species. For better understanding of the phylogenetic diversity and evolution of FSH molecule, we have isolated and sequenced the complementary DNA (cDNA) encoding the Chinese soft-shell turtle (Pelodiscus sinensis, Family of Trionychidae) FSHbeta precursor molecule by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) methods. The cloned Chinese soft-shell turtle FSHbeta cDNA consists of 602-bp nucleotides, including 34-bp nucleotides of the 5'-untranslated region (UTR), 396-bp of the open reading frame, and 3'-UTR of 206-bp nucleotides. It encodes a 131-amino acid precursor molecule of FSHbeta subunit with a signal peptide of 20 amino acids followed by a mature protein of 111 amino acids. Twelve cysteine residues, forming six disulfide bonds within beta-subunit and two putative asparagine-linked glycosylation sites, are also conserved in the Chinese soft-shell turtle FSHbeta subunit. The deduced amino acid sequence of the Chinese soft-shell turtle FSHbeta shares identities of 97% with Reeves's turtle (Family of Bataguridae), 83-89% with birds, 61-70% with mammals, 63-66% with amphibians and 40-58% with fish. By contrast, when comparing the FSHbeta with the beta-subunits of the Chinese soft-shell turtle luteinizing hormone and thyroid stimulating hormone, the homologies are as low as 38 and 39%, respectively. A phylogenetic tree including reptilian species of FSHbeta subunits, is presented for the first time. Out of various tissues examined, FSHbeta mRNA was only expressed in the pituitary gland and can be up-regulated by gonadotropin-releasing hormone in pituitary tissue culture as estimated by fluorescence real-time PCR analysis.

A novel pseudo-complementary PNA G-C base pair

DEFF Research Database (Denmark)

Olsen, Anne G.; Dahl, Otto; Petersen, Asger Bjørn

2011-01-01

Pseudo-complementary oligonucleotide analogues and mimics provide novel opportunities for targeting duplex structures in RNA and DNA. Previously, a pseudo-complementary A-T base pair has been introduced. Towards sequence unrestricted targeting, a pseudo-complementary G-C base pair consisting...

Molecular cloning and functional expression of a human cDNA encoding the antimutator enzyme 8-hydroxyguanine-DNA glycosylase

Science.gov (United States)

Roldán-Arjona, Teresa; Wei, Ying-Fei; Carter, Kenneth C.; Klungland, Arne; Anselmino, Catherine; Wang, Rui-Ping; Augustus, Meena; Lindahl, Tomas

1997-01-01

The major mutagenic base lesion in DNA caused by exposure to reactive oxygen species is 8-hydroxyguanine (8-oxo-7,8-dihydroguanine). In bacteria and Saccharomyces cerevisiae, this damaged base is excised by a DNA glycosylase with an associated lyase activity for chain cleavage. We have cloned, sequenced, and expressed a human cDNA with partial sequence homology to the relevant yeast gene. The encoded 47-kDa human enzyme releases free 8-hydroxyguanine from oxidized DNA and introduces a chain break in a double-stranded oligonucleotide specifically at an 8-hydroxyguanine residue base paired with cytosine. Expression of the human protein in a DNA repair-deficient E. coli mutM mutY strain partly suppresses its spontaneous mutator phenotype. The gene encoding the human enzyme maps to chromosome 3p25. These results show that human cells have an enzyme that can initiate base excision repair at mutagenic DNA lesions caused by active oxygen. PMID:9223306

DNA microarrays : a molecular cloning manual

National Research Council Canada - National Science Library

Sambrook, Joseph; Bowtell, David

2002-01-01

.... This manual, designed to extend and to complement the information in the best-selling Molecular Cloning, is a synthesis of the expertise and experience of more than 30 contributors all innovators in a fast moving field...

The Dermatophagoides farinae group 22 allergen: cloning and expression in Escherichia coli.

Science.gov (United States)

Cui, Yu-bao; Cai, Hong-xing; Zhou, Ying; Wang, Nan; Yu, Li-li; Yang, Li; Zhang, Cheng-bo

2015-09-01

Dermatophagoides farinae (Hughes) (Acari: Pyroglyphidae) and other domestic mites produce allergens that affect people worldwide. Here, the complementary DNA (cDNA) coding for group 22 allergen of D. farinae (Der f 22) from China was cloned, sequenced, and expressed successfully. The cDNA encoding Der f 22 was synthesized by reverse transcription polymerase chain reaction (RT-PCR), then ligated to the pCold-TF for expression in Escherichia coli BL21 cells. The purified recombinant fusion protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western-blotting, and tandem matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF/TOF). The full-length cDNA comprised 468 nucleotides and was 99.57% (466/468) identical with the reference sequence (GenBank: DQ643992). After the plasmid pCold-TF-Der f 22 was transformed into E. coli BL21 and expressed with the induction of IPTG, SDS-PAGE showed a specific band for the recombinant fusion protein. The recombinant fusion protein, which was purified by chromatography, bound with a His-tagged antibody by Western blotting. MALDI-TOF/TOF mass spectrometry revealed that the structure of the recombinant protein was identical to the predicted Der f 22 structure. The hydrophilic protein contains a signal peptide of 20 amino acids, and the mature Der f 22 consists of 135 amino acid residues with a molecular weight of 14.7 kDa and theoretical isoelectric points (pI) of 6.38. Its secondary structure comprises an alpha helix (38.5%), beta-sheet (45.9%), random coils (11.85%), and beta-turns (11.1%). This work represents the first reported full-length sequence and successful cloning of Der f 22 from D. farinae in China; bioinformatics analysis can be used to further study the allergenicity and clinical utility of the recombinant Der f 22. © 2015 ARS-AAOA, LLC.

Array patterns and clones - RMOS | LSDB Archive [Life Science Database Archive metadata

Lifescience Database Archive (English)

Full Text Available List Contact us RMOS Array patterns and clones Data detail Data name Array patterns and clones DOI 10.18908/...lsdba.nbdc00194-002 Description of data contents Static files of array patterns and cDNA clones. Data file F...h rice cDNA comprises a pair of glass slides. The microarray patterns are shown i...escription Download License Update History of This Database Site Policy | Contact Us Array patterns and clones - RMOS | LSDB Archive ...

Purification of MUC1 from Bovine Milk-Fat Globules and Characterization of a Corresponding Full-Length cDNA Clone

DEFF Research Database (Denmark)

Pallesen, Lone Tjener; Andersen, Mikkel Holmen; Nielsen, Rune

2001-01-01

acid sequences obtained by peptide mapping. The complete amino acid sequence of MUC1 was determined by cloning and sequencing the corresponding bovine mammary gland cDNA, which was shown to encode a protein of 580 amino acid residues comprising a cleavable signal peptide of 22 residues. The deduced...

Molecular cloning and characterization of recA-like gene from Schizosaccharomyces pombe

International Nuclear Information System (INIS)

Lee, J.S.; Kang, J.K.; Yoon, S.M.; Park, Y.; Yang, Y.K.; Kim, S.W.; Park, J.K.; Park, J.G.; Hong, S.H.; Park, S.D.

1996-01-01

We have previously purified and characterized a RecA-like protein from Schizosaccharomyces pombe (S. pombe). In the present study, we have cloned a gene encoding the RecA-like protein. The S. pombe recA-like gene was isolated by immunological screening of the expression library of S. pombe using anti-Escherichia coli (E. coli) RecA antibody as a probe. From 10(6) plaques screened, 6 putative clones were finally isolated. Five of the clones screened contained the same kinds of DNA inserts, as determined by crosshybridization analysis. Among the clones, TC-2 was selected for further studies. The pGEM3Zf(-)Delta 17 vector harboring the 4.3 kb DNA insert of TC-2 clone was capable of producing abeta-gal/RecA-like fusion protein, suggesting that the cloned gene encodes the RecA-like protein of S. pombe. It was also revealed by Southern hybridization analysis that the same DNA sequence as the cloned recA-like gene is located within the S. pombe chromosomal DNA. In addition, the cloned recA-like gene was transcribed into a 3.0 kb RNA transcript, as judged by Northern blot analysis. The level of the RNA transcript of recA-like gene was increased approximately 1.6 to 2.4-fold upon treatment with DNA damaging agents such as ultraviolet (UV)-light, methyl methanesulfonate (MMS), and mitomycin-C (MMC). This data suggests that the cloned S. pombe recA-like gene is slightly inducible to DNAdamage as in E. coli recA gene. These results suggest that an inducible repair mechanism analogous to that of E. coli may exist in fission yeast S. pombe

Cloning and restriction enzyme mapping of ribosomal DNA of Giardia duodenalis, Giardia ardeae and Giardia muris.

Science.gov (United States)

van Keulen, H; Campbell, S R; Erlandsen, S L; Jarroll, E L

1991-06-01

In an attempt to study Giardia at the DNA sequence level, the rRNA genes of three species, Giardia duodenalis, Giardia ardeae and Giardia muris were cloned and restriction enzyme maps were constructed. The rDNA repeats of these Giardia show completely different restriction enzyme recognition patterns. The size of the rDNA repeat ranges from approximately 5.6 kb in G. duodenalis to 7.6 kb in both G. muris and G. ardeae. These size differences are mainly attributable to the variation in length of the spacer. Minor differences exist among these Giardia in the sizes of their small subunit rRNA and the internal transcribed spacer between small and large subunit rRNA. The genetic maps were constructed by sequence analysis of the DNA around the 5' and 3' ends of the mature rRNA genes and between the rRNA covering the 5.8S rRNA gene and internal transcribed spacer. Comparison of the 5.8S rDNA and 3' end of large subunit rDNA from these three Giardia species showed considerable sequence variation, but the rDNA sequences of G. duodenalis and G. ardeae appear more closely related to each other than to G. muris.

cDNA cloning of chicken orexin receptor and tissue distribution: sexually dimorphic expression in chicken gonads.

Science.gov (United States)

Ohkubo, T; Tsukada, A; Shamoto, K

2003-12-01

Orexin-A and -B are known to stimulate food intake in mammals. However, the critical roles of orexins in birds are not fully understood, since orexins have no stimulatory effect on food intake in the chicken. To understand the physiological role(s) of orexins in birds, we have cloned chicken orexin receptor (cOXR) cDNA by RT-PCR, and analysed the tIssue distribution of OXR mRNA in the chicken. The cOXR cDNA is 1869 bp long and encodes 501 amino acids. The cloned cDNA for cOXR corresponds to the type 2 OXR in mammals, and shows approximately 80% similarity to those of mammals at the amino acid level. Expression analysis by RNase protection assay revealed OXR mRNA was distributed widely in brain regions, and expression in the cerebrum, hypothalamus and optic tectum were abundant. In peripheral tIssues, OXR mRNA was expressed in the pituitary gland, adrenal gland and testis, but no mRNA expression was observed in other tIssues examined. Furthermore, we found that the amount of cOXR mRNA was different between testis and ovary, while prepro-orexin mRNA is equally expressed in the gonads of both sexes in the chicken. These data indicate that the orexins have neuroendocrine actions in chickens, which are mediated through hypothalamic receptors as has been observed in mammals. In addition, orexin may have specific role(s) in the regulation of gonadal function in which sex-dependent mechanisms could be involved.

Familial hypercholesterolemia and atherosclerosis in cloned minipigs created by DNA transposition of a human PCSK9 gain-of-function mutant

DEFF Research Database (Denmark)

Al-Mashhadi, Rozh Husain; Sørensen, Charlotte Brandt; Kragh, Peter M.

2013-01-01

dominant hypercholesterolemia and accelerates atherosclerosis in humans. Using Sleeping Beauty DNA transposition and cloning by somatic cell nuclear transfer, we created Yucatan minipigs with liver-specific expression of human D374Y-PCSK9. D374Y-PCSK9 transgenic pigs displayed reduced hepatic low...

Nucleotide sequence of cloned cDNA for human sphingolipid activator protein 1 precursor

International Nuclear Information System (INIS)

Dewji, N.N.; Wenger, D.A.; O'Brien, J.S.

1987-01-01

Two cDNA clones encoding prepro-sphingolipid activator protein 1 (SAP-1) were isolated from a λ gt11 human hepatoma expression library using polyclonal antibodies. These had inserts of ≅ 2 kilobases (λ-S-1.2 and λ-S-1.3) and both were both homologous with a previously isolated clone (λ-S-1.1) for mature SAP-1. The authors report here the nucleotide sequence of the longer two EcoRI fragments of S-1.2 and S-1.3 that were not the same and the derived amino acid sequences of mature SAP-1 and its prepro form. The open reading frame encodes 19 amino acids, which are colinear with the amino-terminal sequence of mature SAP-1, and extends far beyond the predicted carboxyl terminus of mature SAP-1, indicating extensive carboxyl-terminal processing. The nucleotide sequence of cDNA encoding prepro-SAP-1 includes 1449 bases from the assigned initiation codon ATG at base-pair 472 to the stop codon TGA at base-pair 1921. The first 23 amino acids coded after the initiation ATG are characteristic of a signal peptide. The calculated molecular mass for a polypeptide encoded by 1449 bases is ≅ 53 kDa, in keeping with the reported value for pro-SAP-1. The data indicate that after removal of the signal peptide mature SAP-1 is generated by removing an additional 7 amino acids from the amino terminus and ≅ 373 amino acids from the carboxyl terminus. One potential glycosylation site was previously found in mature SAP-1. Three additional potential glycosylation sites are present in the processed carboxyl-terminal polypeptide, which they designate as P-2

Constructing and detecting a cDNA library for mites.

Science.gov (United States)

Hu, Li; Zhao, YaE; Cheng, Juan; Yang, YuanJun; Li, Chen; Lu, ZhaoHui

2015-10-01

RNA extraction and construction of complementary DNA (cDNA) library for mites have been quite challenging due to difficulties in acquiring tiny living mites and breaking their hard chitin. The present study is to explore a better method to construct cDNA library for mites that will lay the foundation on transcriptome and molecular pathogenesis research. We selected Psoroptes cuniculi as an experimental subject and took the following steps to construct and verify cDNA library. First, we combined liquid nitrogen grinding with TRIzol for total RNA extraction. Then, switching mechanism at 5' end of the RNA transcript (SMART) technique was used to construct full-length cDNA library. To evaluate the quality of cDNA library, the library titer and recombination rate were calculated. The reliability of cDNA library was detected by sequencing and analyzing positive clones and genes amplified by specific primers. The results showed that the RNA concentration was 836 ng/μl and the absorbance ratio at 260/280 nm was 1.82. The library titer was 5.31 × 10(5) plaque-forming unit (PFU)/ml and the recombination rate was 98.21%, indicating that the library was of good quality. In the 33 expressed sequence tags (ESTs) of P. cuniculi, two clones of 1656 and 1658 bp were almost identical with only three variable sites detected, which had an identity of 99.63% with that of Psoroptes ovis, indicating that the cDNA library was reliable. Further detection by specific primers demonstrated that the 553-bp Pso c II gene sequences of P. cuniculi had an identity of 98.56% with those of P. ovis, confirming that the cDNA library was not only reliable but also feasible.

cDNA Cloning, expression and characterization of an allergenic 60s ribosomal protein of almond (prunus dulcis).

Science.gov (United States)

Abolhassani, Mohsen; Roux, Kenneth H

2009-06-01

Tree nuts, including almond (prunus dulcis) are a source of food allergens often associated with life-threatening allergic reactions in susceptible individuals. Although the proteins in almonds have been biochemically characterized, relatively little has been reported regarding the identity of the allergens involved in almond sensitivity. The present study was undertaken to identify the allergens of the almond by cDNA library approach. cDNA library of almond seeds was constructed in Uni-Zap XR lamda vector and expressed in E. coli XL-1 blue. Plaques were immunoscreened with pooled sera of allergic patients. The cDNA clone reacting significantly with specific IgE antibodies was selected and subcloned and subsequently expressed in E. coli. The amino acids deducted from PCR product of clone showed homology to 60s acidic ribosomal protein of almond. The expressed protein was 11,450 Dalton without leader sequence. Immunoreactivity of the recombinant 60s ribosomal protein (r60sRP) was evaluated with dot blot analysis using pooled and individual sera of allergic patients. The data showed that r60sRP and almond extract (as positive control) possess the ability to bind the IgE antibodies. The results showed that expressed protein is an almond allergen.Whether this r60sRP represents a major allergen of almond needs to be further studied which requires a large number of sera from the almond atopic patients and also need to determine the IgE-reactive frequencies of each individual allergen.

Use of homologous recombination in yeast to create chimeric bovine viral diarrhea virus cDNA clones

Directory of Open Access Journals (Sweden)

Sandra Arenhart

Full Text Available Abstract The open reading frame of a Brazilian bovine viral diarrhea virus (BVDV strain, IBSP4ncp, was recombined with the untranslated regions of the reference NADL strain by homologous recombination in Saccharomyces cerevisiae, resulting in chimeric full-length cDNA clones of BVDV (chi-NADL/IBSP4ncp#2 and chi-NADL/IBSP4ncp#3. The recombinant clones were successfully recovered, resulting in viable viruses, having the kinetics of replication, focus size, and morphology similar to those of the parental virus, IBSP4ncp. In addition, the chimeric viruses remained stable for at least 10 passages in cell culture, maintaining their replication efficiency unaltered. Nucleotide sequencing revealed a few point mutations; nevertheless, the phenotype of the rescued viruses was nearly identical to that of the parental virus in all experiments. Thus, genetic stability of the chimeric clones and their phenotypic similarity to the parental virus confirm the ability of the yeast-based homologous recombination to maintain characteristics of the parental virus from which the recombinant viruses were derived. The data also support possible use of the yeast system for the manipulation of the BVDV genome.

Cloning an expressed gene shared by the human sex chromosomes

International Nuclear Information System (INIS)

Darling, S.M.; Banting, G.S.; Pym, B.; Wolfe, J.; Goodfellow, P.N.

1986-01-01

The existence of genes shared by mammalian sex chromosomes has been predicted on both evolutionary and functional grounds. However, the only experimental evidence for such genes in humans is the cell-surface antigen encoded by loci on the X and Y chromosomes (MIC2X and MIC2Y, respectively), which is recognized by the monoclonal antibody 12E7. Using the bacteriophage λgt11 expression system in Escherichia coli and immunoscreening techniques, the authors have isolated a cDNA clone whose primary product is recognized by 12E7. Southern blot analysis using somatic cell hybrids containing only the human X or Y chromosomes shows that the sequences reacting with the cDNA clone are localized to the sex chromosomes. In addition, the clone hybridizes to DNAs isolated from mouse cells that have been transfected with human DNA and selected for 12E7 expression on the fluorescence-activated cell sorter. The authors conclude that the cDNA clone encodes the 12E7 antigen, which is the primary product of the MIC2 loci. The clone was used to explore sequence homology between MIC2X and MIC2Y; these loci are closely related, if not identical

Virulence in pigs of vPader10 rescued from an infectious cDNA clone of the CSFV strain Paderborn

DEFF Research Database (Denmark)

Friis, Martin Barfred; Nielsen, Jens; Uttenthal, Åse

The BAC clone, pBeloPader10, contains a complete cDNA of the CSFV strain Paderborn. Virus, named vPader10, was rescued from this construct by electroporation of RNA transcripts into porcine PK15 cells. To further study the characteristics of vPader10, we evaluated the virulence of this virus...

cDNA cloning, sequence analysis, and chromosomal localization of the gene for human carnitine palmitoyltransferase

International Nuclear Information System (INIS)

Finocchiaro, G.; Taroni, F.; Martin, A.L.; Colombo, I.; Tarelli, G.T.; DiDonato, S.; Rocchi, M.

1991-01-01

The authors have cloned and sequenced a cDNA encoding human liver carnitine palmitoyltransferase an inner mitochondrial membrane enzyme that plays a major role in the fatty acid oxidation pathway. Mixed oligonucleotide primers whose sequences were deduced from one tryptic peptide obtained from purified CPTase were used in a polymerase chain reaction, allowing the amplification of a 0.12-kilobase fragment of human genomic DNA encoding such a peptide. A 60-base-pair (bp) oligonucleotide synthesized on the basis of the sequence from this fragment was used for the screening of a cDNA library from human liver and hybridized to a cDNA insert of 2255 bp. This cDNA contains an open reading frame of 1974 bp that encodes a protein of 658 amino acid residues including 25 residues of an NH 2 -terminal leader peptide. The assignment of this open reading frame to human liver CPTase is confirmed by matches to seven different amino acid sequences of tryptic peptides derived from pure human CPTase and by the 82.2% homology with the amino acid sequence of rat CPTase. The NH 2 -terminal region of CPTase contains a leucine-proline motif that is shared by carnitine acetyl- and octanoyltransferases and by choline acetyltransferase. The gene encoding CPTase was assigned to human chromosome 1, region 1q12-1pter, by hybridization of CPTase cDNA with a DNA panel of 19 human-hanster somatic cell hybrids

Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality.

Science.gov (United States)

Buemo, Carla Paola; Gambini, Andrés; Moro, Lucia Natalia; Hiriart, María Inés; Fernández-Martín, Rafael; Collas, Philippe; Salamone, Daniel Felipe

2016-01-01

In this study, we analyzed the effects of the cloned embryo aggregation on in vitro embryo development and embryo quality by measuring blastocyst diameter and cell number, DNA fragmentation levels and the expression of genes associated with pluripotency, apoptosis, trophoblast and DNA methylation in the porcine. Zona-free reconstructed cloned embryos were cultured in the well of the well system, placing one (1x non aggregated group) or three (3x group) embryos per microwell. Our results showed that aggregation of three embryos increased blastocyst formation rate and blastocyst diameter of cloned pig embryos. DNA fragmentation levels in 3x aggregated cloned blastocysts were significantly decreased compared to 1x blastocysts. Levels of Oct4, Klf4, Igf2, Bax and Dnmt 1 transcripts were significantly higher in aggregated embryos, whereas Nanog levels were not affected. Transcripts of Cdx2 and Bcl-xl were essentially non-detectable. Our study suggests that embryo aggregation in the porcine may be beneficial for cloned embryo development and embryo quality, through a reduction in apoptotic levels and an improvement in cell reprogramming.

Production of a full-length infectious GFP-tagged cDNA clone of Beet mild yellowing virus for the study of plant-polerovirus interactions.

Science.gov (United States)

Stevens, Mark; Viganó, Felicita

2007-04-01

The full-length cDNA of Beet mild yellowing virus (Broom's Barn isolate) was sequenced and cloned into the vector pLitmus 29 (pBMYV-BBfl). The sequence of BMYV-BBfl (5721 bases) shared 96% and 98% nucleotide identity with the other complete sequences of BMYV (BMYV-2ITB, France and BMYV-IPP, Germany respectively). Full-length capped RNA transcripts of pBMYV-BBfl were synthesised and found to be biologically active in Arabidopsis thaliana protoplasts following electroporation or PEG inoculation when the protoplasts were subsequently analysed using serological and molecular methods. The BMYV sequence was modified by inserting DNA that encoded the jellyfish green fluorescent protein (GFP) into the P5 gene close to its 3' end. A. thaliana protoplasts electroporated with these RNA transcripts were biologically active and up to 2% of transfected protoplasts showed GFP-specific fluorescence. The exploitation of these cDNA clones for the study of the biology of beet poleroviruses is discussed.

A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering.

Science.gov (United States)

Nørholm, Morten H H

2010-03-16

The combined use of restriction enzymes with PCR has revolutionized molecular cloning, but is inherently restricted by the content of the manipulated DNA sequences. Uracil-excision based cloning is ligase and sequence independent and allows seamless fusion of multiple DNA sequences in simple one-tube reactions, with higher accuracy than overlapping PCR. Here, the addition of a highly efficient DNA polymerase and a low-background-, large-insertion- compatible site-directed mutagenesis protocol is described, largely expanding the versatility of uracil-excision DNA engineering. The different uracil-excision based molecular tools that have been developed in an open-source fashion, constitute a comprehensive, yet simple and inexpensive toolkit for any need in molecular cloning.

Molecular cloning of transcripts induced by UV-radiation in rodent cells

International Nuclear Information System (INIS)

Fornace, A.J. Jr.; Mitchell, J.B.

1987-01-01

Several inducible DNA repair genes have been well characterized in bacteria. In eukaryotes including mammalian cells, there is increasing evidence that similar events may occur. Recently, the authors have shown that hybridization subtraction can be used to enrich for sequences induced only several fold by a particular cell treatment such as heat shock. Chinese hamster V79 cells were UV-irradiated with 17 Jm/sup -2/ and cDNA was synthesized from the polyadenylated (poly A) RNA. This ''UV'' cDNA was hybridized with a 3 fold excess of polyA RNA from unirradiated cells and the nonhybridizing cDNA was isolated. With this approach, UV-induced sequences were enriched over 20 fold. This enriched cDNA was cloned into a high copy number plasmid and a cDNA library was constructed. By RNA dot blot and northern analysis, 42 clones from this library were found to represent transcripts induced 3 to 25 fold by UV. The most common isolates were found to be metallothionein transcripts by DNA sequencing. The metallothionein transcripts were found to be induced 10 to 25 fold by UV with maximum induction at 4-8 h after 10 Jm/sup -2/. A similar approach was also used with a Chinese hamster ovary line which does not express metallothionein and multiple clones were isolated which represented transcripts induced 3-15 fold by UV. Except for the metallothionein clones, the other Chinese hamster cDNA clones have not been identified, but it is probable that the protein products of at least some of these transcripts play a role in the cellular response to UV damage

Nucleic acids in mummified plant seeds: screening of twelve specimens by gel-electrophoresis, molecular hybridization and DNA cloning.

Science.gov (United States)

Rollo, F; La Marca, A; Amici, A

1987-02-01

Twelve seed specimens of varying ages and from different archaeological sites were analyzed for the presence of polymerized DNA and RNA. Amongst the samples tested, one of Vitis vinifera from an archaeological site in Iran (2,000-3,000 B.C.) was found to be completely devoid of nucleic acids. Zea mais seeds of Precolumbial age from Peru (about 800 A.D.) contained depolymerized DNA and RNA. Samples of Vitis vinifera and Rubus sp. from a Lombard archaeological site (800 A.D.) as well as radiocarbon dated seeds from the site of the "Spring Sanctuary" near Metaponto (I-IV century B.C.) were found to contain polymerized DNA and rRNA bands. However the electrophoretic properties of the rRNAs in one case and hybridization experiments performed with cloned seed DNA in the other, clearly demonstrated that the polymerized nucleic acids were not of plant origin.

DNA Probe for Lactobacillus delbrueckii

Science.gov (United States)

Delley, Michèle; Mollet, Beat; Hottinger, Herbert

1990-01-01

From a genomic DNA library of Lactobacillus delbrueckii subsp. bulgaricus, a clone was isolated which complements a leucine auxotrophy of an Escherichia coli strain (GE891). Subsequent analysis of the clone indicated that it could serve as a specific DNA probe. Dot-blot hybridizations with over 40 different Lactobacillus strains showed that this clone specifically recognizes L. delbrueckii subsp. delbrueckii, bulgaricus, and lactis. The sensitivity of the method was tested by using an α-32P-labeled DNA probe. Images PMID:16348233

Molecular cloning and characterization of a Streptococcus sanguis DNase necessary for repair of DNA damage induced by UV light and methyl methanesulfonate

International Nuclear Information System (INIS)

Lindler, L.E.; Macrina, F.L.

1987-01-01

We developed a method for cloning cellular nucleases from streptococci. Recombinant lambda gt11 bacteriophage containing streptococcal nuclease determinants were identified by the production of pink plaques on toluidine blue O DNase plates. We used this technique to clone a 3.2-kilobase-pair EcoRI fragment with DNase activity from the chromosome of Streptococcus sanguis. The locus was designated don (DNase one) and could be subcloned and stably maintained on plasmid vectors in Escherichia coli. Minicell analyses of various subclones of the don locus allowed us to determine the coding region and size of the Don nuclease in E. coli. The don gene product had an apparent molecular mass of 34 kilodaltons and degraded native DNA most efficiently, with lesser activity against denatured DNA and no detectable activity against RNA. S. sanguis don deletion mutants were constructed by transformation of competent cells with in vitro-prepared plasmid constructs. S. sanguis don deletion mutants retained normal transformation frequencies for exogenously added donor DNA. However, when compared with Don+ wild-type cells, these mutants were hypersensitive to DNA damage induced by UV light and methyl methanesulfonate. An S. sanguis don-specific DNA probe detected homology to chromosomal DNA isolated from Streptococcus pneumoniae and Streptococcus mutans Bratthall serogroups d and g. Our results suggested that the don locus was the S. sanguis allele of the previously described S. pneumoniae major exonuclease and was involved in repair of DNA damage. Furthermore, hybridization studies suggested that the don locus was conserved among species of oral streptococci

Cloning and characterization of a pathogen-induced chitinase in Brassica napus

DEFF Research Database (Denmark)

Rasmussen, U.; Bojsen, K.; Collinge, D.B.

1992-01-01

A chitinase cDNA clone from rapeseed (Brassica napus L. ssp. oleifera) was isolated. The cDNA clone, ChB4, represents a previously purified and characterized basic chitinase isozyme. The longest open reading frame in ChB4 encodes a polypeptide of 268 amino acids. This polypeptide consists of a 24...

Growth hormone and prolactin in Andrias davidianus: cDNA cloning, tissue distribution and phylogenetic analysis.

Science.gov (United States)

Yang, Liping; Meng, Zining; Liu, Yun; Zhang, Yong; Liu, Xiaochun; Lu, Danqi; Huang, Junhai; Lin, Haoran

2010-01-15

The Chinese giant salamander (Andrias davidianus) is one of the largest and 'living fossil' species of amphibian. To obtain genetic information for this species, the cDNAs encoding growth hormone (adGH) and prolactin (adPRL) were cloned from a pituitary cDNA library. The isolated adGH cDNA consisted of 864 bp and encoded a propeptide of 215 amino acids, while the cDNA of adPRL was 1106 bp in length and encoded a putative peptide of 229 amino acids. Expression of the GH and PRL mRNA was only detected in the pituitary. Phylogenetic analyses were performed based on the isolated pituitary hormone sequences using maximum parsimony and neighbor-joining algorithms. The clustering results are similar to that based on the morphological characteristics or the rRNA genes, which indicate that the two orders (Anura and Caudata) of amphibian were monophyletic, and that A. davidianus was diverged early in the Caudate clade. These results indicated that both the GH and PRL sequence might be useful to study the phylogenies of relatively moderate evolved groups.

High-throughput cloning and expression in recalcitrant bacteria

NARCIS (Netherlands)

Geertsma, Eric R.; Poolman, Bert

We developed a generic method for high-throughput cloning in bacteria that are less amenable to conventional DNA manipulations. The method involves ligation-independent cloning in an intermediary Escherichia coli vector, which is rapidly converted via vector-backbone exchange (VBEx) into an

Aberrant epigenetic changes and gene expression in cloned cattle dying around birth

Directory of Open Access Journals (Sweden)

Zhao Dingsheng

2008-02-01

Full Text Available Abstract Background Aberrant reprogramming of donor somatic cell nuclei may result in many severe problems in animal cloning. To assess the extent of abnormal epigenetic modifications and gene expression in clones, we simultaneously examined DNA methylation, histone H4 acetylation and expression of six genes (β-actin, VEGF, oct4, TERT, H19 and Igf2 and a repetitive sequence (art2 in five organs (heart, liver, spleen, lung and kidney from two cloned cattle groups that had died at different stages. In the ED group (early death, n = 3, the cloned cattle died in the perinatal period. The cattle in the LD group (late death, n = 3 died after the perinatal period. Normally reproduced cattle served as a control group (n = 3. Results Aberrant DNA methylation, histone H4 acetylation and gene expression were observed in both cloned groups. The ED group showed relatively fewer severe DNA methylation abnormalities (p Conclusion Deaths of clones may be ascribed to abnormal expression of a very limited number of genes.

