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Sample records for beta globin gene

  1. Design of retrovirus vectors for transfer and expression of the human. beta. -globin gene

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    Miller, A.D.; Bender, M.A.; Harris, E.A.S.; Kaleko, M.; Gelinas, R.E.

    1988-11-01

    Regulated expression of the human ..beta..-globin gene has been demonstrated in cultured murine erythroleukemia cells and in mice after retrovirus-mediated gene transfer. However, the low titer of recombinant viruses described to date results in relatively inefficient gene transfer, which limits their usefulness for animal studies and for potential gene therapy in humans for diseases involving defective ..beta..-globin genes. The authors found regions that interfered with virus production within intron 2 of the ..beta..-globin gene and on both sides of the gene. The flanking regions could be removed, but intron 2 was required for ..beta..-globin expression. Inclusion of ..beta..-globin introns necessitates an antisense orientation of the gene within the retrovirus vector. However, they found no effect of the antisense ..beta..-globin transcription on virus production. A region downstream of the ..beta..-globin gene that stimulates expression of the gene in transgenic mice was included in the viruses without detrimental effects on virus titer. Virus titers of over 10/sup 6/ CFU/ml were obtained with the final vector design, which retained the ability to direct regulated expression of human ..beta..-globin in murine erythroleukemia cells. The vector also allowed transfer and expression of the human ..beta..-globin gene in hematopoietic cells (CFU-S cells) in mice.

  2. Structure of the human G gamma-A gamma-delta-beta-globin gene locus

    NARCIS (Netherlands)

    Bernards, R.A.; Little, P.F.R.; Annison, F.; Williamson, R.; Flavell, R.A.

    1979-01-01

    We have constructed a physical map of the human G gamma-, A gamma-, delta-, and beta-globin genes. The previously described maps of the fetal and adult beta-like globin genes have been linked to one another by identification of a DNA fragment, generated by BamHI, that contains part of each of the A

  3. The role of EKLF in human beta-globin gene competition.

    Science.gov (United States)

    Wijgerde, M; Gribnau, J; Trimborn, T; Nuez, B; Philipsen, S; Grosveld, F; Fraser, P

    1996-11-15

    We have investigated the role of erythroid Kruppel-like factor (EKLF) in expression of the human beta-globin genes in compound EKLF knockout/human beta-locus transgenic mice. EKLF affects only the adult mouse beta-globin genes in homozygous knockout mice; heterozygous mice are unaffected. Here we show that EKLF knockout mice express the human epsilon and gamma-globin genes normally in embryonic red cells. However, fetal liver erythropoiesis, which is marked by a period of gamma- and beta-gene competition in which the genes are alternately transcribed, exhibits an altered ratio of gamma- to beta-gene transcription. EKLF heterozygous fetal livers display a decrease in the number of transcriptionally active beta genes with a reciprocal increase in the number of transcriptionally active gamma genes. beta-Gene transcription is absent in homozygous knockout fetuses with coincident changes in chromatin structure at the beta promoter. There is a further increase in the number of transcriptionally active gamma genes and accompanying gamma gene promoter chromatin alterations. These results indicate that EKLF plays a major role in gamma- and beta-gene competition and suggest that EKLF is important in stabilizing the interaction between the Locus Control Region and the beta-globin gene. In addition, these findings provide further evidence that developmental modulation of globin gene expression within individual cells is accomplished by altering the frequency and/or duration of transcriptional periods of a gene rather than changing the rate of transcription.

  4. Cis-cotranscription of two beta globin genes during chicken primitive hematopoiesis.

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    Hiroki Nagai

    Full Text Available Chicken beta globin locus contains four genes, two of which, rho and epsilon, are expressed from the earliest stage of primitive hematopoiesis. Here we show that the transcription of these two genes in the nucleus engages in "on/off" phases. During each "on" phase, cotranscription of rho and epsilon in cis is favored. We propose that these two chicken beta globin genes are transcribed not by competing for a transcription initiation complex, but in a cooperative way.

  5. Detection of Rare Beta Globin Gene Mutations in Northwestern Iran

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    M Haghi

    2007-04-01

    Full Text Available Introduction: Recent molecular studies on Iranian β-thalassemia genes revealed the presence of eight common mutations associated with thalassemia. Although these mutations are frequent, there are other rare and unknown mutations that can create large problems in designing preventive programs. We detected and explained the common mutations in north-western Iran previously and detection of the rare and unknown mutations could be useful in diagnosis and design of future preventive programs. Methods: In this study, 5ml peripheral blood from 20 Azari- β-thalassemia patients whose mutation was not revealed in the previous study was collected and DNA extraction was done by isopropanol and proteinase k method. Initially, samples were examined for the rare mutations: Codon6, Codon16, Codon41/42, Codon36/37, -88 and Codon22 by ARMS – PCR techniques and then the unknown cases were directly sequenced. Results: According to our results, Codon15(TGG-TGA, Codon16(-C, Codon36/37(-T, IVSII-848(C-A, IVSII-745(C-G, -28(A-C( and Codon25/26(+T were recognized and added to the spectrom of beta globin gene mutations in Azerbaijan and Iran. Also, we detected four SNP sites: 5’UTR+20(C-T, Codon2 (CAC-CAT , IVSII-16(C-G and IVSII-666(T-C in β-thalassemia genes. Conclusion: Our results could be useful for developing molecular screening plans and help prenatal diagnosis of beta thalassemia in Azerbaijan , Iran and other neighboring countries.

  6. Transformation of frog embryos with a rabbit beta-globin gene.

    OpenAIRE

    Rusconi, S; Schaffner, W

    1981-01-01

    In order to study the fate and possible expression of foreign DNA during embryogenesis of the frog Xenopus laevis, we have injected a rabbit beta-globin gene into fertilized Xenopus eggs. Frog embryo DNA was extracted at various stages of development, fractionated by agarose gel electrophoresis, transferred to nitrocellulose filters, and hybridized to labeled beta-globin recombinant plasmid DNA. It was found that the injected DNA replicated extrachromosomally, reaching, at gastrula stage, a l...

  7. The beta-globin gene in Sardinian delta beta 0-thalassemia carries a C----T nonsense mutation at codon 39.

    OpenAIRE

    Guida, S; Giglioni, B; Comi, P; Ottolenghi, S; Camaschella, C.; Saglio, G

    1984-01-01

    Sardinian delta beta 0-thalassemia is an inherited syndrome characterized by the inactivity of the beta-globin gene and the persistent activity of the fetal gamma-globin genes, particularly the A gamma-globin gene. Previous mapping studies with restriction enzymes failed to show any abnormality in the non-alpha globin gene cluster. We have now examined the possibility that this syndrome might result from a single rather than two different defects. Restriction enzyme polymorphisms linked to th...

  8. Molecular cloning and characterization of the human beta-like globin gene cluster.

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    Fritsch, E F; Lawn, R M; Maniatis, T

    1980-04-01

    The genes encoding human embryonic (epsilon), fetal (G gamma, A gamma) and adult (delta, beta) beta-like globin polypeptides were isolated as a set of overlapping cloned DNA fragments from bacteriophage lambda libraries of high molecular weight (15-20 kb) chromosomal DNA. The 65 kb of DNA represented in these overlapping clones contains the genes for all five beta-like polypeptides, including the embryonic epsilon-globin gene, for which the chromosomal location was previously unknown. All five genes are transcribed from the same DNA strand and are arranged in the order 5'-epsilon-(13.3 kb)-G gamma-(3.5 kb)-A gamma-(13.9 kb)-delta-(5.4 kb)-beta-3'. Thus the genes are positioned on the chromosome in the order of their expression during development. In addition to the five known beta-like globin genes, we have detected two other beta-like globin sequences which do not correspond to known polypeptides. One of these sequences has been mapped to the A gamma-delta intergenic region while the other is located 6-9 kb 5' to the epsilon gene. Cross hybridization experiments between the intergenic sequences of the gene cluster have revealed a nonglobin repeat sequence (*) which is interspersed with the globin genes in the following manner: 5'-**epsilon-*G gamma-A gamma*-**delta-beta*-3'. Fine structure mapping of the region located 5' to the delta-globin gene revealed two repeats with a maximum size of 400 bp, which are separated by approximately 700 bp of DNA not repeated within the cluster. Preliminary experiments indicate that this repeat family is also repeated many times in the human genome.

  9. Globin gene transfer for treatment of the beta-thalassemias and sickle cell disease.

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    Sadelain, Michel; Rivella, Stefano; Lisowski, Leszek; Samakoglu, Selda; Rivière, Isabelle

    2004-09-01

    The beta-thalassemias and sickle cell disease are severe congenital anemias that are caused by mutations that alter the production of the beta chain of hemoglobin. Allogeneic hematopoietic stem cell (HSC) transplantation is curative, but this therapeutic option is not available to the majority of patients. The transfer of a functional globin gene in autologous HCSs thus represents a highly attractive alternative treatment. This strategy, simple in principle, raises major challenges in terms of controlling the expression of the globin transgene, which ideally should be erythroid specific, differentiation-stage restricted, elevated, position independent, and sustained over time. Using lentiviral vectors, we have demonstrated that an optimised combination of proximal and distal transcriptional control elements permits lineage-specific, elevated expression of the beta-globin gene, resulting in therapeutic hemoglobin production and correction of anemia in beta-thalassemic mice. Several groups have now confirmed and extended these findings in various mouse models of severe hemoglobinopathies, thus generating enthusiasm for a genetic treatment based on globin gene transfer. Furthermore, globin vectors represent a general paradigm for the regulation of transgene function and the improvement of vector safety by restricting transgene expression to the differentiated progeny within a single lineage, thereby reducing the risk of activating oncogenes in hematopoietic progenitors. Here we review the principles underlying the genesis of regulated vectors for stem cell therapy.

  10. Identification and molecular characterization of four new large deletions in the beta-globin gene cluster.

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    Joly, Philippe; Lacan, Philippe; Garcia, Caroline; Couprie, Nicole; Francina, Alain

    2009-01-01

    Despite the fact that mutations in the human beta-globin gene cluster are essentially point mutations, a significant number of large deletions have also been described. We present here four new large deletions in the beta-globin gene cluster that have been identified on patients displaying an atypical hemoglobin phenotype (high HbF) at routine analysis. The first deletion, which spreads over 2.0 kb, removes the entire beta-globin gene, including its promoter, and is associated with a typical beta-thal minor phenotype. The three other deletions are larger (19.7 to 23.9 kb) and remove both the delta and beta-globin genes. Phenotypically, they look like an HPFH-deletion as they are associated with normal hematological parameters. The precise localization of their 5' and 3' breakpoints gives new insights about the differences between HPFH and (deltabeta)(0)-thalassemia at the molecular level. The importance of detection of these deletions in prenatal diagnosis and newborn screening of hemoglobinopathies is also discussed.

  11. Meiotic recombination in the beta globin gene cluster causing an error in prenatal diagnosis of beta thalassaemia.

    OpenAIRE

    Camaschella, C.; Serra, A.; Saglio, G; Bertero, M T; Mazza, U; Terzoli, S; Brambati, B; Cremonesi, L.; Travi, M; Ferrari, M

    1988-01-01

    In the course of a prenatal diagnosis for beta thalassaemia by linkage analysis of restriction fragment length polymorphisms, a homozygous beta thalassaemia fetus was misdiagnosed as beta thalassaemia trait. Extensive studies of the polymorphic sites within the beta globin gene cluster in all the members of the family resulted in the conclusion that the paternal chromosome 11 of the newborn was different from that expected. Paternity was confirmed by HLA typing and blood group studies. The an...

  12. Inter-MAR association contributes to transcriptionally active looping events in human beta-globin gene cluster.

    Science.gov (United States)

    Wang, Li; Di, Li-Jun; Lv, Xiang; Zheng, Wei; Xue, Zheng; Guo, Zhi-Chen; Liu, De-Pei; Liang, Chi-Chuan

    2009-01-01

    Matrix attachment regions (MARs) are important in chromatin organization and gene regulation. Although it is known that there are a number of MAR elements in the beta-globin gene cluster, it is unclear that how these MAR elements are involved in regulating beta-globin genes expression. Here, we report the identification of a new MAR element at the LCR (locus control region) of human beta-globin gene cluster and the detection of the inter-MAR association within the beta-globin gene cluster. Also, we demonstrate that SATB1, a protein factor that has been implicated in the formation of network like higher order chromatin structures at some gene loci, takes part in beta-globin specific inter-MAR association through binding the specific MARs. Knocking down of SATB1 obviously reduces the binding of SATB1 to the MARs and diminishes the frequency of the inter-MAR association. As a result, the ACH establishment and the alpha-like globin genes and beta-like globin genes expressions are affected either. In summary, our results suggest that SATB1 is a regulatory factor of hemoglobin genes, especially the early differentiation genes at least through affecting the higher order chromatin structure.

  13. Beta-thalassemia and beta[A] globin gene haplotypes in Mexican mestizos.

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    Villalobos-Arámbula, A R; Bustos, R; Casas-Castañeda, M; Gutiérrez, E; Perea, F J; Thein, S L; Ibarra, B

    1997-04-01

    B-globin haplotypes of 20 beta-thalassemia (beta-thal) and 87 beta(A) Mexican mestizo chromosomes were analyzed to ascertain the origin of the beta-thal alleles and the frequencies and distribution of the beta(A) haplotypes among northwestern Mexican mestizos. Sixteen beta-thal chromosomes carried six Mediterranean alleles [five codon 39 C-->T; two IVS1:1 G-->A; two IVS1:5 G-->A; three IVS1:110 G(A; one codon 11 (-T) and three (deltabeta)zero-thal]; the remaining four were linked to three rare alleles (two -28 A-->C and one each: -87 C-->T and initiation codon ATG-->GTG). Among the 87 beta(A) chromosomes, 17 different 5' haplotypes with frequencies for 1, 3, 2 and 5 of 39.0%, 17. 2%, 9.2% and 6.9%, respectively, were observed. The beta-haplotype analysis showed that 13 out of 16 Mediterranean chromosomes could easily be explained by gene migration; however, one codon 39 associated with haplotype 4 (----+ +-), one IVS1:1 with haplotype 1(+----++) and one IVS1:5 G-->A, may represent separate mutational events. Analysis of the rare alleles showed that the -28 A-->C mutation was associated with the commonest beta(A) haplotype in Mexican mestizos, Mediterraneans and the total world population; therefore an independent origin cannot be ruled out. The -87 C-->T and initiation codon ATG-->GTG were found with beta-haplotypes different from the reported ones, suggesting an indigenous origin.

  14. Total beta-globin gene deletion has high frequency in Filipinos

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    Patrick, N.; Miyakawa, F.; Hunt, J.A. [Univ. of Hawaii, Honolulu, HI (United States)] [and others

    1994-09-01

    The distribution of {beta}-thalassemia [{beta}{sup Th}] mutations is unique to each ethnic group. Most mutations affect one or a few bases; large deletions have been rare. Among families screened in Hawaii, [{beta}{sup Th}] heterozygotes were diagnosed by microcytosis, absence of abnormal hemoglobins on isoelectric focusing, and raised Hb A{sub 2} by chromatography. Gene frequency for {beta}{sup Th} was 0.02 in Filipinos. In Filipinos, polymerase chain reaction [PCR] with denaturing gradient gel electrophoresis for {beta}{sup Th} mutations detected a mutation in only 6 of 42 {beta}{sup Th} heterozygotes; an IVS2-666 C/T polymorphism showed non-heterozygosity in 37 and heterozygosity in only 5 of these {beta}{sup Th} heterozygotes. One {beta}{sup Th}/{beta}{sup Th} major patient and his mother had no mutation detected by allele-specific oligomer hybridization; PCR failed to amplify any DNA from his {beta}-globin gene. After a total {beta}-globin gene deletion [{beta}{sup Del}] was found in a Filipino family in Ontario, specific PCR amplification for {beta}{sup Del} detected this in 43 of 53 {beta}{sup Th} Filipino samples tested; the above {beta}{sup Th}/{beta}{sup Th} patient was a ({beta}{sup Del}/{beta}{sup Del}) homozygote. The {beta}{sup Del} may account for over 60% of all {beta}{sup Th} alleles in Filipinos; this is the highest proportion of a deletion {beta}{sup Th} mutation reported from any population. Most but not all {beta}{sup Del} heterozygotes had high Hb F [5.13 {plus_minus} 3.94 mean {plus_minus} 1 s.d.] compared to the codon 41/42 four base deletion common in Chinese [2.30 {plus_minus} 0.86], or to {beta}{sup Th} heterozygotes with normal {alpha}-globin genes [2.23 {plus_minus} 0.80].

  15. Successful treatment of murine beta-thalassemia intermedia by transfer of the human beta-globin gene.

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    May, Chad; Rivella, Stefano; Chadburn, Amy; Sadelain, Michel

    2002-03-15

    The beta-thalassemias are caused by more than 200 mutations that reduce or abolish beta-globin production. The severity of the resulting anemia can lead to lifelong transfusion dependency. A genetic treatment based on globin gene transfer would require that transgene expression be erythroid specific, elevated, and sustained over time. We report here that long-term synthesis of chimeric hemoglobin (mualpha(2):hubeta(A)(2)) could be achieved in mice with beta-thalassemia intermedia following engraftment with bone marrow cells transduced with a lentiviral vector encoding the human beta-globin gene. In the absence of any posttransduction selection, the treated chimeras exhibit durably increased hemoglobin levels without diminution over 40 weeks. Ineffective erythropoiesis and extramedullary hematopoiesis (EMH) regress, as reflected by normalization of spleen size, architecture, hematopoietic colony formation, and disappearance of liver EMH. These findings establish that a sustained increase of 3 to 4 g/dL hemoglobin is sufficient to correct ineffective erythropoiesis. Hepatic iron accumulation is markedly decreased in 1-year-old chimeras, indicating persistent protection from secondary organ damage. These results demonstrate for the first time that viral-mediated globin gene transfer in hematopoietic stem cells effectively treats a severe hemoglobin disorder.

  16. beta(S)-Globin gene cluster haplotypes in the West Bank of Palestine.

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    Samarah, Fekri; Ayesh, Suhail; Athanasiou, Miranda; Christakis, John; Vavatsi, Norma

    2009-01-01

    Sickle cell disease is an inherited autosomal recessive disorder of the beta-globin chain. In Palestine it is accompanied by a low level of Hb F (mean 5.14%) and a severe clinical presentation. In this study, 59 Palestinian patients, homozygotes for Hb S were studied for their haplotype background. Eight polymorphic sites in the beta-globin gene cluster were examined. The Benin haplotype was predominant with a frequency of 88.1%, followed by a frequency of 5.1% for the Bantu haplotype. One chromosome was found to carry the Cameroon haplotype (0.85%). Three atypical haplotypes were also found (5.95%). Heterogeneity was observed in Hb F production, ranging between 1.5 and 17.0%, whereas the (G)gamma ratio was homogeneous among all haplotypes with a normal amount of about 41%. Our results are in agreement with previous reports of the Benin haplotype origin in the Mediterranean.

  17. A mutation of the beta-globin gene initiation codon, ATG-->AAG, found in a French Caucasian man.

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    Lacan, Philippe; Aubry, Martine; Couprie, Nicole; Francina, Alain

    2005-01-01

    A new mutation of the beta-globin gene initiation codon, ATG-->AAG (Met-->Tyr), is reported in a man originating from the southeast of France. Typical hematological findings of beta-thalassemia (thal) trait were found. We emphasize the importance of characterizing uncommon beta-thal mutations for genetic counseling.

  18. Origin and ascendancy of a chimeric fusion gene: the beta/delta-globin gene of paenungulate mammals.

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    Opazo, Juan C; Sloan, Angela M; Campbell, Kevin L; Storz, Jay F

    2009-07-01

    The delta-globin gene (HBD) of eutherian mammals exhibits a propensity for recombinational exchange with the closely linked beta-globin gene (HBB) and has been independently converted by the HBB gene in multiple lineages. Here we report the presence of a chimeric beta/delta fusion gene in the African elephant (Loxodonta africana) that was created by unequal crossing-over between misaligned HBD and HBB paralogs. The recombinant chromosome that harbors the beta/delta fusion gene in elephants is structurally similar to the "anti-Lepore" duplication mutant of humans (the reciprocal exchange product of the hemoglobin Lepore deletion mutant). However, the situation in the African elephant is unique in that the chimeric beta/delta fusion gene supplanted the parental HBB gene and is therefore solely responsible for synthesizing the beta-chain subunits of adult hemoglobin. A phylogenetic survey of beta-like globin genes in afrotherian and xenarthran mammals revealed that the origin of the chimeric beta/delta fusion gene and the concomitant inactivation of the HBB gene predated the radiation of "Paenungulata," a clade of afrotherian mammals that includes three orders: Proboscidea (elephants), Sirenia (dugongs and manatees), and Hyracoidea (hyraxes). The reduced fitness of the human Hb Lepore deletion mutant helps to explain why independently derived beta/delta fusion genes (which occur on an anti-Lepore chromosome) have been fixed in a number of mammalian lineages, whereas the reciprocal delta/beta fusion gene (which occurs on a Lepore chromosome) has yet to be documented in any nonhuman mammal. This illustrates how the evolutionary fates of chimeric fusion genes can be strongly influenced by their recombinational mode of origin.

  19. Molecular analysis of beta-globin gene mutations among Thai beta-thalassemia children: results from a single center study

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    Boonyawat B

    2014-12-01

    Full Text Available Boonchai Boonyawat,1 Chalinee Monsereenusorn,2 Chanchai Traivaree2 1Division of Genetics, Department of Pediatrics, Phramongkutklao Hospital and College of Medicine, Bangkok, Thailand; 2Division of Hematology/Oncology, Department of Pediatrics, Phramongkutklao Hospital and College of Medicine, Bangkok, Thailand Background: Beta-thalassemia is one of the most common genetic disorders in Thailand. Clinical phenotype ranges from silent carrier to clinically manifested conditions including severe beta-thalassemia major and mild beta-thalassemia intermedia. Objective: This study aimed to characterize the spectrum of beta-globin gene mutations in pediatric patients who were followed-up in Phramongkutklao Hospital. Patients and methods: Eighty unrelated beta-thalassemia patients were enrolled in this study including 57 with beta-thalassemia/hemoglobin E, eight with homozygous beta-thalassemia, and 15 with heterozygous beta-thalassemia. Mutation analysis was performed by multiplex amplification refractory mutation system (M-ARMS, direct DNA sequencing of beta-globin gene, and gap polymerase chain reaction for 3.4 kb deletion detection, respectively. Results: A total of 13 different beta-thalassemia mutations were identified among 88 alleles. The most common mutation was codon 41/42 (-TCTT (37.5%, followed by codon 17 (A>T (26.1%, IVS-I-5 (G>C (8%, IVS-II-654 (C>T (6.8%, IVS-I-1 (G>T (4.5%, and codon 71/72 (+A (2.3%, and all these six common mutations (85.2% were detected by M-ARMS. Six uncommon mutations (10.2% were identified by DNA sequencing including 4.5% for codon 35 (C>A and 1.1% initiation codon mutation (ATG>AGG, codon 15 (G>A, codon 19 (A>G, codon 27/28 (+C, and codon 123/124/125 (-ACCCCACC, respectively. The 3.4 kb deletion was detected at 4.5%. The most common genotype of beta-thalassemia major patients was codon 41/42 (-TCTT/codon 26 (G>A or betaE accounting for 40%. Conclusion: All of the beta-thalassemia alleles have been characterized by

  20. Beta-globin LCR and intron elements cooperate and direct spatial reorganization for gene therapy.

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    Alla Buzina

    2008-04-01

    Full Text Available The Locus Control Region (LCR requires intronic elements within beta-globin transgenes to direct high level expression at all ectopic integration sites. However, these essential intronic elements cannot be transmitted through retrovirus vectors and their deletion may compromise the therapeutic potential for gene therapy. Here, we systematically regenerate functional beta-globin intron 2 elements that rescue LCR activity directed by 5'HS3. Evaluation in transgenic mice demonstrates that an Oct-1 binding site and an enhancer in the intron cooperate to increase expression levels from LCR globin transgenes. Replacement of the intronic AT-rich region with the Igmu 3'MAR rescues LCR activity in single copy transgenic mice. Importantly, a combination of the Oct-1 site, Igmu 3'MAR and intronic enhancer in the BGT158 cassette directs more consistent levels of expression in transgenic mice. By introducing intron-modified transgenes into the same genomic integration site in erythroid cells, we show that BGT158 has the greatest transcriptional induction. 3D DNA FISH establishes that induction stimulates this small 5'HS3 containing transgene and the endogenous locus to spatially reorganize towards more central locations in erythroid nuclei. Electron Spectroscopic Imaging (ESI of chromatin fibers demonstrates that ultrastructural heterochromatin is primarily perinuclear and does not reorganize. Finally, we transmit intron-modified globin transgenes through insulated self-inactivating (SIN lentivirus vectors into erythroid cells. We show efficient transfer and robust mRNA and protein expression by the BGT158 vector, and virus titer improvements mediated by the modified intron 2 in the presence of an LCR cassette composed of 5'HS2-4. Our results have important implications for the mechanism of LCR activity at ectopic integration sites. The modified transgenes are the first to transfer intronic elements that potentiate LCR activity and are designed to facilitate

  1. Beta-globin gene cluster haplotypes in Venezuelan sickle cell patients from the State of Aragua

    OpenAIRE

    Nancy Moreno; Martínez, José A.; Zorella Blanco; Leidys Osorio; Patrick Hackshaw

    2002-01-01

    Seven polymorphic sites in the beta-globin gene cluster were analyzed on a sample of 96 chromosomes of Venezuelan sickle cell patients from the State of Aragua. The Benin haplotype was predominant with a frequency of 0.479, followed by the Bantu haplotype (0.406); a minority of cases with other haplotypes was also identified: atypical Bantu A2 (0.042), Senegal (0.031), atypical Bantu A7 (0.021) and Saudi Arabia/Indian (0.021) haplotypes; however, the Cameroon haplotype was not identified in t...

  2. Therapeutic globin gene delivery using lentiviral vectors.

    Science.gov (United States)

    Rivella, Stefano; Sadelain, Michel

    2002-10-01

    The severe hemoglobinopathies, including beta-thalassemia major and sickle cell anemia, are candidate diseases for a genetic treatment based on the transfer of a regulated globin gene in autologous hematopoietic stem cells. Two years ago, May et al reported that an optimized beta-globin transcription unit containing multiple proximal and distal regulatory elements harbored by a recombinant lentiviral vector could efficiently integrate into murine hematopoietic stem cells and express therapeutic levels of the human beta-globin gene. Here, we review the advantages afforded by lentivirus-mediated globin gene transfer and recent studies based on this strategy.

  3. Characterization of a large deletion in the {beta}-globin gene cluster in a newborn with hemoglobin FE

    Energy Technology Data Exchange (ETDEWEB)

    Louie, E.; Dietz, L.; Shafer, F. [Children`s Hosptial, Oakland, CA (United States)] [and others

    1994-09-01

    A sample on a newborn with hemoglobin FE screen results was obtained to investigate whether E/E or B/{beta}{degrees} thalassemia was present using polymerase chain reaction (PCR) methodology. The newborn appeared homozygous for the hemoglobin E mutation in our initial study, but the parents` genotypes did not support this diagnosis. The father is homozygous for the absence of the hemoglobin E mutation (non E/non E) and the mother is heterozygous (E/non E) for this mutation. The limitation of PCR analysis is an assumption that the amplification of the two {beta}-globin alleles is equivalent. A large deletion on one {beta}-globin gene, which would produce E/{beta}{degrees} thalassemia, would be missed if it included part or the entire region subjected to amplification. The family results were consistent with either non-paternity, sample mix-up or such a deletion of the {beta}-globin gene in the father and child. To rule out the possibility of non-paternity, two polymorphic loci (HLA on chromosome 6 and a VNTR system of chromosome 17) that are outside of the {beta}-globin gene were analyzed and show that inheritance is consistent and the likelihood of a sample mix-up is then reduced. We therefore believe there is a gene deletion in this family. At the present time, analyses of the RFLPs that are 5{prime} of the {beta}-globin gene cluster show that the polymorphisms most distal from the 5{prime} {beta}-globin gene are not being inherited as expected. These results support our interpretation that a deletion exists in the father and was inherited by the child. The father`s clinical picture of possible HPFH (the father has 12% hemoglobin F) also supports the interpretation of a deletion in this family. Deletions of the {beta}-globin gene within this ethnic group are rare. Currently, Southern blots on the family are being probed to determine the extent of the putative deletion.

  4. Inter-ethnic polymorphism of the beta-globin gene locus control region (LCR) in sickle-cell anemia patients.

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    Périchon, B; Ragusa, A; Lapouméroulie, C; Romand, A; Moi, P; Ikuta, T; Labie, D; Elion, J; Krishnamoorthy, R

    1993-06-01

    Sequence polymorphisms within the 5'HS2 segment of human locus control region is described among sickle cell anemia patients. Distinct polymorphic patterns of a simple sequence repeat are observed in strong linkage disequilibrium with each of the five major beta s haplotypes. Potential functional relevance of this polymorphic region in globin gene expression is discussed.

  5. Correction of a splice-site mutation in the beta-globin gene stimulated by triplex-forming peptide nucleic acids

    DEFF Research Database (Denmark)

    Chin, Joanna Y; Kuan, Jean Y; Lonkar, Pallavi S

    2008-01-01

    Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin, causing beta-thalassemia. Triplex-forming DNA oligonucleotides (TFOs) and peptide nucleic acids (PNAs) have been shown to stimulate recombination in reporter gene loci in mammalian...... DNA fragments, can promote single base-pair modification at the start of the second intron of the beta-globin gene, the site of a common thalassemia-associated mutation. This single base pair change was detected by the restoration of proper splicing of transcripts produced from a green fluorescent...

  6. Beta-globin gene cluster haplotypes in Venezuelan sickle cell patients from the State of Aragua

    Directory of Open Access Journals (Sweden)

    Nancy Moreno

    2002-01-01

    Full Text Available Seven polymorphic sites in the beta-globin gene cluster were analyzed on a sample of 96 chromosomes of Venezuelan sickle cell patients from the State of Aragua. The Benin haplotype was predominant with a frequency of 0.479, followed by the Bantu haplotype (0.406; a minority of cases with other haplotypes was also identified: atypical Bantu A2 (0.042, Senegal (0.031, atypical Bantu A7 (0.021 and Saudi Arabia/Indian (0.021 haplotypes; however, the Cameroon haplotype was not identified in this study. Our results are in agreement with the historical records that establish Sudanese and Bantu origins for the African slaves brought into Venezuela.

  7. A review on the origin and spread of deleterious mutants of the beta-globin gene in Indian populations.

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    Das, S K; Talukder, G

    2001-01-01

    Deleterious mutations of the human beta-globin gene are responsible for beta-thalassaemia and other haemoglobinopathies, which are the most common genetic diseases in Indian populations. A highly heterogeneous distribution of those mutations is observed in India and certain mutations are restricted to some extent to particular groups only. The reasons behind the geographical clustering and origin of the mutations in India is a highly debated issue and the evidence is conflicting. Our present article aims at tracing the origin of the deleterious beta-globin mutation and evaluates the role of different evolutionary forces responsible for the spread and present distribution of those mutations in Indian populations, using data from molecular biology and statistical methods. Mutations are generated essentially randomly, but "hot-spot" sites for mutation are reported for the beta-globin gene cluster, indicating sequence dependency of mutation. A single origin of a deleterious beta-globin mutation, followed by recombination (in a hot spot region) and/or interallelic gene conversion (within beta-globin gene) through time is the most plausible hypothesis to explain the association of those mutations with multiple haplotype backgrounds and frameworks. It is suggested that India is the place of origin of HbE and HbD mutations and that they dispersed to other parts of the would by migration. HbS mutants present in Indian populations are not of Middle East origin but rather a fresh mutation is the probable explanation for the prevalence among tribal groups. beta-thalassaemia represents a heterogeneous group of mutant alleles in India. Five common and twelve rare mutations have been reported in variable frequencies among different Indian populations. Gene flow of those mutant alleles from different populations of the world by political, military and commercial interactions possibly accounts for the heterogenous nature of beta-thalassaemia among Indians. A multiple allelic

  8. A novel murine model of Cooley anemia and its rescue by lentiviral-mediated human beta-globin gene transfer.

    Science.gov (United States)

    Rivella, Stefano; May, Chad; Chadburn, Amy; Rivière, Isabelle; Sadelain, Michel

    2003-04-15

    Patients affected by beta-thalassemia major require lifelong transfusions because of insufficient or absent production of the beta chain of hemoglobin (Hb). A minority of patients are cured by allogeneic bone marrow transplantation. In the most severe of the hitherto available mouse models of beta-thalassemia, a model for human beta-thalassemia intermedia, we previously demonstrated that globin gene transfer in bone marrow cells is curative, stably raising Hb levels from 8.0-8.5 to 11.0-12.0 g/dL in long-term chimeras. To fully assess the therapeutic potential of gene therapy in the context of a lethal anemia, we now have created an adult model of beta(0)-thalassemia major. In this novel model, mice engrafted with beta-globin-null (Hbb(th3/th3)) fetal liver cells succumb to ineffective erythropoiesis within 60 days. These mice rapidly develop severe anemia (2-4 g/dL), massive splenomegaly, extramedullary hematopoiesis (EMH), and hepatic iron overload. Remarkably, most mice (11 of 13) treated by lentivirus-mediated globin gene transfer were rescued. Long-term chimeras with an average 1.0-2.4 copies of the TNS9 vector in their hematopoietic and blood cells stably produced up to 12 g/dL chimeric Hb consisting of mu alpha(2):hu beta(2) tetramers. Pathologic analyses indicated that erythroid maturation was restored, while EMH and iron overload dramatically decreased. Thus, we have established an adult animal model for the most severe of the hemoglobinopathies, Cooley anemia, which should prove useful to investigate both genetic and pharmacologic treatments. Our findings demonstrate the remarkable efficacy of lentivirus-mediated globin gene transfer in treating a fulminant blood disorder and strongly support the efficacy of gene therapy in the severe hemoglobinopathies.

  9. Beta-globin gene cluster haplotypes and alpha-thalassemia in sickle cell disease patients from Trinidad.

    Science.gov (United States)

    Jones-Lecointe, Altheia; Smith, Erskine; Romana, Marc; Gilbert, Marie-Georges; Charles, Waveney P; Saint-Martin, Christian; Kéclard, Lisiane

    2008-01-01

    In this study, we have determined the frequency of beta(S) haplotypes in 163 sickle cell disease patients from Trinidad. The alpha(3.7) globin gene deletion status was also studied with an observed gene frequency of 0.17. Among the 283 beta(S) chromosomes analyzed, the Benin haplotype was the most prevalent (61.8%) followed by Bantu (17.3%), Senegal (8.5%), Cameroon (3.5%), and Arab-Indian (3.2%), while 5.7% of them were atypical. This beta(S) haplotypes distribution differed from those previously described in other Caribbean islands (Jamaica, Guadeloupe, and Cuba), in agreement with the known involvement of the major colonial powers (Spain, France, and Great Britain) in the slave trade in Trinidad and documented an Indian origin of the beta(S) gene.

  10. Sequential changes in chromatin structure during transcriptional activation in the beta globin LCR and its target gene.

    Science.gov (United States)

    Kim, Kihoon; Kim, AeRi

    2010-09-01

    Chromatin structure is modulated during transcriptional activation. The changes include the association of transcriptional activators, formation of hypersensitive sites and covalent modifications of histones. To understand the order of the various changes accompanying transcriptional activation, we analyzed the mouse beta globin gene, which is transcriptionally inducible in erythroid MEL cells over a time course of HMBA treatment. Transcription of the globin genes requires the locus control region (LCR) consisting of several hypersensitive sites (HSs). Erythroid specific transcriptional activators such as NF-E2, GATA-1, TAL1 and EKLF were associated with the LCR in the uninduced state before transcriptional activation. The HSs of the LCR were formed in this state as revealed by high sensitivity to DNase I and MNase attack. However the binding of transcriptional activators and the depletion of histones were observed in the promoter of the beta globin gene only after transcriptional activation. In addition, various covalent histone modifications were sequentially detected in lysine residues of histone H3 during the activation. Acetylation of K9, K36 and K27 was notable in both LCR HSs and gene after induction but before transcriptional initiation. Inactive histone marks such as K9me2, K36me2 and K27me2 were removed coincident with transcriptional initiation in the gene region. Taken together, these results indicate that LCR has a substantially active structure in the uninduced state while transcriptional activation serially adds active marks, including histone modifications, and removes inactive marks in the target gene of the LCR.