Preparation of Proper Immunogen by Cloning and Stable Expression of cDNA coding for Human Hematopoietic Stem Cell Marker CD34 in NIH-3T3 Mouse Fibroblast Cell Line

Science.gov (United States)

Shafaghat, Farzaneh; Abbasi-Kenarsari, Hajar; Majidi, Jafar; Movassaghpour, Ali Akbar; Shanehbandi, Dariush; Kazemi, Tohid

2015-01-01

Purpose: Transmembrane CD34 glycoprotein is the most important marker for identification, isolation and enumeration of hematopoietic stem cells (HSCs). We aimed in this study to clone the cDNA coding for human CD34 from KG1a cell line and stably express in mouse fibroblast cell line NIH-3T3. Such artificial cell line could be useful as proper immunogen for production of mouse monoclonal antibodies. Methods: CD34 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy TA-cloning vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. Results: 1158 bp specific band was aligned completely to reference sequence in NCBI database corresponding to long isoform of human CD34. Transient and stable expression of human CD34 on transfected NIH-3T3 mouse fibroblast cells was achieved (25% and 95%, respectively) as shown by flow cytometry. Conclusion: Cloning and stable expression of human CD34 cDNA was successfully performed and validated by standard flow cytometric analysis. Due to murine origin of NIH-3T3 cell line, CD34-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD34. This approach could bypass the need for purification of recombinant proteins produced in eukaryotic expression systems. PMID:25789221

High-Throughput Cloning and Expression Library Creation for Functional Proteomics

Science.gov (United States)

Festa, Fernanda; Steel, Jason; Bian, Xiaofang; Labaer, Joshua

2013-01-01

The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particular important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single gene experiments, creating the need for fast, flexible and reliable cloning systems. These collections of open reading frame (ORF) clones can be coupled with high-throughput proteomics platforms, such as protein microarrays and cell-based assays, to answer biological questions. In this tutorial we provide the background for DNA cloning, discuss the major high-throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and Creator™ DNA Cloning System) and compare them side-by-side. We also report an example of high-throughput cloning study and its application in functional proteomics. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP12). Details can be found at http://www.proteomicstutorials.org. PMID:23457047

The proviral genome of radiation leukemia virus (RadLV): molecular cloning, restriction analysis and integration sites in tumor cell DNA

International Nuclear Information System (INIS)

Janowski, M.; Merregaert, J.; Nuyten, J.M.; Maisin, J.R.

1984-01-01

An infectious clone of the linear, unintegrated RadLV provirus was obtained by insertion in the plasmid pBR322. Its restriction map was indistinguishable from that of the majority of the multiple proviral copies, which are found apparently at random sites in the DNA of RadLV-induced rat thymic lymphomas [fr

Stable expression and replication of hepatitis B virus genome in an integrated state in a human hepatoma cell line transfected with the cloned viral DNA

International Nuclear Information System (INIS)

Tsurimoto, T.; Fujiyama, A.; Matsubara, K.

1987-01-01

A human hepatocellular carcinoma cell line (Huh6-c15) was transfected with a recombinant DNA molecule that consists of tandemly arranged hepatitis B virus (HBV) genome and a neomycin-resistant gene. One clone resistant to G-418 produces and releases surface antigen and e antigen into medium at a high level and accumulates core particles intracellularly. This clone has a chromosomally integrated set of the original recombinant DNA and produces a 3.5-kilobase transcript corresponding to the pregenome RNA as well as HBV DNAs in an extrachromosomal form. Most of these DNAs were in single-stranded or partially double-stranded form and were packaged in the intracellular core particles. In the medium, particles were detected that contained HBV DNA and were morphologically indistinguishable from Dane particles. These results demonstrate that the HBV genome in an integrated state acted as a template for viral gene expression and replication. The cells were maintained for more than 6 months without losing the ability to produce the extrachromosomal HBV DNA and Dane-like particles. Thus, the cells can be used as a model system for analyses of gene expression and DNA replication of HBV in human hepatocytes

cDNA cloning of porcine brain prolyl endopeptidase and identification of the active-site seryl residue

Energy Technology Data Exchange (ETDEWEB)

Rennex, D.; Hemmings, B.A.; Hofsteenge, J.; Stone, S.R. (Friedrich Miescher-Institut, Basel (Switzerland))

1991-02-26

Prolyl endopeptidase is a cytoplasmic serine protease. The enzyme was purified from porcine kidney, and oligonucleotides based on peptide sequences from this protein were used to isolate a cDNA clone from a porcine brain library. This clone contained the complete coding sequence of prolyl endopeptidase and encoded a polypeptide with a molecular mass of 80751 Da. The deduced amino acid sequence of prolyl endopeptidase showed no sequence homology with other known serine proteases. ({sup 3}H)Diisopropyl fluorophosphate was used to identify the active-site serine of prolyl endopeptidase. One labeled peptide was isolated and sequenced. The sequence surrounding the active-site serine was Asn-Gly-Gly-Ser-Asn-Gly-Gly. This sequence is different from the active-site sequences of other known serine proteases. This difference and the lack of overall homology with the known families of serine proteases suggest that prolyl endopeptidase represents a new type of serine protease.

Molecular cloning of a cDNA encoding human calumenin, expression in Escherichia coli and analysis of its Ca2+-binding activity

DEFF Research Database (Denmark)

Vorum, H; Liu, X; Madsen, Peder

1998-01-01

By microsequencing and cDNA cloning we have identified the transformation-sensitive protein No. IEF SSP 9302 as the human homologue of calumenin. The nucleotide sequence predicts a 315 amino acid protein with high identity to murine and rat calumenin. The deduced protein contains a 19 amino acid N...

Molecular cloning of a human glycophorin B cDNA: nucleotide sequence and genomic relationship to glycophorin A

International Nuclear Information System (INIS)

Siebert, P.D.; Fukuda, M.

1987-01-01

The authors describe the isolation and nucleotide sequence of a human glycophorin B cDNA. The cDNA was identified by differential hybridization of synthetic oligonucleotide probes to a human erythroleukemic cell line (K562) cDNA library constructed in phage vector λgt10. The nucleotide sequence of the glycophorin B cDNA was compared with that of a previously cloned glycophorin A cDNA. The nucleotide sequences encoding the NH 2 -terminal leader peptide and first 26 amino acids of the two proteins are nearly identical. This homologous region is followed by areas specific to either glycophorin A or B and a number of small regions of homology, which in turn are followed by a very homologous region encoding the presumed membrane-spanning portion of the proteins. They used RNA blot hybridization with both cDNA and synthetic oligonucleotide probes to prove our previous hypothesis that glycophorin B is encoded by a single 0.5- to 0.6-kb mRNA and to show that glycophorins A and B are negatively and coordinately regulated by a tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate. They established the intron/exon structure of the glycophorin A and B genes by oligonucleotide mapping; the results suggest a complex evolution of the glycophorin genes

Paramyosin from the parasitic mite Sarcoptes scabiei: cDNA cloning and heterologous expression.

Science.gov (United States)

Mattsson, J G; Ljunggren, E L; Bergström, K

2001-05-01

The burrowing mite Sarcoptes scabiei is the causative agent of the highly contagious disease sarcoptic mange or scabies. So far, there is no in vitro propagation system for S. scabiei available, and mites used for various purposes must be isolated from infected hosts. Lack of parasite-derived material has limited the possibilities to study several aspects of scabies, including pathogenesis and immunity. It has also hampered the development of high performance serological assays. We have now constructed an S. scabiei cDNA expression library with mRNA purified from mites isolated from red foxes. Immunoscreening of the library enabled us to clone a full-length cDNA coding for a 102.5 kDa protein. Sequence similarity searches identified the protein as a paramyosin. Recombinant S. scabiei paramyosin expressed in Escherichia coli was recognized by sera from dogs and swine infected with S. scabiei. We also designed a small paramyosin construct of about 17 kDa that included the N-terminal part, an evolutionary variable part of the helical core, and the C-terminal part of the molecule. The miniaturized protein was efficiently expressed in E. coli and was recognized by sera from immunized rabbits. These data demonstrate that the cDNA library can assist in the isolation of important S. scabiei antigens and that recombinant proteins can be useful for the study of scabies.

Cloning and characterization of transferrin cDNA and rapid detection of transferrin gene polymorphism in rainbow trout (Oncorhynchus mykiss).

Science.gov (United States)

Tange, N; Jong-Young, L; Mikawa, N; Hirono, I; Aoki, T

1997-12-01

A cDNA clone of rainbow trout (Oncorhynchus mykiss) transferrin was obtained from a liver cDNA library. The 2537-bp cDNA sequence contained an open reading frame encoding 691 amino acids and the 5' and 3' noncoding regions. The amino acid sequences at the iron-binding sites and the two N-linked glycosylation sites, and the cysteine residues were consistent with known, conserved vertebrate transferrin cDNA sequences. Single N-linked glycosylation sites existed on the N- and C-lobe. The deduced amino acid sequence of the rainbow trout transferrin cDNA had 92.9% identities with transferrin of coho salmon (Oncorhynchus kisutch); 85%, Atlantic salmon (Salmo salar); 67.3%, medaka (Oryzias latipes); 61.3% Atlantic cod (Gadus morhua); and 59.7%, Japanese flounder (Paralichthys olivaceus). The long and accurate polymerase chain reaction (LA-PCR) was used to amplify approximately 6.5 kb of the transferrin gene from rainbow trout genomic DNA. Restriction fragment length polymorphisms (RFLPs) of the LA-PCR products revealed three digestion patterns in 22 samples.

Cloning and characterization of a cDNA encoding topoisomerase II in pea and analysis of its expression in relation to cell proliferation.

Science.gov (United States)

Reddy, M K; Nair, S; Tewari, K K; Mudgil, Y; Yadav, B S; Sopory, S K

1999-09-01

We have isolated and sequenced four overlapping cDNA clones to identify the full-length cDNA for topoisomerase II (PsTopII) from pea. Using degenerate primers, based on the conserved amino acid sequences of other eukaryotic type II topoisomerases, a 680 bp fragment was PCR-amplified with pea cDNA as template. This fragment was used as a probe to screen an oligo-dT-primed pea cDNA library. A partial cDNA clone was isolated that was truncated at the 3' end. RACE-PCR was employed to isolate the remaining portion of the gene. The total size of PsTopII is 4639 bp with an open reading frame of 4392 bp. The deduced amino acid sequence shows a strong homology to other eukaryotic topoisomerase II (topo II) at the N-terminus end. The topo II transcript was abundant in proliferative tissues. We also show that the level of topo II transcripts could be stimulated by exogenous application of growth factors that induced proliferation in vitro cultures. Light irradiation to etiolated tissue strongly stimulated the expression of topo II. These results suggest that topo II gene expression is up-regulated in response to light and hormones and correlates with cell proliferation. Besides, we have also isolated and analysed the 5'-flanking region of the pea TopII gene. This is first report on the isolation of a putative promoter for topoisomerase II from plants.

Molecular cloning, expression analysis and sequence prediction of ...

African Journals Online (AJOL)

CCAAT/enhancer-binding protein beta as an essential transcriptional factor, regulates the differentiation of adipocytes and the deposition of fat. Herein, we cloned the whole open reading frame (ORF) of bovine C/EBPβ gene and analyzed its putative protein structures via DNA cloning and sequence analysis. Then, the ...

16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development - Poster

Science.gov (United States)

We examined the bacterial composition of chlorinated drinking water using 16S rRNA gene clone libraries derived from RNA and DNA extracted from twelve water samples collected in three different months (June, August, and September of 2007). Phylogenetic analysis of 1234 and 1117 ...

Sequencing and characterization of asclepain f: the first cysteine peptidase cDNA cloned and expressed from Asclepias fruticosa latex.

Science.gov (United States)

Trejo, Sebastián A; López, Laura M I; Caffini, Néstor O; Natalucci, Claudia L; Canals, Francesc; Avilés, Francesc X

2009-07-01

Asclepain f is a papain-like protease previously isolated and characterized from latex of Asclepias fruticosa. This enzyme is a member of the C1 family of cysteine proteases that are synthesized as preproenzymes. The enzyme belongs to the alpha + beta class of proteins, with two disulfide bridges (Cys22-Cys63 and Cys56-Cys95) in the alpha domain, and another one (Cys150-Cys201) in the beta domain, as was determined by molecular modeling. A full-length 1,152 bp cDNA was cloned by RT-RACE-PCR from latex mRNA. The sequence was predicted as an open reading frame of 340 amino acid residues, of which 16 residues belong to the signal peptide, 113 to the propeptide and 211 to the mature enzyme. The full-length cDNA was ligated to pPICZalpha vector and expressed in Pichia pastoris. Recombinant asclepain f showed endopeptidase activity on pGlu-Phe-Leu-p-nitroanilide and was identified by PMF-MALDI-TOF MS. Asclepain f is the first peptidase cloned and expressed from mRNA isolated from plant latex, confirming the presence of the preprocysteine peptidase in the latex.

Bovine viral diarrhea virus: molecular cloning of genomic RNA and its diagnostic application

International Nuclear Information System (INIS)

Brock, K.V.

1987-01-01

Molecular cloning of a field isolate of bovine viral diarrhea virus (BVDV) strain 72 RNA was done in this study. The sensitivity and specificity of cloned cDNA sequences in hybridization assays with various BVDV strains were determined. cDNA was synthesized from polyadenylated BVDV RNA templates with oligo-dT primers, reverse transcriptase, and DNA polymerase I. The newly synthesized double-stranded BVDV cDNA was C-tailed with terminal deoxytransferase and annealed into G-tailed, Pst-1-cut pUC9 plasmid. Escherichia coli was transformed with the recombinant plasmids and a library of approximately 200 BVDV specific cDNA clones varying in length from 0.5 to 2.6 kilobases were isolated. The sensitivity and specificity of hybridization between the labelled cDNA and BVDV target sequences were determined. Cloned BVDV sequences were isolated from pUC9 plasmid DNA and labelled with 32 P by nick translation. The detection limit by dot blot hybridization assay was 20 pg of purified genomic BVDV RNA. cDNA hybridization probes were specific for all strains of BVDV tested, regardless of whether they were noncytopathic and cytopathic, but did not hybridize with heterologous bovine viruses tested. Probes did not hybridize with uninfected cell culture or cellular RNA. Hybridization probes were at least as sensitive as infectivity assays in detecting homologous virus

Development and analysis of a tick-borne encephalitis virus infectious clone using a novel and rapid strategy.

Science.gov (United States)

Gritsun, T S; Gould, E A

1998-12-01

In less than 1 month we have constructed an infectious clone of attenuated tick-borne encephalitis virus (strain Vasilchenko) from 100 microl of unpurified virus suspension using long high fidelity PCR and a modified bacterial cloning system. Optimization of the 3' antisense primer concentration was essential to achieve PCR synthesis of an 11 kb cDNA copy of RNA from infectious virus. A novel system utilising two antisense primers, a 14-mer for reverse transcription and a 35-mer for long PCR, produced high yields of genomic length cDNA. Use of low copy number Able K cells and an incubation temperature of 28 degrees C increased the genetic stability of cloned cDNA. Clones containing 11 kb cDNA inserts produced colonies of reduced size, thus providing a positive selection system for full length clones. Sequencing of the infectious clone emphasised the improved fidelity of the method compared with conventional PCR and cloning methods. A simple and rapid strategy for genetic manipulation of the infectious clone is also described. These developments represent a significant advance in recombinant technology and should be applicable to positive stranded RNA viruses which cannot easily be purified or genetically manipulated.

Isolation, cDNA cloning and gene expression of an antibacterial protein from larvae of the coconut rhinoceros beetle, Oryctes rhinoceros.

Science.gov (United States)

Yang, J; Yamamoto, M; Ishibashi, J; Taniai, K; Yamakawa, M

1998-08-01

An antibacterial protein, designated rhinocerosin, was purified to homogeneity from larvae of the coconut rhinoceros beetle, Oryctes rhinoceros immunized with Escherichia coli. Based on the amino acid sequence of the N-terminal region, a degenerate primer was synthesized and reverse-transcriptase PCR was performed to clone rhinocerosin cDNA. As a result, a 279-bp fragment was obtained. The complete nucleotide sequence was determined by sequencing the extended rhinocerosin cDNA clone by 5' rapid amplification of cDNA ends. The deduced amino acid sequence of the mature portion of rhinocerosin was composed of 72 amino acids without cystein residues and was shown to be rich in glycine (11.1%) and proline (11.1%) residues. Comparison of the deduced amino acid sequence of rhinocerosin with those of other antibacterial proteins indicated that it has 77.8% and 44.6% identity with holotricin 2 and coleoptrecin, respectively. Rhinocerosin had strong antibacterial activity against E. coli, Streptococcus pyogenes, Staphylococcus aureus but not against Pseudomonas aeruginosa. Results of reverse-transcriptase PCR analysis of gene expression in different tissues indicated that the rhinocerosin gene is strongly expressed in the fat body and the Malpighian tubule, and weakly expressed in hemocytes and midgut. In addition, gene expression was inducible by bacteria in the fat body, the Malpighian tubule and hemocyte but constitutive expression was observed in the midgut.

Cloning of a cDNA encoding a surface antigen of Schistosoma mansoni schistosomula recognized by sera of vassinated mice

International Nuclear Information System (INIS)

Dalton, J.P.; Tom, T.D.; Strand, M.

1987-01-01

Spleen cells of mice vaccinated with radiation-attenuated Schistosoma mansoni cercariae were used to produce monoclonal antibodies directed against newly transformed schistosomular surface antigens. One of these monoclonal antibodies recognized a polypeptide of 18 kDa. Binding was measured by radioimmunoassay. This glycoprotein was purified by monoclonal antibody immunoaffinity chromatography and a polyclonal antiserum was prepared against it. Immunofluorescence assays showed that the polyclonal antiserum bound to the surface of newly transformed schistosomula and lung-stage organisms but not to the surface of liver-stage and adult worms. Using this polyclonal antiserum we isolated recombinant clones from an adult worm cDNA expression library constructed in λgt11. Clone 654.2 contained an insert of 0.52 kilobase and hybridized to a 1.2-kilobase mRNA species from adult worms. Most importantly, clone 654.2 produced a fusion protein of 125 kDa that was reactive with sera of vaccinated mice that are capable of transferring resistance. This result encourages future vaccination trials with the fusion protein

Detection of a new submicroscopic Norrie disease deletion interval with a novel DNA probe isolated by differential Alu PCR fingerprint cloning

NARCIS (Netherlands)

Bergen, A. A.; Wapenaar, M. C.; Schuurman, E. J.; Diergaarde, P. J.; Lerach, H.; Monaco, A. P.; Bakker, E.; Bleeker-Wagemakers, E. M.; van Ommen, G. J.

1993-01-01

Differential Alu PCR fingerprint cloning was used to isolate a DNA probe from the Xp11.4-->p11.21 region of the human X chromosome. This novel sequence, cpXr318 (DXS742), detects a new submicroscopic deletion interval at the Norrie disease locus (NDP). Combining our data with the consensus genetic

Development of intra-strain self-cloning procedure for breeding baker's yeast strains.

Science.gov (United States)

Nakagawa, Youji; Ogihara, Hiroyuki; Mochizuki, Chisato; Yamamura, Hideki; Iimura, Yuzuru; Hayakawa, Masayuki

2017-03-01

Previously reported self-cloning procedures for breeding of industrial yeast strains require DNA from other strains, plasmid DNA, or mutagenesis. Therefore, we aimed to construct a self-cloning baker's yeast strain that exhibits freeze tolerance via an improved self-cloning procedure. We first disrupted the URA3 gene of a prototrophic baker's yeast strain without the use of any marker gene, resulting in a Δura3 homozygous disruptant. Then, the URA3 gene of the parental baker's yeast strain was used as a selection marker to introduce the constitutive TDH3 promoter upstream of the PDE2 gene encoding high-affinity cyclic AMP phosphodiesterase. This self-cloning procedure was performed without using DNA from other Saccharomyces cerevisiae strains, plasmid DNA, or mutagenesis and was therefore designated an intra-strain self-cloning procedure. Using this self-cloning procedure, we succeeded in producing self-cloning baker's yeast strains that harbor the TDH3p-PDE2 gene heterozygously and homozygously, designated TDH3p-PDE2 hetero and TDH3p-PDE2 homo strains, respectively. These self-cloning strains expressed much higher levels of PDE2 mRNA than the parental strain and exhibited higher viability after freeze stress, as well as higher fermentation ability in frozen dough, when compared with the parental strain. The TDH3p-PDE2 homo strain was genetically more stable than the TDH3p-PDE2 hetero strain. These results indicate that both heterozygous and homozygous strains of self-cloning PDE2-overexpressing freeze-tolerant strains of industrial baker's yeast can be prepared using the intra-strain self-cloning procedure, and, from a practical viewpoint, the TDH3p-PDE2 homo strain constructed in this study is preferable to the TDH3p-PDE2 hetero strain for frozen dough baking. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Human cDNA clones for an α subunit of G/sub i/ signal-transduction protein

International Nuclear Information System (INIS)

Bray, P.; Carter, A.; Guo, V.; Puckett, C.; Kamholz, J.; Spiegel, A.; Nirenberg, M.

1987-01-01

Two cDNA clones were obtained from a λgt11 cDNA human brain library that correspond to α/sub i/ subunits of G signal-transduction proteins (where α/sub i/ subunits refer to the α subunits of G proteins that inhibit adenylate cyclase). The nucleotide sequence of human brain α/sub i/ is highly homologous to that of bovine brain α/sub i/ and the predicted amino acid sequences are identical. However, human and bovine brain α/sub i/ cDNAs differ significantly from α/sub i/ cDNAs from human monocytes, rat glioma, and mouse macrophages in amino acid (88% homology) and nucleotide (71-75% homology) sequences. In addition, the nucleotide sequences of the 3' untranslated regions of human and bovine brain α/sub i/ cDNAs differ markedly from the sequences of human monocyte, rat glioma, and mouse macrophage α/sub i/ cDNAs. These results suggest there are at least two classes of α/sub i/ mRNA

Cloning of the relative genes of endocrine exophthalmos

International Nuclear Information System (INIS)

Zheng, JG

2004-01-01

Aim: In order to clarify the pathogenesis of endocrine exophthalmos, and lay foundations for finding the new functions of its relative genes, the cloning of its relative genes was carried out. Methods: The thyroid tissues of 10 hyperthyroidism patients, 5 of them with endocrine exophthalmos and 5 without that, were obtained. Their mRNA were collected respectively by using Quick Prep Micro mRNA purification kit. Then the same amount of the mRNA from 5 patients with endocrine exophthalmos was added into an eppendorf tube to form a mRNA pool. And that of the 5 patients without endocrine exophthalmos was also prepared as the other pool. As a model, the pool was used to synthesize the single and double chains of cDNA through SMART Tm PCR cDNA Synthesis Kit. The double chains cDNA from the endocrine exophthalmos patients, being used as tester, and that from the patients without endocrine exophthalmos, being used as driver, were digested by restriction endonucleases Hae III to get the fragments which was less than 500 bases. The tester cDNA was ligated with adapt or 1 or 2 respectively. Then the subtractive suppressive hybridization was performed between tester and driver cDNA. And the efficacies of subtraction were measured. The differential genes between the thyroid tissues of endocrine exophthalmos and the thyroid tissues without endocrine exophthalmos were obtained through two cycles of subtractive hybridization and two cycles PCR. The differential genes were cloned into the vector of pT-Adv, and then transformed into E.coliDH5a. 48 white clonies were selected to build the subtractive suppressive library of the relative genes of endocrine exophthalmos. The primer 2 was applied for the colony PCR of the relative genes. The amplified genes were obtained and purified by using Quaqwich Spine PCR Purification Kit. According to the principle of random primer, the double chains cDNA from the thyroid tissues with or without endocrine exophthalmos were digested by Hae III

Molecular Profiling of Microbial Communities from Contaminated Sources: Use of Subtractive Cloning Methods and rDNA Spacer Sequences; FINAL

International Nuclear Information System (INIS)

Robb, Frank T.

2001-01-01

The major objective of this research was to provide appropriate sequences and assemble a DNA array of oligonucleotides to be used for rapid profiling of microbial populations from polluted areas and other areas of interest. The sequences to be assigned to the DNA array were chosen from cloned genomic DNA taken from groundwater sites having well characterized pollutant histories at Hanford Nuclear Plant and Lawrence Livermore Site 300. Glass-slide arrays were made and tested; and a new multiplexed, bead-based method was developed that uses nucleic acid hybridization on the surface of microscopic polystyrene spheres to identify specific sequences in heterogeneous mixtures of DNA sequences. The test data revealed considerable strain variation between sample sites showing a striking distribution of sequences. It also suggests that diversity varies greatly with bioremediation, and that there are many bacterial intergenic spacer region sequences that can indicate its effects. The bead method exhibited superior sequence discrimination and has features for easier and more accurate measurement

[Cloning of human CD45 gene and its expression in Hela cells].

Science.gov (United States)

Li, Jie; Xu, Tianyu; Wu, Lulin; Zhang, Liyun; Lu, Xiao; Zuo, Daming; Chen, Zhengliang

2015-11-01

To clone human CD45 gene PTPRC and establish Hela cells overexpressing recombinant human CD45 protein. The intact cDNA encoding human CD45 amplified using RT-PCR from the total RNA extracted from peripheral blood mononuclear cells (PBMCs) of a healthy donor was cloned into pMD-18T vector. The CD45 cDNA fragment amplified from the pMD-18T-CD45 by PCR was inserted to the coding region of the PcDNA3.1-3xflag vector, and the resultant recombinant expression vector PcDNA3.1-3xflag-CD45 was transfected into Hela cells. The expression of CD45 in Hela cells was detected by flow cytometry and Western blotting, and the phosphastase activity of CD45 was quantified using an alkaline phosphatase assay kit. The cDNA fragment of about 3 900 bp was amplified from human PBMCs and cloned into pMD-18T vector. The recombinant expression vector PcDNA3.1-3xflag-CD45 was constructed, whose restriction maps and sequence were consistent with those expected. The expression of CD45 in transfected Hela cells was detected by flow cytometry and Western blotting, and the expressed recombinant CD45 protein in Hela cells showed a phosphastase activity. The cDNA of human CD45 was successfully cloned and effectively expressed in Hela cells, which provides a basis for further exploration of the functions of CD45.

Artificial cloning of domestic animals.

Science.gov (United States)

Keefer, Carol L

2015-07-21

Domestic animals can be cloned using techniques such as embryo splitting and nuclear transfer to produce genetically identical individuals. Although embryo splitting is limited to the production of only a few identical individuals, nuclear transfer of donor nuclei into recipient oocytes, whose own nuclear DNA has been removed, can result in large numbers of identical individuals. Moreover, clones can be produced using donor cells from sterile animals, such as steers and geldings, and, unlike their genetic source, these clones are fertile. In reality, due to low efficiencies and the high costs of cloning domestic species, only a limited number of identical individuals are generally produced, and these clones are primarily used as breed stock. In addition to providing a means of rescuing and propagating valuable genetics, somatic cell nuclear transfer (SCNT) research has contributed knowledge that has led to the direct reprogramming of cells (e.g., to induce pluripotent stem cells) and a better understanding of epigenetic regulation during embryonic development. In this review, I provide a broad overview of the historical development of cloning in domestic animals, of its application to the propagation of livestock and transgenic animal production, and of its scientific promise for advancing basic research.

Recovery of infectious pariacoto virus from cDNA clones and identification of susceptible cell lines.

Science.gov (United States)

Johnson, K N; Ball, L A

2001-12-01

Pariacoto virus (PaV) is a nodavirus that was recently isolated in Peru from the Southern armyworm, Spodoptera eridania. Virus particles are non enveloped and about 30 nm in diameter and have T=3 icosahedral symmetry. The 3.0-A crystal structure shows that about 35% of the genomic RNA is icosahedrally ordered, with the RNA forming a dodecahedral cage of 25-nucleotide (nt) duplexes that underlie the inner surface of the capsid. The PaV genome comprises two single-stranded, positive-sense RNAs: RNA1 (3,011 nt), which encodes the 108-kDa catalytic subunit of the RNA-dependent RNA polymerase, and RNA2 (1,311 nt), which encodes the 43-kDa capsid protein precursor alpha. In order to apply molecular genetics to the structure and assembly of PaV, we identified susceptible cell lines and developed a reverse genetic system for this virus. Cell lines that were susceptible to infection by PaV included those from Spodoptera exigua, Helicoverpa zea and Aedes albopictus, whereas cells from Drosophila melanogaster and Spodoptera frugiperda were refractory to infection. To recover virus from molecular clones, full-length cDNAs of PaV RNAs 1 and 2 were cotranscribed by T7 RNA polymerase in baby hamster kidney cells that expressed T7 RNA polymerase. Lysates of these cells were infectious both for cultured cells from Helicoverpa zea (corn earworm) and for larvae of Galleria mellonella (greater wax moth). The combination of infectious cDNA clones, cell culture infectivity, and the ability to produce milligram amounts of virus allows the application of DNA-based genetic methods to the study of PaV structure and assembly.

Mammalian α-polymerase: cloning of partial complementary DNA and immunobinding of catalytic subunit in crude homogenate protein blots

International Nuclear Information System (INIS)

SenGupta, D.N.; Kumar, P.; Zmudzka, B.Z.; Coughlin, S.; Vishwanatha, J.K.; Robey, F.A.; Parrott, C.; Wilson, S.H.

1987-01-01

A new polyclonal antibody against the α-polymerase catalytic polypeptide was prepared by using homogeneous HeLa cellα-polymerase. The antibody neutralized α-polymerase activity and was strong and specific for the α-polymerase catalytic polypeptide (M/sub r/ 183,000) in Western blot analysis of crude extracts of HeLa cells. The antibody was used to screen a cDNA library of newborn rat brain poly(A+) RNA in λgt11. A positive phage was identified and plaque purified. This phage, designated λpolα1.2, also was found to be positive with an antibody against Drosophila α-polymerase. The insert in λpolα1.2 (1183 base pairs) contained a poly(A) sequence at the 3' terminus and a short in-phase open reading frame at the 5' terminus. A synthetic oligopeptide (eight amino acids) corresponding to the open reading frame was used to raise antiserum in rabbits. Antibody affinity purified from this serum was found to be immunoreactive against purified α-polymerase by enzyme-linked immunosorbent assay and was capable of immunoprecipitating α-polymerase. This indicated the λpolα1.2 insert encoded an α-polymerase epitope and suggested that the cDNA corresponded to an α-polymerase mRNA. This was confirmed in hybrid selection experiments using pUC9 containing the cDNA insert and poly(A+) RNA from newborn rat brain; the insert hybridized to mRNA capable of encoding α-polymerase catalytic polypeptides. Northern blot analysis of rat brain poly(A+) RNA revealed that this mRNA is ∼5.4 kilobases

DNA probe for lactobacillus delbrueckii

Energy Technology Data Exchange (ETDEWEB)

Delley, M.; Mollet, B.; Hottinger, H. (Nestle Research Centre, Lausanne (Switzerland))

1990-06-01

From a genomic DNA library of Lactobacillus delbrueckii subsp. bulgaricus, a clone was isolated which complements a leucine auxotrophy of an Escherichia coli strain (GE891). Subsequent analysis of the clone indicated that it could serve as a specific DNA probe. Dot-blot hybridizations with over 40 different Lactobacillus strains showed that this clone specifically recognized L. delbrueckii subsp. delbrueckii, bulgaricus, and lactis. The sensitivity of the method was tested by using an {alpha}-{sup 32}P-labeled probe.

DNA probe for lactobacillus delbrueckii

International Nuclear Information System (INIS)

Delley, M.; Mollet, B.; Hottinger, H.

1990-01-01

From a genomic DNA library of Lactobacillus delbrueckii subsp. bulgaricus, a clone was isolated which complements a leucine auxotrophy of an Escherichia coli strain (GE891). Subsequent analysis of the clone indicated that it could serve as a specific DNA probe. Dot-blot hybridizations with over 40 different Lactobacillus strains showed that this clone specifically recognized L. delbrueckii subsp. delbrueckii, bulgaricus, and lactis. The sensitivity of the method was tested by using an α- 32 P-labeled probe

DNA Probe for Lactobacillus delbrueckii

OpenAIRE

Delley, Michèle; Mollet, Beat; Hottinger, Herbert

1990-01-01

From a genomic DNA library of Lactobacillus delbrueckii subsp. bulgaricus, a clone was isolated which complements a leucine auxotrophy of an Escherichia coli strain (GE891). Subsequent analysis of the clone indicated that it could serve as a specific DNA probe. Dot-blot hybridizations with over 40 different Lactobacillus strains showed that this clone specifically recognizes L. delbrueckii subsp. delbrueckii, bulgaricus, and lactis. The sensitivity of the method was tested by using an α-32P-l...

Cloning of the cDNA for a human homologue of the Drosophila white gene and mapping to chromosome 21q22.3.

OpenAIRE

Chen, H.; Rossier, C.; Lalioti, M. D.; Lynn, A.; Chakravarti, A.; Perrin, G.; Antonarakis, S. E.

1996-01-01

In an effort to contribute to the transcript map of human chromosome 21 and the understanding of the pathophysiology of trisomy 21, we have used exon trapping to identify fragments of chromosome 21 genes. Two trapped exons, from pools of chromosome 21-specific cosmids, showed homology to the Drosophila white (w) gene. We subsequently cloned the corresponding cDNA for a human homologue of the Drosophila w gene (hW) from human retina and fetal brain cDNA libraries. The gene belongs to the ATP-b...

Novel infectious cDNA clones of hepatitis C virus genotype 3a (strain S52) and 4a (strain ED43): genetic analyses and in vivo pathogenesis studies

DEFF Research Database (Denmark)

Gottwein, Judith; Scheel, Troels; Callendret, Benoit

2010-01-01

Previously, RNA transcripts of cDNA clones of hepatitis C virus (HCV) genotypes 1a (strains H77, HCV-1, and HC-TN), 1b (HC-J4, Con1, and HCV-N), and 2a (HC-J6 and JFH1) were found to be infectious in chimpanzees. However, only JFH1 was infectious in human hepatoma Huh7 cells. We performed genetic...... analysis of HCV genotype 3a (strain S52) and 4a (strain ED43) prototype strains and generated full-length consensus cDNA clones (pS52 and pED43). Transfection of Huh7.5 cells with RNA transcripts of these clones did not yield cells expressing HCV Core. However, intrahepatic transfection of chimpanzees...... resulted in robust infection with peak HCV RNA titers of approximately 5.5 log(10) international units (IU)/ml. Genomic consensus sequences recovered from serum at the times of peak viral titers were identical to the sequences of the parental plasmids. Both chimpanzees developed acute hepatitis...

A novel nucleo-cytoplasmic hybrid clone formed via androgenesis in polyploid gibel carp

Directory of Open Access Journals (Sweden)

Zhou Li

2011-03-01

Full Text Available Abstract Background Unisexual vertebrates have been demonstrated to reproduce by gynogenesis, hybridogenesis, parthenogenesis, or kleptogenesis, however, it is uncertain how the reproduction mode contributes to the clonal diversity. Recently, polyploid gibel carp has been revealed to possess coexisting dual modes of unisexual gynogenesis and sexual reproduction and to have numerous various clones. Using sexual reproduction mating between clone D female and clone A male and subsequent 7 generation multiplying of unisexual gynogenesis, we have created a novel clone strain with more than several hundred millions of individuals. Here, we attempt to identify genetic background of the novel clone and to explore the significant implication for clonal diversity contribution. Methods Several nuclear genome markers and one cytoplasmic marker, the mitochondrial genome sequence, were used to identify the genetic organization of the randomly sampled individuals from different generations of the novel clone. Results Chromosome number, Cot-1 repetitive DNA banded karyotype, microsatellite patterns, AFLP profiles and transferrin alleles uniformly indicated that nuclear genome of the novel clone is identical to that of clone A, and significantly different from that of clone D. However, the cytoplasmic marker, its complete mtDNA genome sequence, is same to that of clone D, and different from that of clone A. Conclusions The present data indicate that the novel clone is a nucleo-cytoplasmic hybrid between the known clones A and D, because it originates from the offspring of gonochoristic sexual reproduction mating between clone D female and clone A male, and contains an entire nuclear genome from the paternal clone A and a mtDNA genome (cytoplasm from the maternal clone D. It is suggested to arise via androgenesis by a mechanism of ploidy doubling of clone A sperm in clone D ooplasm through inhibiting the first mitotic division. Significantly, the selected nucleo

A novel nucleo-cytoplasmic hybrid clone formed via androgenesis in polyploid gibel carp

Science.gov (United States)

2011-01-01

Background Unisexual vertebrates have been demonstrated to reproduce by gynogenesis, hybridogenesis, parthenogenesis, or kleptogenesis, however, it is uncertain how the reproduction mode contributes to the clonal diversity. Recently, polyploid gibel carp has been revealed to possess coexisting dual modes of unisexual gynogenesis and sexual reproduction and to have numerous various clones. Using sexual reproduction mating between clone D female and clone A male and subsequent 7 generation multiplying of unisexual gynogenesis, we have created a novel clone strain with more than several hundred millions of individuals. Here, we attempt to identify genetic background of the novel clone and to explore the significant implication for clonal diversity contribution. Methods Several nuclear genome markers and one cytoplasmic marker, the mitochondrial genome sequence, were used to identify the genetic organization of the randomly sampled individuals from different generations of the novel clone. Results Chromosome number, Cot-1 repetitive DNA banded karyotype, microsatellite patterns, AFLP profiles and transferrin alleles uniformly indicated that nuclear genome of the novel clone is identical to that of clone A, and significantly different from that of clone D. However, the cytoplasmic marker, its complete mtDNA genome sequence, is same to that of clone D, and different from that of clone A. Conclusions The present data indicate that the novel clone is a nucleo-cytoplasmic hybrid between the known clones A and D, because it originates from the offspring of gonochoristic sexual reproduction mating between clone D female and clone A male, and contains an entire nuclear genome from the paternal clone A and a mtDNA genome (cytoplasm) from the maternal clone D. It is suggested to arise via androgenesis by a mechanism of ploidy doubling of clone A sperm in clone D ooplasm through inhibiting the first mitotic division. Significantly, the selected nucleo-cytoplasmic hybrid female

Design of a Sensitive and Selective Electrochemical Aptasensor for the Determination of the Complementary cDNA of miRNA-145 Based on the Intercalation and Electrochemical Reduction of Doxorubicin.

Science.gov (United States)

Mohamadi, Maryam; Mostafavi, Ali; Torkzadeh-Mahani, Masoud

2017-11-01

The aim of this research was the determination of a microRNA (miRNA) using a DNA electrochemical aptasensor. In this biosensor, the complementary complementary DNA (cDNA) of miRNA-145 (a sense RNA transcript) was the target strand and the cDNA of miRNA-145 was the probe strand. Both cDNAs can be the product of the reverse transcriptase-polymerase chain reaction of miRNA. The proposed aptasensor's function was based on the hybridization of target strands with probes immobilized on the surface of a working electrode and the subsequent intercalation of doxorubicin (DOX) molecules functioning as the electroactive indicators of any double strands that formed. Electrochemical transduction was performed by measuring the cathodic current resulting from the electrochemical reduction of the intercalated molecules at the electrode surface. In the experiment, because many DOX molecules accumulated on each target strand on the electrode surface, amplification was inherently easy, without a need for enzymatic or complicated amplification strategies. The proposed aptasensor also had the excellent ability to regenerate as a result of the melting of the DNA duplex. Moreover, the use of DNA probe strands obviated the challenges of working with an RNA probe, such as sensitivity to RNase enzyme. In addition to the linear relationship between the electrochemical signal and the concentration of the target strands that ranged from 2.0 to 80.0 nM with an LOD of 0.27 nM, the proposed biosensor was clearly capable of distinguishing between complementary (target strand) and noncomplementary sequences. The presented biosensor was successfully applied for the quantification of DNA strands corresponding to miRNA-145 in human serum samples.

Cloning, expression, and chromosome mapping of human galectin-7

DEFF Research Database (Denmark)

Madsen, Peder; Rasmussen, H H; Flint, T

1995-01-01

The galectins are a family of beta-galactoside-binding proteins implicated in modulating cell-cell and cell-matrix interactions. Here we report the cloning and expression of a novel member of this family (galectin-7) that correspond to IEF (isoelectric focusing) 17 (12,700 Da; pI, 7.6) in the human...... keratinocyte protein data base, and that is strikingly down-regulated in SV40 transformed keratinocytes (K14). The cDNA was cloned from a lambda gt11 cDNA expression library using degenerated oligodeoxyribonucleotides back-translated from an IEF 17 peptide sequence. The protein encoded by the galectin-7 clone......14 keratinocytes imply a role in cell-cell and/or cell-matrix interactions necessary for normal growth control. The galectin-7 gene was mapped to chromosome 19. Udgivelsesdato: 1995-Mar-17...

Sequence of human protamine 2 cDNA

Energy Technology Data Exchange (ETDEWEB)

Domenjoud, L; Fronia, C; Uhde, F; Engel, W [Universitaet Goettingen (West Germany)

1988-08-11

The authors report the cloning and sequencing of a cDNA clone for human protamine 2 (hp2), isolated from a human testis cDNA library cloned in the vector {lambda}-gt11. A 66mer oligonucleotide, that corresponds to an amino acid sequence which is highly conserved between hp2 and mouse protamine 2 (mp2) served as hybridization probe. The homology between the amino acid sequence deduced from our cDNA and the published amino acid sequence for hp2 is 100%.

Amino acid sequence of bovine muzzle epithelial desmocollin derived from cloned cDNA: a novel subtype of desmosomal cadherins.

Science.gov (United States)

Koch, P J; Goldschmidt, M D; Walsh, M J; Zimbelmann, R; Schmelz, M; Franke, W W

1991-05-01

Desmosomes are cell-type-specific intercellular junctions found in epithelium, myocardium and certain other tissues. They consist of assemblies of molecules involved in the adhesion of specific cell types and in the anchorage of cell-type-specific cytoskeletal elements, the intermediate-size filaments, to the plasma membrane. To explore the individual desmosomal components and their functions we have isolated DNA clones encoding the desmosomal glycoprotein, desmocollin, using antibodies and a cDNA expression library from bovine muzzle epithelium. The cDNA-deduced amino-acid sequence of desmocollin (presently we cannot decide to which of the two desmocollins, DC I or DC II, this clone relates) defines a polypeptide with a calculated molecular weight of 85,000, with a single candidate sequence of 24 amino acids sufficiently long for a transmembrane arrangement, and an extracellular aminoterminal portion of 561 amino acid residues, compared to a cytoplasmic part of only 176 amino acids. Amino acid sequence comparisons have revealed that desmocollin is highly homologous to members of the cadherin family of cell adhesion molecules, including the previously sequenced desmoglein, another desmosome-specific cadherin. Using riboprobes derived from cDNAs for Northern-blot analyses, we have identified an mRNA of approximately 6 kb in stratified epithelia such as muzzle epithelium and tongue mucosa but not in two epithelial cell culture lines containing desmosomes and desmoplakins. The difference may indicate drastic differences in mRNA concentration or the existence of cell-type-specific desmocollin subforms. The molecular topology of desmocollin(s) is discussed in relation to possible functions of the individual molecular domains.

Cloning and functional expression of a plant voltage-dependent chloride channel.

Science.gov (United States)

Lurin, C; Geelen, D; Barbier-Brygoo, H; Guern, J; Maurel, C

1996-01-01

Plant cell membrane anion channels participate in basic physiological functions, such as cell volume regulation and signal transduction. However, nothing is known about their molecular structure. Using a polymerase chain reaction strategy, we have cloned a tobacco cDNA (CIC-Nt1) encoding a 780-amino acid protein with several putative transmembrane domains. CIC-Nt1 displays 24 to 32% amino acid identity with members of the animal voltage-dependent chloride channel (CIC) family, whose archetype is CIC-0 from the Torpedo marmorata electric organ. Injection of CIC-Nt1 complementary RNA into Xenopus oocytes elicited slowly activating inward currents upon membrane hyperpolarization more negative than -120 mV. These currents were carried mainly by anions, modulated by extracellular anions, and totally blocked by 10 mM extracellular calcium. The identification of CIC-Nt1 extends the CIC family to higher plants and provides a molecular probe for the study of voltage-dependent anion channels in plants. PMID:8624442

DNA fragmentation: manifestation of target cell destruction mediated by cytotoxic T-cell lines, lymphotoxin-secreting helper T-cell clones, and cell-free lymphotoxin-containing supernatant

International Nuclear Information System (INIS)

Schmid, D.S.; Tite, J.P.; Ruddle, N.H.

1986-01-01

A Lyt-2 + , trinitrophenyl-specific, lymphotoxin-secreting, cytotoxic T-cell line, PCl 55, mediates the digestion of target cell DNA into discretely sized fragments. This phenomenon manifests itself within 30 min after effector cell encounter as measured by the release of 3 H counts from target cells prelabeled with [ 3 H]deoxythymidine and occurs even at very low effector to target cell ratios (0.25:1). A Lyt-1 + , ovalbumin-specific, lymphotoxin-secreting T-helper cell clone, 5.9.24, is also able to mediate fragmentation of target cell DNA over a time course essentially indistinguishable from the cytotoxic T lymphocyte-mediated hit. Cell-free lymphotoxin-containing supernatants also cause release of DNA from targets, although they require a longer time course, on the order of 24 hr. In contrast, lysis of cells by antibody plus complement or Triton X-100 does not result in DNA release even after extended periods of incubation (24 hr). All three treatments that result in the release of DNA from cells cause fragmentation of that DNA into discretely sized pieces that are multiples of 200 base pairs. The results thus suggest that cytotoxic T cells, lymphotoxin-secreting helper clones with cytolytic activity, and lymphotoxin all effect target cell destruction by means of a similar mechanism and that observed differences in time course and the absence of target cell specificity in killing mediated by lymphotoxin may simply reflect differences in the mode of toxin delivery

Assessment of mitochondrial functions in Daphnia pulex clones using high-resolution respirometry.

Science.gov (United States)

Kake-Guena, Sandrine A; Touisse, Kamal; Vergilino, Roland; Dufresne, France; Blier, Pierre U; Lemieux, Hélène

2015-06-01

The objectives of our study were to adapt a method to measure mitochondrial function in intact mitochondria from the small crustacean Daphnia pulex and to validate if this method was sensitive enough to characterize mitochondrial metabolism in clones of the pulex complex differing in ploidy levels, mitochondrial DNA haplotypes, and geographic origins. Daphnia clones belonging to the Daphnia pulex complex represent a powerful model to delineate the link between mitochondrial DNA evolution and mitochondrial phenotypes, as single genotypes with divergent mtDNA can be grown under various experimental conditions. Our study included two diploid clones from temperate environments and two triploid clones from subarctic environments. The whole animal permeabilization and measurement of respiration with high-resolution respirometry enabled the measurement of the functional capacity of specific mitochondrial complexes in four clones. When expressing the activity as ratios, our method detected significant interclonal variations. In the triploid subarctic clone from Kuujjurapik, a higher proportion of the maximal physiological oxidative phosphorylation (OXPHOS) capacity of mitochondria was supported by complex II, and a lower proportion by complex I. The triploid subarctic clone from Churchill (Manitoba) showed the lowest proportion of the maximal OXPHOS supported by complex II. Additional studies are required to determine if these differences in mitochondrial functions are related to differences in mitochondrial haplotypes or ploidy level and if they might be associated with fitness divergences and therefore selective value. © 2015 Wiley Periodicals, Inc.

[Telomere lengthening by trichostatin A treatment in cloned pigs].

Science.gov (United States)

Xie, Bing-Teng; Ji, Guang-Zhen; Kong, Qing-Ran; Mao, Jian; Shi, Yong-Qian; Liu, Shi-Chao; Wu, Mei-Ling; Wang, Juan; Liu, Lin; Liu, Zhong-Hua

2012-12-01

Telomeres are repeated GC rich sequences at the end of chromosomes, and shorten with each cell division due to DNA end replication problem. Previously, reprogrammed somatic cells of cloned animals display variable telomere elongation. However, it was reported that the cloned animals including Dolly do not reset telomeres and show premature aging. In this study, we investigated telomere function in cloned or transgenic cloned pigs, including the cloned Northeast Min pigs, eGFP, Mx, and PGC1α transgenic cloned pigs, and found that the telomere lengths of cloned pigs were significantly shorter than the nuclear donor adult fibroblasts and age-matched noncloned pigs (Pstage for 24 h. Consistent with previous reports, the developmental rate of SCNT embryos to the blastocyst stage was significantly increased compared with those of the control group (16.35% vs. 27.09%, 21.60% vs. 34.90%, Plengthen the telomere lengths of cloned pigs.

Biophysical characterization of the association of histones with single-stranded DNA.

Science.gov (United States)

Wang, Ying; van Merwyk, Luis; Tönsing, Katja; Walhorn, Volker; Anselmetti, Dario; Fernàndez-Busquets, Xavier

2017-11-01

Despite the profound current knowledge of the architecture and dynamics of nucleosomes, little is known about the structures generated by the interaction of histones with single-stranded DNA (ssDNA), which is widely present during replication and transcription. Non-denaturing gel electrophoresis, transmission electron microscopy, atomic force microscopy, magnetic tweezers. Histones have a high affinity for ssDNA in 0.15M NaCl ionic strength, with an apparent binding constant similar to that calculated for their association with double-stranded DNA (dsDNA). The length of DNA (number of nucleotides in ssDNA or base pairs in dsDNA) associated with a fixed core histone mass is the same for both ssDNA and dsDNA. Although histone-ssDNA complexes show a high tendency to aggregate, nucleosome-like structures are formed at physiological salt concentrations. Core histones are able to protect ssDNA from digestion by micrococcal nuclease, and a shortening of ssDNA occurs upon its interaction with histones. The purified (+) strand of a cloned DNA fragment of nucleosomal origin has a higher affinity for histones than the purified complementary (-) strand. At physiological ionic strength histones have high affinity for ssDNA, possibly associating with it into nucleosome-like structures. In the cell nucleus histones may spontaneously interact with ssDNA to facilitate their participation in the replication and transcription of chromatin. Copyright © 2017 Elsevier B.V. All rights reserved.

Structure and characterization of a cDNA clone for phenylalanine ammonia-lyase from cut-injured roots of sweet potato

International Nuclear Information System (INIS)

Tanaka, Yoshiyuki; Matsuoka, Makoto; Yamanoto, Naoki; Ohashi, Yuko; Kano-Murakami, Yuriko; Ozeki, Yoshihiro

1989-01-01

A cDNA clone for phenylalanine ammonia-lyase (PAL) induced in wounded sweet potato (Ipomoea batatas Lam.) root was obtained by immunoscreening a cDNA library. The protein produced in Escherichia coli cells containing the plasmid pPAL02 was indistinguishable from sweet potato PAL as judged by Ouchterlony double diffusion assays. The M r of its subunit was 77,000. The cells converted [ 14 C]-L-phenylalanine into [ 14 C]-t-cinnamic acid and PAL activity was detected in the homogenate of the cells. The activity was dependent on the presence of the pPAL02 plasmid DNA. The nucleotide sequence of the cDNA contained a 2,121-base pair (bp) open-reading frame capable of coding for a polypeptide with 707 amino acids (M r 77,137), a 22-bp 5'-noncoding region and a 207-bp 3'-noncoding region. The results suggest that the insert DNA fully encoded the amino acid sequence for sweet potato PAL that is induced by wounding. Comparison of the deduced amino acid sequence with that of a PAL cDNA fragment from Phaseolus vulgaris revealed 78.9% homology. The sequence from amino acid residues 258 to 494 was highly conserved, showing 90.7% homology

Delineating Rearrangements in Single Yeast Artificial Chromosomes by Quantitative DNA Fiber Mapping

Energy Technology Data Exchange (ETDEWEB)

Weier, Heinz-Ulrich G.; Greulich-Bode, Karin M.; Wu, Jenny; Duell, Thomas

2009-09-18

Cloning of large chunks of human genomic DNA in recombinant systems such as yeast or bacterial artificial chromosomes has greatly facilitated the construction of physical maps, the positional cloning of disease genes or the preparation of patient-specific DNA probes for diagnostic purposes. For this process to work efficiently, the DNA cloning process and subsequent clone propagation need to maintain stable inserts that are neither deleted nor otherwise rearranged. Some regions of the human genome; however, appear to have a higher propensity than others to rearrange in any host system. Thus, techniques to detect and accurately characterize such rearrangements need to be developed. We developed a technique termed 'Quantitative DNA Fiber Mapping (QDFM)' that allows accurate tagging of sequence elements of interest with near kilobase accuracy and optimized it for delineation of rearrangements in recombinant DNA clones. This paper demonstrates the power of this microscopic approach by investigating YAC rearrangements. In our examples, high-resolution physical maps for regions within the immunoglobulin lambda variant gene cluster were constructed for three different YAC clones carrying deletions of 95 kb and more. Rearrangements within YACs could be demonstrated unambiguously by pairwise mapping of cosmids along YAC DNA molecules. When coverage by YAC clones was not available, distances between cosmid clones were estimated by hybridization of cosmids onto DNA fibers prepared from human genomic DNA. In addition, the QDFM technology provides essential information about clone stability facilitating closure of the maps of the human genome as well as those of model organisms.

Cloning of the cDNA for U1 small nuclear ribonucleoprotein particle 70K protein from Arabidopsis thaliana

Science.gov (United States)

Reddy, A. S.; Czernik, A. J.; An, G.; Poovaiah, B. W.

1992-01-01

We cloned and sequenced a plant cDNA that encodes U1 small nuclear ribonucleoprotein (snRNP) 70K protein. The plant U1 snRNP 70K protein cDNA is not full length and lacks the coding region for 68 amino acids in the amino-terminal region as compared to human U1 snRNP 70K protein. Comparison of the deduced amino acid sequence of the plant U1 snRNP 70K protein with the amino acid sequence of animal and yeast U1 snRNP 70K protein showed a high degree of homology. The plant U1 snRNP 70K protein is more closely related to the human counter part than to the yeast 70K protein. The carboxy-terminal half is less well conserved but, like the vertebrate 70K proteins, is rich in charged amino acids. Northern analysis with the RNA isolated from different parts of the plant indicates that the snRNP 70K gene is expressed in all of the parts tested. Southern blotting of genomic DNA using the cDNA indicates that the U1 snRNP 70K protein is coded by a single gene.

Cloning and characterization of the major histone H2A genes completes the cloning and sequencing of known histone genes of Tetrahymena thermophila.

Science.gov (United States)

Liu, X; Gorovsky, M A

1996-01-01

A truncated cDNA clone encoding Tetrahymena thermophila histone H2A2 was isolated using synthetic degenerate oligonucleotide probes derived from H2A protein sequences of Tetrahymena pyriformis. The cDNA clone was used as a homologous probe to isolate a truncated genomic clone encoding H2A1. The remaining regions of the genes for H2A1 (HTA1) and H2A2 (HTA2) were then isolated using inverse PCR on circularized genomic DNA fragments. These partial clones were assembled into intact HTA1 and HTA2 clones. Nucleotide sequences of the two genes were highly homologous within the coding region but not in the noncoding regions. Comparison of the deduced amino acid sequences with protein sequences of T. pyriformis H2As showed only two and three differences respectively, in a total of 137 amino acids for H2A1, and 132 amino acids for H2A2, indicating the two genes arose before the divergence of these two species. The HTA2 gene contains a TAA triplet within the coding region, encoding a glutamine residue. In contrast with the T. thermophila HHO and HTA3 genes, no introns were identified within the two genes. The 5'- and 3'-ends of the histone H2A mRNAs; were determined by RNase protection and by PCR mapping using RACE and RLM-RACE methods. Both genes encode polyadenylated mRNAs and are highly expressed in vegetatively growing cells but only weakly expressed in starved cultures. With the inclusion of these two genes, T. thermophila is the first organism whose entire complement of known core and linker histones, including replication-dependent and basal variants, has been cloned and sequenced. PMID:8760889

Characterization of the env gene and long terminal repeat of molecularly cloned Friend mink cell focus-inducing virus DNA.

OpenAIRE

Adachi, A; Sakai, K; Kitamura, N; Nakanishi, S; Niwa, O; Matsuyama, M; Ishimoto, A

1984-01-01

The highly oncogenic erythroleukemia-inducing Friend mink cell focus-inducing (MCF) virus was molecularly cloned in phage lambda gtWES.lambda B, and the DNA sequences of the env gene and the long terminal repeat were determined. The nucleotide sequences of Friend MCF virus and Friend spleen focus-forming virus were quite homologous, supporting the hypothesis that Friend spleen focus-forming virus might be generated via Friend MCF virus from an ecotropic Friend virus mainly by some deletions. ...

Cloning and study of the pectate lyase gene of Erwinia carotovora

International Nuclear Information System (INIS)

Bukanov, N.O.; Fonshtein, M.Yu.; Evtushenkov, A.N.; Syarinskii, M.A.; Strel'chenko, P.P.; Yankovski, N.K.; Alikhanyan, S.I.; Fomichev, Yu.K.; Debabov, V.G.

1986-01-01

The cloning of the gene of a secretable protein of Erwinia carotovora, pectate lyase, in Escherichia coli was described. Primary cloning was conducted using the phage vector λ 47.1. In the gene library of E. carotovora obtained, eight phages carrying the gene sought were identified according to the appearance of enzymatic activity of the gene product, pectate lyase, in situ. The BamHI fragment of DNA, common to all these phages, was recloned on the plasmid pUC19. It was shown that the cloned pectate lyase gene is represented on the E. carotovora chromosome in one copy. Methods of production of representative gene libraries on phage vectors from no less than 1 μg of cloned DNA even for the genomes of eukaryotes have now been developed. Vectors have been created, for example, λ 47.1, permitting the selection only of hybrid molecules. A number of methods have been developed for the search for a required gene in the library, depending on whether the cloned gene can be expressed or not, and if it can, what properties it will impart to the hybrid clone containing it

Generation of species-specific DNA probes for Leishmania aethiopica

NARCIS (Netherlands)

Laskay, T.; Kiessling, R.; Rinke deWit, T. F.; Wirth, D. F.

1991-01-01

We report here the cloning of kinetoplast DNA (kDNA) sequences from Leishmania aethiopica in order to develop a specific and sensitive method for the identification of the parasite. Analysis of the cloned kDNA sequences showed different taxonomic specificities demonstrating sequence diversity within

Creation of Functional Viruses from Non-Functional cDNA Clones Obtained from an RNA Virus Population by the Use of Ancestral Reconstruction

DEFF Research Database (Denmark)

Fahnøe, Ulrik; Pedersen, Anders Gorm; Dräger, Carolin

2015-01-01

necessarily be the descendant of a functional ancestor, we hypothesized that it should be possible to produce functional clones by reconstructing ancestral sequences. To test this we used phylogenetic methods to infer two ancestral sequences, which were then reconstructed as cDNA clones. Viruses rescued from...... the reconstructed cDNAs were tested in cell culture and pigs. Both reconstructed ancestral genomes proved functional, and displayed distinct phenotypes in vitro and in vivo. We suggest that reconstruction of ancestral viruses is a useful tool for experimental and computational investigations of virulence and viral...... evolution. Importantly, ancestral reconstruction can be done even on the basis of a set of sequences that all correspond to non-functional variants....

Construction and characterization of a full-length infectious cDNA clone of foot-and-mouth disease virus strain O/JPN/2010 isolated in Japan in 2010.

Science.gov (United States)

Nishi, Tatsuya; Onozato, Hiroyuki; Ohashi, Seiichi; Fukai, Katsuhiko; Yamada, Manabu; Morioka, Kazuki; Kanno, Toru

2016-06-01

A full-length infectious cDNA clone of the genome of a foot-and-mouth disease virus isolated from the 2010 epidemic in Japan was constructed and designated pSVL-f02. Transfection of Cos-7 or IBRS-2 cells with this clone allowed the recovery of infectious virus. The recovered virus had the same in vitro characterization as the parental virus with regard to antigenicity in neutralization and indirect immunofluorescence tests, plaque size and one-step growth. Pigs were experimentally infected with the parental virus or the recombinant virus recovered from pSVL-f02 transfected cells. There were no significant differences in clinical signs or antibody responses between the two groups, and virus isolation and viral RNA detection from clinical samples were similar. Virus recovered from transfected cells therefore retained the in vitro characteristics and the in vivo pathogenicity of their parental strain. This cDNA clone should be a valuable tool to analyze determinants of pathogenicity and mechanisms of virus replication, and to develop genetically engineered vaccines against foot-and-mouth disease virus. Copyright © 2016 Elsevier Ltd. All rights reserved.

Molecular cloning of a cDNA and chromosomal localization of a human theta-class glutathione S-transferase gene (GSTT2) to chromosome 22

Energy Technology Data Exchange (ETDEWEB)

Tan, K.L.; Baker, R.T.; Board, P.G. [Australian National Univ., Canberra (Australia)] [and others

1995-01-20

Until recently the Theta-class glutathione S-transferases (GSTs) were largely overlooked due to their low activity with the model substrate 1-chloro-2,4-dinitrobenzene (CDNB) and their failure to bind to immobilized glutathione affinity matrices. Little is known about the number of genes in this class. Recently, Pemble et al. reported the cDNA cloning of a human Theta-class GST, termed GSTT1. In this study, we describe the molecular cloning of a cDNA encoding a second human Theta-class GST (GSTT2) from a {lambda}gt11 human liver 5{prime}-stretch cDNA library. The encoded protein contains 244 amino acids and has 78.3% sequence identity with the rat subunit 12 and only 55.0% identity with human GSTT1. GSTT2 has been mapped to chromosome 22 by somatic cell hybrid analysis. The precise position of the gene was localized to subband 22q11.2 by in situ hybridization. The absence of other regions of hybridization suggests that there are no closely related sequences (e.g., reverse transcribed pseudogenes) scattered throughout the genome and that if there are closely related genes, they must be clustered near GSTT2. Southern blot analysis of human DNA digested with BamHI shows that the size of the GSTT2 gene is relatively small, as the coding sequence falls within a 3.6-kb BamHI fragment. 35 refs., 6 figs.