  11. Molecular analysis of the beta-globin gene cluster in the Niokholo Mandenka population reveals a recent origin of the beta(S) Senegal mutation.

    Science.gov (United States)

    Currat, Mathias; Trabuchet, Guy; Rees, David; Perrin, Pascale; Harding, Rosalind M; Clegg, John B; Langaney, André; Excoffier, Laurent

    2002-01-01

    A large and ethnically well-defined Mandenka sample from eastern Senegal was analyzed for the polymorphism of the beta-globin gene cluster on chromosome 11. Five RFLP sites of the 5' region were investigated in 193 individuals revealing the presence of 10 different haplotypes. The frequency of the sickle-cell anemia causing mutation (beta(S)) in the Mandenka estimated from this sample is 11.7%. This mutation was found strictly associated with the single Senegal haplotype. Approximately 600 bp of the upstream region of the beta-globin gene were sequenced for a subset of 94 chromosomes, showing the presence of four transversions, five transitions, and a composite microsatellite polymorphism. The sequence of 22 beta(S) chromosomes was also identical to the previously defined Senegal haplotype, suggesting that this mutation is very recent. Monte Carlo simulations (allowing for a specific balancing selection model, a logistic growth of the population, and variable initial frequencies of the Senegal haplotype) were used to estimate the age of the beta(S) mutation. Resulting maximum-likelihood estimates are 45-70 generations (1,350-2,100 years) for very different demographic scenarios. Smallest confidence intervals (25-690 generations) are obtained under the hypothesis that the Mandenka population is large (N(e) >5,000) and stationary or that it has undergone a rapid demographic expansion to a current size of >5,000 reproducing individuals, which is quite likely in view of the great diversity found on beta(A) chromosomes.

  12. Generation of a genomic reporter assay system for analysis of gamma- and beta-globin gene regulation

    NARCIS (Netherlands)

    Chan, Kasey S. K.; Xu, Jian; Wardan, Hady; McColl, Bradley; Orkin, Stuart; Vadolas, Jim

    2012-01-01

    A greater understanding of the regulatory mechanisms that govern gamma-globin expression in humans, especially the switching from gamma- to beta-globin, which occurs after birth, would help to identify new therapeutic targets for patients with beta-hemoglobinopathy. To further elucidate the mechanis

  13. Identification and characterization of adult alpha-and beta-globin genes and their genomic arrangement in Pseudosciaena crocea

    Institute of Scientific and Technical Information of China (English)

    CHU Wuying; QIAN Ronghua; WANG Lianshen; YU Xiameng; YOU Zhenqiang; YU Lian

    2006-01-01

    The α- and β-globin genes from Pseudosciaena crocea were cloned by rapid amplification of cDNA 3'-end (3'-RACE). The cDNA of the α-globin is 595 bp with the ATG start codon located at Position 37, the TAA stop codon at Position 469 and the AATAAA polyadenylation signal at Position 560, which codifies 145 amino acids. The entire open reading frame of the β-globin gene is 447 bp long, which encodes 148 amino acids. Amino acid identity of the α- globin or β-globin gene compared with those reported in other fish species, ranged from 31.9% to 76.4%. When comparing with human α- and β-globins, three important alterations in the structural regions can be noted: α39 Thr→Gln, α113 His→Tyr and β117 His→Lys. The α-globin has a unique inserted amino acid residue in the 47th position. To understand the process of globin gene duplication and identify the regulatory elements present in the intergenic and intragenic regions of globin genes, the genomic arrangement of α- and β-globin genes was investigated. The results showed that the orientation of the two genes was head-to-head relative to each other. The intergenic region between the translation initiation codons of the linked α- and β-globin genes contains classical promoter elements and the length of it is much shorter than that reported in other fish.

  14. Comparative evolutionary analyses of beta globin gene in eutherian, dinosaurian and neopterygii taxa

    Directory of Open Access Journals (Sweden)

    Gauri Awasthi, Garima Srivastava & Aparup Das

    2011-03-01

    Full Text Available Background & objectives: Comparative genomics and evolutionary analyses of conserved genes have enabled usto understand the complexity of genomes of closely related species. For example: -globin gene present inhuman hemoglobin is one such gene that has experienced many genetic changes in many related taxa andproduced more than 600 variants. One of the variant, HBS causes sickle-cell anemia in humans but offersprotection against severe malaria due to Plasmodium falciparum. In the present study, we characterized andperformed evolutionary comparative analyses of the -globin gene in different related and unrelated taxa tohave a comprehensive view of its evolution.Methods: DNA and protein sequences of -globin gene were downloaded from NCBI and characterized in detailin nine eutherian (Homo sapiens, Pan troglodytes, Macaca mulatta, Mus musculus, Rattus norvegicus, Bostaurus, Canis familiaris, Equus caballus, Oryctolagus cuniculus, a dinosaurian (Gallus gallus and a neopterygii(Danio rerio taxa. Three more eutherian (Papio anubis, Ovis aries and Sus scrofa taxa were included for ananalysis at the protein level but not included at the gene level owing to lack of genomic information. Computationaland phylogenetic analyses were performed using evolutionary comparative approach.Results: Results of comparative and phylogenetic analyses revealed less conservation of genetic architecture of-globin compared to its protein architecture in all eutherian taxa. Both dinosaurian and neopterygii taxa servedas outgroups and varied at gene and protein levels.Interpretation & conclusion: Most remarkably, all primates from eutherian taxa including P. anubis showedonly nine codon position differences and an absolute similarity between H. sapiens and P. troglodytes. Absoluteconservation of coding region in Equus caballus (horse was observed. The results were discussed with aninference on the role of evolutionary forces in maintaining such close similarities

  15. Definition of the minimal requirements within the human beta-globin gene and the dominant control region for high level expression.

    Science.gov (United States)

    Collis, P; Antoniou, M; Grosveld, F

    1990-01-01

    The human beta-globin dominant control region (DCR) was previously identified as a region from the 5' end of the human beta-globin locus which directs high level, site of integration-independent, copy number-dependent expression on a linked human beta-globin gene in transgenic mice and stably transfected mouse erythroleukaemia (MEL) cells. We have now analysed the elements comprising the DCR by systematic deletion mutagenesis in stable MEL transfectants. We have identified two independent elements within the DNase I hypersensitive sites 2 and 3, containing fragments which direct strong transcriptional inducibility of a beta-globin gene. Whilst the remaining two hypersensitive sites do not direct significant transcriptional induction, our data suggest that all four sites may be necessary for the fully regulated expression conferred by the DCR. We have also tested a number of beta-globin minigene constructs under the control of the DCR to assess if any of the local sequences from the gene may be removed without loss of expression. We find that the 3' enhancer may be removed without affecting expression, but there is an absolute requirement for the presence of the second intron, not related to the enhancer present in that intron. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:2295312

  16. An analysis of fetal hemoglobin variation in sickle cell disease: the relative contributions of the X-linked factor, beta-globin haplotypes, alpha-globin gene number, gender, and age.

    Science.gov (United States)

    Chang, Y C; Smith, K D; Moore, R D; Serjeant, G R; Dover, G J

    1995-02-15

    Five factors have been shown to influence the 20-fold variation of fetal hemoglobin (Hb F) levels in sickle cell anemia (SS): age, sex, the alpha-globin gene number, beta-globin haplotypes, and an X-linked locus that regulates the production of Hb F-containing erythrocytes (F cells), ie, the F-cell production (FCP) locus. To determine the relative importance of these factors, we studied 257 Jamaican SS subjects from a Cohort group identified by newborn screening and from a Sib Pair study. Linear regression analyses showed that each variable, when analyzed alone, had a significant association with Hb F levels (P haplotypes, alpha-globin genes, and age accounted for less than 10% of the variation. The association between the beta-globin haplotypes and Hb F levels becomes apparent if the influence of the FCP locus is removed by analyzing only individuals with the same FCP phenotype. Thus, the FCP locus is the most important factor identified to date in determining Hb F levels. The variation within each FCP phenotype is modulated by factors associated with the three common beta-globin haplotypes and other as yet unidentified factor(s).

  17. Gamma rays and bleomycin nick DNA and reverse the DNase I sensitivity of beta-globin gene chromatin in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Villeponteau, B.; Martinson, H.G.

    1987-05-01

    The active beta-globin genes in chicken erythrocytes, like all active genes, reside in large chromatin domains which are preferentially sensitive to digestion by DNase I. We have recently proposed that the special structure of chromatin in active domains is maintained by torsional stress in the DNA. This hypothesis predicts that nicking of the DNA within any such chromosomal domain in vivo will relax the DNA and lead to loss of the special DNase I-sensitive state. Here we have tested this prediction by using gamma irradiation and bleomycin treatment to cleave DNA within intact chicken embryo erythrocytes. Both treatments cause reversal of DNase I sensitivity. Moreover, reversal occurs at approximately one nick per 150 kilobase pairs for both agents despite their entirely unrelated modes of cell penetration and DNA attack. These results suggest that the domain of DNase I sensitivity surrounding the beta-globin genes comprises 150 kilobase pairs of chromatin under torsional stress and that a single DNA nick in this region is sufficient to reverse the DNase I sensitivity throughout the entire domain.

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

    Science.gov (United States)

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

    2001-12-01

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

  19. The beta-globin gene cluster haplotypes in sickle cell anemia patients from Northeast Brazil: a clinical and molecular view.

    Science.gov (United States)

    Adorno, Elisângela Vitória; Zanette, Angela; Lyra, Isa; Souza, Cyntia Cajado; Santos, Leandro Ferraz; Menezes, Joelma Figueiredo; Dupuit, Marie France; Almeida, Mari Ney Tavares; Reis, Mitermayer Galvão; Gonçalves, Marilda Souza

    2004-08-01

    The beta(S)-globin haplotypes were studied in 78 sickle cell Brazilian patients from Bahia, Northeast Brazil, that has a large population of African origin. Hemoglobin (Hb) profiles were developed by high-performance liquid chromatography (HPLC), and beta(S)-globin gene haplotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques. We identified 44 (55.0%) patients with the CAR/Ben (Central African Republic/Benin) genotype, 16 (20.0%) Ben/Ben, 13 (16.2%) CAR/CAR and seven (8.8%) with other genotypes. Analyses of the phenotypes showed clinical differences related only to Hb F levels and blood transfusion therapy; the presence of -alpha(-3.7)-thalassemia (thal) demonstrated statistical significance when associated with hematocrit (p=0.044), MCV (p=0.0007), MCH (p=0.012) and spleen sequestration events. The haplotype diversity found in the present study can be justified by information about the origin of the slave traffic period in Bahia during the 19th century. The specific characteristics described among the Bahian sickle cell patients could be confirmed by increasing the number of patients with specific genotypes and further studies of genetic markers.

  20. Concordance of a point mutation 5' to the A gamma-globin gene with A gamma beta + hereditary persistence of fetal hemoglobin in Greeks.

    Science.gov (United States)

    Waber, P G; Bender, M A; Gelinas, R E; Kattamis, C; Karaklis, A; Sofroniadou, K; Stamatoyannopoulos, G; Collins, F S; Forget, B G; Kazazian, H H

    1986-02-01

    In the Greek A gamma beta + type of hereditary persistence of fetal hemoglobin (HPFH), adult heterozygotes produce about 20% fetal hemoglobin (HbF), which is predominantly of the A gamma chain variety. The affected beta-globin gene cluster produces near normal amounts of beta-like globin, but in a A gamma to beta ratio of 20:80 instead of 0.5:99.5. Gelinas et al and Collins et al have shown a G to A change 117 nucleotides 5' to the A gamma gene in two Greeks with A gamma beta + HPFH. To demonstrate that this change is not a neutral polymorphism, we carried out hybridization with oligonucleotide probes (19mers) specific for the normal and the mutant sequences. While normal probe identified the A gamma fragment in genomic DNA of all subjects studied, mutant probe was positive only in Greeks with A gamma beta + HPFH. In sum, 108 beta-globin gene clusters of individuals without HPFH were negative when tested with mutant probe, but all 11 affected individuals of six families with Greek A gamma beta + HPFH (two previously sequenced and four new families) were positive with mutant probe. These data support the conclusion that the -117 mutation is causative of A gamma beta + HPFH in Greeks.

  1. beta (+)-Thalassaemia in the Po river delta region (northern Italy): genotype and beta globin synthesis.

    Science.gov (United States)

    Del Senno, L; Pirastu, M; Barbieri, R; Bernardi, F; Buzzoni, D; Marchetti, G; Perrotta, C; Vullo, C; Kan, Y W; Conconi, F

    1985-01-01

    Six beta(+)-thalassaemic patients from the Po river delta region have been studied. Using synthetic oligonucleotides as specific hybridisation probes, the beta(+) IVS I mutation (G----A at position 108) was demonstrated. This lesion and the enzyme polymorphism pattern in the subjects examined are the same as have been described for other Mediterranean beta(+)-thalassaemias. Antenatal diagnosis through DNA analysis of beta(+)-thalassaemia is therefore possible. The production of beta globin in a beta(+), homozygote and in a beta (+), beta(0) 39 (nonsense mutation at codon 39) double heterozygote is approximately 20% and 10% respectively of total non-alpha globin synthesis. Despite some overlapping of the results, similar beta globin synthesis levels have been obtained in 43 beta(+)-thalassaemia patients. This suggests that in the Po river delta region the most common thalassaemic genes are beta(0) 39 and beta(+) IVS I. Images PMID:2580095

  2. Effect of beta-globin gene cluster haplotype on the hematological and clinical features of sickle cell anemia.

    Science.gov (United States)

    Rieder, R F; Safaya, S; Gillette, P; Fryd, S; Hsu, H; Adams, J G; Steinberg, M H

    1991-03-01

    In 113 black American adults with sickle cell anemia (HbSS), we examined nine polymorphic restriction sites, including the Xmnl site 5' to the G gamma gene, to see whether haplotype is related to the level of HbF and the proportion of G gamma chains or if it influences the hematological and clinical features of the disease. Seventy-five percent of the patients were homozygous or heterozygous for the Benin (no. 19) or Central African Republic (Bantu, no. 20) haplotypes; 13.3% were homozygous or heterozygous for the Senegal (no. 3) haplotype, while 11.5% had other genotypes. Of the subjects, 14.2% were either homozygous or heterozygous for the Xmnl restriction site 5' to the G gamma gene. We found no effect of haplotype on HbF levels. The level of G gamma chains was 60.5% +/- 17.0% in individuals heterozygous or homozygous for haplotype no. 3 and was 46.9% +/- 11.6% in individuals with other haplotypes. Subjects with the Xmnl site 5' to the G gamma gene had G gamma globin levels of 59.5% +/- 16.7% while those lacking that site had an average of 47.2% +/- 12.1%. There were no significant differences among these groups in hemoglobin concentration, packed cell volume, mean cell volume, or clinical indicators of vaso-occlusive severity, including crises, hospitalizations per year, aseptic bone necrosis, acute chest syndrome, or leg ulcers. While the presence of haplotype 3 and the 5' G gamma Xmnl site were associated with increased G gamma chains, there was no effect on HbF level or other hematological and clinical features that might reflect disease severity. It is likely that determinants unrelated to haplotype, linked or unlinked to the beta-globin gene cluster, are the major effectors of differences in the levels of HbF in American patients with sickle cell anemia.

  3. Origin and spread of beta-globin gene mutations in India, Africa, and Mediterranea: analysis of the 5' flanking and intragenic sequences of beta S and beta C genes.

    Science.gov (United States)

    Trabuchet, G; Elion, J; Baudot, G; Pagnier, J; Bouhass, R; Nigon, V M; Labie, D; Krishnamoorthy, R

    1991-06-01

    Nucleotide polymorphisms of both the 5' flanking and intragenic regions of the human beta-globin gene were investigated by directly sequencing genomic DNA after amplification by the polymerase chain reaction in 47 subjects homozygous for the beta S or the beta C mutation. The sickle-cell mutation was found in the context of five different haplotypes defined by eight nucleotide substitutions and various structures of a region of the simple repeated sequence (AT) chi Ty. All subjects from the same geographic origin bear an identical chromosomal structure, defining the Senegal-, Bantu-, Benin-, Cameroon-, and Indian-type chromosomes. These results strengthen our previous conclusions about the multiple occurrence of the sickle-cell mutation. The Benin-type chromosome was also found among Algerian and Sicilian sickle-cell patients, whereas the Indian-type chromosome was observed in two geographically distant tribes, illustrating the spread of these sickle-cell genes. We also found that the intragenic sequence polymorphisms (frameworks) are not always in linkage disequilibrium with the BamH I polymorphism downstream from the beta-globin gene, as had been previously observed. Finally, we present a tentative phylogenetic tree of the different alleles at this locus. Some polymorphisms of this sequence might be contemporary with our last common ancestor, the great apes, that is, about 4-6 millions years old.

  4. Therapeutic haemoglobin synthesis in beta-thalassaemic mice expressing lentivirus-encoded human beta-globin.

    Science.gov (United States)

    May, C; Rivella, S; Callegari, J; Heller, G; Gaensler, K M; Luzzatto, L; Sadelain, M

    2000-07-06

    The stable introduction of a functional beta-globin gene in haematopoietic stem cells could be a powerful approach to treat beta-thalassaemia and sickle-cell disease. Genetic approaches aiming to increase normal beta-globin expression in the progeny of autologous haematopoietic stem cells might circumvent the limitations and risks of allogeneic cell transplants. However, low-level expression, position effects and transcriptional silencing hampered the effectiveness of viral transduction of the human beta-globin gene when it was linked to minimal regulatory sequences. Here we show that the use of recombinant lentiviruses enables efficient transfer and faithful integration of the human beta-globin gene together with large segments of its locus control region. In long-term recipients of unselected transduced bone marrow cells, tetramers of two murine alpha-globin and two human betaA-globin molecules account for up to 13% of total haemoglobin in mature red cells of normal mice. In beta-thalassaemic heterozygous mice higher percentages are obtained (17% to 24%), which are sufficient to ameliorate anaemia and red cell morphology. Such levels should be of therapeutic benefit in patients with severe defects in haemoglobin production.