Molecular cloning of L-methylmalonyl-CoA mutase: Gene transfer and analysis of mut cell lines

International Nuclear Information System (INIS)

Ledley, F.D.; Lumetta, M.; Nguyen, P.N.; Kolhouse, J.F.; Allen, R.H.

1988-01-01

L-Methylmalonyl-CoA mutase (MCM, EC 5.4.99.2) is a mitochondrial adenosylcobalamin-requiring enzyme that catalyzes the isomerization of L-methylmalonyl-CoA to succinyl-CoA. This enzyme is deficient in methylmalonic acidemia, an often fatal disorder of organic acid metabolism. Antibody against human placental MCM was used to screen human placenta and liver cDNA expression libraries for MCM cDNA clones. One clone expressed epitopes that could affinity-purify antibodies against MCM. A cDNA corresponding in length to the mRNA was obtained and introduced into COS cells by DNA-mediated gene transfer. Cells transformed with this clone expressed increased levels of MCM enzymatic activity. RNA blot analysis of cells genetically deficient in MCM indicates that several deficient cell lines have a specific decrease in the amount of hybridizable mRNA. These data confirm the authenticity of the MCM cDNA clone, establish the feasibility of constituting MCM activity by gene transfer for biochemical analysis and gene therapy, and provide a preliminary picture of the genotypic spectrum underlying MCM deficiency

Cloning and characterization of cDNA encoding xyloglucan ...

African Journals Online (AJOL)

Jane

2011-08-22

Aug 22, 2011 ... plays important role in growth and development of plants. XETs are a family of enzymes .... cloned into pGEM-T Easy vector (Promega Corporation, WI, USA). The recombinant ..... wall modification in the poaceae. Protein Sci.

Comparison of the genetic organization of the early salt-stress-response gene system in salt-tolerant Lophopyrum elongatum and salt-sensitive wheat

OpenAIRE

Dubcovsky, J; Galvez, AF; Dvořák, J

1994-01-01

Lophopyrum elongatum is a facultative halophyte related to wheat. Eleven unique clones corresponding to genes showing enhanced mRNA accumulation in the early stages of salt stress were previously isolated from a L. elongatum salt-stressed-root cDNA library. The chromosomal distribution of genes complementary to these clones in several genomes of the tribe Triticeae and their copy number in the L. elongatum and wheat genomes are reported. Genes complementary to clones pESI4, pESI14, pESI15, pE...

Fidelity of DNA Replication in Normal and Malignant Human Breast Cells.

Science.gov (United States)

1997-08-01

enzyme) into the multiple cloning site (MCS). This template will not only replicate inside a mammalian cell (utilizing the E-B virus origin), and...Maniatis, T. Commonly used techniques in molecular cloning . In: Molecular cloning : REFERENCES a laboratory manual, 2nd edition. Cold Spring Harbor...A vatit"Y Of DNA synthesis and the typt of DNA replica~tion Products " celular prca including DNA rsplicatlon. DNA repsair. R~NA formed in experiments

Radioactive and enzymatic cloned cDNA probes for bovine enteric coronavirus detection by molecular hybridization

International Nuclear Information System (INIS)

Collomb, J.; Finance, C.; Alabouch, S.; Laporte, J.

1992-01-01

Genomic RNA of F15 strain bovine enteric coronavirus (BECV) was cloned in E. coli. Three clones (174, 160, PG 78), selected in the cDNA library, including a large portion of the nucleocapsid (N), matrix (M) and peplomeric (S) protein genes , were used as probes for a slot blot hybridization assay. Two probe labelling techniques were compared, radiolabelling with 32 P and enzymatic labelling through covalent linkage to peroxidase and chemiluminescence detection. The radioactive probe 174 detected as little as 1 to 3 pg of viral RNA, while the less sensitive enzymatic probe could not reveal more than 100 pg of RNA. No significant detection amplification was achieved when a mixture of the three probes was used. Probe 174 allowed specific identification for BECV. No hybridization was noticed either with rotaviruses or even with other antigenically unrelated members of the family Coronaviridae such as transmissible gastroenteritis virus. The test proved valid for detection of BECV in the supernatant of infected HRT-18 cells: genomic RNA could be detected after direct spotting of samples, but prior nucleic acid extraction after proteinase K treatment improved virus detection. BECV diagnosis in faecal samples using enzymatic probe was compared with conventional diagnostic methods. (authors)

Radioactive and enzymatic cloned cDNA probes for bovine enteric coronavirus detection by molecular hybridization

Energy Technology Data Exchange (ETDEWEB)

Collomb, J; Finance, C; Alabouch, S [Lab. de Microbiologie Moleculaire, Faculte des Sciences Pharmaceutiques et Biologiques, Univ. de Nancy I, Nancy (France); Laporte, J [Station de Virologie et d' Immunologie Moleculaires, INRA, Jouy-en-Josas (France)

1992-01-01

Genomic RNA of F15 strain bovine enteric coronavirus (BECV) was cloned in E. coli. Three clones (174, 160, PG 78), selected in the cDNA library, including a large portion of the nucleocapsid (N), matrix (M) and peplomeric (S) protein genes , were used as probes for a slot blot hybridization assay. Two probe labelling techniques were compared, radiolabeled with [sup 32]P and enzymatic labeled through covalent linkage to peroxidase for chemiluminescence detection. The radioactive probe 174 detected as little as 1-3 pg of viral RNA, while the less sensitive enzymatic probe could not reveal more than 100 pg of RNA. No significant detection amplification was achieved when a mixture of the three probes was used. Probe 174 allowed specific identification for BECV. No hybridization was noticed either with rotaviruses or even with other antigenically unrelated members of the family Coronaviridae such as transmissible gastroenteritis virus. The test proved valid for detection of BECV in the supernatant of infected HRT-18 cells: genomic RNA could be detected after direct spotting of samples, but prior nucleic acid extraction after proteinase K treatment improved virus detection. BECV diagnosis in fecal samples using enzymatic probe was compared with conventional diagnostic methods. (authors).

Rapid cloning and bioinformatic analysis of spinach Y chromosome ...

Indian Academy of Sciences (India)

(DOP-PCR) products were hybridized with cDNA of the male spinach flowers in florescence. The female .... China), 1 μL (3.5 U/μL) T4 ligase and sterile double- distilled ... Cloning of hybrid PCR products and screening of cDNA sequence.

Cloning of an endangered species (Bos gaurus) using interspecies nuclear transfer.

Science.gov (United States)

Lanza, R P; Cibelli, J B; Diaz, F; Moraes, C T; Farin, P W; Farin, C E; Hammer, C J; West, M D; Damiani, P

2000-01-01

Approximately 100 species become extinct a day. Despite increasing interest in using cloning to rescue endangered species, successful interspecies nuclear transfer has not been previously described, and only a few reports of in vitro embryo formation exist. Here we show that interspecies nuclear transfer can be used to clone an endangered species with normal karyotypic and phenotypic development through implantation and the late stages of fetal growth. Somatic cells from a gaur bull (Bos gaurus), a large wild ox on the verge of extinction, (Species Survival Plan cloned animals was gaurus in origin. The gaur nuclei were shown to direct normal fetal development, with differentiation into complex tissue and organs, even though the mitochondrial DNA (mtDNA) within all the tissue types evaluated was derived exclusively from the recipient bovine oocytes. These results suggest that somatic cell cloning methods could be used to restore endangered, or even extinct, species and populations.

Frame-Insensitive Expression Cloning of Fluorescent Protein from Scolionema suvaense

Directory of Open Access Journals (Sweden)

Yuki Horiuchi

2018-01-01

Full Text Available Expression cloning from cDNA is an important technique for acquiring genes encoding novel fluorescent proteins. However, the probability of in-frame cDNA insertion following the first start codon of the vector is normally only 1/3, which is a cause of low cloning efficiency. To overcome this issue, we developed a new expression plasmid vector, pRSET-TriEX, in which transcriptional slippage was induced by introducing a DNA sequence of (dT14 next to the first start codon of pRSET. The effectiveness of frame-insensitive cloning was validated by inserting the gene encoding eGFP with all three possible frames to the vector. After transformation with one of these plasmids, E. coli cells expressed eGFP with no significant difference in the expression level. The pRSET-TriEX vector was then used for expression cloning of a novel fluorescent protein from Scolionema suvaense. We screened 3658 E. coli colonies transformed with pRSET-TriEX containing Scolionema suvaense cDNA, and found one colony expressing a novel green fluorescent protein, ScSuFP. The highest score in protein sequence similarity was 42% with the chain c of multi-domain green fluorescent protein like protein “ember” from Anthoathecata sp. Variations in the N- and/or C-terminal sequence of ScSuFP compared to other fluorescent proteins indicate that the expression cloning, rather than the sequence similarity-based methods, was crucial for acquiring the gene encoding ScSuFP. The absorption maximum was at 498 nm, with an extinction efficiency of 1.17 × 105 M−1·cm−1. The emission maximum was at 511 nm and the fluorescence quantum yield was determined to be 0.6. Pseudo-native gel electrophoresis showed that the protein forms obligatory homodimers.

Complementary specificity of restriction endonucleases of Diplococcus pneumoniae with respect to DNA methylation. [Haemophilus influenzae, Escherichia coli, Paramecium aurelia

Energy Technology Data Exchange (ETDEWEB)

Lacks, S.; Greenberg, B.

1977-01-01

Restriction endonucleases Dpn I and Dpn II are produced by two distinct strains of Diplococcus pneumoniae. The two enzymes show complementary specificity with respect to methylation of sites in DNA. From the identity of its cleavage site with that of Mbo I, it appears that Dpn II cleaves at the unmodified sequence 5'-G-A-T-C-3'. Dpn I cleaves at the same sequence when the adenine residue is methylated. Both enzymes produce only double-strand breaks in susceptible DNA. Their susceptibility to Dpn I and not Dpn II shows that essentially all the G-A-T-C sequences are methylated in DNA from the pneumococcal strain that produces Dpn II as well as in DNA from Hemophilus influenzae and Escherichia coli. In the dam-3 mutant of E. coli none of these sequences appear to be methylated. Residual adenine methylation in the dam-3 mutant DNA most likely occurs at different sites. Different but characteristic degrees of methylation at G-A-T-C sites are found in the DNA of bacterial viruses grown in E. coli. DNAs from mammalian cells and viruses are not methylated at this sequence. Mitochondrial DNA from Paramecium aurelia is not methylated, but a small proportion of G-A-T-C sequences in the macronuclear DNA of this eukaryote appear to be methylated. Possible roles of sequence-specific methylation in the accommodation of plasmids, in the replication of DNA, in the regulation of gene function and in the restriction of viral infection are discussed.

DNA-mediated gene transfer into ataxia-telangiectasia cells

International Nuclear Information System (INIS)

Crescenzi, M.; Pulciani, S.; Carbonari, M.; Tedesco, L.; Russo, G.; Gaetano, C.; Fiorilli, M.

1986-01-01

The complete description of the genetic lesion(s) underlying the AT mutation might, therefore, highlight not only a DNA-repair pathwa, but also an important aspect of the physiology of lymphocytes. DNA-mediated gene transfer into eukaryotic cells has proved a powerful tool for the molecular cloning of certain mammalian genes. The possibility to clone a given gene using this technology depends, basically, on the availability of a selectable marker associated with the expression of the transfected gene in the recipient cell. Recently, a human DNA repair gene has been cloned in CHO mutant cells by taking advantage of the increased resistance to ultraviolet radiation of the transformants. As a preliminary step toward the molecular cloning of the AT gene(s), the authors have attempted to confer radioresistance to AT cells by transfection with normal human DNA

Molecular cloning and biological characterization of the human excision repair gene ERCC-3

International Nuclear Information System (INIS)

Weeda, G.; van Ham, R.C.; Masurel, R.; Westerveld, A.; Odijk, H.; de Wit, J.; Bootsma, D.; van der Eb, A.J.; Hoeijmakers, J.H.

1990-01-01

In this report we present the cloning, partial characterization, and preliminary studies of the biological activity of a human gene, designated ERCC-3, involved in early steps of the nucleotide excision repair pathway. The gene was cloned after genomic DNA transfection of human (HeLa) chromosomal DNA together with dominant marker pSV3gptH to the UV-sensitive, incision-defective Chinese hamster ovary (CHO) mutant 27-1. This mutant belongs to complementation group 3 of repair-deficient rodent mutants. After selection of UV-resistant primary and secondary 27-1 transformants, human sequences associated with the induced UV resistance were rescued in cosmids from the DNA of a secondary transformant by using a linked dominant marker copy and human repetitive DNA as probes. From coinheritance analysis of the ERCC-3 region in independent transformants, we deduce that the gene has a size of 35 to 45 kilobases, of which one essential segment has so far been refractory to cloning. Conserved unique human sequences hybridizing to a 3.0-kilobase mRNA were used to isolate apparently full-length cDNA clones. Upon transfection to 27-1 cells, the ERCC-3 cDNA, inserted in a mammalian expression vector, induced specific and (virtually) complete correction of the UV sensitivity and unscheduled DNA synthesis of mutants of complementation group 3 with very high efficiency. Mutant 27-1 is, unlike other mutants of complementation group 3, also very sensitive toward small alkylating agents. This unique property of the mutant is not corrected by introduction of the ERCC-3 cDNA, indicating that it may be caused by an independent second mutation in another repair function. By hybridization to DNA of a human x rodent hybrid cell panel, the ERCC-3 gene was assigned to chromosome 2, in agreement with data based on cell fusion

Partial structure of the phylloxin gene from the giant monkey frog, Phyllomedusa bicolor: parallel cloning of precursor cDNA and genomic DNA from lyophilized skin secretion.

Science.gov (United States)

Chen, Tianbao; Gagliardo, Ron; Walker, Brian; Zhou, Mei; Shaw, Chris

2005-12-01

Phylloxin is a novel prototype antimicrobial peptide from the skin of Phyllomedusa bicolor. Here, we describe parallel identification and sequencing of phylloxin precursor transcript (mRNA) and partial gene structure (genomic DNA) from the same sample of lyophilized skin secretion using our recently-described cloning technique. The open-reading frame of the phylloxin precursor was identical in nucleotide sequence to that previously reported and alignment with the nucleotide sequence derived from genomic DNA indicated the presence of a 175 bp intron located in a near identical position to that found in the dermaseptins. The highly-conserved structural organization of skin secretion peptide genes in P. bicolor can thus be extended to include that encoding phylloxin (plx). These data further reinforce our assertion that application of the described methodology can provide robust genomic/transcriptomic/peptidomic data without the need for specimen sacrifice.

[Product safety analysis of somatic cell cloned bovine].

Science.gov (United States)

Hua, Song; Lan, Jie; Song, Yongli; Lu, Chenglong; Zhang, Yong

2010-05-01

Somatic cell cloning (nuclear transfer) is a technique through which the nucleus (DNA) of a somatic cell is transferred into an enucleated oocyte for the generation of a new individual, genetically identical to the somatic cell donor. It could be applied for the enhancement of reproduction rate and the improvement of food products involving quality, yield and nutrition. In recent years, the United States, Japan and Europe as well as other countries announced that meat and milk products made from cloned cattle are safe for human consumption. Yet, cloned animals are faced with a wide range of health problems, with a high death rate and a high incidence of disease. The precise causal mechanisms for the low efficiency of cloning remain unclear. Is it safe that any products from cloned animals were allowed into the food supply? This review focuses on the security of meat, milk and products from cloned cattle based on the available data.

Molecular cloning of cDNAs of human liver and placenta NADH-cytochrome b5 reductase

International Nuclear Information System (INIS)

Yubisui, T.; Naitoh, Y.; Zenno, S.; Tamura, M.; Takeshita, M.; Sakaki, Y.

1987-01-01

A cDNA coding for human liver NADH-cytochrome b 5 reductase was cloned from a human liver cDNA library constructed in phage λgt11. The library was screened by using an affinity-purified rabbit antibody against NADH-cytochrome b 5 reductase of human erythrocytes. A cDNA about 1.3 kilobase pairs long was isolated. By using the cDNA as a probe, another cDNA (pb 5 R141) of 1817 base pairs was isolated that hybridized with a synthetic oligonucleotide encoding Pro-Asp-Ile-Lys-Tyr-Pro, derived from the amino acid sequence at the amino-terminal region of the enzyme from human erythrocytes. Furthermore, by using the pb 5 R141 as a probe, cDNA clones having more 5' sequence were isolated from a human placenta cDNA library. The amino acid sequences deduced from the nucleotide sequences of these cDNA clones overlapped each other and consisted of a sequence that completely coincides with that of human erythrocytes and a sequence of 19 amino acid residues extended at the amino-terminal side. The latter sequence closely resembles that of the membrane-binding domain of steer liver microsomal enzyme

Binding proteins for the regulatory subunit (RII-B) of brain cAMP-dependent protein kinase II: isolation and initial characterization of cDNA clones

International Nuclear Information System (INIS)

Bregman, D.B.; Hu, E.; Rubin, C.S.

1987-01-01

In mammalian brain several proteins bind RII-B with high affinity. An example is P75, which co-purifies with RII-B and also complexes Ca 2+ -calmodulin. Thus, RII-B binding proteins (RBPs) might play a role in integrating the Ca 2+ and cAMP signalling pathways in the CNS. In order to study the structure and function of these polypeptides they have isolated cloned cDNAs for RBPs by screening brain λgt11 expression libraries using a functional assay: the binding of 32 P-labeled RII to fusion proteins produced by recombinants expressing RII binding domains. Inserts from rat brain recombinant clones λ7B and λ10B both hybridize to a brain mRNA of 7000 nucleotides. Northern gel analyses indicate that the putative RBP mRNA is also expressed in lung, but not in several other tissues. The λ7B insert was subcloned into the expression plasmid pINIA. A 50 kDa high affinity RII-B binding polypeptide accumulated in E. coli transformed with pINIA-7B. Two RBP cDNAs (λ77, λ100A) have been retrieved from a bovine λgt 11 library using a monoclonal antibody directed against P75 and the binding assay respectively. On Southern blots the insert from λ100A hybridizes to the cDNA insert from clones λ77, suggesting that λ 77 cDNA might contain sequences coding for both an RII binding domain and a P75 epitope. The bovine λ100A insert also hybridizes with the rat λ7B clone indicating that an RII binding domain is conserved in the two species

Cloning and characterization of a Candida albicans maltase gene involved in sucrose utilization.

Science.gov (United States)

Geber, A; Williamson, P R; Rex, J H; Sweeney, E C; Bennett, J E

1992-01-01

In order to isolate the structural gene involved in sucrose utilization, we screened a sucrose-induced Candida albicans cDNA library for clones expressing alpha-glucosidase activity. The C. albicans maltase structural gene (CAMAL2) was isolated. No other clones expressing alpha-glucosidase activity. were detected. A genomic CAMAL2 clone was obtained by screening a size-selected genomic library with the cDNA clone. DNA sequence analysis reveals that CAMAL2 encodes a 570-amino-acid protein which shares 50% identity with the maltase structural gene (MAL62) of Saccharomyces carlsbergensis. The substrate specificity of the recombinant protein purified from Escherichia coli identifies the enzyme as a maltase. Northern (RNA) analysis reveals that transcription of CAMAL2 is induced by maltose and sucrose and repressed by glucose. These results suggest that assimilation of sucrose in C. albicans relies on an inducible maltase enzyme. The family of genes controlling sucrose utilization in C. albicans shares similarities with the MAL gene family of Saccharomyces cerevisiae and provides a model system for studying gene regulation in this pathogenic yeast. Images PMID:1400249

[Cloning of cDNA for RNA polymerase subunit from the fission yeast Schizosaccharomyces pombe by heterospecific complementation in Saccharomyces cerevisiae].

Science.gov (United States)

Shpakovskiĭ, G V; Lebedenko, E N; Thuriaux, P

1997-02-01

The rpb10 cDNA of the fission yeast Schizosaccharomyces pombe, encoding one of the five small subunits common to all three nuclear DNA-dependent RNA polymerases, was isolated from an expression cDNA library by two independent approaches: PCR-based screening and direct suppression by means of heterospecific complementation of a temperature-sensitive mutant defective in the corresponding gene of Saccharomyces cerevisiae. The cloned Sz. pombe cDNA encodes a protein Rpb10 of 71 amino acids with an M of 8,275 Da, sharing 51 amino acids (71% identity) with the subunit ABC10 beta of RNA polymerases I-III from S. cerevisiae. All eukaryotic members of this protein family have the same general organization featuring two highly conserved motifs (RCFT/SCGK and RYCCRRM) around an atypical zinc finger and an additional invariant HVDLIEK motif toward the C-terminal end. The last motif is only characteristics for homologs from eukaryotes. In keeping with this remarkable structural conservation, the Sz. pombe cDNA also fully complemented a S. cerevisiae deletion mutant lacking subunit ABC10 beta (null allele rpb10-delta 1::HIS3).

Skewed X-inactivation in cloned mice

International Nuclear Information System (INIS)

Senda, Sho; Wakayama, Teruhiko; Yamazaki, Yukiko; Ohgane, Jun; Hattori, Naka; Tanaka, Satoshi; Yanagimachi, Ryuzo; Shiota, Kunio

2004-01-01

In female mammals, dosage compensation for X-linked genes is accomplished by inactivation of one of two X chromosomes. The X-inactivation ratio (a percentage of the cells with inactivated maternal X chromosomes in the whole cells) is skewed as a consequence of various genetic mutations, and has been observed in a number of X-linked disorders. We previously reported that phenotypically normal full-term cloned mouse fetuses had loci with inappropriate DNA methylation. Thus, cloned mice are excellent models to study abnormal epigenetic events in mammalian development. In the present study, we analyzed X-inactivation ratios in adult female cloned mice (B6C3F1). Kidneys of eight naturally produced controls and 11 cloned mice were analyzed. Although variations in X-inactivation ratio among the mice were observed in both groups, the distributions were significantly different (Ansary-Bradley test, P < 0.01). In particular, 2 of 11 cloned mice showed skewed X-inactivation ratios (19.2% and 86.8%). Similarly, in intestine, 1 of 10 cloned mice had a skewed ratio (75.7%). Skewed X-inactivation was observed to various degrees in different tissues of different individuals, suggesting that skewed X-inactivation in cloned mice is the result of secondary cell selection in combination with stochastic distortion of primary choice. The present study is the first demonstration that skewed X-inactivation occurs in cloned animals. This finding is important for understanding both nuclear transfer technology and etiology of X-linked disorders

Accurate Dna Assembly And Direct Genome Integration With Optimized Uracil Excision Cloning To Facilitate Engineering Of Escherichia Coli As A Cell Factory

DEFF Research Database (Denmark)

Cavaleiro, Mafalda; Kim, Se Hyeuk; Nørholm, Morten

2015-01-01

Plants produce a vast diversity of valuable compounds with medical properties, but these are often difficult to purify from the natural source or produce by organic synthesis. An alternative is to transfer the biosynthetic pathways to an efficient production host like the bacterium Escherichia co......-excision-based cloning and combining it with a genome-engineering approach to allow direct integration of whole metabolic pathways into the genome of E. coli, to facilitate the advanced engineering of cell factories........ Cloning and heterologous gene expression are major bottlenecks in the metabolic engineering field. We are working on standardizing DNA vector design processes to promote automation and collaborations in early phase metabolic engineering projects. Here, we focus on optimizing the already established uracil...

Disease-causing mitochondrial heteroplasmy segregated within induced pluripotent stem cell clones derived from a patient with MELAS.

Science.gov (United States)

Folmes, Clifford D L; Martinez-Fernandez, Almudena; Perales-Clemente, Ester; Li, Xing; McDonald, Amber; Oglesbee, Devin; Hrstka, Sybil C; Perez-Terzic, Carmen; Terzic, Andre; Nelson, Timothy J

2013-07-01

Mitochondrial diseases display pathological phenotypes according to the mixture of mutant versus wild-type mitochondrial DNA (mtDNA), known as heteroplasmy. We herein examined the impact of nuclear reprogramming and clonal isolation of induced pluripotent stem cells (iPSC) on mitochondrial heteroplasmy. Patient-derived dermal fibroblasts with a prototypical mitochondrial deficiency diagnosed as mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) demonstrated mitochondrial dysfunction with reduced oxidative reserve due to heteroplasmy at position G13513A in the ND5 subunit of complex I. Bioengineered iPSC clones acquired pluripotency with multilineage differentiation capacity and demonstrated reduction in mitochondrial density and oxygen consumption distinguishing them from the somatic source. Consistent with the cellular mosaicism of the original patient-derived fibroblasts, the MELAS-iPSC clones contained a similar range of mtDNA heteroplasmy of the disease-causing mutation with identical profiles in the remaining mtDNA. High-heteroplasmy iPSC clones were used to demonstrate that extended stem cell passaging was sufficient to purge mutant mtDNA, resulting in isogenic iPSC subclones with various degrees of disease-causing genotypes. On comparative differentiation of iPSC clones, improved cardiogenic yield was associated with iPSC clones containing lower heteroplasmy compared with isogenic clones with high heteroplasmy. Thus, mtDNA heteroplasmic segregation within patient-derived stem cell lines enables direct comparison of genotype/phenotype relationships in progenitor cells and lineage-restricted progeny, and indicates that cell fate decisions are regulated as a function of mtDNA mutation load. The novel nuclear reprogramming-based model system introduces a disease-in-a-dish tool to examine the impact of mutant genotypes for MELAS patients in bioengineered tissues and a cellular probe for molecular features of individual

Assessment of genetic diversity in different clones of Dalbergia ...

African Journals Online (AJOL)

Genetic diversity of forty (40) clones of Dalbergia sissoo Roxb was analyzed using randomly amplified polymorphic DNA (RAPD) markers by selecting 30 decamer primers, which were later reduced to 10 based on the preliminary PCR amplification. A total of 129 distinct DNA fragments (bands) were amplified, of which 104 ...

Cloning and characterization of the rec2 gene of Ustilago maydis

International Nuclear Information System (INIS)

Bauchwitz, R.P.; Holloman, W.K.

1989-01-01

The authors are exploring the molecular basis for genetic recombination using the corn smut fungus Ustilago maydis, from which the first two eucaryotic DNA repair and recombination mutants, rec1 and rec2, were described. Cells mutant at the rec2 locus are unable to repair lethal damage to their DNA from UV and X irradiation or from chemical alkylating agents such as N-methyl-nitrosoguanidine. Rec2 mutants retain only a residual level of DNA-damage inducible mitotic recombination, and are unable to complete meiosis. Using an autonomously replicating plasmid vector for Ustilago, they established the first nonintegrating plasmid library of the Ustilago genome. The rec2 locus was cloned by complementation of the rec2 mutation in vivo. One clone was found to restore all of the deficient activities. Although this rec2 complementing clone is present on a multicopy plasmid, the authors observed that it fully restored but did not further increase the fifty-fold inducibility of heteroallelic recombination at the nitrate reductase and inositol loci of rec2 or wild type cells. Northern blot analysis using the rec2 complementing clone revealed three UV inducible transcripts, one of which is absent in a rec2 mutant strain. This transcript organization resembles that of the yeast rad10 and the human ERCC-1 genes (MCB 9:1794), but sequence obtained to date from rec2 does not show homology with these genes. They have also observed that the rec2 mutation may alter the level of homologous integration of transformed DNA markers. Integration of a Leu1 complementing plasmid by Scott Fotheringham of the lab has shown that while much of plasmid integration in wild type Ustilago is nonhomologous, integration in at least some rec2 strains is entirely homologous. They are using the cloned rec2 gene to confirm that rec2 is indeed involved in altering the level of homologous integration in Ustilago, and if so, they plan to clone a mammalian analogue of rec2

Cloning and characterization of cDNA encoding xyloglucan ...

African Journals Online (AJOL)

Evolutionary studies using phylogenetic tree indicated its grouping with XETs from maize (with >95% bootstrap support), barley, rice, etc. This is the first report on cloning and characterization of an XET (PgXET1) from pearl millet, an important dual-purpose crop. Key words: Xyloglucan endotransglucosylase, Pennisetum ...

Semi-conservative synthesis of DNA in UV-sensitive mutant cells of Chinese hamster after UV-irradiation

International Nuclear Information System (INIS)

Vikhanskaya, F.L.; Khrebtukova, I.A.; Manuilova, E.S.

1985-01-01

A study was made of the rate of semi-conservative DNA synthesis in asynchronous UV-resistant (clone V79) and UV-sensitive clones (VII and XII) of Chinese hamster cells after UV-irradiation. In all 3 clones studied, UV-irradiation (5-30 J/m 2 ) induced a decrease in the rate of DNA synthesis during the subsequent 1-2 h. In the resistant clone (V79) recovery of DNA synthesis rate started after the first 2 h post-irradiation (5 J/m 2 ) and by the 3rd hour reached its maximum value, which constituted 70% of that observed in control, non-irradiated cells. The UV-sensitive mutant clones VII and XII showed no recovery in the rate of DNA synthesis during 6-7 h post-irradiation. The results obtained show that the survival of cells is correlated with the ability of DNA synthesis to recover after UV-irradiation in 3 clones studied. The observed recovery of UV-inhibited DNA synthesis in mutant clones may be due to certain defects in DNA repair. (orig.)

YAC clone information - RGP physicalmap | LSDB Archive [Life Science Database Archive metadata

Lifescience Database Archive (English)

Full Text Available 08/lsdba.nbdc00318-06-002 Description of data contents YAC clones selected with DNA markers Data file File name: rgp_physical...map_yac_clones.zip File URL: ftp://ftp.biosciencedbc.jp/archive/rgp-physicalmap/LATEST/rgp_physical...sciencedbc.jp/togodb/view/rgp_physicalmap_yac_clones#en Data acquisition method YAC clones selected with RGP...rom. No. Chromosome number Region Region number Physical map image The file name of rice physical map Order ...bout This Database Database Description Download License Update History of This Database Site Policy | Contact Us YAC clone information - RGP physicalmap | LSDB Archive ...

阴道毛滴虫Rac1蛋白的cDNA克隆和序列分析%Molecular Cloning and Characterization of a Rac1 Homologue cDNA from Trichomonas vaginalis

Institute of Scientific and Technical Information of China (English)

傅玉才; 章家新; 郑晓虹; 刘红

2004-01-01

Objective To clone and characterize a Racl homologue from Trichomonas vaginalis for studying cell cycle of the organism. Methods A cDNA library derived from T. vaginalis mRNA was constructed into λ TriplEx2 phage vector. An expression sequence tag program was launched. Sequences of cDNA clones were analyzed using NCBI BLAST algorithms, and ClustalW and Treeview programs. Results A cDNA clone with a length of 714 base pairs was isolated. The sequence analysis showed that the cDNA clone has an open reading frame with 600 bp. The deduced amino acid sequence from the open reading frame contains 200 residuals and is most homologous to Rac1 subfamily of Rho GTPases with ＞ 60% identity. The conserved sequence elements of Rho GTPases, such as GTP-binding sites, GTPase-activating protein (GAP) interaction motifs, GTP-dissociation inhibitors (GDI) interaction motifs, guanine nucleotide exchange factor (GEF) interaction elements, etc, were detected in the amino acid sequence. The phylogenetic analysis showed that the cDNA clone is grouped in the Rac subfamily and is more closely related to Rac1 proteins of protozoa. Conclusion The cDNA clone isolated belongs to Rac subfamily of Rho GTPases and is probably a Rac1 protein of T. vaginalis.%目的获得阴道毛滴虫Rac1蛋白的cDNA克隆,研究其在细胞周期中的调解作用.方法提取阴道毛滴虫总RNA,构建cDNA表达文库,随机分离cDNA克隆并测序.用在线生物分析软件NCBI BLAST、ClustalW以及Treeview等程序进行序列分析.结果获得一株有714 bp的cDNA克隆.序列分析表明,该克隆开放阅读框具600 bp,推测肽链具200个氨基酸.该肽链与Rho家族中Rac1鸟苷三磷酸(GTP)酶同源性最高(＞60%),并具多种Rho GTP酶的保守基序,如GTP结合部位、GTP酶激活蛋白作用基序、GTP分离抑制因子作用基序、鸟嘌呤核苷酸交换因子作用基序等.进化树分析显示该克隆属于Rac亚家族GTP酶,与原虫Rac1蛋白最接近.结论该克隆�

Molecular cloning and expression of Corynebacterium glutamicum genes for amino acid synthesis in Escherichia coli cells

International Nuclear Information System (INIS)

Beskrovnaya, O.Yu.; Fonshtein, M.Yu.; Kolibaba, L.G.; Yankovskii, N.K.; Debabov, V.G.

1989-01-01

Molecular cloning of Corynebacterium glutamicum genes for threonine and lysine synthesis has been done in Escherichia coli cells. The clonal library of EcoRI fragments of chromosomal DNA of C. glutamicum was constructed on the plasmid vector λpSL5. The genes for threonine and lysine synthesis were identified by complementation of E. coli mutations in thrB and lysA genes, respectively. Recombinant plasmids, isolated from independent ThrB + clone have a common 4.1-kb long EcoRI DNA fragment. Hybrid plasmids isolated from LysA + transductants of E. coli have common 2.2 and 3.3 kb long EcoRI fragments of C. glutamicum DNA. The hybrid plasmids consistently transduced the markers thrB + and lysA + . The Southern hybridization analysis showed that the cloned DNA fragments hybridized with the fragments of identical length in C. glutamicum chromosomes

Cloning, molecular characterization and expression of a cDNA encoding a functional NADH-cytochrome b5 reductase from Mucor racemosus PTCC 5305 in E. coli

Directory of Open Access Journals (Sweden)

NED A SETAYESH

2009-01-01

Full Text Available The present work aims to study a new NADH-cytochrome b5 reductase (cb5r from Mucor racemosus PTCC 5305. A cDNA coding for cb s r was isolated from a Mucor racemosus PTCC 5305 cDNA library. The nucleotide sequence of the cDNA including coding and sequences flanking regions was determined. The open reading frame starting from ATG and ending with TAG stop codon encoded 228 amino acids and displayed the closest similarity (73% with Mortierella alpina cb s r. Lack of hydrophobic residues in the N-terminal sequence was apparent, suggesting that the enzyme is a soluble isoform. The coding sequence was then cloned in the pET16b transcription vector carrying an N-terminal-linked His-Tag® sequence and expressed in Escherichia coli BL21 (DE3. The enzyme was then homogeneously purified by a metal affinity column. The recombinant Mucor enzyme was shown to have its optimal activity at pH and temperature of about 7.5 and 40 °C, respectively. The apparent Km value was calculated to be 13 μM for ferricyanide. To our knowledge, this is the first report on cloning and expression of a native fungal soluble isoform of NADH-cytochrome b5 reductase in E. coli.

Molecular transformation, gene cloning, and gene expression systems for filamentous fungi

Science.gov (United States)

Gold, Scott E.; Duick, John W.; Redman, Regina S.; Rodriguez, Rusty J.

2001-01-01

This chapter discusses the molecular transformation, gene cloning, and gene expression systems for filamentous fungi. Molecular transformation involves the movement of discrete amounts of DNA into cells, the expression of genes on the transported DNA, and the sustainable replication of the transforming DNA. The ability to transform fungi is dependent on the stable replication and expression of genes located on the transforming DNA. Three phenomena observed in bacteria, that is, competence, plasmids, and restriction enzymes to facilitate cloning, were responsible for the development of molecular transformation in fungi. Initial transformation success with filamentous fungi, involving the complementation of auxotrophic mutants by exposure to sheared genomic DNA or RNA from wt isolates, occurred with low transformation efficiencies. In addition, it was difficult to retrieve complementing DNA fragments and isolate genes of interest. This prompted the development of transformation vectors and methods to increase efficiencies. The physiological studies performed with fungi indicated that the cell wall could be removed to generate protoplasts. It was evident that protoplasts could be transformed with significantly greater efficiencies than walled cells.

A rapid method for screening arrayed plasmid cDNA library by PCR

International Nuclear Information System (INIS)

Hu Yingchun; Zhang Kaitai; Wu Dechang; Li Gang; Xiang Xiaoqiong

1999-01-01

Objective: To develop a PCR-based method for rapid and effective screening of arrayed plasmid cDNA library. Methods: The plasmid cDNA library was arrayed and screened by PCR with a particular set of primers. Results: Four positive clones were obtained through about one week. Conclusion: This method can be applied to screening not only normal cDNA clones, but also cDNA clones-containing small size fragments. This method offers significant advantages over traditional screening method in terms of sensitivity, specificity and efficiency

Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus niger in Escherichia coli

International Nuclear Information System (INIS)

Woon, J. S. K.; Murad, A. M. A.; Abu Bakar, F. D.

2015-01-01

A cellobiohydrolase B (CbhB) from Aspergillus niger ATCC 10574 was cloned and expressed in E. coli. CbhB has an open reading frame of 1611 bp encoding a putative polypeptide of 536 amino acids. Analysis of the encoded polypeptide predicted a molecular mass of 56.2 kDa, a cellulose binding module (CBM) and a catalytic module. In order to obtain the mRNA of cbhB, total RNA was extracted from A. niger cells induced by 1% Avicel. First strand cDNA was synthesized from total RNA via reverse transcription. The full length cDNA of cbhB was amplified by PCR and cloned into the cloning vector, pGEM-T Easy. A comparison between genomic DNA and cDNA sequences of cbhB revealed that the gene is intronless. Upon the removal of the signal peptide, the cDNA of cbhB was cloned into the expression vector pET-32b. However, the recombinant CbhB was expressed in Escherichia coli Origami DE3 as an insoluble protein. A homology model of CbhB predicted the presence of nine disulfide bonds in the protein structure which may have contributed to the improper folding of the protein and thus, resulting in inclusion bodies in E. coli

Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus niger in Escherichia coli

Energy Technology Data Exchange (ETDEWEB)

Woon, J. S. K., E-mail: jameswoon@siswa.ukm.edu.my; Murad, A. M. A., E-mail: munir@ukm.edu.my; Abu Bakar, F. D., E-mail: fabyff@ukm.edu.my [School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor (Malaysia)

2015-09-25

A cellobiohydrolase B (CbhB) from Aspergillus niger ATCC 10574 was cloned and expressed in E. coli. CbhB has an open reading frame of 1611 bp encoding a putative polypeptide of 536 amino acids. Analysis of the encoded polypeptide predicted a molecular mass of 56.2 kDa, a cellulose binding module (CBM) and a catalytic module. In order to obtain the mRNA of cbhB, total RNA was extracted from A. niger cells induced by 1% Avicel. First strand cDNA was synthesized from total RNA via reverse transcription. The full length cDNA of cbhB was amplified by PCR and cloned into the cloning vector, pGEM-T Easy. A comparison between genomic DNA and cDNA sequences of cbhB revealed that the gene is intronless. Upon the removal of the signal peptide, the cDNA of cbhB was cloned into the expression vector pET-32b. However, the recombinant CbhB was expressed in Escherichia coli Origami DE3 as an insoluble protein. A homology model of CbhB predicted the presence of nine disulfide bonds in the protein structure which may have contributed to the improper folding of the protein and thus, resulting in inclusion bodies in E. coli.

Isolation, molecular cloning and expression of cellobiohydrolase B (CbhB) from Aspergillus niger in Escherichia coli

Science.gov (United States)

Woon, J. S. K.; Murad, A. M. A.; Abu Bakar, F. D.

2015-09-01

A cellobiohydrolase B (CbhB) from Aspergillus niger ATCC 10574 was cloned and expressed in E. coli. CbhB has an open reading frame of 1611 bp encoding a putative polypeptide of 536 amino acids. Analysis of the encoded polypeptide predicted a molecular mass of 56.2 kDa, a cellulose binding module (CBM) and a catalytic module. In order to obtain the mRNA of cbhB, total RNA was extracted from A. niger cells induced by 1% Avicel. First strand cDNA was synthesized from total RNA via reverse transcription. The full length cDNA of cbhB was amplified by PCR and cloned into the cloning vector, pGEM-T Easy. A comparison between genomic DNA and cDNA sequences of cbhB revealed that the gene is intronless. Upon the removal of the signal peptide, the cDNA of cbhB was cloned into the expression vector pET-32b. However, the recombinant CbhB was expressed in Escherichia coli Origami DE3 as an insoluble protein. A homology model of CbhB predicted the presence of nine disulfide bonds in the protein structure which may have contributed to the improper folding of the protein and thus, resulting in inclusion bodies in E. coli.

Clonal diversity and clone formation in the parthenogenetic Caucasian rock Lizard Darevskia dahli [corrected].

Science.gov (United States)

Vergun, Andrey A; Martirosyan, Irena A; Semyenova, Seraphima K; Omelchenko, Andrey V; Petrosyan, Varos G; Lazebny, Oleg E; Tokarskaya, Olga N; Korchagin, Vitaly I; Ryskov, Alexey P

2014-01-01

The all-female Caucasian rock lizard species Darevskia dahli and other parthenogenetic species of this genus reproduce normally via true parthenogenesis. Previously, the genetic diversity of this species was analyzed using allozymes, mitochondrial DNA, and DNA fingerprint markers. In the present study, variation at three microsatellite loci was studied in 111 specimens of D. dahli from five populations from Armenia, and new information regarding clonal diversity and clone formation in D. dahli was obtained that suggests a multiple hybridization origin. All individuals but one were heterozygous at the loci studied. Based on specific allele combinations, 11 genotypes were identified among the individuals studied. Individuals with the same genotypes formed distinct clonal lineages: one major clone was represented by 72 individuals, an intermediate clone was represented by 21 individuals, and nine other clones were rare and represented by one or several individuals. A new approach based on the detection and comparison of genotype-specific markers formed by combinations of parental-specific markers was developed and used to identify at least three hybridization founder events that resulted in the initial formation of one major and two rare clones. All other clones, including the intermediate and seven rare clones, probably arose through postformation microsatellite mutations of the major clone. This approach can be used to identify hybridization founder events and to study clone formation in other unisexual taxa.

Molecular cloning and functional analysis of the gene encoding ...

African Journals Online (AJOL)

Here we report for the first time the cloning of a full-length cDNA encoding GGPPS (Jc-GGPPS) from Jatropha curcas L. The full-length cDNA was 1414 base pair (bp), with an 1110-bp open reading frame (ORF) encoding a 370- amino-acids polypeptide. Bioinformatic analysis revealed that Jc-GGPPS is a member of the ...

Cloning and Expression Vector Construction of Glutamate Decarboxylase Gene from Lactobacillus Plantarum

Directory of Open Access Journals (Sweden)

B Arabpour

2016-06-01

Full Text Available BACKGROUND AND OBJECTIVE: Gamma-aminobutyric acid (GABA is a four-carbon non-protein amino acid used in the treatment of hypertension, diabetes, inflammation, and depression. GABA is synthesized by glutamic acid decarboxylase (GAD enzyme in many organisms, including bacteria. Therefore, cloning of this enzyme is essential to the optimization of GABA production. This study aimed to clone and construct the expression vector of GAD gene from Lactobacillus plantarum PTCC 1058 bacterium. METHODS: In this experimental study, we investigated the morphological, biochemical, genetic and 16s rDNA sequencing of L. plantarum PTCC 1058 strain. Genomic DNA of the bacterium was isolated and amplified using the GAD gene via polymerase chain reaction (PCR. Afterwards, the gene was inserted into the pJET1.2/blunt cloning vector and subcloned in vector pET32a. Plasmid pET32a-gad expression vector was transformed in Escherichia coli BL21 strain, and protein expression was assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE. FINDINGS: Morphological, biochemical and genetic analyses of 16s rDNA sequencing indicated that the studied substrain was of the L. plantarum strain. In addition, results of nucleotide sequencing of the fragmented segment via PCR showed the presence of GAD gene. Results of colony PCR and SDS-PAGE analysis confirmed the accuracy of the cloning and gene expression of the recombinant Escherichia coli BL21 strain. CONCLUSION: According to the results of this study, cloning of GAD gene from L. plantarum PTCC 1058 was successful. These cloned genes could grow rapidly in prokaryotic and eukaryotic systems and be used in cost-effective culture media and even non-recyclable waste.

Cloning of radiation-induced new gene RS1 expressed in mouse intestinal epithelium by enhanced RACE

International Nuclear Information System (INIS)

Wang Fengchao; Wang Junping; Su Yongping; Gao Jinsheng; Lou Shufen; Liu Xiaohong; Ren Jiong; Zhang Bo

2003-01-01

Objective: To obtain full-length cDNA of radiation-induced new gene RS1 expressed in mouse intestinal epithelium. Methods: The tissue expression profile of RS1 was analyzed by semi-quantitative RT-PCR to find the target tissue which highly expresses RS1. The total RNA extracted from the corresponding tissue was taken as the template for reverse-transcription. Enhanced RACE PCR was used to clone the full-length cDNA of RS1, including enrichment of the target gene through biotin-labeled probe for magnetic bead purification and nested PCR. Results: About a 2 kb long 3' end was successfully cloned and cloning of the 5' end proceeded well. Conclusion: The result is consistent with our experiment design. The set of combined techniques has been identified with the cloning of full-length cDNA from EST sequence especially when the optimal gene-specific primers are not available or the expression level of target gene is low

Human beta 2 chain of laminin (formerly S chain): cDNA cloning, chromosomal localization, and expression in carcinomas

DEFF Research Database (Denmark)

Wewer, U M; Gerecke, D R; Durkin, M E

1994-01-01

or other known laminin genes. Immunostaining showed that the beta 2 chain is localized to the smooth muscle basement membranes of the arteries, while the homologous beta 1 chain is confined to the subendothelial basement membranes. The beta 2 chain was found in the basement membranes of ovarian carcinomas......Overlapping cDNA clones that encode the full-length human laminin beta 2 chain, formerly called the S chain, were isolated. The cDNA of 5680 nt contains a 5391-nt open reading frame encoding 1797 amino acids. At the amino terminus is a 32-amino-acid signal peptide that is followed by the mature...... beta 2 chain polypeptide of 1765 amino acids with a calculated molecular mass of 192,389 Da. The human beta 2 chain is predicted to have all of the seven structural domains typical of the beta chains of laminin, including the short cysteine-rich alpha region. The amino acid sequence of human beta 2...

Development of identification process for insect group using radiation marker DNA

International Nuclear Information System (INIS)

Muraji, M.; Tamura, T.

2004-01-01

Detection of a band pattern for insect groups was tried by using radiation marked DNA clone. A rapid segregation process for poly-type DNA segment was investigated. A band pattern of silkworm was detected by analysis using DNA type transposon, K1.4. The exon regions on genes of hemiptera insect were segregated by in vitro cloning. Band patterns of the silkworm and the other insects were detected by identification process of DNA clone and radiation marker. Family singularity mutation existed in the inserted position of transposon. The family of insect was identified easily by the difference of the detection band patterns. Effective band pattern for family discrimination were obtained by analysis for a part of mitochondria DNA and ribosomal DNA. DNA segregation process was investigated by using the enriched library, also. (M. Suetake)

Isolation and characterisation of cDNA clones representing the genes encoding the major tuber storage protein (dioscorin) of yam (Dioscorea cayenensis Lam.).

Science.gov (United States)

Conlan, R S; Griffiths, L A; Napier, J A; Shewry, P R; Mantell, S; Ainsworth, C

1995-06-01

cDNA clones encoding dioscorins, the major tuber storage proteins (M(r) 32,000) of yam (Dioscorea cayenesis) have been isolated. Two classes of clone (A and B, based on hybrid release translation product sizes and nucleotide sequence differences) which are 84.1% similar in their protein coding regions, were identified. The protein encoded by the open reading frame of the class A cDNA insert is of M(r) 30,015. The difference in observed and calculated molecular mass might be attributed to glycosylation. Nucleotide sequencing and in vitro transcription/translation suggest that the class A dioscorin proteins are synthesised with signal peptides of 18 amino acid residues which are cleaved from the mature peptide. The class A and class B proteins are 69.6% similar with respect to each other, but show no sequence identity with other plant proteins or with the major tuber storage proteins of potato (patatin) or sweet potato (sporamin). Storage protein gene expression was restricted to developing tubers and was not induced by growth conditions known to induce expression of tuber storage protein genes in other plant species. The codon usage of the dioscorin genes suggests that the Dioscoreaceae are more closely related to dicotyledonous than to monocotyledonous plants.

DNA fragments assembly based on nicking enzyme system.

Directory of Open Access Journals (Sweden)

Rui-Yan Wang

Full Text Available A couple of DNA ligation-independent cloning (LIC methods have been reported to meet various requirements in metabolic engineering and synthetic biology. The principle of LIC is the assembly of multiple overlapping DNA fragments by single-stranded (ss DNA overlaps annealing. Here we present a method to generate single-stranded DNA overlaps based on Nicking Endonucleases (NEases for LIC, the method was termed NE-LIC. Factors related to cloning efficiency were optimized in this study. This NE-LIC allows generating 3'-end or 5'-end ss DNA overlaps of various lengths for fragments assembly. We demonstrated that the 10 bp/15 bp overlaps had the highest DNA fragments assembling efficiency, while 5 bp/10 bp overlaps showed the highest efficiency when T4 DNA ligase was added. Its advantage over Sequence and Ligation Independent Cloning (SLIC and Uracil-Specific Excision Reagent (USER was obvious. The mechanism can be applied to many other LIC strategies. Finally, the NEases based LIC (NE-LIC was successfully applied to assemble a pathway of six gene fragments responsible for synthesizing microbial poly-3-hydroxybutyrate (PHB.

Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA

Energy Technology Data Exchange (ETDEWEB)

Greulich-Bode, Karin; Wang, Mei; Rhein, Andreas; Weier, Jingly; Weier, Heinz-Ulli

2008-12-16

Fluorescence in situ hybridization (FISH) is a sensitive and rapid procedure to detect gene rearrangements in tumor cells using non-isotopically labeled DNA probes. Large insert recombinant DNA clones such as bacterial artificial chromosome (BAC) or P1/PAC clones have established themselves in recent years as preferred starting material for probe preparations due to their low rates of chimerism and ease of use. However, when developing probes for the quantitative analysis of rearrangements involving genomic intervals of less than 100kb, careful probe selection and characterization are of paramount importance. We describe a sensitive approach to quality control probe clones suspected of carrying deletions or for measuring clone overlap with near kilobase resolution. The method takes advantage of the fact that P1/PAC/BAC's can be isolated as circular DNA molecules, stretched out on glass slides and fine-mapped by multicolor hybridization with smaller probe molecules. Two examples demonstrate the application of this technique: mapping of a gene-specific {approx}6kb plasmid onto an unusually small, {approx}55kb circular P1 molecule and the determination of the extent of overlap between P1 molecules homologous to the human NF-?B2 locus. The relatively simple method presented here does not require specialized equipment and may thus find widespread applications in DNA probe preparation and characterization, the assembly of physical maps for model organisms or in studies on gene rearrangements.

Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA

Energy Technology Data Exchange (ETDEWEB)

Greulich-Bode, Karin M.; Wang, Mei; Rhein, Andreas P.; Weier, Jingly F.; Weier, Heinz-Ulli G.

2008-12-04

Fluorescence in situ hybridization (FISH) is a sensitive and rapid procedure to detect gene rearrangements in tumor cells using non-isotopically labeled DNA probes. Large insert recombinant DNA clones such as bacterial artificial chromosome (BAC) or P1/PAC clones have established themselves in recent years as preferred starting material for probe preparations due to their low rates of chimerism and ease of use. However, when developing probes for the quantitative analysis of rearrangements involving genomic intervals of less than 100kb, careful probe selection and characterization are of paramount importance. We describe a sensitive approach to quality control probe clones suspected of carrying deletions or for measuring clone overlap with near kilobase resolution. The method takes advantage of the fact that P1/PAC/BAC's can be isolated as circular DNA molecules, stretched out on glass slides and fine-mapped by multicolor hybridization with smaller probe molecules. Two examples demonstrate the application of this technique: mapping of a gene-specific {approx}6kb plasmid onto an unusually small, {approx}55kb circular P1 molecule and the determination of the extent of overlap between P1 molecules homologous to the human NF-{kappa}B2 locus. The relatively simple method presented here does not require specialized equipment and may thus find widespread applications in DNA probe preparation and characterization, the assembly of physical maps for model organisms or in studies on gene rearrangements.

Cloning and sequencing of a cellobiohydrolase gene from Trichoderma harzianum FP108

Science.gov (United States)

Patrick Guilfoile; Ron Burns; Zu-Yi Gu; Matt Amundson; Fu-Hsian Chang

1999-01-01

A cbbl cellobiohydrolase gene was cloned and sequenced from the fungus Trichoderrna harzianum FP108. The cloning was performed by PCR amplification of T. harzianum genomic DNA, using PCR primers whose sequence was based on the cbbl gene from Tricboderma reesei. The 3' end of the gene was isolated by inverse...

[cDNA library construction from panicle meristem of finger millet].

Science.gov (United States)

Radchuk, V; Pirko, Ia V; Isaenkov, S V; Emets, A I; Blium, Ia B

2014-01-01

The protocol for production of full-size cDNA using SuperScript Full-Length cDNA Library Construction Kit II (Invitrogen) was tested and high quality cDNA library from meristematic tissue of finger millet panicle (Eleusine coracana (L.) Gaertn) was created. The titer of obtained cDNA library comprised 3.01 x 10(5) CFU/ml in avarage. In average the length of cDNA insertion consisted about 1070 base pairs, the effectivity of cDNA fragment insertions--99.5%. The selective sequencing of cDNA clones from created library was performed. The sequences of cDNA clones were identified with usage of BLAST-search. The results of cDNA library analysis and selective sequencing represents prove good functionality and full length character of inserted cDNA clones. Obtained cDNA library from meristematic tissue of finger millet panicle represents good and valuable source for isolation and identification of key genes regulating metabolism and meristematic development and for mining of new molecular markers to conduct out high quality genetic investigations and molecular breeding as well.

Stimulation of albumin gene transcription by insulin in primary cultures of rat hepatocytes

International Nuclear Information System (INIS)

Lloyd, C.E.; Kalinyak, J.E.; Hutson, S.M.; Jefferson, L.S.

1987-01-01

The first goal of the work reported here was to prepare single-stranded DNA sequences for use in studies on the regulation of albumin gene expression. A double-stranded rat albumin cDNA clone was subcloned into the bacteriophage vector M13mp7. Single-stranded recombinant clones were screened for albumin sequences containing either the mRNA strand or the complementary strand. Two clones were selected that contained the 1200 nucleotide long 3' end of the albumin sequence. DNA from the clone containing the mRNA strand was used as a template for DNA polymerase I to prepare a radiolabeled, single-stranded cDNA to albumin mRNA. This radiolabeled cDNA probe was used to quantitate the relative abundance of albumin mRNA in samples of total cellular RNA. DNA from the clone containing the complementary strand was used to measure relative rates of albumin gene transcription in isolated nuclei. The second goal was to use the single-stranded DNA probes to investigate the mechanism of the insulin-mediated stimulation of albumin synthesis in primary cultures of rat hepatocytes. Addition of insulin to hepatocytes maintained in a chemically defined, serum-free medium for 40 h in the absence of any hormones resulted in a specific 1.5- to 2.5-fold stimulation of albumin gene transcription that was maximal at 3 h and was maintained above control values for at least 24 h. The rate of albumin gene transcription in nuclei isolated from livers of diabetic rats was reduced to 50% of the value recorded in control nuclei. Taken together, these findings demonstrate that insulin regulates synthesis of albumin at the level of gene transcription

Cloning of cellulase genes using pUC18 and lambda 2001 vectors

International Nuclear Information System (INIS)

Bashir, A.; Ashfaq, S.R.; Rajoka, M.I.; Malik, K.A.; Batt, C.A.

1991-01-01

Chromosomal DNA from cellulomonas biazotea NIAB 442 was used for isolation and cloning of cellulase genes. For this purpose plasmid pUC18 was used for cloning fragments in the range of 109 Kb and phase vector lambda 2001 for cloning fragments in the range of 15-20 Kb respectively. Three restriction enzymes BamHI, Sau3AI and SaII were used for partial restriction of chromosomal DNA to obtain fragment size in the range of 0.5 - 20 Kb. BamHI and SaII were used to linearize pUC18 to obtain compatible ends against the three enzymes used in chromosomal DNA restriction. Linearized pUC18 was then ligated to respective compatible chromosomal DNA fragments and transformed to JM109 competent cells. A total of 6781 recombinants were tested for the production of B-glucosidase and carboxy methyl cellulase (CMC-ase) production. Only one of the recombinants was found to be positive for B-glucosidase production in solid culture. One of the recombinants was found positive for CMC-ase production in solid culture and is being verified and characterized. Larger DNA fragments in the range of 15-20 Kilobase were obtained by partial restriction of chromosomal DNA with BamHI, SaII and Xhol. Lambda 2001 was double digested with BamHI/EcoRI and Xhol/EcoRI for removal of stuffer fragment. Ligation of respective compatible ends was performed between Lambda DNA and chromosomal DNA. Ligation mixture was used for packaging and infection of P2 lysogen. No plaques could be obtained on P2 lysogen due to inefficient packaging. (author)

Cloning and sequence analysis of serine proteinase of Gloydius ussuriensis venom gland

International Nuclear Information System (INIS)

Sun Dejun; Liu Shanshan; Yang Chunwei; Zhao Yizhuo; Chang Shufang; Yan Weiqun

2005-01-01

Objective: To construct a cDNA library by using mRNA from Gloydius ussuriensis (G. Ussuriensis) venom gland, to clone and analyze serine proteinase gene from the cDNA library. Methods: Total RNA was isolated from venom gland of G. ussuriensis, mRNA was purified by using mRNA isolation Kit. The whole length cDNA was synthesized by means of smart cDNA synthesis strategy, and amplified by long distance PCR procedure, lately cDAN was cloned into vector pBluescrip-sk. The recombinant cDNA was transformed into E. coli DH5α. The cDNA of serine proteinase gene in the venom gland of G. ussuriensis was detected and amplified using the in situ hybridization. The cDNA fragment was inserted into pGEMT vector, cloned and its nucleotide sequence was determined. Results: The capacity of cDNA library of venom gland was above 2.3 x 10 6 . Its open reading frame was composed of 702 nucleotides and coded a protein pre-zymogen of 234 amino acids. It contained 12 cysteine residues. The sequence analysis indicated that the deduced amino acid sequence of the cDNA fragment shared high identity with the thrombin-like enzyme genes of other snakes in the GenBank. the query sequence exhibited strong amino acid sequence homology of 85% to the serine proteas of T. gramineus, thrombin-like serine proteinase I of D. acutus and serine protease catroxase II of C. atrox respectively. Based on the amino acid sequences of other thrombin-like enzymes, the catalytic residues and disulfide bridges of this thrombin-like enzyme were deduced as follows: catalytic residues, His 41 , Asp 86 , Ser 180 ; and six disulfide bridges Cys 7 -Cys 139 , Cys 26 -Cys 42 , Cys 74 -Cys 232 , Cys 118 -Cys 186 , Cys 150 -Cys 165 , Cys 176 -Cys 201 . Conclusion: The capacity of cDNA library of venom gland is above 2.3 x 10 6 , overtop the level of 10 5 capicity. The constructed cDNA library of G. ussuriensis venom gland would be helpful platform to detect new target genes and further gene manipulate. The cloned serine

Intraclonal genome diversity of Pseudomonas aeruginosa clones CHA and TB

Science.gov (United States)

2013-01-01

Background Adaptation of Pseudomonas aeruginosa to different living conditions is accompanied by microevolution resulting in genomic diversity between strains of the same clonal lineage. In order to detect the impact of colonized habitats on P. aeruginosa microevolution we determined the genomic diversity between the highly virulent cystic fibrosis (CF) isolate CHA and two temporally and geographically unrelated clonal variants. The outcome was compared with the intraclonal genome diversity between three more closely related isolates of another clonal complex. Results The three clone CHA isolates differed in their core genome in several dozen strain specific nucleotide exchanges and small deletions from each other. Loss of function mutations and non-conservative amino acid replacements affected several habitat- and lifestyle-associated traits, for example, the key regulator GacS of the switch between acute and chronic disease phenotypes was disrupted in strain CHA. Intraclonal genome diversity manifested in an individual composition of the respective accessory genome whereby the highest number of accessory DNA elements was observed for isolate PT22 from a polluted aquatic habitat. Little intraclonal diversity was observed between three spatiotemporally related outbreak isolates of clone TB. Although phenotypically different, only a few individual SNPs and deletions were detected in the clone TB isolates. Their accessory genome mainly differed in prophage-like DNA elements taken up by one of the strains. Conclusions The higher geographical and temporal distance of the clone CHA isolates was associated with an increased intraclonal genome diversity compared to the more closely related clone TB isolates derived from a common source demonstrating the impact of habitat adaptation on the microevolution of P. aeruginosa. However, even short-term habitat differentiation can cause major phenotypic diversification driven by single genomic variation events and uptake of phage

RTA, a candidate G protein-coupled receptor: Cloning, sequencing, and tissue distribution

International Nuclear Information System (INIS)

Ross, P.C.; Figler, R.A.; Corjay, M.H.; Barber, C.M.; Adam, N.; Harcus, D.R.; Lynch, K.R.

1990-01-01

Genomic and cDNA clones, encoding a protein that is a member of the guanine nucleotide-binding regulatory protein (G protein)-coupled receptor superfamily, were isolated by screening rat genomic and thoracic aorta cDNA libraries with an oligonucleotide encoding a highly conserved region of the M 1 muscarinic acetylcholine receptor. Sequence analyses of these clones showed that they encode a 343-amino acid protein (named RTA). The RTA gene is single copy, as demonstrated by restriction mapping and Southern blotting of genomic clones and rat genomic DNA. RTA RNA sequences are relatively abundant throughout the gut, vas deferens, uterus, and aorta but are only barely detectable (on Northern blots) in liver, kidney, lung, and salivary gland. In the rat brain, RTA sequences are markedly abundant in the cerebellum. TRA is most closely related to the mas oncogene (34% identity), which has been suggested to be a forebrain angiotensin receptor. They conclude that RTA is not an angiotensin receptor; to date, they have been unable to identify its ligand

Cloning a T-DNA-Linked Phosphate Gene that mediates Salt Tolerance on Mutant of Arabidopsis thaliana

International Nuclear Information System (INIS)

Njoroge, N.C; Tremblay, L.; Lefebvre, D.D.