  5. Genomic organization and differential signature of positive selection in the alpha and beta globin gene clusters in two cetacean species.

    Science.gov (United States)

    Nery, Mariana F; Arroyo, José Ignacio; Opazo, Juan C

    2013-01-01

    The hemoglobin of jawed vertebrates is a heterotetramer protein that contains two α- and two β-chains, which are encoded by members of α- and β-globin gene families. Given the hemoglobin role in mediating an adaptive response to chronic hypoxia, it is likely that this molecule may have experienced a selective pressure during the evolution of cetaceans, which have to deal with hypoxia tolerance during prolonged diving. This selective pressure could have generated a complex history of gene turnover in these clusters and/or changes in protein structure themselves. Accordingly, we aimed to characterize the genomic organization of α- and β-globin gene clusters in two cetacean species and to detect a possible role of positive selection on them using a phylogenetic framework. Maximum likelihood and Bayesian phylogeny reconstructions revealed that both cetacean species had retained a similar complement of putatively functional genes. For the α-globin gene cluster, the killer whale presents a complement of genes composed of HBZ, HBK, and two functional copies of HBA and HBQ genes, whereas the dolphin possesses HBZ, HBK, HBA and HBQ genes, and one HBA pseudogene. For the β-globin gene cluster, both species retained a complement of four genes, two early expressed genes-HBE and HBH-and two adult expressed genes-HBD and HBB. Our natural selection analysis detected two positively selected sites in the HBB gene (56 and 62) and four in HBA (15, 21, 49, 120). Interestingly, only the genes that are expressed during the adulthood showed the signature of positive selection.

  6. Beta-globin gene cluster haplotypes and HbF levels are not the only modulators of sickle cell disease in Lebanon.

    Science.gov (United States)

    Inati, A; Taher, A; Bou Alawi, W; Koussa, S; Kaspar, H; Shbaklo, H; Zalloua, P A

    2003-02-01

    Sickle cell disease (SCD) is an inherited autosomal recessive disorder of the beta-globin chain. Despite the fact that all subjects with SCD have the same single base pair mutation, the severity of the clinical and hematological manifestations is extremely variable. This study examined for the first time in Lebanon the correlation between the clinical manifestation of SCD and the beta-globin gene haplotypes. The haplotypes of 50 patients diagnosed with SCD were determined using polymerase chain reaction amplification of fragments containing nine polymorphic restriction sites around and within the epsilon-Ggamma-Agamma-psibeta-delta-beta-globin gene complex. Most reported haplotypes were found in our population with the Benin haplotype as the most prevalent one. When the patients were divided according to their HbF levels into three groups (Group A: HbF 15%), surprisingly, the highest levels of HbF were associated with the most severe clinical cases. Our findings suggest that fetal hemoglobin levels are important but not the only parameters that affect the severity of the disease. In addition, the high levels of HbF in patients with CAR haplotypes did not seem to ameliorate the severity of symptoms, suggesting that genetic factors other than haplotypes are the major determinants of increased HbF levels in Lebanon.

  7. Non-random association of the Rsa I polymorphic site 5' to the beta-globin gene with major sickle cell haplotypes.

    Science.gov (United States)

    Sharon, B; Poncz, M; Surrey, S; Schwartz, E

    1988-01-01

    There are three main African haplotypes associated with the sickle mutation on chromosome 11. We have examined an Rsa I polymorphism 550 bp 5' to the beta-globin gene to study the degree of linkage disequilibrium between this Rsa I site and the three haplotypes. This Rsa I site is contained within the 10.3 kb or less area of randomization separating the 5'- and 3'-haplotype clusters. The beta S-containing chromosomes of the Benin and Senegal haplotypes are not cut, while those of the Central African Republic are cleaved by Rsa I at this site. Possible explanations of these findings are discussed.

  8. Beta-globin gene cluster haplotypes in sickle cell patients from southwest Iran.

    Science.gov (United States)

    Rahimi, Z; Karimi, M; Haghshenass, M; Merat, A

    2003-11-01

    Sickle cell anemia in Iran is accompanied by a high level of HbF and mild clinical presentation. Here we report haplotypes of the beta gene cluster found in 81 randomly selected sickle cell patients, including 47 sickle cell anemia (SS), 17 sickle cell trait (AS), and 17 sickle/thalassemia (S/thal) from southwest Iran. We found all five common typical haplotypes as well as five atypical haplotypes in our patients. Except for four patients with homozygous Benin haplotype, none of the other African typical haplotypes were found in a homozygous state. Arab-Indian was found to be the most prevalent haplotype in the study population. This haplotype accounted for 51.1% as the homozygous form in SS patients, where 69.1% of chromosomes in these patients had the Arab-Indian haplotype. Bantu A2 was the second most prevalent haplotype among all patients. The mean %HbF in SS patients was 27.83 and in the homozygous Arab-Indian haplotype it was still higher (30.40%), while in AS patients the %HbF was only 1.20. The high %Ggamma chain (71.81) in the Arab-Indian homozygous haplotype was concomitant with the presence of an Xmn I site in both chromosomes. The presence of the Arab-Indian haplotype as the predominant haplotype might be suggestive of a gene flow to/from Saudi Arabia or India. More haplotype investigations of a normal population can clarify the high incidence of Bantu A2 haplotype in our population.

  9. Morbidity, beta S haplotype and alpha-globin gene patterns among sickle cell anemia patients in Kuwait.

    Science.gov (United States)

    Adekile, A D; Haider, M Z

    1996-01-01

    Admission records of children with sickle cell anemia (SS), in the two main teaching hospitals in Kuwait, were reviewed for a 1-year period. The haplotypes of 92 beta s chromosomes (from 39 SS, 11 AS, 2 S beta-thalassemia [S beta-thal] and 1 SD individuals) were determined using an allele-specific oligonucleotide (ASO) hybridization technique, while the alpha-globin gene status of 27 SS and 33 AS individuals, i.e. 120 chromosomes, was determined with a combination of polymerase chain reaction and AS techniques. A vasooclusive crisis was the most common (60.0%) cause of hospitalization, followed by infections (20%). Hospital admissions were most common during the hottest month of the year (July). Few complications of the disease were seen among patients on follow-up; however, splenomegaly was present in 24.0%, hepatomegaly in 15.2%, gallstones in 15.2% and aseptic necrosis of the femoral head in 6.1%. Haplotype 31 (Saudi Arabia/India) is the most frequent in this community, being present in 80.4% of the chromosomes tested; Benin haplotype 19 was found in 12.0% and Bantu haplotype 20 in 6.5%. Hb F in the haplotype 31 homozygotes and heterozygotes ranged from 11.4 to 35.1% (mean 22.5 +/- 5.2%). The frequency of alpha-thal determinants in the study was 40.0%, the commonest being the -alpha 3.7-kb deletion (27.5%), the alpha 2 polyadenylation signal (AATAAA-> AATAAG) mutation (10.2%) and the IVS-I 5' end GAGGT-GAGG->GAGG pentanucleotide (5 nt) deletion (3.3%). SS patients with coexistent alpha-thal trait did not have severe recurrent infections and none had gallstones. The high frequencies of the Saudi Arabia/India beta s haplotype and alpha-thalassemia trait contribute to the mild nature of SS disease among Kuwaiti Arabs comparable to that in eastern Saudi Arabia.

  10. The role of EKLF in human β-globin gene competition.

    NARCIS (Netherlands)

    M.G.J.M. Wijgerde (Mark); J.H. Gribnau (Joost); T. Trimborn (Tolleiv); B. Nuez (Beatriz); J.N.J. Philipsen (Sjaak); F.G. Grosveld (Frank); P.J. Fraser (Peter)

    1996-01-01

    textabstractWe have investigated the role of erythroid Kruppel-like factor (EKLF) in expression of the human beta-globin genes in compound EKLF knockout/human beta-locus transgenic mice. EKLF affects only the adult mouse beta-globin genes in homozygous knockout mice; heterozygous mice are unaffected

  11. Mechanisms of developmental control of transcription in the murine alpha- and beta-globin loci

    NARCIS (Netherlands)

    T. Trimborn (Tolleiv); J.H. Gribnau (Joost); P.J. Fraser (Peter); F.G. Grosveld (Frank)

    1999-01-01

    textabstractWe have characterized mRNA expression and transcription of the mouse alpha- and beta-globin loci during development. S1 nuclease and primary transcript in situ hybridization analyses demonstrate that all seven murine globin genes (zeta, alpha1, alpha2, epsil

  12. Common haplotype dependency of high G gamma-globin gene expression and high Hb F levels in beta-thalassemia and sickle cell anemia patients.

    OpenAIRE

    Labie, D; Pagnier, J.; Lapoumeroulie, C; Rouabhi, F; Dunda-Belkhodja, O; Chardin, P; Beldjord, C; Wajcman, H; Fabry, M E; Nagel, R L

    1985-01-01

    We have studied 42 homozygous beta-thalassemia patients from Algeria and 34 sickle cell anemia patients from Senegal and Benin, determining the relationship between haplotypes, Hb F, and G gamma-globin/A gamma-globin ratios. Populations selected have a high frequency of haplotype homozygotes because of consanguinity (Algeria) and geographic homogeneity (West Africa). We find in beta-thalassemia patients, that haplotype IX in haplotypic homozygotes and heterozygotes, haplotype III in heterozyg...

  13. β-globin gene promoter generates 5' truncated transcripts in the embryonic foetal erythroid environment.

    NARCIS (Netherlands)

    K. Khazaie; F. Gounari; M. Antoniou (Michael); E. de Boer (Ernie); F.G. Grosveld (Frank)

    1987-01-01

    textabstractWe report here the localisation of sequences responsible for the faulty expression of human beta-globin gene in Putko and K562 cells. Complete beta-globin gene introduced into these cells produces transcripts with abnormal 5' ends, while cotransfected mouse H2 gene is expressed correctly

  14. Familial Polycythemia Caused by a Novel Mutation in the Beta Globin Gene: Essential Role of P50 in Evaluation of Familial Polycythemia

    Directory of Open Access Journals (Sweden)

    Neeraj Agarwal, Mariluz P. Mojica-Henshaw, Elizabeth. D. Simmons, Dottie Hussey, Ching N. Ou, Josef T. Prchal

    2007-01-01

    Full Text Available Two polycythemic subjects from a family with multiple polycythemic subjects were evaluated. Estimation of oxygen affinity of Hb from venous blood gas parameters (P50 revealed low P50 suggesting a high affinity Hb variant. Further work up, which included beta globin gene sequencing, revealed a novel mutation changing a codon to the previously reported high affinity Hb - Hb Johnstown (beta109 Val->Leu. Polycythemic subjects with high affinity Hb variant are asymptomatic with normal life expectancy. Their differentiation from polycythemia vera (PV is crucial to avoid therapy which is otherwise reserved for PV patients. We provide an electronic version (in Microsoft excel program of a previously reported mathematical formula for rapid calculation of P50 from venous blood gases. Estimation of P50 is an essential initial step in the evaluation of a subject with personal and family history of polycythemia.

  15. Molecular variations linked to the grouping of beta- and alpha-globin genes in neonatal patients with sickle cell disease in the State of Pernambuco, Brazil.

    Science.gov (United States)

    Bezerra, Marcos André C; Santos, Magnun N N; Araújo, Aderson S; Gomes, Yara M; Abath, Frederico G C; Bandeira, Flavia M G C

    2007-01-01

    Various factors have been described as phenotypic modulators of sickle cell disease, such as levels of fetal hemoglobin (Hb F), presence of alpha-thalassemia (thal), and haplotypes of the beta-globin genes. In order to characterize and determine the frequency of the betaS and betaC mutations and the prevalence of -alpha3.7-thal, 74 patients with sickle cell disease detected during neonatal screening in the State of Pernambuco, Brazil, were studied. The haplotypes of the beta gene and -alpha3.7-thal were determined using polymerase chain reaction (PCR), and specific restriction endonucleases were used to establish the polymorphic sites of the haplotypes. The results showed the high frequency of the Central African Republic (CAR) or Bantu haplotype in the State of Pernambuco, Brazil. The low frequency of the Benin haplotype recorded in this study, in comparison with other states in northeast Brazil, suggests the diversity of origins of Afro-Brazilians in this region.

  16. The entire β-globin gene cluster is deleted in a form of τδβ-thalassemia.

    NARCIS (Netherlands)

    E.R. Fearon; H.H.Jr. Kazazian; P.G. Waber (Pamela); J.I. Lee (Joseph); S.E. Antonarakis; S.H. Orkin (Stuart); E.F. Vanin; P.S. Henthorn; F.G. Grosveld (Frank); A.F. Scott; G.R. Buchanan

    1983-01-01

    textabstractWe have used restriction endonuclease mapping to study a deletion involving the beta-globin gene cluster in a Mexican-American family with gamma delta beta-thalassemia. Analysis of DNA polymorphisms demonstrated deletion of the beta-globin gene from the affected chromosome. Using a DNA f

  17. Diversity of [beta]-globin mutations in Israeli ethnic groups reflects recent historic events

    Energy Technology Data Exchange (ETDEWEB)

    Filon, D.; Oron, V.; Krichevski, S.; Shaag, A.; Goldfarb, A.; Aker, M.; Rachmilewitz, E.A.; Rund, D.; Oppenheim, A. (Hebrew Univ. Hadassah-Medical School, Jerusalem (Israel)) (and others)

    1994-05-01

    The authors characterized nearly 500 [beta]-thalassemia genes from the Israeli population representing a variety of ethnic subgroups. They found 28 different mutations in the [beta]-globin gene, including three mutations ([beta][sup S], [beta][sup C], and [beta][sup O-Arab]) causing hemoglobinopathies. Marked genetic heterogeneity was observed in both the Arab (20 mutations) and Jewish (17 mutations) populations. On the other hand, two ethnic isolates - Druze and Samaritans - had a single mutation each. Fifteen of the [beta]-thalassemia alleles are Mediterranean in type, 5 originated in Kurdistan, 2 are of Indian origin, and 2 sporadic alleles came from Europe. Only one mutant allele-nonsense codon 37-appears to be indigenous to Israel. While human habitation in Israel dates back to early prehistory, the present-day spectrum of [beta]-globin mutations can be largely explained by migration events that occurred in the past millennium. 26 refs., 2 figs., 3 tabs.

  18. Confirmation of the potential usefulness of two human beta globin pseudogene markers to estimate gene flows to and from sub-Saharan Africans.

    Science.gov (United States)

    Ciminelli, Bianca Maria; Pompei, Fiorenza; Relucenti, Michela; Lum, J Koji; Simporé, Jacques; Spedini, Gabriella; Martínez-Labarga, Cristina; Pardo, Miguel G

    2002-04-01

    Two polymorphic sites, -107 and -100 with respect to the "cap" site of the human beta globin pseudogene, recently discovered in our laboratory, turned out to have an ethnically complementary distribution. The first site is polymorphic in Europeans, North Africans, Indians (Hindu), and Oriental Asians, and monomorphic in sub-Saharan Africans. Conversely, the second site is polymorphic in sub-Saharan African populations and monomorphic in the aforementioned populations. Here we report the gene frequencies of these two polymorphic sites in nine additional populations (Egyptians, Spaniards, Japanese, Chinese, Filipinos, Vietnamese, Africans from Togo and from Benin, and Pygmies), confirming their ethnospecificity and, through the analysis of these two markers in Oromo and Amhara of Ethiopia (two mixed populations), their usefulness in genetic admixture studies. Moreover, we studied another marker polymorphic in sub-Saharan African populations only, a TaqI restriction fragment length polymorphism located in the same region as the present markers, demonstrating the absence of linkage disequilibrium between it and the -100 site, so that we can exclude that the information they provide is redundant.

  19. High-density SNP genotyping to define beta-globin locus haplotypes.

    Science.gov (United States)

    Liu, Li; Muralidhar, Shalini; Singh, Manisha; Sylvan, Caprice; Kalra, Inderdeep S; Quinn, Charles T; Onyekwere, Onyinye C; Pace, Betty S

    2009-01-01

    Five major beta-globin locus haplotypes have been established in individuals with sickle cell disease (SCD) from the Benin, Bantu, Senegal, Cameroon, and Arab-Indian populations. Historically, beta-haplotypes were established using restriction fragment length polymorphism (RFLP) analysis across the beta-locus, which consists of five functional beta-like globin genes located on chromosome 11. Previous attempts to correlate these haplotypes as robust predictors of clinical phenotypes observed in SCD have not been successful. We speculate that the coverage and distribution of the RFLP sites located proximal to or within the globin genes are not sufficiently dense to accurately reflect the complexity of this region. To test our hypothesis, we performed RFLP analysis and high-density single nucleotide polymorphism (SNP) genotyping across the beta-locus using DNA samples from healthy African Americans with either normal hemoglobin A (HbAA) or individuals with homozygous SS (HbSS) disease. Using the genotyping data from 88 SNPs and Haploview analysis, we generated a greater number of haplotypes than that observed with RFLP analysis alone. Furthermore, a unique pattern of long-range linkage disequilibrium between the locus control region and the beta-like globin genes was observed in the HbSS group. Interestingly, we observed multiple SNPs within the HindIII restriction site located in the Ggamma-globin intervening sequence II which produced the same RFLP pattern. These findings illustrated the inability of RFLP analysis to decipher the complexity of sequence variations that impacts genomic structure in this region. Our data suggest that high-density SNP mapping may be required to accurately define beta-haplotypes that correlate with the different clinical phenotypes observed in SCD.

  20. Deletion of a region that is a candidate for the difference between the deletion forms of hereditary persistence of fetal hemoglobin and deltabeta-thalassemia affects beta- but not gamma-globin gene expression.