2006-01-01

T-DNA insertionally mutagenized seeds of Arabidopsis thaliana were used to unravel genetic mechanisms underlying salt tolerance in plants. Over a period of two weeks, kanamycin homozygous (KK) seeds of the mutant NN143 attain germination levels of 65% and 77% on 175mM Nacl and 300mM mannitol respectively. Under these conditions of osmotic stress, the wild type seeds were incapable of germination. The mutant was also capable of germination on a medium containing 2μM abscisic acid (ABA). After two weeks on 2μM ABA, it attained 100% germination and the wild type did not germinate. The ABA level in the mutant was 40% higher than the wild type. Segregation analysis indicated that salt tolerance in the mutant is T-DNA linked. Genetic analysis of the F1 and F2 generations indicated that the salt tolerance trait in the mutant is dominant. The putative salt tolerance gene of mutant NN143 was cloned by plasmid rescue and sequence data indicated involvement of a protein phosphatase. The possible mechanism underlying salt tolerance in the mutant is discussed.(author)

cDNA cloning and sequencing of human fibrillarin, a conserved nucleolar protein recognized by autoimmune antisera

International Nuclear Information System (INIS)

Aris, J.P.; Blobel, G.

1991-01-01

The authors have isolated a 1.1-kilobase cDNA clone that encodes human fibrillarin by screening a hepatoma library in parallel with DNA probes derived from the fibrillarin genes of Saccharomyces cerevisiae (NOP1) and Xenopus laevis. RNA blot analysis indicates that the corresponding mRNA is ∼1,300 nucleotides in length. Human fibrillarin expressed in vitro migrates on SDS gels as a 36-kDa protein that is specifically immunoprecipitated by antisera from humans with scleroderma autoimmune disease. Human fibrillarin contains an amino-terminal repetitive domain ∼75-80 amino acids in length that is rich in glycine and arginine residues and is similar to amino-terminal domains in the yeast and Xenopus fibrillarins. The occurrence of a putative RNA-binding domain and an RNP consensus sequence within the protein is consistent with the association of fibrillarin with small nucleolar RNAs. Protein sequence alignments show that 67% of amino acids from human fibrillarin are identical to those in yeast fibrillarin and that 81% are identical to those in Xenopus fibrillarin. This identity suggests the evolutionary conservation of an important function early in the pathway for ribosome biosynthesis

Cloning and DNA sequence of the mercuric- and organomercurial-resistance determinants of plasmid pDU1358

International Nuclear Information System (INIS)

Griffin, H.G.; Foster, T.J.; Silver, S.; Misra, T.K.

1987-01-01

The broad-spectrum mercurial-resistance plasmid pDU1358 was analyzed by cloning the resistance determinants and preparing a physical and genetic map of a 45-kilobase (kb) region of the plasmid that contains two separate mercurial-resistance operons that mapped about 20 kb apart. One encoded narrow-spectrum mercurial resistance to Hg 2+ and a few organomercurials; the other specified broad-spectrum resistance to phenylmercury and additional organomercurials. Each determinant governed mercurial transport functions. Southern DNA x DNA hybridization experiments using gene-specific probes from the plasmid R100 mer operon indicated close homology with the R100 deteminant. The 2153 base pairs of the promoter-distal part of the broad-spectrum Hg 2+ -resistance operon of pDU1358 were sequenced. This region included the 3'-terminal part of the merA gene, merD, unidentified reading frame URF1, and a part of URF2 homologous to previously sequenced determinants of plasmid R100. Between the merA and merD genes, an open reading frame encoding a 212 amino acid polypeptide was identified as the merB gene that determines the enzyme organomercurial lyase that cleaves the C-Hg bond of phenylmercury

Cloning of gibberellin 3 beta-hydroxylase cDNA and analysis of endogenous gibberellins in the developing seeds in watermelon.

Science.gov (United States)

Kang, Hong-Gyu; Jun, Sung-Hoon; Kim, Joonyul; Kawaide, Hiroshi; Kamiya, Yuji; An, Gynheung

2002-02-01

We have isolated Cv3h, a cDNA clone from the developing seeds of watermelon, and have demonstrated significant amino acid homology with gibberellin (GA) 3 beta-hydroxylases. This cDNA clone was expressed in Escherichia coli as a fusion protein that oxidized GA(9) and GA(12) to GA(4) and GA(14), respectively. The Cv3h protein had the highest similarity with pumpkin GA 2 beta,3 beta-hydroxylase, but did not possess 2 beta-hydroxylation function. RNA blot analysis showed that the gene was expressed primarily in the inner parts of developing seeds, up to 10 d after pollination (DAP). In the parthenocarpic fruits induced by treatment with 1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU), the embryo and endosperm of the seeds were undeveloped, whereas the integumental tissues, of maternal origin, showed nearly normal development. Cv3h mRNA was undetectable in the seeds of CPPU-treated fruits, indicating that the GA 3 beta-hydroxylase gene was expressed in zygotic cells. In our analysis of endogenous GAs from developing seeds, GA(9) and GA(4) were detected at high levels but those of GA(20) and GA(1) were very low. This demonstrates that GA biosynthesis in seeds prefers a non-13-hydroxylation pathway over an early 13-hydroxylation pathway. We also analyzed endogenous GAs from seeds of the parthenocarpic fruits. The level of bioactive GA(4 )was much lower there than in normal seeds, indicating that bioactive GAs, unconnected with Cv3h, exist in integumental tissues during early seed development.

Distinct forms of the β subunit of GTP-binding regulatory proteins identified by molecular cloning

International Nuclear Information System (INIS)

Fong, H.K.W.; Amatruda, T.T. III; Birren, B.W.; Simon, M.I.

1987-01-01

Two distinct β subunits of guanine nucleotide-binding regulatory proteins have been identified by cDNA cloning and are referred to as β 1 and β 1 subunits. The bovine transducin β subunit (β 1 ) has been cloned previously. The author now isolated and analyzed cDNA clones that encode the β 2 subunit from bovine adrenal, bovine brain, and a human myeloid leukemia cell line, HL-60. The 340-residue M/sub r/ 37,329 Β 2 protein is 90% identical with β 1 in predicted amino acid sequence, and it is also organized as a series of repetitive homologous segments. The major mRNA that encodes the bovine β 2 subunit is 1.7 kilobases in length. It is expressed at lower levels than β 1 subunit mRNA in all tissues examined. The β 1 and β 2 messages are expressed in cloned human cell lines. Hybridization of cDNA probes to bovine DNA showed that β 1 and β 2 are encoded by separate genes. The amino acid sequences for the bovine and human β 2 subunit are identical, as are the amino acid sequences for the bovine and human β 1 subunit. This evolutionary conservation suggests that the two β subunits have different roles in the signal transduction process

Intra-strain polymorphisms are detected but no genomic alteration is found in cloned mice

International Nuclear Information System (INIS)

Gotoh, Koshichi; Inoue, Kimiko; Ogura, Atsuo; Oishi, Michio

2006-01-01

In-gel competitive reassociation (IGCR) is a method for differential subtraction of polymorphic (RFLP) DNA fragments between two DNA samples of interest without probes or specific sequence information. Here, we applied the IGCR procedure to two cloned mice derived from an F1 hybrid of the C57BL/6Cr and DBA/2 strains, in order to investigate the possibility of genomic alteration in the cloned mouse genomes. Each of the five of the genomic alterations we detected between the two cloned mice corresponded to the 'intra-strain' polymorphisms in the C57BL/6Cr and DBA/2 mouse strains. Our result suggests that no severe aberration of genome sequences occurs due to somatic cell nuclear transfer

Cloning and sequencing of Lol pI, the major allergenic protein of rye-grass pollen.

Science.gov (United States)

Griffith, I J; Smith, P M; Pollock, J; Theerakulpisut, P; Avjioglu, A; Davies, S; Hough, T; Singh, M B; Simpson, R J; Ward, L D

1991-02-25

We have isolated a full length cDNA clone encoding the major glycoprotein allergen Lol pI. The clone was selected using a combination of immunological screening of a cDNA expression library and PCR amplification of Lol pI-specific transcripts. Lol pI expressed in bacteria as a fusion protein shows recognition by specific IgE antibodies present in sera of grass pollen-allergic subjects. Northern analysis has shown that the Lol pI transcripts are expressed only in pollen of rye-grass. Molecular cloning of Lol pI provides a molecular genetic approach to study the structure-function relationship of allergens.

Genomic clones of bovine parvovirus: Construction and effect of deletions and terminal sequence inversions on infectivity

International Nuclear Information System (INIS)

Shull, B.C.; Chen, K.C.; Lederman, M.; Stout, E.R.; Bates, R.C.

1988-01-01

Genomic clones of the autonomous parvovirus bovine parvovirus (BPV) were constructed by blunt-end ligation of reannealed virion plus and minus DNA strands into the plasmid pUC8. These clones were stable during propagation in Escherichia coli JM107. All clones tested were found to be infectious by the criteria of plaque titer and progressive cytophathic effect after transfection into bovine fetal lung cells. Sequencing of the recombinant plasmids demonstrated that all of the BPV inserts had left-end (3')-terminal deletions of up to 34 bases. Defective genomes could also be detected in the progeny DNA even though the infection was initiated with homogeneous, cloned DNA. Full-length genomic clones with 3' flip and 3' flop conformations were constructed and were found to have equal infectivity. Expression of capsid proteins from tranfected genomes was demonstrated by hemagglutination, indirect immunofluorescence, and immunoprecipitation of [ 35 S]methionine-labeled cell lysates. Use of appropriate antiserum for immunoprecipitation showed the synthesis of BPV capsid and noncapsid proteins after transfection. Independently, a series of genomic clones with increasingly larger 3'-terminal deletions was prepared from separately subcloned 3'-terminal fragments. Transfection of these clones into bovine fetal lung cells revealed that deletions of up to 34 bases at the 3' end lowered but did not abolish infectivity, while deletions of greater than 52 bases were lethal. End-label analysis showed that the 34-base deletion was repaired to wild-type length in the progeny virus

Glucocorticoids inhibit the proliferation of IL-2-dependent T cell clones

International Nuclear Information System (INIS)

Fresno, M.; Redondo, J.M.; Lopez-Rivas, A.

1986-01-01

It has been shown that glucocorticoids inhibit mitogen or antigen-induced lymphocyte proliferation by decreasing the production of interleukin-2 (IL-2). They have studied the effect of dexamethasone (Dx) on the proliferation of IL-2-dependent T cell clones. They have found that preincubation of these clones with Dx inhibits ( 3 H) thymidine incorporation and cell proliferation in a dose-dependent manner (ID 50 % 5 x 10 -10 M). The inhibition of DNA synthesis by Dx was dependent on the concentration of IL-2. High concentration of IL-2 reversed completely this inhibition. The action of Dx seems to be mediated through the induction of a protein since the simultaneous presence of cycloheximide and Dx prevented the inhibitory effect of the latter. Moreover, dialyzed conditioned medium of Dx treated cells inhibited DNA synthesis by T cell clones. The biochemical characterization of this protein is in progress

Molecular cloning of the human gene for von Willebrand factor and identification of the transcription initiation site

International Nuclear Information System (INIS)

Collins, C.J.; Underdahl, J.P.; Levene, R.B.; Ravera, C.P.; Morin, M.J.; Dombalagian, M.J.; Ricca, G.; Livingston, D.M.; Lynch, D.C.

1987-01-01

A series of overlapping cosmid genomic clones have been isolated that contain the entire coding unit of the human gene for van Willebrand factor (vWf), a major component of the hemostatic system. The cloned segments span ≅ 175 kilobases of human DNA sequence, and hybridization analysis suggest that the vWf coding unit is ≅150 kilobases in length. Within one of these clones, the vWF transcription initiation site has been mapped and a portion of the vWf promoter region has been sequenced, revealing a typical TATA box, a downstream CCAAT box, and a perfect downstream repeat of the 8 base pairs containing the transcription start site. Sequencing of a segment of another genomic clone has revealed the vWF translation termination codon. Where tested, comparative restriction analysis of cloned and chromosomal DNA segments strongly suggests that no major alterations occurred during cloning and that there is only one complete copy of the vWf gene in the human haploid genome. Similar analyses of DNA from vWf-producing endothelial cells and nonexpressing leukocytes suggest that vWf gene expression is not accompanied by gross genomic rearrangements. In addition, there is significant homology of C-terminal coding sequences among the vWf genes of several vertebrate species

Molecular cloning and characterization of a cDNA encoding ...

African Journals Online (AJOL)

enoh

2012-03-29

Mar 29, 2012 ... Cloning and Functional. Expression of Cycloartenol Synthases from Mangrove Species. Rhizophora stylosa Griff. And Kandelia candel (L.) Druce. Biosci. Biotechnol. Biochem. 71(7): 1788-1792. Felsenstein J (1985). Confidence limits on phylogenies: An approach using the bootstrap. Evolution. 39:783-791 ...

Restriction enzyme body doubles and PCR cloning: on the general use of type IIs restriction enzymes for cloning.

Science.gov (United States)

Tóth, Eszter; Huszár, Krisztina; Bencsura, Petra; Kulcsár, Péter István; Vodicska, Barbara; Nyeste, Antal; Welker, Zsombor; Tóth, Szilvia; Welker, Ervin

2014-01-01

The procedure described here allows the cloning of PCR fragments containing a recognition site of the restriction endonuclease (Type IIP) used for cloning in the sequence of the insert. A Type IIS endonuclease--a Body Double of the Type IIP enzyme--is used to generate the same protruding palindrome. Thus, the insert can be cloned to the Type IIP site of the vector without digesting the PCR product with the same Type IIP enzyme. We achieve this by incorporating the recognition site of a Type IIS restriction enzyme that cleaves the DNA outside of its recognition site in the PCR primer in such a way that the cutting positions straddle the desired overhang sequence. Digestion of the PCR product by the Body Double generates the required overhang. Hitherto the use of Type IIS restriction enzymes in cloning reactions has only been used for special applications, the approach presented here makes Type IIS enzymes as useful as Type IIP enzymes for general cloning purposes. To assist in finding Body Double enzymes, we summarised the available Type IIS enzymes which are potentially useful for Body Double cloning and created an online program (http://group.szbk.u-szeged.hu/welkergr/body_double/index.html) for the selection of suitable Body Double enzymes and the design of the appropriate primers.

Procedure for normalization of cDNA libraries

Science.gov (United States)

Bonaldo, Maria DeFatima; Soares, Marcelo Bento

1997-01-01

This invention provides a method to normalize a cDNA library constructed in a vector capable of being converted to single-stranded circles and capable of producing complementary nucleic acid molecules to the single-stranded circles comprising: (a) converting the cDNA library in single-stranded circles; (b) generating complementary nucleic acid molecules to the single-stranded circles; (c) hybridizing the single-stranded circles converted in step (a) with complementary nucleic acid molecules of step (b) to produce partial duplexes to an appropriate Cot; (e) separating the unhybridized single-stranded circles from the hybridized single-stranded circles, thereby generating a normalized cDNA library.

Comparative evaluation of prokaryotic 16S rDNA clone libraries and SSCP in groundwater samples.

Science.gov (United States)

Larentis, Michael; Alfreider, Albin

2011-06-01

A comparison of ribosomal RNA sequence analysis methods based on clone libraries and single-strand conformational polymorphism technique (SSCP) was performed with groundwater samples obtained between 523-555 meters below surface. The coverage of analyzed clones by phylotype-richness estimates was between 88-100%, confirming that the clone libraries were adequately examined. Analysis of individual bands retrieved from SSCP gels identified 1-6 different taxonomic units per band, suggesting that a single SSCP band does often represent more than one single prokaryotic species. The prokaryotic diversity obtained by both methods showed an overall difference of 42-80%. In comparison to SSCP, clone libraries underestimated the phylogenetic diversity and only 36-66% of the phylotypes observed with SSCP were also detected with the clone libraries. An exception was a sample where the SSCP analysis of Archaea identified only half of the phylotypes retrieved by the clone library. Overall, this study suggests that the clone library and the SSCP approach do not provide an identical picture of the prokaryotic diversity in groundwater samples. The results clearly show that the SSCP method, although this approach is prone to generate methodological artifacts, was able to detect significantly more phylotypes than microbial community analysis based on clone libraries. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Recombinant pollen allergens from Dactylis glomerata: preliminary evidence that human IgE cross-reactivity between Dac g II and Lol p I/II is increased following grass pollen immunotherapy

NARCIS (Netherlands)

Roberts, A. M.; van Ree, R.; Cardy, S. M.; Bevan, L. J.; Walker, M. R.

1992-01-01

We previously described the isolation of three identical complementary DNA (cDNA) clones, constructed from Orchard/Cocksfoot grass (Dactylis glomerata) anther messenger RNA (mRNA), expressing a 140,000 MW beta-galactosidase fusion protein recognized by IgE antibodies in atopic sera. Partial

Author template for journal articles

African Journals Online (AJOL)

USER

2012-04-10

Apr 10, 2012 ... and Ac-ubiquitin were cloned from pineapple in this study. The four genes .... 15, 30 and 60 days of flower bloom; and 3) stem, calyx, petal, stylus and ovary ... The first-strand complementary DNA (cDNA) was synthesized by.

Identification of a mammalian nuclear factor and human cDNA-encoded proteins that recognize DNA containing apurinic sites

International Nuclear Information System (INIS)

Lenz, J.; Okenquist, S.A.; LoSardo, J.E.; Hamilton, K.K.; Doetsch, P.W.

1990-01-01

Damage to DNA can have lethal or mutagenic consequences for cells unless it is detected and repaired by cellular proteins. Repair depends on the ability of cellular factors to distinguish the damaged sites. Electrophoretic binding assays were used to identify a factor from the nuclei of mammalian cells that bound to DNA containing apurinic sites. A binding assay based on the use of β-galactosidase fusion proteins was subsequently used to isolate recombinant clones of human cDNAs that encoded apurinic DNA-binding proteins. Two distinct human cDNAs were identified that encoded proteins that bound apurinic DNA preferentially over undamaged, methylated, or UV-irradiated DNA. These approaches may offer a general method for the detection of proteins that recognize various types of DNA damage and for the cloning of genes encoding such proteins

A Simple Method for the Extraction, PCR-amplification, Cloning, and Sequencing of Pasteuria 16S rDNA from Small Numbers of Endospores.

Science.gov (United States)

Atibalentja, N; Noel, G R; Ciancio, A

2004-03-01

For many years the taxonomy of the genus Pasteuria has been marred with confusion because the bacterium could not be cultured in vitro and, therefore, descriptions were based solely on morphological, developmental, and pathological characteristics. The current study sought to devise a simple method for PCR-amplification, cloning, and sequencing of Pasteuria 16S rDNA from small numbers of endospores, with no need for prior DNA purification. Results show that DNA extracts from plain glass bead-beating of crude suspensions containing 10,000 endospores at 0.2 x 10 endospores ml(-1) were sufficient for PCR-amplification of Pasteuria 16S rDNA, when used in conjunction with specific primers. These results imply that for P. penetrans and P. nishizawae only one parasitized female of Meloidogyne spp. and Heterodera glycines, respectively, should be sufficient, and as few as eight cadavers of Belonolaimus longicaudatus with an average number of 1,250 endospores of "Candidatus Pasteuria usgae" are needed for PCR-amplification of Pasteuria 16S rDNA. The method described in this paper should facilitate the sequencing of the 16S rDNA of the many Pasteuria isolates that have been reported on nematodes and, consequently, expedite the classification of those isolates through comparative sequence analysis.

Spontaneous mutation rate in Chinese hamster cell clones differing in UV-sensitivity

International Nuclear Information System (INIS)

Manuilova, E.S.; Bagrova, A.M.; Moskovskij Gosudarstvennyj Univ.

1983-01-01

The spontaneous rate of appearance of mutations to 6-mercaptopurine (6 MP) resistence in the cells of CHR2 and CHs2 clones dofferent in sensitivity to lethal and matagenous effect of UV-rays, is investigated. Increased UV-sensitivity of CHs2 clone is caused by the violation of postreplicative DNA reparation. It is established that the purity of spontaneously occuring mutations in both clones turns out to be similar, i.e. (1.5-1.8)x10 -5 for the cell pergeneration. It is shown that the effect of postreplicative DNA reparation in the cells of chinese hamster is not connected with the increase of spontaneous mutation ability. The problem on the possible role of reparation in the mechanism of appearance of spontaneous and induced mutations in the cells of Chinese hamster with increased UV-sensitivity is discussed

DNA damage-inducible transcripts in mammalian cells

International Nuclear Information System (INIS)

Fornace, A.J. Jr.; Alamo, I. Jr.; Hollander, M.C.

1988-01-01

Hybridization subtraction at low ratios of RNA to cDNA was used to enrich for the cDNA of transcripts increased in Chinese hamster cells after UV irradiation. Forty-nine different cDNA clones were isolated. Most coded for nonabundant transcripts rapidly induced 2- to 10-fold after UV irradiation. Only 2 of the 20 cDNA clones sequenced matched known sequences (metallothionein I and II). The predicted amino acid sequence of one cDNA had two localized areas of homology with the rat helix-destabilizing protein. These areas of homology were at the two DNA-binding sites of this nucleic acid single-strand-binding protein. The induced transcripts were separated into two general classes. Class I transcripts were induced by UV radiation and not by the alkylating agent methyl methanesulfonate. Class II transcripts were induced by UV radiation and by methyl methanesulfonate. Many class II transcripts were induced also by H2O2 and various alkylating agents but not by heat shock, phorbol 12-tetradecanoate 13-acetate, or DNA-damaging agents which do not produce high levels of base damage. Since many of the cDNA clones coded for transcripts which were induced rapidly and only by certain types of DNA-damaging agents, their induction is likely a specific response to such damage rather than a general response to cell injury

Molecular cloning and characterization of the plasma membrane ...

African Journals Online (AJOL)

O. violaceus) was cloned. The full-length cDNA of O. violaceus gene (OvPIP) was 1314 bp and contained 1188 open reading frame encoding a protein of 395 amino acids. Homology analysis revealed that OvPIP strongly resembled other PIP ...

Solid-phase cloning for high-throughput assembly of single and multiple DNA parts

DEFF Research Database (Denmark)

Lundqvist, Magnus; Edfors, Fredrik; Sivertsson, Åsa

2015-01-01

We describe solid-phase cloning (SPC) for high-throughput assembly of expression plasmids. Our method allows PCR products to be put directly into a liquid handler for capture and purification using paramagnetic streptavidin beads and conversion into constructs by subsequent cloning reactions. We ...

Cloning and expression of calmodulin gene in Scoparia dulcis.

Science.gov (United States)

Saitoh, Daisuke; Asakura, Yuki; Nkembo, Marguerite Kasidimoko; Shite, Masato; Sugiyama, Ryuji; Lee, Jung-Bum; Hayashi, Toshimitsu; Kurosaki, Fumiya

2007-06-01

A homology-based cloning strategy yielded a cDNA clone, designated Sd-cam, encoding calmodulin protein from Scoparia dulcis. The restriction digests of genomic DNA of S. dulcis showed a single hybridized signal when probed with the fragment of this gene in Southern blot analyses, suggesting that Sd-cam occurs as a sole gene encoding calmodulin in the plant. The reverse-transcription polymerase chain reaction analysis revealed that Sd-cam was appreciably expressed in leaf, root and stem tissues. It appeared that transcription of this gene increased transiently when the leaf cultures of S. dulcis were treated with methyl jasmonate and calcium ionophore A23187. These results suggest that transcriptional activation of Sd-cam is one of the early cellular events of the methyl jasmonate-induced responses of S. dulcis.

Construction of cDNA libraries from Pseudocercospora fijiensis Morelet infected leaves of the cultivars Calcutta 4 and Niyarma Yik

Directory of Open Access Journals (Sweden)

Milady Mendoza-Rodríguez

2004-01-01

Full Text Available Molecular studies of plant-pathogen interaction are very important for the identification of gene (s related with the pathogenic process, as well as with the plant resistance. These gene (s could be use for the genetic improvement programs in order to obtain resistant cultivars. The aim of this work was to construct complementary DNA (cDNA libraries from infected leaves with Pseudocercospora fijiensis CCIBP-Pf1 isolated of two banana cultivars (a resistant one Calcutta4 and another one susceptible Niyarma Yik. First-strand cDNA synthesis, was made beginning with one microgram of total RNA by using oligo dT primer and cDNA quality was checked by Polimerase chain reaction (PCR with cytochrome b specific primers. Second-strand cDNA synthesis was performed by using the homopolymeric tailing with dC-BamH I + dT-Not I primer combination. Four cDNA libraries of infected plants at different times of infection with the pathogen were obtained. Forty one clones of one of the libraries of Niyarma Yik were sequenced and the obtained sequences correspond with genes related to fungi. Key words: Banana-Mycosphaerella fijiensis interaction,Black Sigatoka, Musa spp.

Cloning Expeditions: Risky but Rewarding

Science.gov (United States)

2013-01-01

In the 1980s, a good part of my laboratory was using the then-new recombinant DNA techniques to clone and characterize many important cell surface membrane proteins: GLUT1 (the red cell glucose transporter) and then GLUT2 and GLUT4, the red cell anion exchange protein (Band 3), asialoglycoprotein receptor subunits, sucrase-isomaltase, the erythropoietin receptor, and two of the subunits of the transforming growth factor β (TGF-β) receptor. These cloned genes opened many new fields of basic research, including membrane insertion and trafficking of transmembrane proteins, signal transduction by many members of the cytokine and TGF-β families of receptors, and the cellular physiology of glucose and anion transport. They also led to many insights into the molecular biology of several cancers, hematopoietic disorders, and diabetes. This work was done by an exceptional group of postdocs and students who took exceptionally large risks in developing and using novel cloning technologies. Unsurprisingly, all have gone on to become leaders in the fields of molecular cell biology and molecular medicine. PMID:24061478

TA-GC cloning: A new simple and versatile technique for the directional cloning of PCR products for recombinant protein expression.

Science.gov (United States)

Niarchos, Athanasios; Siora, Anastasia; Konstantinou, Evangelia; Kalampoki, Vasiliki; Lagoumintzis, George; Poulas, Konstantinos

2017-01-01

During the last few decades, the recombinant protein expression finds more and more applications. The cloning of protein-coding genes into expression vectors is required to be directional for proper expression, and versatile in order to facilitate gene insertion in multiple different vectors for expression tests. In this study, the TA-GC cloning method is proposed, as a new, simple and efficient method for the directional cloning of protein-coding genes in expression vectors. The presented method features several advantages over existing methods, which tend to be relatively more labour intensive, inflexible or expensive. The proposed method relies on the complementarity between single A- and G-overhangs of the protein-coding gene, obtained after a short incubation with T4 DNA polymerase, and T and C overhangs of the novel vector pET-BccI, created after digestion with the restriction endonuclease BccI. The novel protein-expression vector pET-BccI also facilitates the screening of transformed colonies for recombinant transformants. Evaluation experiments of the proposed TA-GC cloning method showed that 81% of the transformed colonies contained recombinant pET-BccI plasmids, and 98% of the recombinant colonies expressed the desired protein. This demonstrates that TA-GC cloning could be a valuable method for cloning protein-coding genes in expression vectors.

Fiscal 2000 report on result of the full-length cDNA structure analysis; 2000 nendo kanzen cho cDNA kozo kaiseki seika hokokusho

Energy Technology Data Exchange (ETDEWEB)

NONE

2001-03-01

This paper explains the results of research on full-length cDNA structure analysis for the period from April, 2000 to March, 2001. The outline of human genome sequence was published in June, 2000. In Japan, human gene analysis was such that, as the basic technology of the bio industry, a millennium project was decided in the budget of fiscal 2000. The full-length cDNA structure analysis is the core of the project. The libraries of cDNA were prepared using full-length and more than 4-5kbp-long cDNAs by oligo-capping method. It began from determining partial sequence data at end cDNA, and then, with new clones selected therefrom, full-length human cDNA sequence data were determined. The partial sequence data determined by fiscal 2000 were 1,035,000 clones while the full-length sequence data were 12,144 clones. The sequence data obtained were analyzed by homology search and translated into amino acid coding sequences, with predictions conducted on protein functions. A clustering method was examined that selects new clones from partial sequences. Database was constructed on gene expression profiles and disease-related gene sequence data. (NEDO)

Design and Construction of a Cloning Vector Containing the hspX Gene of Mycobacterium tuberculosis.

Science.gov (United States)

Yaghoubi, Atieh; Aryan, Ehsan; Zare, Hosna; Alami, Shadi; Teimourpour, Roghayeh; Meshkat, Zahra

2016-10-01

Tuberculosis (TB) is a major cause of death worldwide. Finding an effective vaccine against TB is the best way to control it. Several vaccines against this disease have been developed but none are completely protective. The aim of this study was to design and construct a cloning vector containing the Mycobacterium tuberculosis (M. tuberculosis) heat shock protein X (hspX) . First, an hspX fragment was amplified by PCR and cloned into plasmid pcDNA3.1(+) and recombinant vector was confirmed. A 435 bp hspX fragment was isolated. The fragment was 100% homologous with hspX of M. tuberculosis strain H37Rv in GenBank. In this study, the cloning vector pcDNA3.1(+), containing a 435-bp hspX fragment of M. tuberculosis , was constructed. This could be used as a DNA vaccine to induce immune responses in animal models in future studies.

Cloning of the γ-aminobutyric acid (GABA) ρ1 cDNA: A GABA receptor subunit highly expressed in the retina

International Nuclear Information System (INIS)

Cutting, G.R.; Lu, Luo; Kasch, L.M.; Montrose-Rafizadeh, C.; Antonarakis, S.E.; Guggino, W.B.; Kazazian, H.H. Jr.; O'Hara, B.F.; Donovan, D.M.; Shimada, Shoichi; Uhl, G.R.

1991-01-01

Type A γ-aminobutyric acid (GABA A ) receptors are a family of ligand-gated chloride channels that are the major inhibitory neurotransmitter receptors in the nervous system. Molecular cloning has revealed diversity in the subunits that compose this heterooligomeric receptor, but each previously elucidated subunit displays amino acid similarity in conserved structural elements. The authors have used these highly conserved regions to identify additional members of this family by using the polymerase chain reaction (PCR). One PCR product was used to isolate a full-length cDNA from a human retina cDNA library. The mature protein predicted from this cDNA sequence is 458 amino acids long and displays between 30 and 38% amino acid similarity to the previously identified GABA A subunits. This gene is expressed primarily in the retina but transcripts are also detected in the brain, lung, and thymus. Injection of Xenopus oocytes with RNA transcribed in vitro produces a GABA-responsive chloride conductance and expression of the cDNA in COS cells yields GABA-displaceable muscimol binding. These features are consistent with our identification of a GABA subunit, GABA ρ 1 , with prominent retinal expression that increases the diversity and tissue specificity of this ligand-gated ion-channel receptor family

[TSA improve transgenic porcine cloned embryo development and transgene expression].