    NARCIS (Netherlands)

    R. Calzolari (Roberta); T. McMorrow (Tara); N. Yannoutsos (Nikos); A. Langeveld (An); F.G. Grosveld (Frank)

    1999-01-01

    textabstractThe analysis of a number of cases of beta-globin thalassemia and hereditary persistence of fetal hemoglobin (HPFH) due to large deletions in the beta-globin locus has led to the identification of several DNA elements that have been implicated in the switch f

  1. Production of beta-globin and adult hemoglobin following G418 treatment of erythroid precursor cells from homozygous beta(0)39 thalassemia patients.

    Science.gov (United States)

    Salvatori, Francesca; Breveglieri, Giulia; Zuccato, Cristina; Finotti, Alessia; Bianchi, Nicoletta; Borgatti, Monica; Feriotto, Giordana; Destro, Federica; Canella, Alessandro; Brognara, Eleonora; Lampronti, Ilaria; Breda, Laura; Rivella, Stefano; Gambari, Roberto

    2009-11-01

    In several types of thalassemia (including beta(0)39-thalassemia), stop codon mutations lead to premature translation termination and to mRNA destabilization through nonsense-mediated decay. Drugs (for instance aminoglycosides) can be designed to suppress premature termination, inducing a ribosomal readthrough. These findings have introduced new hopes for the development of a pharmacologic approach to the cure of this disease. However, the effects of aminoglycosides on globin mRNA carrying beta-thalassemia stop mutations have not yet been investigated. In this study, we have used a lentiviral construct containing the beta(0)39-thalassemia globin gene under control of the beta-globin promoter and a LCR cassette. We demonstrated by fluorescence-activated cell sorting (FACS) analysis the production of beta-globin by K562 cell clones expressing the beta(0)39-thalassemia globin gene and treated with G418. More importantly, after FACS and high-performance liquid chromatography (HPLC) analyses, erythroid precursor cells from beta(0)39-thalassemia patients were demonstrated to be able to produce beta-globin and adult hemoglobin after treatment with G418. This study strongly suggests that ribosomal readthrough should be considered a strategy for developing experimental strategies for the treatment of beta(0)-thalassemia caused by stop codon mutations. Am. J. Hematol., 2009. (c) 2009 Wiley-Liss, Inc.

  2. Common 5' beta-globin RFLP haplotypes harbour a surprising level of ancestral sequence mosaicism.

    Science.gov (United States)

    Webster, Matthew T; Clegg, John B; Harding, Rosalind M

    2003-07-01

    Blocks of linkage disequilibrium (LD) in the human genome represent segments of ancestral chromosomes. To investigate the relationship between LD and genealogy, we analysed diversity associated with restriction fragment length polymorphism (RFLP) haplotypes of the 5' beta-globin gene complex. Genealogical analyses were based on sequence alleles that spanned a 12.2-kb interval, covering 3.1 kb around the psibeta gene and 6.2 kb of the delta-globin gene and its 5' flanking sequence known as the R/T region. Diversity was sampled from a Kenyan Luo population where recent malarial selection has contributed to substantial LD. A single common sequence allele spanning the 12.2-kb interval exclusively identified the ancestral chromosome bearing the "Bantu" beta(s) (sickle-cell) RFLP haplotype. Other common 5' RFLP haplotypes comprised interspersed segments from multiple ancestral chromosomes. Nucleotide diversity was similar between psibeta and R/T-delta-globin but was non-uniformly distributed within the R/T-delta-globin region. High diversity associated with the 5' R/T identified two ancestral lineages that probably date back more than 2 million years. Within this genealogy, variation has been introduced into the 3' R/T by gene conversion from other ancestral chromosomes. Diversity in delta-globin was found to lead through parts of the main genealogy but to coalesce in a more recent ancestor. The well-known recombination hotspot is clearly restricted to the region 3' of delta-globin. Our analyses show that, whereas one common haplotype in a block of high LD represents a long segment from a single ancestral chromosome, others are mosaics of short segments from multiple ancestors related in genealogies of unsuspected complexity.

  3. Structure and in vitro transcription of human globin genes.

    Science.gov (United States)

    Proudfoot, N J; Shander, M H; Manley, J L; Gefter, M L; Maniatis, T

    1980-09-19

    The alpha-like and beta-like subunits of human hemoglobin are encoded by a small family of genes that are differentially expressed during development. Through the use of molecular cloning procedures, each member of this gene family has been isolated and extensively characterized. Although the alpha-like and beta-like globin genes are located on different chromosomes, both sets of genes are arranged in closely linked clusters. In both clusters, each of the genes is transcribed from the same DNA strand, and the genes are arranged in the order of their expressions during development. Structural comparisons of immediately adjacent genes within each cluster have provided evidence for the occurrence of gene duplication and correction during evolution and have led to the discovery of pseudogenes, genes that have acquired numerous mutations that prevent their normal expression. Recently, in vivo and in vitro systems for studying the expression of cloned eukaryotic genes have been developed as a means of identifying DNA sequences that are necessary for normal gene function. This article describes the application of an in vitro transcription procedure to the study of human globin gene expression.

  4. Evaluation of β-globin gene therapy constructs in single-copy transgenic mice.

    NARCIS (Netherlands)

    J. Ellis (James); K.C. Tan-Un; P. Pasceri; A. Harper; X. Wu; P.J. Fraser (Peter); F.G. Grosveld (Frank)

    1997-01-01

    textabstractEffective gene therapy constructs based on retrovirus or adeno-associated virus vectors will require regulatory elements that direct expression of genes transduced at single copy. Most beta-globin constructs designed for therapy of beta-thalassemias are regulated by the 5'HS2 component o

  5. Análise dos haplótipos do gene da betaS-globina no Ceará Analysis of betaS-globin gene haplotypes in Ceará, Brazil

    Directory of Open Access Journals (Sweden)

    Gentil Claudino de Galiza Neto

    2005-10-01

    Full Text Available No presente trabalho abordam-se vários aspectos relacionados à natureza molecular da anemia falciforme (AF, desordem hematológica de caráter hereditário. A descoberta do polimorfismo do DNA no grupamento do gene betaS, originando diferentes haplótipos da doença, permitiu ampliar o conhecimento em torno da heterogeneidade clínica observada nos pacientes falcêmicos nas mais diversas regiões do mundo. Analisaram-se os diferentes haplótipos e seus parâmetros hematológicos, presentes em um grupo de 22 pacientes naturais e procedentes do estado do Ceará. A distribuição das freqüências dos haplótipos encontrados foi de 55,9% para Benin; 41,2% para República Centro-Africana (CAR; e de 2,9% para o haplótipo Senegal. Esses dados, em comparação com os demais estudos realizados no Brasil, mostram associação entre os seus valores para um alfa de 5% (p The present work deals with numerous aspects related to the molecular nature of sickle cell anemia. The discovery of the DNA polymorphism in the betas-globin gene cluster, gave origin to different haplotypes of the disease, making possible to enlarge the knowledge about the clinical heterogenity observed on the patients with sickle cell disease, in the various regions of the world. The different haplotypes and its hematological parameters were analysed in a group of 22 patients born in the State of Ceará, northeast of Brazil. The distribution found in the haplotypes frequency was of 55.9% for the Benin, of 41.2% for the CAR, and of 2.9% for Senegal haplotype. The data, compared to the others works done in Brazil, show relations among their values to alpha of 5% (p < 0,05. The results make possible a full understanding of the pathophisiology of the illness and of its clinical complexity in our State, as well as it allows a better knowledge of the sickle cell disease in our country.

  6. Targeted correction of a thalassemia-associated beta-globin mutation induced by pseudo-complementary peptide nucleic acids

    DEFF Research Database (Denmark)

    Lonkar, Pallavi; Kim, Ki-Hyun; Kuan, Jean Y

    2009-01-01

    Beta-thalassemia is a genetic disorder caused by mutations in the beta-globin gene. Triplex-forming oligonucleotides and triplex-forming peptide nucleic acids (PNAs) have been shown to stimulate recombination in mammalian cells via site-specific binding and creation of altered helical structures...

  7. CP2 binding to the promoter is essential for the enhanced transcription of globin genes in erythroid cells.

    Science.gov (United States)

    Chae, Ji Hyung; Kim, Chul Geun

    2003-02-28

    We have previously reported that the reduced level of CP2 suppresses the mouse alpha- and beta-globin gene expression and hemoglobin synthesis during terminal differentiation of mouse erythroleukemia (MEL) cells in vitro [Chae et al. (1999)]. As an extension of this study, we demonstrated that human alpha-, epsilon-, and gamma- globin genes were also suppressed by the reduced expression of CP2 in K562 cells. To address how much CP2 contributes in the regulation of globin gene expression, we measured transcriptional activities of the wild type alpha-globin promoter and its various factor-binding sites mutants in erythroid and nonerythroid cells. Interestingly, CP2 site dependent transcriptional activation occurred in an erythroid-cell specific manner, even though CP2 is ubiquitously expressed. In addition, CP2 site mutation within the alpha-promoter severely suppressed promoter activity in differentiated, but not in undifferentiated MEL cells, suggesting that the CP2 binding site is needed for the enhanced transcription of globin genes during erythroid differentiation. When the human beta-globin locus control region was linked to the alpha-promoter, suppression was more severe in the CP2 site mutant in differentiated MEL cells. Overall data indicate that CP2 is a major factor in the regulation of globin expression in human and mouse erythroid cells, and CP2 binding to the globin gene promoter is essential for the enhanced transcription of globin genes in erythroid differentiation.

  8. Structural analysis of the 5 prime flanking region of the. beta. -globin gene in African sickle cell anemia patients: Further evidence for three origins of the sickle cell mutation in Africa

    Energy Technology Data Exchange (ETDEWEB)

    Chebloune, Y.; Pagnier, J.; Trabuchet, G.; Faure, C.; Verdier, G.; Labie, D.; Nigon, V. (Universite Claude Bernard-Lyon, Villeurbane (France))

    1988-06-01

    Haplotype analysis of the {beta}-globin gene cluster shows two regions of DNA characterized by nonrandom association of restriction site polymorphisms. These regions are separated by a variable segment containing the repeated sequences (ATTTT){sub n} and (AT){sub x}T{sub y}, which might be involved in recombinational events. Studies of haplotypes linked to the sickle cell gene in Africa provide strong argument for three origins of the mutation: Benin, Senegal, and the Central African Republic. The structure of the variable segment in the three African populations was studied by S1 nuclease mapping of genomic DNA, which allows a comparison of several samples. A 1080-base-pair DNA segment was sequenced for one sample from each population. S1 nuclease mapping confirmed the homogeneity of each population with regard to both (ATTTT){sub n} and (AT){sub x}T{sub y} repeats. The authors found three additional structures for (AT){sub x}T{sub y} correlating with the geographic origin of the patients. Ten other nucleotide positions, 5{prime} and 3{prime} to the (AT){sub x}T{sub y} copies, were found to be variable when compared to homologous sequences from human and monkey DNAs. These results allow us to propose an evolutionary scheme for the polymorphisms in the 5{prime} flanking region of the {beta}-globin gene. The results strongly support the hypothesis of three origins for the sickle mutation in Africa.

  9. beta (+)-Thalassaemia in the Po river delta region (northern Italy): genotype and beta globin synthesis.

    OpenAIRE

    del Senno, L; Pirastu, M; Barbieri, R.; De Bernardi, F.; Buzzoni, D; Marchetti, G.; Perrotta, C; Vullo, C; Kan, Y W; Conconi, F

    1985-01-01

    Six beta(+)-thalassaemic patients from the Po river delta region have been studied. Using synthetic oligonucleotides as specific hybridisation probes, the beta(+) IVS I mutation (G----A at position 108) was demonstrated. This lesion and the enzyme polymorphism pattern in the subjects examined are the same as have been described for other Mediterranean beta(+)-thalassaemias. Antenatal diagnosis through DNA analysis of beta(+)-thalassaemia is therefore possible. The production of beta globin in...

  10. Axolotl hemoglobin: cDNA-derived amino acid sequences of two alpha globins and a beta globin from an adult Ambystoma mexicanum.

    Science.gov (United States)

    Shishikura, Fumio; Takeuchi, Hiro-aki; Nagai, Takatoshi

    2005-11-01

    Erythrocytes of the adult axolotl, Ambystoma mexicanum, have multiple hemoglobins. We separated and purified two kinds of hemoglobin, termed major hemoglobin (Hb M) and minor hemoglobin (Hb m), from a five-year-old male by hydrophobic interaction column chromatography on Alkyl Superose. The hemoglobins have two distinct alpha type globin polypeptides (alphaM and alpham) and a common beta globin polypeptide, all of which were purified in FPLC on a reversed-phase column after S-pyridylethylation. The complete amino acid sequences of the three globin chains were determined separately using nucleotide sequencing with the assistance of protein sequencing. The mature globin molecules were composed of 141 amino acid residues for alphaM globin, 143 for alpham globin and 146 for beta globin. Comparing primary structures of the five kinds of axolotl globins, including two previously established alpha type globins from the same species, with other known globins of amphibians and representatives of other vertebrates, we constructed phylogenetic trees for amphibian hemoglobins and tetrapod hemoglobins. The molecular trees indicated that alphaM, alpham, beta and the previously known alpha major globin were adult types of globins and the other known alpha globin was a larval type. The existence of two to four more globins in the axolotl erythrocyte is predicted.

  11. Sequence variations in the 5' flanking and IVS-II regions of the G gamma- and A gamma-globin genes of beta S chromosomes with five different haplotypes.

    Science.gov (United States)

    Lanclos, K D; Oner, C; Dimovski, A J; Gu, Y C; Huisman, T H

    1991-06-01

    We have amplified and sequenced the 5' flanking and the second intervening sequence (IVS-II) regions of both the G gamma- and A gamma-globin genes of the beta S chromosomes from sickle cell anemia (SS) patients with homozygosities for five different haplotypes. The sequencing data, compared with previously published sequences for the normal chromosomes A and B, show many similarities to chromosome B for haplotypes 19, 20, and 17, while haplotypes 3 and 31 are remarkably similar to chromosome A and also similar to each other. Several unique mutations were found in the 5' flanking regions (G gamma and A gamma) of haplotypes 19 and 20 and in the IVS-II segments of the same genes of haplotypes 19, 20, and 17; the IVS-II of haplotypes 3 and 31 were identical to those of chromosome A. Dot-blot analyses of amplified DNA from additional SS patients with specific probes have confirmed that these mutations are unique for each haplotype. The two general patterns that have been observed among the five haplotypes have most probably arisen by gene conversion events between the A and B type chromosomes in the African population. These patterns correlate with high and low fetal hemoglobin expression, and it is speculated that these and other yet unknown gene conversions may contribute to the variations in hemoglobin F and G gamma levels observed among SS patients. In vitro expression experiments involving the approximately 1.3-kb 5' flanking regions of the G gamma- and A gamma-globin genes of the beta S chromosomes with the five different haplotypes failed to detect differences between the levels of expression, suggesting that the sequence variations observed between these segments of DNA are not the primary cause of the differences in hemoglobin F levels among the SS patients.

  12. Frequency and origin of haplotypes associated with the beta-globin gene cluster in individuals with trait and sickle cell anemia in the Atlantic and Pacific coastal regions of Colombia.

    Science.gov (United States)

    Fong, Cristian; Lizarralde-Iragorri, María Alejandra; Rojas-Gallardo, Diana; Barreto, Guillermo

    2013-12-01

    Sickle cell anemia is a genetic disease with high prevalence in people of African descent. There are five typical haplotypes associated with this disease and the haplotypes associated with the beta-globin gene cluster have been used to establish the origin of African-descendant people in America. In this work, we determined the frequency and the origin of haplotypes associated with hemoglobin S in a sample of individuals with sickle cell anemia (HbSS) and sickle cell hemoglobin trait (HbAS) in coastal regions of Colombia. Blood samples from 71 HbAS and 79 HbSS individuals were obtained. Haplotypes were determined based on the presence of variable restriction sites within the β-globin gene cluster. On the Pacific coast of Colombia the most frequent haplotype was Benin, while on the Atlantic coast Bantu was marginally higher than Benin. Eight atypical haplotypes were observed on both coasts, being more diverse in the Atlantic than in the Pacific region. These results suggest a differential settlement of the coasts, dependent on where slaves were brought from, either from the Gulf of Guinea or from Angola, where the haplotype distributions are similar. Atypical haplotypes probably originated from point mutations that lost or gained a restriction site and/or by recombination events.

  13. Frequency and origin of haplotypes associated with the beta-globin gene cluster in individuals with trait and sickle cell anemia in the Atlantic and Pacific coastal regions of Colombia

    Directory of Open Access Journals (Sweden)

    Cristian Fong

    2013-01-01

    Full Text Available Sickle cell anemia is a genetic disease with high prevalence in people of African descent. There are five typical haplotypes associated with this disease and the haplotypes associated with the beta-globin gene cluster have been used to establish the origin of African-descendant people in America. In this work, we determined the frequency and the origin of haplotypes associated with hemoglobin S in a sample of individuals with sickle cell anemia (HbSS and sickle cell hemoglobin trait (HbAS in coastal regions of Colombia. Blood samples from 71 HbAS and 79 HbSS individuals were obtained. Haplotypes were determined based on the presence of variable restriction sites within the β-globin gene cluster. On the Pacific coast of Colombia the most frequent haplotype was Benin, while on the Atlantic coast Bantu was marginally higher than Benin. Eight atypical haplotypes were observed on both coasts, being more diverse in the Atlantic than in the Pacific region. These results suggest a differential settlement of the coasts, dependent on where slaves were brought from, either from the Gulf of Guinea or from Angola, where the haplotype distributions are similar. Atypical haplotypes probably originated from point mutations that lost or gained a restriction site and/or by recombination events.

  14. Reactivation of developmentally silenced globin genes by forced chromatin looping.

    Science.gov (United States)

    Deng, Wulan; Rupon, Jeremy W; Krivega, Ivan; Breda, Laura; Motta, Irene; Jahn, Kristen S; Reik, Andreas; Gregory, Philip D; Rivella, Stefano; Dean, Ann; Blobel, Gerd A

    2014-08-14

    Distal enhancers commonly contact target promoters via chromatin looping. In erythroid cells, the locus control region (LCR) contacts β-type globin genes in a developmental stage-specific manner to stimulate transcription. Previously, we induced LCR-promoter looping by tethering the self-association domain (SA) of Ldb1 to the β-globin promoter via artificial zinc fingers. Here, we show that targeting the SA to a developmentally silenced embryonic globin gene in adult murine erythroblasts triggers its transcriptional reactivation. This activity depends on the LCR, consistent with an LCR-promoter looping mechanism. Strikingly, targeting the SA to the fetal γ-globin promoter in primary adult human erythroblasts increases γ-globin promoter-LCR contacts, stimulating transcription to approximately 85% of total β-globin synthesis, with a reciprocal reduction in adult β-globin expression. Our findings demonstrate that forced chromatin looping can override a stringent developmental gene expression program and suggest a novel approach to control the balance of globin gene transcription for therapeutic applications.

  15. Unexpected expression of alpha- and beta-globin in mesencephalic dopaminergic neurons and glial cells.

    Science.gov (United States)

    Biagioli, Marta; Pinto, Milena; Cesselli, Daniela; Zaninello, Marta; Lazarevic, Dejan; Roncaglia, Paola; Simone, Roberto; Vlachouli, Christina; Plessy, Charles; Bertin, Nicolas; Beltrami, Antonio; Kobayashi, Kazuto; Gallo, Vittorio; Santoro, Claudio; Ferrer, Isidro; Rivella, Stefano; Beltrami, Carlo Alberto; Carninci, Piero; Raviola, Elio; Gustincich, Stefano

    2009-09-08

    The mesencephalic dopaminergic (mDA) cell system is composed of two major groups of projecting cells in the substantia nigra (SN) (A9 neurons) and the ventral tegmental area (VTA) (A10 cells). A9 neurons form the nigrostriatal pathway and are involved in regulating voluntary movements and postural reflexes. Their selective degeneration leads to Parkinson's disease. Here, we report that gene expression analysis of A9 dopaminergic neurons (DA) identifies transcripts for alpha- and beta-chains of hemoglobin (Hb). Globin immunoreactivity decorates the majority of A9 DA, a subpopulation of cortical and hippocampal astrocytes and mature oligodendrocytes. This pattern of expression was confirmed in different mouse strains and in rat and human. We show that Hb is expressed in the SN of human postmortem brain. By microarray analysis of dopaminergic cell lines overexpressing alpha- and beta-globin chains, changes in genes involved in O(2) homeostasis and oxidative phopshorylation were observed, linking Hb expression to mitochondrial function. Our data suggest that the most famed oxygen-carrying globin is not exclusively restricted to the blood, but it may play a role in the normal physiology of the brain and neurodegenerative diseases.

  16. Developmental effect of the XmnI site on Ggamma-globin gene expression among newborn Hb F-Malta-I [Ggamma117(G19)His-->Arg, CAT-->CGT] heterozygotes and adult beta+ -Thalassemia homozygotes.

    Science.gov (United States)

    Pulis, Svetlana; Scerri, Christian A; Wismayer, Pierre Schembri; Galdies, Ruth; Wettinger, Stephanie Bezzina; Felice, Alex E

    2007-01-01

    Hb F-Malta-I [Ggamma117(19)His-->Arg, CAT-->CGT] is a stable and benign variant of Hb F found in 1.8% of Maltese newborn. We studied 120 Hb F-Malta-I heterozygotes and four Hb F-Malta-I homozygotes. The mean proportion of Ggamma-F-Malta-I in Hb F was 0.26 +/- 0.03 for the Hb F-Malta-I heterozygotes and 0.58 +/- 0.06 for the Hb F-Malta-I homozygotes. The Hb F-Malta-I allele was shown to occur on a background of the common Mediterranean haplotype Va [+ + - - - - - + + -]. Furthermore, the common Mediterranean haplotypes Va, IIIb [- + + + - + + + + -], I [+ + - - - - - + + +] and II [- + - + + - + + + +] accounted for most (66.2%) of the wild-type alleles among the tested Hb F-Malta-I heterozygotes. Different genotypes at the 5' epsilon HincII, Ggamma and Agamma HindIII, and 3'psibeta HincII sites (but not at the 5' Ggamma XmnI site) were found to be linked to significant variations in the proportion of Ggamma-F-Malta-I and Ggamma-globins in the Hb F of newborn Hb F-Malta-I heterozygotes. Moreover, the 5' Ggamma XmnI site was found to be associated with variations in Hb F and Ggamma-globin levels in a population of adult Maltese beta-thalassemia (thal) homozygotes. This implies that a determinant linked to the XmnI site which effects Ggamma-globin gene expression is active in anemic adults but not in normal infants.

  17. DNA sequence analysis proves Hb M-Milwaukee-2 is due to beta-globin gene codon 92 (CAC-->TAC), the presumed mutation of Hb M-Hyde Park and Hb M-Akita.

    Science.gov (United States)

    Hutt, P J; Pisciotta, A V; Fairbanks, V F; Thibodeau, S N; Green, M M

    1998-01-01

    Among the causes of congenital methemoglobinemia, Hb M-Milwaukee-2 was one of the earliest described, in a patient who also had Hb E trait. The structure of Hb M-Milwaukee-2 has been elusive. DNA sequence analysis, as here reported, proves that this hemoglobin variant is due to the mutation CAC-->TAC at codon 92 of the beta-globin gene, corresponding to the substitution of tyrosine for histidine. This mutation is identical with that presumed to be the cause of Hb M-Hyde Park and Hb M-Akita. In addition, the DNA mutation of Hb E, GAG-->AAG at codon 26, was confirmed in this case.

  18. The effects of old and recent migration waves in the distribution of HBB*S globin gene haplotypes

    OpenAIRE

    Lindenau, Juliana D.; Sandrine C. Wagner; Simone M. Castro; Mara H. Hutz

    2016-01-01

    Abstract Sickle cell hemoglobin is the result of a mutation at the sixth amino acid position of the beta (β) globin chain. The HBB*S gene is in linkage disequilibrium with five main haplotypes in the β-globin-like gene cluster named according to their ethnic and geographic origins: Bantu (CAR), Benin (BEN), Senegal (SEN), Cameroon (CAM) and Arabian-Indian (ARAB). These haplotypes demonstrated that the sickle cell mutation arose independently at least five times in human history. The...

  19. Generation of a high-titer retroviral vector capable of expressing high levels of the human β-globin gene

    NARCIS (Netherlands)

    M. Sadelain (Michel); C.H.J. Wang (Jason); M. Antoniou (Michael); F.G. Grosveld (Frank); R.C. Mulligan

    1995-01-01

    textabstractRetrovirus-mediated gene transfer into hematopoietic cells may provide a means of treating both inherited and acquired diseases involving hematopoietic cells. Implementation of this approach for disorders resulting from mutations affecting the beta-globin gene (e.g., beta-thalassemia and

  20. Beta-globin haplotype analysis suggests that a major source of Malagasy ancestry is derived from Bantu-speaking Negroids.

    OpenAIRE

    Hewitt, R.; Krause, A.; Goldman, A; Campbell, G.; Jenkins, T

    1996-01-01

    The origins of the inhabitants of Madagascar have not been fully resolved. Anthropological studies and preliminary genetic data point to two main sources of ancestry of the Malagasy, namely, Indonesian and African, with additional contributions from India and Arabia. The sickle-cell (beta s) mutation is found in populations of African and Indian origin. The frequency of the beta s-globin gene, derived from 1,425 Malagasy individuals, varies from 0 in some highland populations to .25 in some c...

  1. High-level, erythroid specific, expression of the human α-globin gene in transgenic mice and the production of human haemoglobin in murine erythrocytes.

    NARCIS (Netherlands)

    O. Hanscombe (Olivia); M. Vidal; J. Kaeda; L. Luzzatto; D.R. Greaves (David); F.G. Grosveld (Frank)

    1989-01-01

    textabstractUsing the dominant control region (DCR) sequences that flank the beta-globin gene locus, we have been able to achieve high-level expression of the human alpha-globin gene in transgenic mice. Expression in fetal liver and blood is copy number dependent and at levels comparable to that of

  2. Co-inheritance of novel ATRX gene mutation and globin (α & β) gene mutations in transfusion dependent beta-thalassemia patients.

    Science.gov (United States)

    Al-Nafie, Awatif N; Borgio, J Francis; AbdulAzeez, Sayed; Al-Suliman, Ahmed M; Qaw, Fuad S; Naserullah, Zaki A; Al-Jarrash, Sana; Al-Madan, Mohammed S; Al-Ali, Rudaynah A; AlKhalifah, Mohammed A; Al-Muhanna, Fahad; Steinberg, Martin H; Al-Ali, Amein K

    2015-06-01

    α-Thalassemia X-linked mental retardation syndrome is a rare inherited intellectual disability disorder due to mutations in the ATRX gene. In our previous study of the prevalence of β-thalassemia mutations in the Eastern Province of Saudi Arabia, we confirmed the widespread coinheritance of α-thalassemia mutation. Some of these subjects have a family history of mental retardation, the cause of which is unknown. Therefore, we investigated the presence or absence of mutations in the ATRX gene in these patients. Three exons of the ATRX gene and their flanking regions were directly sequenced. Only four female transfusion dependent β-thalassemia patients were found to be carriers of a novel mutation in the ATRX gene. Two of the ATRX gene mutations, c.623delA and c.848T>C were present in patients homozygous for IVS I-5(G→C) and homozygous for Cd39(C → T) β-thalassemia mutation, respectively. While the other two that were located in the intronic region (flanking regions), were present in patients homozygous for Cd39(C → T) β-thalassemia mutation. The two subjects with the mutations in the coding region had family members with mental retardation, which suggests that the novel frame shift mutation and the missense mutation at coding region of ATRX gene are involved in ATRX syndrome.

  3. Conservation of globin genes in the "living fossil" Latimeria chalumnae and reconstruction of the evolution of the vertebrate globin family.

    Science.gov (United States)

    Schwarze, Kim; Burmester, Thorsten

    2013-09-01

    The (hemo-)globins are among the best-investigated proteins in biomedical sciences. These small heme-proteins play an important role in oxygen supply, but may also have other functions. In addition to well known hemoglobin and myoglobin, six other vertebrate globin types have been identified in recent years: neuroglobin, cytoglobin, globin E, globin X, globin Y, and androglobin. Analyses of the genome of the "living fossil" Latimeria chalumnae show that the coelacanth is the only known vertebrate that includes all eight globin types. Thus, Latimeria can also be considered as a "globin fossil". Analyses of gene synteny and phylogenetic reconstructions allow us to trace the evolution and the functional changes of the vertebrate globin family. Neuroglobin and globin X diverged from the other globin types before the separation of Protostomia and Deuterostomia. The cytoglobins, which are unlikely to be involved in O2 supply, form the earliest globin branch within the jawed vertebrates (Gnathostomata), but do not group with the agnathan hemoglobins, as it has been proposed before. There is strong evidence from phylogenetic reconstructions and gene synteny that the eye-specific globin E and muscle-specific myoglobin constitute a common clade, suggesting a similar role in intracellular O2 supply. Latimeria possesses two α- and two β-hemoglobin chains, of which one α-chain emerged prior to the divergence of Actinopterygii and Sarcopterygii, but has been retained only in the coelacanth. Notably, the embryonic hemoglobin α-chains of Gnathostomata derive from a common ancestor, while the embryonic β-chains - with the exception of a more complex pattern in the coelacanth and amphibians - display a clade-specific evolution. Globin Y is associated with the hemoglobin gene cluster, but its phylogenetic position is not resolved. Our data show an early divergence of distinct globin types in the vertebrate evolution before the emergence of tetrapods. The subsequent loss of

  4. Geographic distribution of 119 alleles of the alpha and beta globin genes detected in 432 French Caucasian carriers of haemoglobin variant.

    Science.gov (United States)

    Chami, B; Braconnier, F; Riou, J; Bardakdjian-Michau, J; Préhu, C; Blouquit, Y; Rosa, J; Wajcman, H; Galactéros, F

    1995-01-01

    The French population is the result of mixing of different peoples including the Celts, Saxons, Germans, Italians and Hispanics. Between 1981 and 1993 patients were selected during investigations in France for haematological disorders associated or otherwise with the presence of a haemoglobin (Hb) variant. Further carriers of abnormal Hb were identified by HPLC measurement of glycated Hb in diabetics and by neonatal screening. Four-hundred and thirty-two subjects were found to be heterozygous for one of the 119 different alpha and the beta gene alleles encountered. These variants were characterised by a combination of 6 electrophoretic methods and in some cases by protein structure determinations. Some mutants reflected the population movements in and into France. A few mutants are frequently described in the French Caucasian population: Hb Lepore Boston, Hb D Punjab, whereas others appear to be anthropological markers. Hb Winnipeg has only been found in the West of France (Normandy); Hb J Baltimore is mainly found in French subjects of Spanish origin. Several cases of sporadic and previously undescribed mutations of Hb were identified. The last immigration waves from Africa and Asia appear to have contributed to the evolution of the pattern of haemoglobinopathies in the French population (Hb S, Hb O Arab or Hb C).

  5. Mutations in the paralogous human α-globin genes yielding identical hemoglobin variants

    NARCIS (Netherlands)

    K. Moradkhani (Kamran); C. Prehu (Claude); J. Old (John); S. Henderson (Shirley); V. Balamitsa (Vera); H-Y. Luo; M-C. Poon (Man-Chiu); D.H. Chui (David); H. Wajcman (Henri); G.P. Patrinos (George)

    2009-01-01

    textabstractThe human α-globin genes are paralogues, sharing a high degree of DNA sequence similarity and producing an identical α-globin chain. Over half of the α-globin structural variants reported to date are only characterized at the amino acid level. It is likely that a fraction of these varian

  6. Search for antisense copies of beta-globin mRNA in anemic mouse spleen

    Directory of Open Access Journals (Sweden)

    Taylor John M

    2001-03-01

    Full Text Available Abstract Background Previous studies by Volloch and coworkers have reported that during the expression of high levels of β-globin mRNA in the spleen of anemic mice, they could also detect small but significant levels of an antisense (AS globin RNA species, which they postulated might have somehow arisen by RNA-directed RNA synthesis. For two reasons we undertook to confirm and possibly extend these studies. First, previous studies in our lab have focussed on what is an unequivocal example of host RNA-directed RNA polymerase activity on the RNA genome of human hepatitis delta virus. Second, if AS globin species do exist they could in turn form double-stranded RNA species which might induce post-transcriptional gene silencing, a phenomenon somehow provoked in eukaryotic cells by AS RNA sequences. Results We reexamined critical aspects of the previous globin studies. We used intraperitoneal injections of phenylhydrazine to induce anemia in mice, as demonstrated by the appearance and ultimate disappearance of splenomegaly. While a 30-fold increase in globin mRNA was detected in the spleen, the relative amount of putative AS RNA could be no more than 0.004%. Conclusions Contrary to earlier reports, induction of a major increase in globin transcripts in the mouse spleen was not associated with a detectable level of antisense RNA to globin mRNA.

  7. Implications of the genetic epidemiology of globin haplotypes linked to the sickle cell gene in southern Iran.

    Science.gov (United States)

    Rahimi, Zohreh; Merat, Ahmad; Gerard, Nathalie; Krishnamoorthy, Rajagopal; Nagel, Ronald L

    2006-12-01

    To determine the origin of sickle cell mutation in different ethnic groups living in southern Iran, we studied the haplotype background of the betaS and betaA genes in subjects from the provinces of Fars, Khuzestan, Bushehr, Hormozgan, and Kerman and from the islands of Khark and Qeshm. beta-globin gene cluster haplotypes were determined using the PCR-RFLP technique. Detection of -alpha 3.7 deletion and beta-thalassemia mutations were defined by PCR and reverse dot blot techniques, respectively. The framework of the beta-globin gene was determined using denaturing gradient gel electrophoresis. We found that the betaS mutation in southern Iran is associated with multiple mutational events. Most of the patients were from two ethnic groups: Farsi speakers (presumably Persian in origin) from Fars province and patients of Arab origin from Khuzestan province. In both ethnic groups the Arab-Indian haplotype was the most prevalent. The frequencies of the Arab-Indian and African haplotypes in sickle cell anemia patients from the provinces of Fars and Khuzestan were similar. Among betaA chromosomes the Bantu A2 haplotype was the most prevalent. The decrease in alpha-globin production in SS patients and AS individuals appeared to be related to the reduction in mean cell volume and mean cell hemoglobin. The Arab-Indian haplotype gene flow into this region of Iran can be traced to the Sassanian Empire. It is likely that the influx of betaS genes linked to the Benin and Bantu haplotypes, of African origin, must have occurred during the Arab slave trade.

  8. The organization of the gamma-delta-beta gene complex in normal and thalassemia cells.

    Science.gov (United States)

    Bank, A; Mears, J G; Ramirez, F; Burns, A L; Spence, S; Feldenzer, J; Baird, M

    1980-01-01

    Restriction enzyme digestion analysis and direct human globin gene cloning have permitted analysis of the physical arrangement of nucleotide sequences within and surrounding the human globin genes. With these methods it has been shown that the linear arrangement 5' to 3' of the globin genes is G gamma-A gamma-delta-beta. The G gamma and A gamma genes are separated by about 3.5 kilobases (kb), while the A gamma and delta genes are 15 kb apart, and the delta and beta 6.5 kb apart. Each of these genes contains a large intervening sequence (IVS) of approximately 1 kb in precisely the same position between condons 104 and 105. In addition, each of these genes has a small IVS between codons 30 and 31. In homozygous delta beta thalassemia DNA, there is deletion of all of the normal delta and beta gene fragments. However, a new fragment 4.2 kb in size containing the 5' end of the delta globin gene is retained. Retention of this fragment in delta beta thalassemia, but not in HPFH is consistent with a role for sequences in this region for limiting gamma globin gene expression. Studies to date suggest that the beta + and beta 0 thalassemias will be due to a heterogeneous group of DNA defects affecting either beta globin gene transcription or beta mRNA processing. In most cases of beta + and beta 0 thalassemia DNA analyzed, there is no detectable deletion of beta or delta genes. In three India beta 0 patients, deletion of the 3' end of the beta gene has been found. Analysis of cloned beta globin genes from a patient with beta + thalasseia shows differences from normal in the fragments generated by restriction enzymes which cut frequently. Whether these differences are responsible for the defect in thalassemia or are polymorphisms unrelated to thalassemia remains to be determined.