Science.gov (United States)

Kong, Qing-Ran; Zhu, Jiang; Huang, Bo; Huan, Yan-Jun; Wang, Feng; Shi, Yong-Qian; Liu, Zhong-Feng; Wu, Mei-Ling; Liu, Zhong-Hua

2011-07-01

Uncompleted epigenetic reprogramming is attributed to the low efficiency of producing transgenic cloned animals. Histone modification associated with epigenetics can directly influence the embryo development and transgene expression. Trichostatin A (TSA), as an inhibitor of histone deacetylase, can change the status of histone acetylation, improve somatic cell reprogramming, and enhance cloning efficiency. TSA prevents the chromatin structure from being condensed, so that transcription factor could binds to DNA sequence easily and enhance transgene expression. Our study established the optimal TSA treatment on porcine donor cells and cloned embryos, 250 nmol/L, 24 h and 40 nmol/L, 24 h, respectively. Furthermore, we found that both the cloned embryo and the donor cell treated by TSA resulted in the highest development efficiency. Meanwhile, TSA can improve transgene expression in donor cell and cloned embryo. In summary, TSA can significantly improve porcine reconstructed embryo development and transgene expression.

Construction and sequencing of an infectious clone of the human parvovirus B19

International Nuclear Information System (INIS)

Zhi Ning; Zadori, Zoltan; Brown, Kevin E.; Tijssen, Peter

2004-01-01

Human parvovirus B19 has a nonenveloped, icosahedral capsid packaging a linear single-stranded DNA genome of 5.6 kb with long inverted terminal repeats (ITR) at both the 5' and 3' end. Previous attempts to construct a full-length B19 clone were unsuccessful due to deletions in the ITR sequences. We cloned the complete parvovirus B19 genome with intact ITRs from an aplastic crisis patient. Sequence analysis of the complete viral genome indicated that both 5' and 3' ITRs have two sequence configurations and several base changes within the ITRs compared to previous published sequences. After transfection of the plasmid into permissive cells, spliced and non-spliced viral transcripts and viral capsid proteins could be detected. Southern blot analysis of the DNA purified from the plasmid-transfected cells confirmed parvovirus B19 DNA replication. Production of infectious virus by the B19 plasmid was shown by inoculation of cell lysate derived from transfected cells into fresh cells. Together, these results indicate the first successful production of an infectious clone for parvovirus B19 virus

cDNA cloning, structural analysis, SNP detection and tissue ...

Indian Academy of Sciences (India)

THOMAS NAICY

detection and tissue expression profile of the IGF1 gene in Malabari and Attappady Black goats of India. J. Genet. ... Keywords. gene cloning; gene expression; goat; insulin-like growth factor 1; mRNA; single-nucleotide ..... cle tenderness (Koohmaraie et al. .... growth factor (IGF) system in the bovine oviduct at oestrus and.

Cloning, DNA sequence, and expression of the Rhodobacter sphaeroides cytochrome c/sub 2/ gene

Energy Technology Data Exchange (ETDEWEB)

Donohue, T.J.; McEwan, A.G.; Kaplan, S.

1986-11-01

The Rhodobacter sphaeroides cytochrome c/sub 2/ functions as a mobile electron carrier in both aerobic and photosynthetic electron transport chains. Synthetic deoxyoligonucleotide probes, based on the known amino acid sequence of this protein (M/sub r/ 14,000), were used to identify and clone the cytochrome c/sub 2/ structural gene (cycA). DNA sequence analysis of the cycA gene indicated the presence of a typical procaryotic 21-residue signal sequence, suggesting that this periplasmic protein is synthesized in vivo as a precursor. Synthesis of an immunoreactive cytochrome c/sub 2/ precursor protein (M/sub r/ 15,500) was observed in vitro when plasmids containing the cycA gene were used as templates in an R. sphaeroides coupled transcription-translation system. Approximately 500 base pairs of DNA upstream of the cycA gene was sufficient to allow expression of this gene product in vitro. Northern blot analysis with an internal cycA-specific probe identified at least two possibly monocistronic transcripts present in both different cellular levels and relative stoichiometries in steady-state cells grown under different physiological conditions. The ratio of the small (740-mucleotide) and large (920-nucleotide) cycA-specific mRNA species was dependent on cultural conditions but was not affected by light intensity under photosynthetic conditions. These results suggest that the increase in the cellular level of the cytochrome c/sub 2/ protein found in photosynthetic cells was due, in part, to increased transcription of the single-copy cyc operon.

Integrative annotation of 21,037 human genes validated by full-length cDNA clones.

Directory of Open Access Journals (Sweden)

Tadashi Imanishi

2004-06-01

Full Text Available The human genome sequence defines our inherent biological potential; the realization of the biology encoded therein requires knowledge of the function of each gene. Currently, our knowledge in this area is still limited. Several lines of investigation have been used to elucidate the structure and function of the genes in the human genome. Even so, gene prediction remains a difficult task, as the varieties of transcripts of a gene may vary to a great extent. We thus performed an exhaustive integrative characterization of 41,118 full-length cDNAs that capture the gene transcripts as complete functional cassettes, providing an unequivocal report of structural and functional diversity at the gene level. Our international collaboration has validated 21,037 human gene candidates by analysis of high-quality full-length cDNA clones through curation using unified criteria. This led to the identification of 5,155 new gene candidates. It also manifested the most reliable way to control the quality of the cDNA clones. We have developed a human gene database, called the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/. It provides the following: integrative annotation of human genes, description of gene structures, details of novel alternative splicing isoforms, non-protein-coding RNAs, functional domains, subcellular localizations, metabolic pathways, predictions of protein three-dimensional structure, mapping of known single nucleotide polymorphisms (SNPs, identification of polymorphic microsatellite repeats within human genes, and comparative results with mouse full-length cDNAs. The H-InvDB analysis has shown that up to 4% of the human genome sequence (National Center for Biotechnology Information build 34 assembly may contain misassembled or missing regions. We found that 6.5% of the human gene candidates (1,377 loci did not have a good protein-coding open reading frame, of which 296 loci are strong candidates for non-protein-coding RNA

Systematic cloning of human minisatellites from ordered array charomid libraries.

Science.gov (United States)

Armour, J A; Povey, S; Jeremiah, S; Jeffreys, A J

1990-11-01

We present a rapid and efficient method for the isolation of minisatellite loci from human DNA. The method combines cloning a size-selected fraction of human MboI DNA fragments in a charomid vector with hybridization screening of the library in ordered array. Size-selection of large MboI fragments enriches for the longer, more variable minisatellites and reduces the size of the library required. The library was screened with a series of multi-locus probes known to detect a large number of hypervariable loci in human DNA. The gridded library allowed both the rapid processing of positive clones and the comparative evaluation of the different multi-locus probes used, in terms of both the relative success in detecting hypervariable loci and the degree of overlap between the sets of loci detected. We report 23 new human minisatellite loci isolated by this method, which map to 14 autosomes and the sex chromosomes.

Molecular and Biological Characterization of an Isolate of Cucumber mosaic virus from Glycine soja by Generating its Infectious Full-genome cDNA Clones

Directory of Open Access Journals (Sweden)

Mi Sa Vo Phan

2014-06-01

Full Text Available Molecular and biological characteristics of an isolate of Cucumber mosaic virus (CMV from Glycine soja (wild soybean, named as CMV-209, was examined in this study. Comparison of nucleotide sequences and phylogenetic analyses of CMV-209 with the other CMV strains revealed that CMV-209 belonged to CMV subgroup I. However, CMV-209 showed some genetic distance from the CMV strains assigned to subgroup IA or subgroup IB. Infectious full-genome cDNA clones of CMV-209 were generated under the control of the Cauliflower mosaic virus 35S promoter. Infectivity of the CMV-209 clones was evaluated in Nicotiana benthamiana and various legume species. Our assays revealed that CMV-209 could systemically infect Glycine soja (wild soybean and Pisum sativum (pea as well as N. benthamiana, but not the other legume species.

Cloning and characterisation of a glucoamylase gene (GlaM) from dimorphic zygomycete Mucor circinelloides

DEFF Research Database (Denmark)

Houghton-Larsen, J.; Pedersen, Per Amstrup

2003-01-01

This article reports a novel strategy for the cloning of glucoamylase genes using conserved sequences and semi-nested PCR and its application in cloning the GlaM glucoamylase gene and cDNA from the dimorphic zygomycete Mucor circinelloides. The deduced 609-amino-acid enzyme (including signal...

Cloning in Escherichia coli of the enterotoxin gene from Clostridium perfringens type A.

Science.gov (United States)

Iwanejko, L A; Routledge, M N; Stewart, G S

1989-04-01

A 26 bp DNA probe has been constructed with minimal degeneracy to the protein sequence for Clostridium perfringens enterotoxin. The probe has been hybridized against a 6-10 kb chromosomal bank from C. perfringens 8239, prepared as a HindIII partial digest in pHG165. From this survey a clone has been identified containing a 6.8 kb DNA insert with strong hybridization to the probe. Direct plasmid sequencing has identified a translational reading frame within this clone which correlates with the known protein sequence for the type A enterotoxin. DNA sequences 5' to this open reading frame and containing the putative transcriptional control regions show areas of significant homology with regions upstream from the ATG codon of the tetanus toxin gene.

DNA microarrays : a molecular cloning manual

National Research Council Canada - National Science Library

Sambrook, Joseph; Bowtell, David

2002-01-01

.... DNA Microarrays provides authoritative, detailed instruction on the design, construction, and applications of microarrays, as well as comprehensive descriptions of the software tools and strategies...

Clone and characterization of photolyase-gene from soybean

International Nuclear Information System (INIS)

Najrana, T.; Hirouchi, T.; Yamamoto, K.

2003-01-01

Full text: Cyclobutane pyrimidine dimer (CPD) and pyrimidine [6-4] pyrimidone photoproduct (6-4pp) are the major products of UV-radiation. Both CPD and 6-4pp posses lethal as well as mutagenic property. Excision repair and photoreactivation are involved as major pathways in repairing those photoproducts. To repair those products plant uses photoreactivation as a major pathway. In photoreactivation process photolyase (enzyme encoded by PHR-gene) catalyzes the splitting of the dimer into a monomer under blue light. Photolyase is specific for damage CPD or 6-4pp. The CPD and 6-4pp photolyases are responsible for repairing CPD and 6-4pp lesions respectively. Several investigators reported that removal of CPD lesion is necessary for survival in higher plants in the early development. Thus one should realize the importance of clone and characterization of CPD-photolyase gene from plants especially from those are lying in the list of foods such as wheat, corn, soybean etc. cDNA library (pSPORT-P) of soybean was amplified using the primers that designated as common for CPD-photolyase gene for plants. These primers gave the desire size of PCR product. Desirable PCR product inserted into TA-cloning vector and sequenced. Amino acid sequence revealed considerable homology with CPD-photolyases of rice, arabidopsis thaliana. Then using dilution-PCR amplification method (Hirouchi et al., MGG in press) I have identified the true clone from cDNA library of soybean that containing the full length of CPD-photolyase gene. Full length of cloned gene is about 1698 bps long and exist start and stop codon. Amino acid sequence of the cloned gene shows more than 70% homology with rice, arabidopsis thaliana. Cloned gene enables to complement the E. coli ( phr-uvrA-recA-) system that is completely defective in photoreactivation. The size of CPD-photolyase of soybean is about 56 KDa as identified by 12% SDS PAGE

Cloning and sequencing of growth hormone gene of Iranian Lori Bakhtiari sheep

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M Dayani-Nia

2010-05-01

Full Text Available Growth hormone (GH is a peptide hormone that stimulates growth and cell reproduction in humans and animals. It is a 191-amino acid, single chain polypeptide hormone which is synthesized, stored, and secreted by the somatotroph cells within the lateral wings of the anterior pituitary gland. The goal of this research was to clone and sequence sheep growth hormone of Lori Bakhtiary breed in Iran. For this purpose, RNA was extracted from the pituitary gland of freshly slaughtered sheep and cDNA of growth hormone produced. The T/A cloning technique was used to clone the cDNA of growth hormone and then the synthesized construct was transferred into E. coli as the host. Once the correct recombinants were further confirmed by colony PCR or restriction enzyme digestion, sequencing was done. The sequencing results showed that, the length of sheep growth hormone cDNA was 690 bp fragments. Comparison of sequence of growth hormone inside the synthesized construct with those recorded in Genebank (NCBI, Blast indicated high degrees of similarity between Iranian native sheep and other sheep breeds of the world.

Cloning of Bacteroides fragilis plasmid genes affecting metronidazole resistance and ultraviolet survival in Escherichia coli

International Nuclear Information System (INIS)

Wehnert, G.U.; Abratt, V.R.; Goodman, H.J.; Woods, D.R.

1990-01-01

Since reduced metronidazole causes DNA damage, resistance to metronidazole was used as a selection method for the cloning of Bacteroides fragilis genes affecting DNA repair mechanisms in Escherichia coli. Genes from B. fragilis Bf-2 were cloned on a recombinant plasmid pMT100 which made E. coli AB1157 and uvrA, B, and C mutant strains more resistant to metronidazole, but more sensitive to far uv irradiation under aerobic conditions. The loci affecting metronidazole resistance and uv sensitivity were linked and located on a 5-kb DNA fragment which originated from the small 6-kb cryptic plasmid pBFC1 present in B. fragilis Bf-2 cells

Pemotongan dan Menyambung DNA dalam Kloning Gen, Studi pada Kloning Gen Prolidase dari Bakteri Asam Laktat

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Ketut Suriasih

2015-03-01

Full Text Available Gene cloning in lactic acid bacteria (LAB is crucial in term to increase their ability to hydrolyze milk protein such as proline. This proline could be hydrolyzed when the LAB undergone cloning on their genome coding the enzyme. The cloning process need technology to separate/isolate the gene capable of proline hydrolyze. Isolation of DNA containing prolidase gene, need DNA genome cutting. After isolation of DNA gene coding prolidase, it is then recombined with other bacterial DNA to obtained recombinant gene. The process need ligase. In gene cloning, knowledge of cutting and joining the DNA should be understood. The enzyme take the role in cutting and joining the DNA were restriction endonuclease and ligase. The restriction enzyme function (1 in inserting a gen into plasmid contained in a vector during gene cloning, and gene expression experiment, and (2 to identify the gene. It is important that the researcher already have standardized  sequenced gene as control. The DNA contained target gene was cut using some restriction enzyme, then the gene was arrayed in electrophoresis gel using southern blot technique. DNA sequence was elucidated by addition of ethydium bromide. To identify/characterize the isolated gene, this DNA sequence was encountered the control DNA.

Endangered wolves cloned from adult somatic cells.

Science.gov (United States)

Kim, Min Kyu; Jang, Goo; Oh, Hyun Ju; Yuda, Fibrianto; Kim, Hye Jin; Hwang, Woo Suk; Hossein, Mohammad Shamim; Kim, Joung Joo; Shin, Nam Shik; Kang, Sung Keun; Lee, Byeong Chun

2007-01-01

Over the world, canine species, including the gray wolf, have been gradually endangered or extinct. Many efforts have been made to recover and conserve these canids. The aim of this study was to produce the endangered gray wolf with somatic cell nuclear transfer (SCNT) for conservation. Adult ear fibroblasts from a female gray wolf (Canis lupus) were isolated and cultured in vitro as donor cells. Because of limitations in obtaining gray wolf matured oocytes, in vivo matured canine oocytes obtained by flushing the oviducts from the isthmus to the infundibulum were used. After removing the cumulus cells, the oocyte was enucleated, microinjected, fused with a donor cell, and activated. The reconstructed cloned wolf embryos were transferred into the oviducts of the naturally synchronized surrogate mothers. Two pregnancies were detected by ultrasonography at 23 days of gestation in recipient dogs. In each surrogate dog, two fetal sacs were confirmed by early pregnancy diagnosis at 23 days, but only two cloned wolves were delivered. The first cloned wolf was delivered by cesarean section on October 18, 2005, 60 days after embryo transfer. The second cloned wolf was delivered on October 26, 2005, at 61 days postembryo transfer. Microsatellite analysis was performed with genomic DNA from the donor wolf, the two cloned wolves, and the two surrogate female recipients to confirm the genetic identity of the cloned wolves. Analysis of 19 microsatellite loci confirmed that the cloned wolves were genetically identical to the donor wolf. In conclusion, we demonstrated live birth of two cloned gray wolves by nuclear transfer of wolf somatic cells into enucleated canine oocyte, indicating that SCNT is a practical approach for conserving endangered canids.

Purification, reactivity with IgE and cDNA cloning of parvalbumin as the major allergen of mackerels.

Science.gov (United States)

Hamada, Y; Tanaka, H; Ishizaki, S; Ishida, M; Nagashima, Y; Shiomi, K

2003-08-01

Three species of mackerels (Scomber japonicus, S. australasicus and S. scombrus) are widely consumed and considered to be most frequently involved in incidents of IgE-mediated fish allergy in Japan. In this study, parvalbumin, a possible candidate for the major allergen, was purified from the white muscle of three species of mackerels by gel filtration on Sephadex G-75 and reverse-phase HPLC on TSKgel ODS-120T. All the purified preparations from three species gave a single band of about 11 kDa and were clearly identified as parvalbumins by analyses of their partial amino acid sequences. In ELISA experiments, four of five sera from fish-allergic patients reacted to all the purified parvalbumins, demonstrating that parvalbumin is the major allergen in common with the mackerels. Antigenic cross-reactivity among the mackerel parvalbumins was also established by ELISA inhibition experiments. A cDNA library was constructed from the white muscle of S. japonicus and the cDNA encoding parvalbumin was cloned. The amino acid sequence translated from the nucleotide sequence revealed that the S. japonicus parvalbumin is composed of 108 residues, being a member of beta-type parvalbumins.

Cloning and mRNA expression pattern analysis under low ...

African Journals Online (AJOL)

Jane

2011-07-13

Jul 13, 2011 ... This research cloned endochitinase-antifreeze protein precursor (EAPP) gene of .... Company, RevertAidTM First Strand cDNA Synthesis Kit from .... different times of low temperature in root, stem and leaf of Dongmu-70 rye.

Cloning and characterization of an insecticidal crystal protein gene ...

Indian Academy of Sciences (India)

Unknown

The sequence of the cloned crystal protein gene showed almost complete homology with a mosquitocidal toxin gene from Bacillus .... diet or by topical application on food substrates as .... has very high similarity (99.74%) at DNA level with.

Human catechol-O-methyltransferase: Cloning and expression of the membrane-associated form

International Nuclear Information System (INIS)

Bertocci, B.; Miggiano, V.; Da Prada, M.; Dembic, Z.; Lahm, H.W.; Malherbe, P.

1991-01-01

A cDNA clone for human catechol-O-methyltransferase was isolated from a human hepatoma cell line (Hep G2) cDNA library by hybridization screening with a porcine cDNA probe. The cDNA clone was sequenced and found to have an insert of 1226 nucleotides. The deduced primary structure of hCOMT is composed of 271 amino acid residues with the predicted molecular mass of 30 kDa. At its N terminus it has a hydrophobic segment of 21 amino acid residues that may be responsible for insertion of hCOMT into the endoplasmic reticulum membrane. The primary structure of hCOMT exhibits high homology to the porcine partial cDNA sequence (93%). The deduced amino acid sequence contains two tryptic peptide sequences (T-22, T-33) found in porcine liver catechol-O-methyltransferase (CEMT). The coding region of hCOMT cDNA was placed under the control of the cytomegalovirus promoter to transfect human kidney 293 cells. The recombinant hCOMT was shown by immunoblot analysis to be mainly associated with the membrane fraction. RNA blot analysis revealed one COMT mRNA transcript of 1.4 kilobases in Hep G2 poly(A) + RNA

A Full-Length Infectious cDNA Clone of Zika Virus from the 2015 Epidemic in Brazil as a Genetic Platform for Studies of Virus-Host Interactions and Vaccine Development

Directory of Open Access Journals (Sweden)

Konstantin A. Tsetsarkin

2016-08-01

Full Text Available An arthropod-borne virus, Zika virus (ZIKV, has recently emerged as a major human pathogen. Associated with complications during perinatal development and Guillain-Barré syndrome in adults, ZIKV raises new challenges for understanding the molecular determinants of flavivirus pathogenesis. This underscores the necessity for the development of a reverse genetic system based on an epidemic ZIKV strain. Here, we describe the generation and characterization in cell cultures of an infectious cDNA clone of ZIKV isolated from the 2015 epidemic in Brazil. The cDNA-derived ZIKV replicated efficiently in a variety of cell lines, including those of both neuronal and placental origin. We observed that the growth of cDNA-derived virus was attenuated compared to the growth of the parental isolate in most cell lines, which correlates with substantial differences in sequence heterogeneity between these viruses that were determined by deep-sequencing analysis. Our findings support the role of genetic diversity in maintaining the replicative fitness of viral populations under changing conditions. Moreover, these results indicate that caution should be exercised when interpreting the results of reverse-genetics experiments in attempts to accurately predict the biology of natural viruses. Finally, a Vero cell-adapted cDNA clone of ZIKV was generated that can be used as a convenient platform for studies aimed at the development of ZIKV vaccines and therapeutics.

Design and Construction of a Cloning Vector Containing the hspX Gene of Mycobacterium tuberculosis

Directory of Open Access Journals (Sweden)

Atieh Yaghoubi

2016-10-01

Full Text Available Background: Tuberculosis (TB is a major cause of death worldwide. Finding an effective vaccine against TB is the best way to control it. Several vaccines against this disease have been developed but none are completely protective. The aim of this study was to design and construct a cloning vector containing the Mycobacterium tuberculosis (M. tuberculosis heat shock protein X (hspX. Methods: First, an hspX fragment was amplified by PCR and cloned into plasmid pcDNA3.1(+ and recombinant vector was confirmed. Results: A 435 bp hspX fragment was isolated. The fragment was 100% homologous with hspX of M. tuberculosis strain H37Rv in GenBank. Conclusions: In this study, the cloning vector pcDNA3.1(+, containing a 435-bp hspX fragment of M. tuberculosis, was constructed. This could be used as a DNA vaccine to induce immune responses in animal models in future studies.

Molecular cloning and expression of bovine kappa-casein in Escherichia coli

International Nuclear Information System (INIS)

Kang, Y.C.; Richardson, T.

1988-01-01

A cDNA library was constructed using poly(A) + RNA from bovine mammary gland. This cDNA library of 6000 clones was screened employing colony hybridization using 32 P-labelled oligonucleotide probes and restriction endonuclease mapping. The cDNA from the selected plasmid, pKR76, was sequenced using the dideoxy-chain termination method. The cDNA insert of pKR76 carries the full-length sequence, which codes for mature kappa-casein protein. The amino acid sequence deduced from the cDNA sequence fits the published amino acid sequence with three exceptions; the reported pyroglutamic acid at position 1, tyrosine at position 35, and aspartic acid at position 81 are, respectively, a glutamine, a histidine, and an asparagine in the clone containing pKR76. The MspI-, NlaIV-cleaved fragment (630 base pair) from the kappa-casein cDNA insert has been subcloned into expression vectors pUC18 and pKK233-2, which contain a lac promoter and a trc promoter, respectively. Escherichia coli cells carrying the recombinant expression plasmids were shown to produce kappa-casein protein having the expected mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and being recognized by specific antibodies raised against natural bovine kappa-casein

Molecular cloning and characterization of Antheraea mylitta cytoplasmic polyhedrosis virus polyhedrin gene and its variant forms

International Nuclear Information System (INIS)

Sinha-Datta, Uma; Chavali, Venkata Ramana Murthy; Ghosh, Ananta K.

2005-01-01

The segments 10 (S10) of the 11 double stranded RNA genomes from Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) encoding a novel polyhedrin polypeptide was converted to cDNA, cloned, and sequenced. Three cDNA clones consisting of 1502 (AmCPV10-1), 1120 (AmCPV10-2), and 1415 (AmCPV10-3) nucleotides encoding polyhedrin of 254, 339, and 319 amino acids with molecular masses of 29, 39, and 37 kDa, respectively, were obtained, and verified by Northern analysis. These clones showed 70-94% sequence identity among them but none with any sequences in databases. The expression of AmCPV10-1 cDNA encoded polyhedrin in Sf-9 cells was detected by immunoblot analysis and formation of polyhedra by electron microscopy, as observed in AmCPV-infected gut cells, but no expression of AmCPV10-2 or AmCPV10-3 cDNA was detected, indicating that during AmCPV replication, along with functional S10 RNA, some defective variant forms of S10 RNAs are packaged in virion particles

Cloning of precursors for two MIH/VIH-related peptides in the prawn, Macrobrachium rosenbergii.

Science.gov (United States)

Yang, W J; Rao, K R

2001-11-30

Two cDNA clones (634 and 1366 bp) encoding MIH/VIH (molt-inhibiting hormone/vitellogenesis-inhibiting hormone)-related peptides were isolated and sequenced from a Macrobrachium rosenbergii eyestalk ganglia cDNA library. The clones contain a 360 and 339 bp open-reading frame, and their conceptually translated peptides consist of a 41 and 34 amino acid signal peptide, respectively, and a 78 amino acid residue mature peptide hormone. The amino acid sequences of the peptides exhibit higher identities with other known MIHs and VIH (44-69%) than with CHHs (28-33%). This is the first report describing the cloning and sequencing of two MIH/VIH-related peptides in a single crustacean species. Transcription of these mRNAs was detected in the eyestalk ganglia, but not in the thoracic ganglia, hepatopancreas, gut, gill, heart, or muscle.

Cloning-free template DNA preparation for cell-free protein synthesis via two-step PCR using versatile primer designs with short 3'-UTR.

Science.gov (United States)

Nomoto, Mika; Tada, Yasuomi

2018-01-01

Cell-free protein synthesis (CFPS) systems largely retain the endogenous translation machinery of the host organism, making them highly applicable for proteomics analysis of diverse biological processes. However, laborious and time-consuming cloning procedures hinder progress with CFPS systems. Herein, we report the development of a rapid and efficient two-step polymerase chain reaction (PCR) method to prepare linear DNA templates for a wheat germ CFPS system. We developed a novel, effective short 3'-untranslated region (3'-UTR) sequence that facilitates translation. Application of the short 3'-UTR to two-step PCR enabled the generation of various transcription templates from the same plasmid, including fusion proteins with N- or C-terminal tags, and truncated proteins. Our method supports the cloning-free expression of target proteins using an mRNA pool from biological material. The established system is a highly versatile platform for in vitro protein synthesis using wheat germ CFPS. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Production of cloned mice and ES cells from adult somatic cells by nuclear transfer: how to improve cloning efficiency?

Science.gov (United States)

Wakayama, Teruhiko

2007-02-01

Although it has now been 10 years since the first cloned mammals were generated from somatic cells using nuclear transfer (NT), most cloned embryos usually undergo developmental arrest prior to or soon after implantation, and the success rate for producing live offspring by cloning remains below 5%. The low success rate is believed to be associated with epigenetic errors, including abnormal DNA hypermethylation, but the mechanism of "reprogramming" is unclear. We have been able to develop a stable NT method in the mouse in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Especially in the mouse, only a few laboratories can make clones from adult somatic cells, and cloned mice are never successfully produced from most mouse strains. However, this technique promises to be an important tool for future research in basic biology. For example, NT can be used to generate embryonic stem (NT-ES) cell lines from a patient's own somatic cells. We have shown that NT-ES cells are equivalent to ES cells derived from fertilized embryos and that they can be generated relatively easily from a variety of mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly. In general, NT-ES cell techniques are expected to be applied to regenerative medicine; however, this technique can also be applied to the preservation of genetic resources of mouse strain instead of embryos, oocytes and spermatozoa. This review describes how to improve cloning efficiency and NT-ES cell establishment and further applications.

Phosphoribosylpyrophosphate synthetase of Bacillus subtilis. Cloning, characterization and chromosomal mapping of the prs gene

DEFF Research Database (Denmark)

Nilsson, Dan; Hove-Jensen, Bjarne

1987-01-01

The gene (prs) encoding phosphoribosylpyrophosphate (PRPP) synthetase has been cloned from a library of Bacillus subtilis DNA by complementation of an Escherichia coli prs mutation. Flanking DNA sequences were pruned away by restriction endonuclease and exonuclease BAL 31 digestions, resulting...... in a DNA fragment of approx. 1.8 kb complementing the E. coli prs mutation. Minicell experiments revealed that this DNA fragment coded for a polypeptide, shown to be the PRPP synthetase subunit, with an Mr of approx. 40,000. B. subtilis strains harbouring the prs gene in a multicopy plasmid contained up...... to nine-fold increased PRPP synthetase activity. The prs gene was cloned in an integration vector and the resulting hybrid plasmid inserted into the B. subtilis chromosome by homologous recombination. The integration site was mapped by transduction and the gene order established as purA-guaA-prs-cysA....

Cloning and sequencing of the bovine gastrin gene

DEFF Research Database (Denmark)

Lund, T; Rehfeld, J F; Olsen, Jørgen

1989-01-01

In order to deduce the primary structure of bovine preprogastrin we therefore sequenced a gastrin DNA clone isolated from a bovine liver cosmid library. Bovine preprogastrin comprises 104 amino acids and consists of a signal peptide, a 37 amino acid spacer-sequence, the gastrin-34 sequence followed...

TA-GC cloning: A new simple and versatile technique for the directional cloning of PCR products for recombinant protein expression.

Directory of Open Access Journals (Sweden)

Athanasios Niarchos

Full Text Available During the last few decades, the recombinant protein expression finds more and more applications. The cloning of protein-coding genes into expression vectors is required to be directional for proper expression, and versatile in order to facilitate gene insertion in multiple different vectors for expression tests. In this study, the TA-GC cloning method is proposed, as a new, simple and efficient method for the directional cloning of protein-coding genes in expression vectors. The presented method features several advantages over existing methods, which tend to be relatively more labour intensive, inflexible or expensive. The proposed method relies on the complementarity between single A- and G-overhangs of the protein-coding gene, obtained after a short incubation with T4 DNA polymerase, and T and C overhangs of the novel vector pET-BccI, created after digestion with the restriction endonuclease BccI. The novel protein-expression vector pET-BccI also facilitates the screening of transformed colonies for recombinant transformants. Evaluation experiments of the proposed TA-GC cloning method showed that 81% of the transformed colonies contained recombinant pET-BccI plasmids, and 98% of the recombinant colonies expressed the desired protein. This demonstrates that TA-GC cloning could be a valuable method for cloning protein-coding genes in expression vectors.

Molecular cloning of cellulase genes from indigenous bacterial isolates

International Nuclear Information System (INIS)

Jong Bor Chyan; Pauline Liew Woan Ying; Mat Rasol Awang

2006-01-01

Indigenous cellulolytic bacterial isolates having high activities in degrading carboxymethyl cellulose (CMC) were isolated from local environments. Identification of these isolates were performed by molecular techniques. By using polymerase chain reaction (PCR) techniques, PCR products encoding cellulase gene were amplified from the total genomic DNAs. Purified PCR product was successfully cloned and expressed in Escherichia coli host system. The complete nucleotide sequences of the cellulase genes determined. The analysis of amino acid sequences deduced from the genes indicated that the cloned DNA fragments show high homology to those of endoglucanase genes of family GH5. All cloned genes consist of an N-terminal signal peptide, a catalytic domain of family 5 glycosyl hydrolase and a cellulose-binding domain of family III. (Author)

Isolation and characterization of human glycophorin A cDNA clones by a synthetic oligonucleotide approach: nucleotide sequence and mRNA structure

International Nuclear Information System (INIS)

Siebert, P.D.; Fukuda, M.

1986-01-01

In an effort to understand the relationships among and the regulation of human glycophorins, the authors have isolated and characterized several glycophorin A-specific cDNA clones obtained from a human erythroleukemic K562 cell cDNA library. This was accomplished by using mixed synthetic oligonucleotides, corresponding to various regions of the known amino acid sequence, to prime the synthesis of the cDNA as well as to screen the cDNA library. They also used synthetic oligonucleotides to sequence the largest of the glycophorin cDNAs. The nucleotide sequence obtained suggests the presence of a potential leader peptide, consistent with the membrane localization of this glycoprotein. Examination of the structure of glycophorin mRNA by blot hybridization revealed the existence of several electrophoretically distinct mRNAs numbering three or four, depending on the size of the glycophorin cDNA used as a hybridization probe. The smaller cDNA hybridized to three mRNAs of approximately 2.8, 1.7, and 1.0 kilobases. In contrast, the larger cDNA hybridized to an additional mRNA of approximately 0.6 kilobases. Further examination of the relationships between these multiple mRNAs by blot hybridization was conducted with the use of exact-sequence oligonucleotide probes constructed from various regions of the cDNA representing portions of the amino acid sequence of glycophorin A with or without known homology with glycophorin B. In total, the results obtained are consistent with the hypothesis that the three larger mRNAs represent glycophorin A gene transcripts and that the smallest (0.6 kilobase) mRNA may be specific for glycophorin B

Stochasticity or the fatal `imperfection' of cloning

Indian Academy of Sciences (India)

2005-01-07

Jan 7, 2005 ... The concept of clone is analysed with the aim of exploring the limits to which a phenotype can be said to be determined geneticaly. First of all, mutations that result from the replication, topological manipulation or lesion of DNA introduce a source of heritable variation in an otherwise identical genetic ...

Clone and expression of human transferrin receptor gene: a marker gene for magnetic resonance imaging

International Nuclear Information System (INIS)

Li Li; Liu Lizhi; Lv Yanchun; Liu Xuewen; Cui Chunyan; Wu Peihong; Liu Qicai; Ou Shanxing

2007-01-01

Objective: To clone human transferrin receptor (hTfR) gene and construct expression vector producing recombination protein. Methods: Human transferrin receptor gene cDNA was amplified by RT-PCR from human embryonic liver and lung tissue. Recombinant pcDNA3-hTfR and pEGFP-Cl-hTfR plasmids were constructed and confirmed by DNA sequencing. These plasmids were stably transfected into the HEK293 cells. The protein expression in vitro was confirmed by Western Blot. The efficiency of expression and the location of hTfR were also investigated by fluorescence microscopy and confocal fluorescence microscopy. Results: The full length cDNA of hTfR gene (2332 bp) was cloned and sequenced. The hTfR (190 000) was overexpressed in transfected HEK293 cells by Western blot analysis. Fluorescence micrographs displayed that the hTfR was expressed at high level and located predominantly in the cell surface. Conclusions: Human transferrin receptor (hTfR) gene has been successfully cloned and obtained high-level expression in HEK293 cells, and the recombination protein of hTfR distributed predominantly in the cell membrane. (authors)

Human Chromosome 21: Mapping of the chromosomes and cloning of cDNAs

Energy Technology Data Exchange (ETDEWEB)

Antonarakis, S.E.

1991-09-01

The objective of the research funded by DOE grant DE-FG02-89ER60857 from 6/15/89 to 8/31/91 was to contribute to the physical mapping of human chromosome 21 (HC21) by cloning large fragments of DNA into Yeast Artificial Chromosomes (YACs) and identify YACs that map on HC21. A total of 54 sequence tagged sites (STS) have been developed and mapped in our laboratory to HC21 and can be used as initial reference points for YAC identification and construction of overlapping clones. A small YAC library was constructed which is HC21 specific. DNA from somatic cell hybrid WAV17 or from flow-sorted HC21 was partially digested with EcoRI, ligated into vectors PJS97, PJS98, and YACs have been obtained with average size insert of more than 300 kb. This library has been deposited in D. Patterson's lab for the Joint YAC screening effort. Additional YAC libraries from ICI Pharmaceuticals or from Los Alamos National Laboratories have been screened with several STS and positive YACs have been identified. Work in progress includes screening of YAC libraries in order to construct overlapping clones, characterization of the cloning ends of YACs, characterization of additional STS and cloning of HC21 specific cDNAs. 15 refs., 2 figs., 5 tabs.

[Construction of fetal mesenchymal stem cell cDNA subtractive library].

Science.gov (United States)

Yang, Li; Wang, Dong-Mei; Li, Liang; Bai, Ci-Xian; Cao, Hua; Li, Ting-Yu; Pei, Xue-Tao

2002-04-01

To identify differentially expressed genes between fetal mesenchymal stem cell (MSC) and adult MSC, especially specified genes expressed in fetal MSC, a cDNA subtractive library of fetal MSC was constructed using suppression subtractive hybridization (SSH) technique. At first, total RNA was isolated from fetal and adult MSC. Using SMART PCR synthesis method, single-strand and double-strand cDNAs were synthesized. After Rsa I digestion, fetal MSC cDNAs were divided into two groups and ligated to adaptor 1 and adaptor 2 respectively. Results showed that the amplified library contains 890 clones. Analysis of 890 clones with PCR demonstrated that 768 clones were positive. The positive rate is 86.3%. The size of inserted fragments in these positive clones was between 0.2 - 1 kb, with an average of 400 - 600 bp. SSH is a convenient and effective method for screening differentially expressed genes. The constructed cDNA subtractive library of fetal MSC cDNA lays solid foundation for screening and cloning new and specific function related genes of fetal MSC.

Detection of antibody against antigen expressed by molecularly cloned hepatitis C virus cDNA: Application to diagnosis and blood screening for posttransfusion hepatitis

Energy Technology Data Exchange (ETDEWEB)

Miyamura, Tatsuo; Saito, Izumu (National Institute of Health, Tokyo (Japan)); Katayama, Tohru (Tokyo National Chest Hospital (Japan)); Kikuchi, Shu; Tateda, Akira (Sendai National Hospital (Japan)); Houghton, M.; Choo, Quilim; Kuo, G. (Chiron Corporation, Emeryville, CA (USA))

1990-02-01

A cDNA clone has been derived from the plasma of a chimpanzee with chronic non-A, non-B viral hepatits (NANBH). The authors have assayed for antibodies reacting with the encoded antigen in sera from posttransfusion hepatitis patients (643 samples from 23 patients) and their corresponding donors collected during the past 10 years in Japan. The antibody was detected in 15 out of 17 (88.2%) posttransfusion NANBH (PT-NANBH) patients whose sera over time displayed multiple alanine aminotransferase (ALT) peaks. In general, the antibody was detected after several peaks of serum ALT elevations and, once detected, it persisted for years. Of the 15 well-defined cases of PT-NANBH that showed multiple ALT peaks and hepatitis C virus seroconversions, 11 (73.3%) were shown to be transfused with at least one unit of blood positive for the antibody. The retrospective analysis showed that all tested donor blood found to be positive for the antibody had been transfused to recipients who afterwards developed NANBH. These data strongly suggest that the cloned cDNA originated from an etiological agent of NANBH termed the hepatitis C virus. Furthermore, the present study demonstrates that had the screening been done with the anti-hepatitis C virus assay, 11 out of 17 (64.7%) cases of chronic PT-NANBH and 1 out of 6 (16.6%) acute PT-NANBH would have been prevented.

Detection of antibody against antigen expressed by molecularly cloned hepatitis C virus cDNA: Application to diagnosis and blood screening for posttransfusion hepatitis

International Nuclear Information System (INIS)

Miyamura, Tatsuo; Saito, Izumu; Katayama, Tohru; Kikuchi, Shu; Tateda, Akira; Houghton, M.; Choo, Quilim; Kuo, G.

1990-01-01

A cDNA clone has been derived from the plasma of a chimpanzee with chronic non-A, non-B viral hepatits (NANBH). The authors have assayed for antibodies reacting with the encoded antigen in sera from posttransfusion hepatitis patients (643 samples from 23 patients) and their corresponding donors collected during the past 10 years in Japan. The antibody was detected in 15 out of 17 (88.2%) posttransfusion NANBH (PT-NANBH) patients whose sera over time displayed multiple alanine aminotransferase (ALT) peaks. In general, the antibody was detected after several peaks of serum ALT elevations and, once detected, it persisted for years. Of the 15 well-defined cases of PT-NANBH that showed multiple ALT peaks and hepatitis C virus seroconversions, 11 (73.3%) were shown to be transfused with at least one unit of blood positive for the antibody. The retrospective analysis showed that all tested donor blood found to be positive for the antibody had been transfused to recipients who afterwards developed NANBH. These data strongly suggest that the cloned cDNA originated from an etiological agent of NANBH termed the hepatitis C virus. Furthermore, the present study demonstrates that had the screening been done with the anti-hepatitis C virus assay, 11 out of 17 (64.7%) cases of chronic PT-NANBH and 1 out of 6 (16.6%) acute PT-NANBH would have been prevented

Cloning and characterization of stress responsive Glp genes and their promotor regions from rice (abstract)

International Nuclear Information System (INIS)

Naqvi, S.M.S.; Mahmood, T.

2005-01-01

Plants respond to a number of environmental stimuli by modulating expression of genes. One such family of genes is now known as germin/germin-like protein genes (Glps). In order to detect any Glp gene response in rice, a pair of degenerate primers was designed based on consensus region from Glp sequences in Genbank. Using these primers a DNA fragment of about 550 bp was obtained by PCR amplification from genomic template. This 550 bp DNA was used as probe in Northern analysis. These studies provided evidence pointing to differential response of Glp expression to salt stress. RNA obtained from the roots was used for synthesis of cDNA. This cDNA was amplifiable with sense primer (RGLP1) from above mentioned pair and oligo-(dt) yielding a fragment of approx. 800 bp. Restriction analysis revealed that the PCR product was heterogeneous. After establishing that 800 bp fragment was the desired product, it was cloned in pCRII-TOPO. Five clones were picked up and analyzed by restriction analysis and sequencing. Two different Glp cDNAs were represented by these partial clones. Remaining sequence of the 5' end for clone 4 and 16 was obtained by Rapid Amplification of cDNA ends (RACE). The resultant sequences have been submitted to Genbank as Oryza sativa Rice Germin-like Protein 1 and 2 (osRGLP1 and 2). When full length genes corresponding to these sequences were amplified from genomic templates, resulting fragments were nearly 150 by larger than cDNAs. Cloning of structural genes for osRGLP1 revealed presence of a 162 bp intron in the coding region near 3' end. Preliminary evidence shows that expression of both osRGLP1 and 2 is severely reduced during salt stress. Another approach to establish both osRGLP1 and 2 genes involvement in stress tolerance is to study the ability of their promotor regions to drive expression of some reporter gene during stress. Promotor regions of about 1100 bp has been amplified and cloned and has been confirmed by restriction analysis and nested

Predictive mutagenesis of ligation-independent cloning (LIC) vectors for protein expression and site-specific chemical conjugation

DEFF Research Database (Denmark)

Vernet, Erik; Sauer, Jørgen; Andersen, Peter Andreas

2011-01-01

Ligation-independent cloning (LIC) allows for cloning of DNA constructs independent of insert restriction sites and ligases. However, any required mutations are typically introduced by additional, time-consuming steps. We present a rapid, inexpensive method for mutagenesis in the 5' LIC site...

Molecular analysis of Toxoplasma gondii Surface Antigen 1 (SAG1) gene cloned from Toxoplasma gondii DNA isolated from Javanese acute toxoplasmosis

Science.gov (United States)

Haryati, Sri; Agung Prasetyo, Afiono; Sari, Yulia; Dharmawan, Ruben

2018-05-01

Toxoplasma gondii Surface Antigen 1 (SAG1) is often used as a diagnostic tool due to its immunodominant-specific as antigen. However, data of the Toxoplasma gondii SAG1 protein from Indonesian isolate is limited. To study the protein, genomic DNA was isolated from a Javanese acute toxoplasmosis blood samples patient. A complete coding sequence of Toxoplasma gondii SAG1 was cloned and inserted into an Escherichia coli expression plasmid and sequenced. The sequencing results were subjected to bioinformatics analysis. The Toxoplasma gondii SAG1 complete coding sequences were successfully cloned. Physicochemical analysis revealed the 336 aa of SAG1 had 34.7 kDa of weight. The isoelectric point and aliphatic index were 8.4 and 78.4, respectively. The N-terminal methionine half-life in Escherichia coli was more than 10 hours. The antigenicity, secondary structure, and identification of the HLA binding motifs also had been discussed. The results of this study would contribute information about Toxoplasma gondii SAG1 and benefits for further works willing to develop diagnostic and therapeutic strategies against the parasite.

Cloning of resistance gene analogs located on the alien chromosome in an addition line of wheat-Thinopyrum intermedium.

Science.gov (United States)

Jiang, Shu-Mei; Hu, Jun; Yin, Wei-Bo; Chen, Yu-Hong; Wang, Richard R-C; Hu, Zan-Min

2005-09-01

Homology-based gene/gene-analog cloning method has been extensively applied in isolation of RGAs (resistance gene analogs) in various plant species. However, serious interference of sequences on homoeologous chromosomes in polyploidy species usually occurred when cloning RGAs in a specific chromosome. In this research, the techniques of chromosome microdissection combined with homology-based cloning were used to clone RGAs from a specific chromosome of Wheat-Thinopyrum alien addition line TAi-27, which was derived from common wheat and Thinopyrum intermedium with a pair of chromosomes from Th. intermedium. The alien chromosomes carry genes for resistance to BYDV. The alien chromosome in TAi-27 was isolated by a glass needle and digested with proteinase K. The DNA of the alien chromosome was amplified by two rounds of Sau3A linker adaptor-mediated PCR. RGAs were amplified by PCR with the degenerated primers designed based on conserved domains of published resistance genes (R genes) by using the alien chromosome DNA, genomic DNA and cDNA of Th. intermedium, TAi-27 and 3B-2 (a parent of TAi-27) as templates. A total of seven RGAs were obtained and sequenced. Of which, a constitutively expressed single-copy NBS-LRR type RGA ACR 3 was amplified from the dissected alien chromosome of TAi-27, TcDR 2 and TcDR 3 were from cDNA of Th. intermedium, AcDR 3 was from cDNA of TAi-27, FcDR 2 was from cDNA of 3B-2, AR 2 was from genomic DNA of TAi-27 and TR 2 was from genomic DNA of Th. intermedium. Sequence homology analyses showed that the above RGAs were highly homologous with known resistance genes or resistance gene analogs and belonged to NBS-LRR type of R genes. ACR 3 was recovered by PCR from genomic DNA and cDNA of Th. intermedium and TAi-27, but not from 3B-2. Southern hybridization using the digested genomic DNA of Th. intermedium, TAi-27 and 3B-2 as the template and ACR 3 as the probe showed that there is only one copy of ACR 3 in the genome of Th. intermedium and TAi

Nonequilibrium Phase Transitions Associated with DNA Replication

Science.gov (United States)

2011-02-11

polymerases) catalyzing the growth of a DNA primer strand (the nascent chain of nucleotides complementary to the template strand) based on the Watson ...the fraction (error rate) of monomers for which y, where y is the correct Watson - Crick complementary base of , can be obtained by ¼ X...Nonequilibrium Phase Transitions Associated with DNA Replication Hyung-June Woo* and Anders Wallqvist Biotechnology High Performance Computing

Characterization of cis-Acting RNA Elements of Zika Virus by Using a Self-Splicing Ribozyme-Dependent Infectious Clone.

Science.gov (United States)

Liu, Zhong-Yu; Yu, Jiu-Yang; Huang, Xing-Yao; Fan, Hang; Li, Xiao-Feng; Deng, Yong-Qiang; Ji, Xue; Cheng, Meng-Li; Ye, Qing; Zhao, Hui; Han, Jian-Feng; An, Xiao-Ping; Jiang, Tao; Zhang, Bo; Tong, Yi-Gang; Qin, Cheng-Feng

2017-11-01

Zika virus (ZIKV) has caused significant outbreaks and epidemics in the Americas recently, raising global concern due to its ability to cause microcephaly and other neurological complications. A stable and efficient infectious clone of ZIKV is urgently needed. However, the instability and toxicity of flavivirus cDNA clones in Escherichia coli hosts has hindered the development of ZIKV infectious clones. Here, using a novel self-splicing ribozyme-based strategy, we generated a stable infectious cDNA clone of a contemporary ZIKV strain imported from Venezuela to China in 2016. The constructed clone contained a modified version of the group II self-splicing intron P.li.LSUI2 near the junction between the E and NS1 genes, which were removed from the RNA transcripts by an easy-to-establish in vitro splicing reaction. Transfection of the spliced RNAs into BHK-21 cells led to the production of infectious progeny virus that resembled the parental virus. Finally, potential cis -acting RNA elements in ZIKV genomic RNA were identified based on this novel reverse genetics system, and the critical role of 5'-SLA promoter and 5'-3' cyclization sequences were characterized by a combination of different assays. Our results provide another stable and reliable reverse genetics system for ZIKV that will help study ZIKV infection and pathogenesis, and the novel self-splicing intron-based strategy could be further expanded for the construction of infectious clones from other emerging and reemerging flaviviruses. IMPORTANCE The ongoing Zika virus (ZIKV) outbreaks have drawn global concern due to the unexpected causal link to fetus microcephaly and other severe neurological complications. The infectious cDNA clones of ZIKV are critical for the research community to study the virus, understand the disease, and inform vaccine design and antiviral screening. A panel of existing technologies have been utilized to develop ZIKV infectious clones. Here, we successfully generated a stable

Cloning of low-temperature induced gene from Morus mongolica CK

African Journals Online (AJOL)

SERVER

2008-03-04

Mar 4, 2008 ... extracted form the stem and was reverse-transcribed into cDNA. Mulberry .... positive clones to obtain engineered Agrobacterium LBA4404/. pIG121/ ..... Comparative experiment for a new cold and drought tolerance mulberry ...

DNA repair genes

International Nuclear Information System (INIS)

Morimyo, Mitsuoki

1995-01-01

Fission yeast S. pombe is assumed to be a good model for cloning of human DNA repair genes, because human gene is normally expressed in S. pombe and has a very similar protein sequence to yeast protein. We have tried to elucidate the DNA repair mechanisms of S. pombe as a model system for those of mammals. (J.P.N.)

Amplification of a transcriptionally active DNA sequence in the human brain

International Nuclear Information System (INIS)

Yakovlev, A.G.; Sazonov, A.E.; Spunde, A.Ya.; Gindilis, V.M.

1986-01-01

The authors present their findings of tissue-specific amplification of a DNA fragment actively transcribed in the human brain. This genome fragment was found in the library complement of cDNA of the human brain and evidently belongs to a new class of moderate repetitions of DNA with an unstable copying capacity in the human genome. The authors isolated total cell RNA from various human tissues (brain, placenta), and rat tissues (brain, liver), by the method of hot phenol extraction with guanidine thiocynate. The poly(A + ) RNA fraction was isolated by chromatography. Synthesis of cDNA was done on a matrix of poly(A + ) RNA of human brain. The cDNA obtained was cloned in plasmid pBR322 for the PstI site using (dC/dG) sequences synthesized on the 3' ends of the vector molecule and cDNA respectively. In cloning 75 ng cDNA, the authors obtained approximately 10 5 recombinant. This library was analyzed by the hybridization method on columns with two radioactive ( 32 P) probes: the total cDNA preparation and the total nuclear DNA from the human brain. The number of copies of the cloned DNA fragment in the genome was determined by dot hybridization. Restricting fragments of human and rat DNA genomes homologous to the cloned cDNA were identified on radio-autographs. In each case, 10 micrograms of EcoRI DNA hydrolyzate was fractionated in 1% agarose gel. The probe was also readied with RNA samples fractionated in agarose gel with formaldehyde and transferred to a nitrocellulose filter under weak vacuum. The filter was hybridized with 0.1 micrograms DNA pAG 02, labeled with ( 32 P) to a specific activity of 0.5-1 x 10 9 counts/min x microgram. The autograph was exposed with amplifying screens at -70 0 C for 2 days

Nucleotide sequence preservation of human mitochondrial DNA

International Nuclear Information System (INIS)

Monnat, R.J. Jr.; Loeb, L.A.

1985-01-01

Recombinant DNA techniques have been used to quantitate the amount of nucleotide sequence divergence in the mitochondrial DNA population of individual normal humans. Mitochondrial DNA was isolated from the peripheral blood lymphocytes of five normal humans and cloned in M13 mp11; 49 kilobases of nucleotide sequence information was obtained from 248 independently isolated clones from the five normal donors. Both between- and within-individual differences were identified. Between-individual differences were identified in approximately = to 1/200 nucleotides. In contrast, only one within-individual difference was identified in 49 kilobases of nucleotide sequence information. This high degree of mitochondrial nucleotide sequence homogeneity in human somatic cells is in marked contrast to the rapid evolutionary divergence of human mitochondrial DNA and suggests the existence of mechanisms for the concerted preservation of mammalian mitochondrial DNA sequences in single organisms

Development of a recombinant DNA assay system for the detection of genetic change in astronauts' cells

International Nuclear Information System (INIS)

Atchley, S.V.; Chen, D.J.C.; Strniste, G.F.; Walters, R.A.; Moyzis, R.K.

1984-01-01

We are developing a new recombinant DNA system for the detection and measurement of genetic change in humans caused by exposure to low level ionizing radiation. A unique feature of the method is the use of cloned repetitive DNA probes to assay human DNA for structural changes during or after irradiation. Repetitive sequences exist in different families. Collectively they constitute over 25% of the DNA in a human cell. Repeat families have between 10 and 500,000 members. We have constructed repetitive DNA sequence libraries using recombinant DNA techniques. From these libraries we have isolated and characterized individual repeats comprising 75 to 90% of the mass of human repetitive DNA. Repeats used in our assay system exist in tandem arrays in the genome. Perturbation of these sequences in a cell, followed by detection with a repeat probe, produces a new, multimeric ''ladder'' pattern on an autoradiogram. The repeat probe used in our initial study is complementary to 1% of human DNA. Therefore, the sensitivity of this method is several orders of magnitude better than existing assays. Preliminary evidence from human skin cells exposed to acute, low-dose x-ray treatments indicates that DNA is affected at a dose as low as 5R. The radiation doses used in this system are well within the range of doses received by astronauts during spaceflight missions. Due to its small material requirements, this technique could easily be adapted for use in space. 16 refs., 1 fig

CDNA cloning, characterization and expression of an endosperm-specific barley peroxidase

DEFF Research Database (Denmark)

Rasmussen, Søren Kjærsgård; Welinder, K.G.; Hejgaard, J.

1991-01-01

A barley peroxidase (BP 1) of pI ca. 8.5 and M(r) 37000 has been purified from mature barley grains. Using antibodies towards peroxidase BP 1, a cDNA clone (pcR7) was isolated from cDNA expression library. The nucleotide sequence of pcR7 gave a derived amino acid sequence identical to the 158 C...

Molecular cloning and characterization of strictosidine synthase, a ...

African Journals Online (AJOL)

Mitragynine is one of the most dominant indole alkaloids present in the leaves of Mitragyna speciosa, a species of Rubiaceae. This alkaloid is believed to be synthesized via condensation of the amino acid derivative, tryptamine and secologanine by the action of strictosidine synthase (STR). The cDNA clone encoding STR ...

Serine Protease Variants Encoded by Echis ocellatus Venom Gland cDNA: Cloning and Sequencing Analysis

Directory of Open Access Journals (Sweden)

S. S. Hasson

2010-01-01

Full Text Available Envenoming by Echis saw-scaled viper is the leading cause of death and morbidity in Africa due to snake bite. Despite its medical importance, there have been few investigations into the toxin composition of the venom of this viper. Here, we report the cloning of cDNA sequences encoding four groups or isoforms of the haemostasis-disruptive Serine protease proteins (SPs from the venom glands of Echis ocellatus. All these SP sequences encoded the cysteine residues scaffold that form the 6-disulphide bonds responsible for the characteristic tertiary structure of venom serine proteases. All the Echis ocellatus EoSP groups showed varying degrees of sequence similarity to published viper venom SPs. However, these groups also showed marked intercluster sequence conservation across them which were significantly different from that of previously published viper SPs. Because viper venom SPs exhibit a high degree of sequence similarity and yet exert profoundly different effects on the mammalian haemostatic system, no attempt was made to assign functionality to the new Echis ocellatus EoSPs on the basis of sequence alone. The extraordinary level of interspecific and intergeneric sequence conservation exhibited by the Echis ocellatus EoSPs and analogous serine proteases from other viper species leads us to speculate that antibodies to representative molecules should neutralise (that we will exploit, by epidermal DNA immunization the biological function of this important group of venom toxins in vipers that are distributed throughout Africa, the Middle East, and the Indian subcontinent.

Molecular characterization of two sweepoviruses from China and evaluation of the infectivity of cloned SPLCV-JS in Nicotiana benthamiana.

Science.gov (United States)

Bi, Huiping; Zhang, Peng

2012-03-01

Sweepoviruses are important begomoviruses that infect Ipomoea plants worldwide and cause sweet potato yield losses and cultivar decline. Two sweepoviruses, sweet potato leaf curl virus-Jiangsu (SPLCV-JS) and sweet potato leaf curl China virus-Zhejiang (SPLCCNV-ZJ), were cloned from diseased sweet potato plants collected in the Jiangsu and Zhejiang provinces of China. Sequence characterization and phylogenetic analysis demonstrated that both are typical monopartite begomoviruses and have close relationships to several reported SPLCV and SPLCCNV isolates, respectively, from Asian countries. Analysis of the protein alignments and subcellular localizations of the six SPLCV-JS proteins was also conducted to verify their putative functions. In Nicotiana benthamiana, an infectivity assay of the infectious SPLCV-JS clone resulted in mild symptoms and weak viral DNA accumulation. Interestingly, SPLCV-JS, together with a heterologous betasatellite DNA (tomato yellow leaf curl China virus isolate Y10 [TYLCCNV-Y10] DNA-β), showed a synergistic effect on enhanced symptom severity and viral DNA accumulation. This is the first reported infectious SPLCV clone.

Cloning and expression of the receptor for human urokinase plasminogen activator, a central molecule in cell surface, plasmin dependent proteolysis

DEFF Research Database (Denmark)

Roldan, A.L.; Cubellis, M.V.; Masucci, M.T.

1990-01-01

, and therefore the capacity of cells to migrate and invade neighboring tissues. We have isolated a 1.4 kb cDNA clone coding for the entire human uPAR. An oligonucleotide synthesized on the basis of the N-terminal sequence of the purified protein was used to screen a cDNA library made from SV40 transformed human......, a size very close to that of the cloned cDNA. Expression of the uPAR cDNA in mouse cells confirms that the clone is complete and expresses a functional uPA binding protein, located on the cell surface and with properties similar to the human uPAR. Caseinolytic plaque assay, immunofluorescence analysis......The surface receptor for urokinase plasminogen activator (uPAR) has been recognized in recent years as a key molecule in regulating plasminogen mediated extracellular proteolysis. Surface plasminogen activation controls the connections between cells, basement membrane and extracellular matrix...

Discovering hidden biodiversity: the use of complementary monitoring of fish diet based on DNA barcoding in freshwater ecosystems.

Science.gov (United States)

Jo, Hyunbin; Ventura, Marc; Vidal, Nicolas; Gim, Jeong-Soo; Buchaca, Teresa; Barmuta, Leon A; Jeppesen, Erik; Joo, Gea-Jae

2016-01-01

Ecological monitoring contributes to the understanding of complex ecosystem functions. The diets of fish reflect the surrounding environment and habitats and may, therefore, act as useful integrating indicators of environmental status. It is, however, often difficult to visually identify items in gut contents to species level due to digestion of soft-bodied prey beyond visual recognition, but new tools rendering this possible are now becoming available. We used a molecular approach to determine the species identities of consumed diet items of an introduced generalist feeder, brown trout (Salmo trutta), in 10 Tasmanian lakes and compared the results with those obtained from visual quantification of stomach contents. We obtained 44 unique taxa (OTUs) belonging to five phyla, including seven classes, using the barcode of life approach from cytochrome oxidase I (COI). Compared with visual quantification, DNA analysis showed greater accuracy, yielding a 1.4-fold higher number of OTUs. Rarefaction curve analysis showed saturation of visually inspected taxa, while the curves from the DNA barcode did not saturate. The OTUs with the highest proportions of haplotypes were the families of terrestrial insects Formicidae, Chrysomelidae, and Torbidae and the freshwater Chironomidae. Haplotype occurrence per lake was negatively correlated with lake depth and transparency. Nearly all haplotypes were only found in one fish gut from a single lake. Our results indicate that DNA barcoding of fish diets is a useful and complementary method for discovering hidden biodiversity.

Construction of a self-cloning system in the unicellular green alga Pseudochoricystis ellipsoidea.

Science.gov (United States)

Kasai, Yuki; Oshima, Kohei; Ikeda, Fukiko; Abe, Jun; Yoshimitsu, Yuya; Harayama, Shigeaki

2015-01-01

Microalgae have received considerable interest as a source of biofuel production. The unicellular green alga Pseudochoricystis ellipsoidea (non-validated scientific name) strain Obi appears to be suitable for large-scale cultivation in outdoor open ponds for biodiesel production because it accumulates lipids to more than 30 % of dry cell weight under nitrogen-depleted conditions. It also grows rapidly under acidic conditions at which most protozoan grazers of microalgae may not be tolerant. The lipid productivity of this alga could be improved using genetic engineering techniques; however, genetically modified organisms are the subject of regulation by specific laws. Therefore, the aim of this study was to develop a self-cloning-based positive selection system for the breeding of P. ellipsoidea. In this study, uracil auxotrophic mutants were isolated after the mutagenesis of P. ellipsoidea using either ultraviolet light or a transcription activator-like effector nuclease (TALEN) system. The cDNA of the uridine monophosphate synthase gene (PeUMPS) of P. ellipsoidea was cloned downstream of the promoter of either a beta-tubulin gene (PeTUBULIN1) or the gene for the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (PeRBCS) to construct the pUT1 or pUT2 plasmid, respectively. These constructs were introduced into uracil auxotroph strains, and genetically complementary transformants were isolated successfully on minimal agar plates. Use of Noble agar as the solidifying agent was essential to avoid the development of false-positive colonies. It took more than 6 weeks for the formation of colonies of pUT1 transformants, whereas pUT2 transformants formed colonies in 2 weeks. Real-time PCR revealed that there were more PeUMPS transcripts in pUT2 transformants than in pUT1 transformants. Uracil synthesis (Ura(+)) transformants were also obtained using a gene cassette consisting solely of PeUMPS flanked by the PeRBCS promoter and terminator. A self-cloning