  9. The relative importance of the X-linked FCP locus and beta-globin haplotypes in determining haemoglobin F levels: a study of SS patients homozygous for beta S haplotypes.

    Science.gov (United States)

    Chang, Y P; Maier-Redelsperger, M; Smith, K D; Contu, L; Ducroco, R; de Montalembert, M; Belloy, M; Elion, J; Dover, G J; Girot, R

    1997-03-01

    Five factors have been hypothesized to influence the 20-fold variation in fetal haemoglobin (Hb F) levels in sickle cell anaemia (SS): age sex, alpha-globin gene number, beta-globin haplotype, and the X-linked F-cell production locus (FCP) that regulates the production of Hb F containing erythrocytes (F cells). We analysed the association of these factors with Hb F levels in 112 SS patients living in France who are homozygous for the three common African beta-globin haplotypes (Benin, Bantu or Central African Republic and Senegal). We found that: (1) FCP accounts for about 40% of the overall variation in Hb F levels, (2) when the FCP influence is removed, beta-globin haplotype is associated with 14% of the remaining Hb F variation, and (3) the other factors have little influence. Comparison with our previous study of SS individuals in Jamaica leads to the following conclusions: (1) the X-linked FCP locus is a major determinant of Hb F levels in SS disease, (2) factors linked to the beta-globin haplotype have only a small effect on the variation in Hb F levels, in either the homozygous or heterozygous state, and (3)approximately half of the variation in Hb F levels still remains to be explained.

  10. Beta-globin gene haplotypes among cameroonians and review of the global distribution: is there a case for a single sickle mutation origin in Africa?

    Science.gov (United States)

    Bitoungui, Valentina J Ngo; Pule, Gift D; Hanchard, Neil; Ngogang, Jeanne; Wonkam, Ambroise

    2015-03-01

    Studies of hemoglobin S haplotypes in African subpopulations have potential implications for patient care and our understanding of genetic factors that have shaped the prevalence of sickle cell disease (SCD). We evaluated HBB gene cluster haplotypes in SCD patients from Cameroon, and reviewed the literature for a global distribution. We reviewed medical records to obtain pertinent socio-demographic and clinical features for 610 Cameroonian SCD patients, including hemoglobin electrophoresis and full blood counts. RFLP-PCR was used to determine the HBB gene haplotype on 1082 chromosomes. A systematic review of the current literature was undertaken to catalogue HBB haplotype frequencies in SCD populations around the world. Benin (74%; n = 799) and Cameroon (19%; n = 207) were the most prevalent haplotypes observed among Cameroonian patients. There was no significant association between HBB haplotypes and clinical life events, anthropometric measures, hematological parameters, or fetal hemoglobin (HbF) levels. The literature review of the global haplotype distributions was consistent with known historical migrations of the people of Africa. Previously reported data from Sudan showed a distinctly unusual pattern; all four classical haplotypes were reported, with an exceptionally high proportion of the Senegal, Cameroon, and atypical haplotypes. We did not observe any significant associations between HBB haplotype and SCD disease course in this cohort. Taken together, the data from Cameroon and from the wider literature suggest that a careful reassessment of African HBB haplotypes may shed further light on the evolutionary dynamics of the sickle allele, which could suggest a single origin of the sickle mutation.

  11. Genome scan identifies a locus affecting gamma-globin expression in human beta-cluster YAC transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Lin, S.D.; Cooper, P.; Fung, J.; Weier, H.U.G.; Rubin, E.M.

    2000-03-01

    Genetic factors affecting post-natal g-globin expression - a major modifier of the severity of both b-thalassemia and sickle cell anemia, have been difficult to study. This is especially so in mice, an organism lacking a globin gene with an expression pattern equivalent to that of human g-globin. To model the human b-cluster in mice, with the goal of screening for loci affecting human g-globin expression in vivo, we introduced a human b-globin cluster YAC transgene into the genome of FVB mice . The b-cluster contained a Greek hereditary persistence of fetal hemoglobin (HPFH) g allele resulting in postnatal expression of human g-globin in transgenic mice. The level of human g-globin for various F1 hybrids derived from crosses between the FVB transgenics and other inbred mouse strains was assessed. The g-globin level of the C3HeB/FVB transgenic mice was noted to be significantly elevated. To map genes affecting postnatal g-globin expression, a 20 centiMorgan (cM) genome scan of a C3HeB/F VB transgenics [prime] FVB backcross was performed, followed by high-resolution marker analysis of promising loci. From this analysis we mapped a locus within a 2.2 cM interval of mouse chromosome 1 at a LOD score of 4.2 that contributes 10.4% of variation in g-globin expression level. Combining transgenic modeling of the human b-globin gene cluster with quantitative trait analysis, we have identified and mapped a murine locus that impacts on human g-globin expression in vivo.

  12. An in vitro globin gene switching model based on differentiated embryonic stem cells.

    NARCIS (Netherlands)

    M.H. Lindenbaum; F.G. Grosveld (Frank)

    1990-01-01

    textabstractWe used mouse embryonic stem (ES) cells to study globin gene expression and switching in vitro. We show that ES-derived embryoid bodies express the full complement of mouse embryonic globin genes in the correct temporal order and that on further differentiation, a switch occurs to the fe

  13. Beta Thalassemia

    Science.gov (United States)

    Beta thalassemia is found in people of Mediterranean, Middle Eastern, African, South Asian (Indian, Pakistani, etc.), Southeast Asian and Chinese descent. 1 Beta Thalassemia ßß Normal beta globin genes found on chromosomes ...

  14. Allele-specific enzymatic amplification of. beta. -globin genomic DNA for diagnosis of sickle cell anemia

    Energy Technology Data Exchange (ETDEWEB)

    Wu, D.Y.; Ugozzoli, L.; Pal, B.K.; Wallace, B. (Beckman Research Institute of the City of Hope, Duarte, CA (USA))

    1989-04-01

    A rapid nonradioactive approach to the diagnosis of sickle cell anemia is described based on an allele-specific polymerase chain reaction (ASPCR). This method allows direct detection of the normal or the sickle cell {beta}-globin allele in genomic DNA without additional steps of probe hybridization, ligation, or restriction enzyme cleavage. Two allele-specific oligonucleotide primers, one specific for the sickle cell allele and one specific for the normal allele, together with another primer complementary to both alleles were used in the polymerase chain reaction with genomic DNA templates. The allele-specific primers differed from each other in their terminal 3{prime} nucleotide. Under the proper annealing temperature and polymerase chain reaction conditions, these primers only directed amplification on their complementary allele. In a single blind study of DNA samples from 12 individuals, this method correctly and unambiguously allowed for the determination of the genotypes with no false negatives or positives. If ASPCR is able to discriminate all allelic variation (both transition and transversion mutations), this method has the potential to be a powerful approach for genetic disease diagnosis, carrier screening, HLA typing, human gene mapping, forensics, and paternity testing.

  15. A preclinical approach for gene therapy of beta-thalassemia.

    Science.gov (United States)

    Breda, Laura; Kleinert, Dorothy A; Casu, Carla; Casula, Laura; Cartegni, Luca; Fibach, Eitan; Mancini, Irene; Giardina, Patricia J; Gambari, Roberto; Rivella, Stefano

    2010-08-01

    Lentiviral-mediated beta-globin gene transfer successfully treated beta-thalassemic mice. Based on this result, clinical trials were initiated. To date, however, no study has investigated the efficacy of gene therapy in relation to the nature of the different beta-globin mutations found in patients. Most mutations can be classified as beta(0) or beta(+), based on the amount of beta-globin protein produced. Therefore, we propose that a screening in vitro is necessary to verify the efficacy of gene transfer prior to treatment of individual patients. We used a two-phase liquid culture system to expand and differentiate erythroid progenitor cells (ErPCs) transduced with lentiviral vectors. We propose the use of this system to test the efficiency of lentiviral vectors carrying the human beta-globin gene, to correct the phenotype of ErPCs from patients preparing for gene therapy. This new approach might have profound implications for designing gene therapy and for understanding the genotype/phenotype variability observed in Cooley's anemia patients.

  16. Erythroid Krüppel-like factor (EKLF) is active in primitive and definitive erythroid cells and is required for the function of 5'HS3 of the beta-globin locus control region.

    Science.gov (United States)

    Tewari, R; Gillemans, N; Wijgerde, M; Nuez, B; von Lindern, M; Grosveld, F; Philipsen, S

    1998-04-15

    Disruption of the gene for transcription factor EKLF (erythroid Krüppel-like factor) results in fatal anaemia caused by severely reduced expression of the adult beta-globin gene, while other erythroid-specific genes, including the embryonic epsilon- and fetal gamma-globin genes, are expressed normally. Thus, EKLF is thought to be a stage-specific factor acting through the CACC box in the beta-gene promoter, even though it is already present in embryonic red cells. Here, we show that a beta-globin gene linked directly to the locus control region (LCR) is expressed at embryonic stages, and that this is only modestly reduced in EKLF-/- embryos. Thus, embryonic beta-globin expression is not intrinsically dependent on EKLF. To investigate whether EKLF functions in the locus control region, we analysed the expression of LCR-driven lacZ reporters. This shows that EKLF is not required for reporter activation by the complete LCR. However, embryonic expression of reporters driven by 5'HS3 of the LCR requires EKLF. This suggests that EKLF interacts directly with the CACC motifs in 5'HS3 and demonstrates that EKLF is also a transcriptional activator in embryonic erythropoiesis. Finally, we show that overexpression of EKLF results in an earlier switch from gamma- to beta-globin expression. Adult mice with the EKLF transgene have reduced platelet counts, suggesting that EKLF levels affect the balance between the megakaryocytic and erythroid lineages. Interestingly, the EKLF transgene rescues the lethal phenotype of EKLF null mice, setting the stage for future studies aimed at the analysis of the EKLF protein and its role in beta-globin gene activation.

  17. A novel approach to rapid determination of betaS-globin haplotypes: sequencing of the Agamma-IVS-II region.

    Science.gov (United States)

    Vinson, Amy E; Walker, Aisha; Elam, Dedrey; Glendenning, Michele; Kutlar, Ferdane; Clair, Betsy; Harbin, Jeanette; Kutlar, Abdullah

    2004-01-01

    beta-Globin gene cluster haplotypes were originally determined by restriction endonuclease mapping with Southern blots of polymorphic sites around the gene cluster. Over the years, haplotyping has been found to be useful, not only in population genetics but also in predicting the severity of hemoglobinopathies such as sickle cell disease. The sickle mutation occurs on five distinct haplotypes. The hitherto used methods are cumbersome and time-consuming, making haplotype determination a tedious procedure. We report our experience with a novel, rapid approach to haplotyping based on sequence polymorphisms in the Agamma-IVS-II region. We provide an algorithm that allows rapid assignment of the four African haplotypes carrying the sickle mutation.

  18. Eos negatively regulates human γ-globin gene transcription during erythroid differentiation.

    Directory of Open Access Journals (Sweden)

    Hai-Chuan Yu

    Full Text Available BACKGROUND: Human globin gene expression is precisely regulated by a complicated network of transcription factors and chromatin modifying activities during development and erythropoiesis. Eos (Ikaros family zinc finger 4, IKZF4, a member of the zinc finger transcription factor Ikaros family, plays a pivotal role as a repressor of gene expression. The aim of this study was to examine the role of Eos in globin gene regulation. METHODOLOGY/PRINCIPAL FINDINGS: Western blot and quantitative real-time PCR detected a gradual decrease in Eos expression during erythroid differentiation of hemin-induced K562 cells and Epo-induced CD34+ hematopoietic stem/progenitor cells (HPCs. DNA transfection and lentivirus-mediated gene transfer demonstrated that the enforced expression of Eos significantly represses the expression of γ-globin, but not other globin genes, in K562 cells and CD34+ HPCs. Consistent with a direct role of Eos in globin gene regulation, chromatin immunoprecipitaion and dual-luciferase reporter assays identified three discrete sites located in the DNase I hypersensitivity site 3 (HS3 of the β-globin locus control region (LCR, the promoter regions of the Gγ- and Aγ- globin genes, as functional binding sites of Eos protein. A chromosome conformation capture (3C assay indicated that Eos may repress the interaction between the LCR and the γ-globin gene promoter. In addition, erythroid differentiation was inhibited by enforced expression of Eos in K562 cells and CD34+ HPCs. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that Eos plays an important role in the transcriptional regulation of the γ-globin gene during erythroid differentiation.

  19. Mutation screening in the human epsilon-globin gene using single-strand conformation polymorphism analysis.

    Science.gov (United States)

    Papachatzopoulou, Adamantia; Menounos, Panagiotis G; Kolonelou, Christina; Patrinos, George P

    2006-02-01

    The human epsilon-globin gene is necessary for primitive human erythropoiesis in the yolk sac. Herein we report a non-radioactive single-strand conformation polymorphism (SSCP) approach to screen the human epsilon-globin gene and its regulatory regions for possible mutations and single-nucleotide polymorphisms in normal adult subjects, in order to determine those genomic regions, which are not necessary for its proper regulation and function. We identified no sequence variations apart from the expected 5'epsilon /HincII polymorphism in the fragments analyzed, suggesting that genomic alterations in the epsilon-globin gene are most likely incompatible with normal erythropoiesis and proper embryonic development.

  20. Gene turnover in the avian globin gene families and evolutionary changes in hemoglobin isoform expression.

    Science.gov (United States)

    Opazo, Juan C; Hoffmann, Federico G; Natarajan, Chandrasekhar; Witt, Christopher C; Berenbrink, Michael; Storz, Jay F

    2015-04-01

    The apparent stasis in the evolution of avian chromosomes suggests that birds may have experienced relatively low rates of gene gain and loss in multigene families. To investigate this possibility and to explore the phenotypic consequences of variation in gene copy number, we examined evolutionary changes in the families of genes that encode the α- and β-type subunits of hemoglobin (Hb), the tetrameric α2β2 protein responsible for blood-O2 transport. A comparative genomic analysis of 52 bird species revealed that the size and membership composition of the α- and β-globin gene families have remained remarkably constant during approximately 100 My of avian evolution. Most interspecific variation in gene content is attributable to multiple independent inactivations of the α(D)-globin gene, which encodes the α-chain subunit of a functionally distinct Hb isoform (HbD) that is expressed in both embryonic and definitive erythrocytes. Due to consistent differences in O2-binding properties between HbD and the major adult-expressed Hb isoform, HbA (which incorporates products of the α(A)-globin gene), recurrent losses of α(D)-globin contribute to among-species variation in blood-O2 affinity. Analysis of HbA/HbD expression levels in the red blood cells of 122 bird species revealed high variability among lineages and strong phylogenetic signal. In comparison with the homologous gene clusters in mammals, the low retention rate for lineage-specific gene duplicates in the avian globin gene clusters suggests that the developmental regulation of Hb synthesis in birds may be more highly conserved, with orthologous genes having similar stage-specific expression profiles and similar functional properties in disparate taxa.

  1. Two new beta0-thalassemic mutations: a deletion (-CC) at codon 142 or overlapping codons 142-143, and an insertion (+T) at codon 45 or overlapping codons 44-45/45-46 of the beta-globin gene.

    Science.gov (United States)

    Lacan, Philippe; Aubry, Martine; Couprie, Nicole; Francina, Alain

    2007-01-01

    We report here two new beta(0)-thalassemic mutations. In the first case, a deletion of two nucleotides (-CC) at codon 142 was found in a French Caucasian woman. In the second case, an insertion of a single nucleotide (+T) at codon 45 was found in a Turkish girl. In both cases, no dominant thalassemia-like phenotype was observed.

  2. Three globin lineages belonging to two structural classes in genomes from the three kingdoms of life.

    Science.gov (United States)

    Vinogradov, Serge N; Hoogewijs, David; Bailly, Xavier; Arredondo-Peter, Raúl; Guertin, Michel; Gough, Julian; Dewilde, Sylvia; Moens, Luc; Vanfleteren, Jacques R

    2005-08-09

    Although most globins, including the N-terminal domains within chimeric proteins such as flavohemoglobins and globin-coupled sensors, exhibit a 3/3 helical sandwich structure, many bacterial, plant, and ciliate globins have a 2/2 helical sandwich structure. We carried out a comprehensive survey of globins in the genomes from the three kingdoms of life. Bayesian phylogenetic trees based on manually aligned sequences indicate the possibility of past horizontal globin gene transfers from bacteria to eukaryotes. blastp searches revealed the presence of 3/3 single-domain globins related to the globin domains of the bacterial and fungal flavohemoglobins in many bacteria, a red alga, and a diatom. Iterated psi-blast searches based on groups of globin sequences found that only the single-domain globins and flavohemoglobins recognize the eukaryote 3/3 globins, including vertebrate neuroglobins, alpha- and beta-globins, and cytoglobins. The 2/2 globins recognize the flavohemoglobins, as do the globin coupled sensors and the closely related single-domain protoglobins. However, the 2/2 globins and the globin-coupled sensors do not recognize each other. Thus, all globins appear to be distributed among three lineages: (i) the 3/3 plant and metazoan globins, single-domain globins, and flavohemoglobins; (ii) the bacterial 3/3 globin-coupled sensors and protoglobins; and (iii) the bacterial, plant, and ciliate 2/2 globins. The three lineages may have evolved from an ancestral 3/3 or 2/2 globin. Furthermore, it appears likely that the predominant functions of globins are enzymatic and that oxygen transport is a specialized development that accompanied the evolution of metazoans.

  3. Beta-globin haplotype analysis suggests that a major source of Malagasy ancestry is derived from Bantu-speaking Negroids.

    Science.gov (United States)

    Hewitt, R; Krause, A; Goldman, A; Campbell, G; Jenkins, T

    1996-06-01

    The origins of the inhabitants of Madagascar have not been fully resolved. Anthropological studies and preliminary genetic data point to two main sources of ancestry of the Malagasy, namely, Indonesian and African, with additional contributions from India and Arabia. The sickle-cell (beta s) mutation is found in populations of African and Indian origin. The frequency of the beta s-globin gene, derived from 1,425 Malagasy individuals, varies from 0 in some highland populations to .25 in some coastal populations. The beta s mutation is thought to have arisen at least five times, on the basis of the presence of five distinct beta s-associated haplotypes, each found in a separate geographic area. Twenty-five of the 35 Malagasy beta s haplotypes were of the typical "Bantu" type, 1 "Senegal" haplotype was found, and 2 rare or atypical haplotypes were observed; the remaining 7 haplotypes were consistent with the Bantu haplotype. The Bantu beta s mutation is thought to have been introduced into Madagascar by Bantu-speaking immigrants (colonists or slaves) from central or east Africa. The Senegal beta s mutation may have been introduced to the island via Portuguese naval explorers. This study provides the first definitive biological evidence that a major component of Malagasy ancestry is derived from African populations, in particular, Bantu-speaking Negroids. beta A haplotypes are also consistent with the claim for a significant African contribution to Malagasy ancestry but are also suggestive of Asian/Oceanic and Caucasoid admixture within the Malagasy population.

  4. Gamma-interferon alters globin gene expression in neonatal and adult erythroid cells

    Energy Technology Data Exchange (ETDEWEB)

    Miller, B.A.; Perrine, S.P.; Antognetti, G.; Perlmutter, D.H.; Emerson, S.G.; Sieff, C.; Faller, D.V.

    1987-06-01

    The effect of gamma-interferon on fetal hemoglobin synthesis by purified cord blood, fetal liver, and adult bone marrow erythroid progenitors was studied with a radioligand assay to measure hemoglobin production by BFU-E-derived erythroblasts. Coculture with recombinant gamma-interferon resulted in a significant and dose-dependent decrease in fetal hemoglobin production by neonatal and adult, but not fetal, BFU-E-derived erythroblasts. Accumulation of fetal hemoglobin by cord blood BFU-E-derived erythroblasts decreased up to 38.1% of control cultures (erythropoietin only). Synthesis of both G gamma/A gamma globin was decreased, since the G gamma/A gamma ratio was unchanged. Picograms fetal hemoglobin per cell was decreased by gamma-interferon addition, but picograms total hemoglobin was unchanged, demonstrating that a reciprocal increase in beta-globin production occurred in cultures treated with gamma-interferon. No toxic effect of gamma-interferon on colony growth was noted. The addition of gamma-interferon to cultures resulted in a decrease in the percentage of HbF produced by adult BFU-E-derived cells to 45.6% of control. Fetal hemoglobin production by cord blood, fetal liver, and adult bone marrow erythroid progenitors, was not significantly affected by the addition of recombinant GM-CSF, recombinant interleukin 1 (IL-1), recombinant IL-2, or recombinant alpha-interferon. Although fetal progenitor cells appear unable to alter their fetal hemoglobin program in response to any of the growth factors added here, the interaction of neonatal and adult erythroid progenitors with gamma-interferon results in an altered expression of globin genes.

  5. Mutations in the paralogous human alpha-globin genes yielding identical hemoglobin variants.

    Science.gov (United States)

    Moradkhani, Kamran; Préhu, Claude; Old, John; Henderson, Shirley; Balamitsa, Vera; Luo, Hong-Yuan; Poon, Man-Chiu; Chui, David H K; Wajcman, Henri; Patrinos, George P

    2009-06-01

    The human alpha-globin genes are paralogues, sharing a high degree of DNA sequence similarity and producing an identical alpha-globin chain. Over half of the alpha-globin structural variants reported to date are only characterized at the amino acid level. It is likely that a fraction of these variants, with phenotypes differing from one observation to another, may be due to the same mutation but on a different alpha-globin gene. There have been very few previous examples of hemoglobin variants that can be found at both HBA1 and HBA2 genes. Here, we report the results of a systematic multicenter study in a large multiethnic population to identify such variants and to analyze their differences from a functional and evolutionary perspective. We identified 14 different Hb variants resulting from identical mutations on either one of the two human alpha-globin paralogue genes. We also showed that the average percentage of hemoglobin variants due to a HBA2 gene mutation (alpha2) is higher than the percentage of hemoglobin variants due to the same HBA1 gene mutation (alpha1) and that the alpha2/alpha1 ratio varied between variants. These alpha-globin chain variants have most likely occurred via recurrent mutations, gene conversion events, or both. Based on these data, we propose a nomenclature for hemoglobin variants that fall into this category.

  6. Ancient Duplications and Expression Divergence in the Globin Gene Superfamily of Vertebrates: Insights from the Elephant Shark Genome and Transcriptome.

    Science.gov (United States)

    Opazo, Juan C; Lee, Alison P; Hoffmann, Federico G; Toloza-Villalobos, Jessica; Burmester, Thorsten; Venkatesh, Byrappa; Storz, Jay F

    2015-07-01

    Comparative analyses of vertebrate genomes continue to uncover a surprising diversity of genes in the globin gene superfamily, some of which have very restricted phyletic distributions despite their antiquity. Genomic analysis of the globin gene repertoire of cartilaginous fish (Chondrichthyes) should be especially informative about the duplicative origins and ancestral functions of vertebrate globins, as divergence between Chondrichthyes and bony vertebrates represents the most basal split within the jawed vertebrates. Here, we report a comparative genomic analysis of the vertebrate globin gene family that includes the complete globin gene repertoire of the elephant shark (Callorhinchus milii). Using genomic sequence data from representatives of all major vertebrate classes, integrated analyses of conserved synteny and phylogenetic relationships revealed that the last common ancestor of vertebrates possessed a repertoire of at least seven globin genes: single copies of androglobin and neuroglobin, four paralogous copies of globin X, and the single-copy progenitor of the entire set of vertebrate-specific globins. Combined with expression data, the genomic inventory of elephant shark globins yielded four especially surprising findings: 1) there is no trace of the neuroglobin gene (a highly conserved gene that is present in all other jawed vertebrates that have been examined to date), 2) myoglobin is highly expressed in heart, but not in skeletal muscle (reflecting a possible ancestral condition in vertebrates with single-circuit circulatory systems), 3) elephant shark possesses two highly divergent globin X paralogs, one of which is preferentially expressed in gonads, and 4) elephant shark possesses two structurally distinct α-globin paralogs, one of which is preferentially expressed in the brain. Expression profiles of elephant shark globin genes reveal distinct specializations of function relative to orthologs in bony vertebrates and suggest hypotheses about

  7. β-globin gene cluster haplotypes in ethnic minority populations of southwest China

    Science.gov (United States)

    Sun, Hao; Liu, Hongxian; Huang, Kai; Lin, Keqin; Huang, Xiaoqin; Chu, Jiayou; Ma, Shaohui; Yang, Zhaoqing

    2017-01-01

    The genetic diversity and relationships among ethnic minority populations of southwest China were investigated using seven polymorphic restriction enzyme sites in the β-globin gene cluster. The haplotypes of 1392 chromosomes from ten ethnic populations living in southwest China were determined. Linkage equilibrium and recombination hotspot were found between the 5′ sites and 3′ sites of the β-globin gene cluster. 5′ haplotypes 2 (+−−−), 6 (−++−+), 9 (−++++) and 3′ haplotype FW3 (−+) were the predominant haplotypes. Notably, haplotype 9 frequency was significantly high in the southwest populations, indicating their difference with other Chinese. The interpopulation differentiation of southwest Chinese minority populations is less than those in populations of northern China and other continents. Phylogenetic analysis shows that populations sharing same ethnic origin or language clustered to each other, indicating current β-globin cluster diversity in the Chinese populations reflects their ethnic origin and linguistic affiliations to a great extent. This study characterizes β-globin gene cluster haplotypes in southwest Chinese minorities for the first time, and reveals the genetic variability and affinity of these populations using β-globin cluster haplotype frequencies. The results suggest that ethnic origin plays an important role in shaping variations of the β-globin gene cluster in the southwestern ethnic populations of China. PMID:28205625

  8. Fetal Haemoglobin and β-globin Gene Cluster Haplotypes among Sickle Cell Patients in Chhattisgarh

    OpenAIRE

    Bhagat, Sanjana; Patra, Pradeep Kumar; Thakur, Amar Singh

    2013-01-01

    Background: Foetal Haemoglobin (HbF) is the best-known genetic modulator of sickle cell anaemia, which varies dramatically in concentration in the blood of these patients. The patients with SCA display a remarkable variability in the disease severity. High HbF levels and the β-globin gene cluster haplotypes influence the clinical presentation of sickle cell disease. To identify the genetic modifiers which influence the disease severity, we conducted a β-globin haplotype analysis in the sickle...

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

    Science.gov (United States)

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

    2014-05-09

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

  10. Development of K562 cell clones expressing beta-globin mRNA carrying the beta039 thalassaemia mutation for the screening of correctors of stop-codon mutations.

    Science.gov (United States)

    Salvatori, Francesca; Cantale, Vera; Breveglieri, Giulia; Zuccato, Cristina; Finotti, Alessia; Bianchi, Nicoletta; Borgatti, Monica; Feriotto, Giordana; Destro, Federica; Canella, Alessandro; Breda, Laura; Rivella, Stefano; Gambari, Roberto

    2009-07-09

    Nonsense mutations, giving rise to UAA, UGA and UAG stop codons within the coding region of mRNAs, promote premature translational termination and are the leading cause of approx. 30% of inherited diseases, including cystic fibrosis, Duchenne muscular dystrophy and thalassaemia. For instance, in beta(0)39-thalassaemia the CAG (glutamine) codon is mutated to the UAG stop codon, leading to premature translation termination and to mRNA destabilization through the well-described NMD (nonsense-mediated mRNA decay). In order to develop an approach facilitating translation and, therefore, protection from NMD, aminoglycoside antibiotics have been tested on mRNAs carrying premature stop codons. These drugs decrease the accuracy in the codon-anticodon base-pairing, inducing a ribosomal read-through of the premature termination codons. Interestingly, recent papers have described drugs designed and produced for suppressing premature translational termination, inducing a ribosomal read-through of premature but not normal termination codons. These findings have introduced new hopes for the development of a pharmacological approach to the therapy of beta(0)39-thalassaemia. In this context, we started the development of a cellular model of the beta(0)39-thalassaemia mutation that could be used for the screening of a high number of aminoglycosides and analogous molecules. To this aim, we produced a lentiviral construct containing the beta(0)39-thalassaemia globin gene under a minimal LCR (locus control region) control and used this construct for the transduction of K562 cells, subsequently subcloned, with the purpose to obtain several K562 clones with different integration copies of the construct. These clones were then treated with Geneticin (also known as G418) and other aminoglycosides and the production of beta-globin was analysed by FACS analysis. The results obtained suggest that this experimental system is suitable for the characterization of correction of the beta(0

  11. ISOBUTYRAMIDE ACTIVATES TRANSCRIPTION OF HUMAN FETAL γ- AND MURINE EMBRYONIC εy-GLOBIN GENES

    Institute of Scientific and Technical Information of China (English)

    张俊武; 张雪青; 陈平

    2001-01-01

    Objective. To examine the effect of isobutyramide synthesized in our laboratory on human and murine globingene expression and to test cell toxicity of the drug.Methods. ME L cells were transfected with the recombinant construct μLCRAγψβδβ3 and the stable transfor-mants were cultured in the medium with different concentrations of isobutyramide. The experimental mice and rab-bit were injected with different doses of isobutyramide. The globin mRNAs were analyzed by RNase protection as-say. The hematologdcal toxicity and electrolyte toxicity of the drug were tested.Results. An inducible and dose-dependent expression of the human γγ-, ββ- and mouse Aa-globin gene was ob-served in the transfected MEL cells. The induction of the human γ-globin gene is significant stronger than that ofthe ββ-globin gene. With 2.5 ~ 5 mmol/L isobutyramide, the induction of the human γ-globin gene is even moreeffective than that of mouse aa-globin gene. After a 15-day injection under the doses of 500 ~ 900mg * kg- 1 * d-1,the level of the mouse embryonic εy-globin mRNA could be significantly induced up to 3 ~ 4 fold of that of uninject-ed controls. The changes of hemoglobin(Hb), RBC, hematocrit(HCT), WBC, derived from mice injected withdifferent doses of isobutyramide at the interval of 24 hours for 2 ~4 weeks, were generally within the normalrange. In rabbits injected with isobutyramide in the same regiment for 2 weeks, the concentration of blood K+,Na +, C1- and CO.2 were all within normal range and serum ionic osmotic pressure remained stable as well.Conclusion. Our results suggested that isobutyramide is a weak inducer of cell differentiation, but it canselectively activate transcription of human γy-globin gene at a certain degree, and it can act on early stages of ery-throid progenitor differentiation in adult mice and activate transcription of embryonic εy-globin gene and have nohematological toxicity. Our results have further proved the potential value of

  12. Cell and Gene Therapy for the Beta-Thalassemias: Advances and Prospects.

    Science.gov (United States)

    Mansilla-Soto, Jorge; Riviere, Isabelle; Boulad, Farid; Sadelain, Michel

    2016-04-01

    The beta-thalassemias are inherited anemias caused by mutations that severely reduce or abolish expression of the beta-globin gene. Like sickle cell disease, a related beta-globin gene disorder, they are ideal candidates for performing a genetic correction in patient hematopoietic stem cells (HSCs). The most advanced approach utilizes complex lentiviral vectors encoding the human β-globin gene, as first reported by May et al. in 2000. Considerable progress toward the clinical implementation of this approach has been made in the past five years, based on effective CD34+ cell mobilization and improved lentiviral vector manufacturing. Four trials have been initiated in the United States and Europe. Of 16 evaluable subjects, 6 have achieved transfusion independence. One of them developed a durable clonal expansion, which regressed after several years without transformation. Although globin lentiviral vectors have so far proven to be safe, this occurrence suggests that powerful insulators with robust enhancer-blocking activity will further enhance this approach. The combined discovery of Bcl11a-mediated γ-globin gene silencing and advances in gene editing are the foundations for another gene therapy approach, which aims to reactivate fetal hemoglobin (HbF) production. Its clinical translation will hinge on the safety and efficiency of gene targeting in true HSCs and the induction of sufficient levels of HbF to achieve transfusion independence. Altogether, the progress achieved over the past 15 years bodes well for finding a genetic cure for severe globin disorders in the next decade.

  13. ß-globin gene regulation and chromatin structure

    NARCIS (Netherlands)

    J.H. Gribnau (Joost)

    1999-01-01

    textabstractOxygen transport in the blood is mediated by highly specialized red cells. The majority of the proteins of a red cell comprises the oxygen carrier protein hemoglobin, which is a hetero-tctrameric protein that consists of two a and two p globin chains. In humans different u- and p-like gl

  14. Treatment of β-Thalassemia With Hydroxyurea (HU)——Effects of HU on Globin Gene Expression

    Institute of Scientific and Technical Information of China (English)

    黄淑帧; 任兆瑞; 陈美珏; 许洪平; 曾溢滔; G.P.Rodgers; 曾凡一; A.N.Schechter

    1994-01-01

    A newly developed method of RT-PCR/competitive PCR for measuring the relative and ab-solute content of globin mRNAs as well as micro-globin chain biosynthetic assay have been used to study thealterations of globin gene expressions in the patients with β-thalassemia pre-and post-hydroxyurea(HU)treatment.It was found for the first time that HU had the effect of enhancing β-globin gene expression insome patients.Two cases with β-thalassemia who were subjected to HU treatment for over two years showeda marked increase in β-globin mRNA level and β-globin chain synthesis,resulting in more effective erythro-poiesis and the alleviation of clinical symptoms.

  15. Distinctive patterns of evolution of the δ-globin gene (HBD in primates.

    Directory of Open Access Journals (Sweden)

    Ana Moleirinho

    Full Text Available In most vertebrates, hemoglobin (Hb is a heterotetramer composed of two dissimilar globin chains, which change during development according to the patterns of expression of α- and β-globin family members. In placental mammals, the β-globin cluster includes three early-expressed genes, ε(HBE-γ(HBG-ψβ(HBBP1, and the late expressed genes, δ (HBD and β (HBB. While HBB encodes the major adult β-globin chain, HBD is weakly expressed or totally silent. Paradoxically, in human populations HBD shows high levels of conservation typical of genes under strong evolutionary constraints, possibly due to a regulatory role in the fetal-to-adult switch unique of Anthropoid primates. In this study, we have performed a comprehensive phylogenetic and comparative analysis of the two adult β-like globin genes in a set of diverse mammalian taxa, focusing on the evolution and functional divergence of HBD in primates. Our analysis revealed that anthropoids are an exception to a general pattern of concerted evolution in placental mammals, showing a high level of sequence conservation at HBD, less frequent and shorter gene conversion events. Moreover, this lineage is unique in the retention of a functional GATA-1 motif, known to be involved in the control of the developmental expression of the β-like globin genes. We further show that not only the mode but also the rate of evolution of the δ-globin gene in higher primates are strictly associated with the fetal/adult β-cluster developmental switch. To gain further insight into the possible functional constraints that have been shaping the evolutionary history of HBD in primates, we calculated dN/dS (ω ratios under alternative models of gene evolution. Although our results indicate that HBD might have experienced different selective pressures throughout primate evolution, as shown by different ω values between apes and Old World Monkeys + New World Monkeys (0.06 versus 0.43, respectively, these estimates

  16. The globin gene repertoire of lampreys: convergent evolution of hemoglobin and myoglobin in jawed and jawless vertebrates.

    Science.gov (United States)

    Schwarze, Kim; Campbell, Kevin L; Hankeln, Thomas; Storz, Jay F; Hoffmann, Federico G; Burmester, Thorsten

    2014-10-01

    Agnathans (jawless vertebrates) occupy a key phylogenetic position for illuminating the evolution of vertebrate anatomy and physiology. Evaluation of the agnathan globin gene repertoire can thus aid efforts to reconstruct the origin and evolution of the globin genes of vertebrates, a superfamily that includes the well-known model proteins hemoglobin and myoglobin. Here, we report a comprehensive analysis of the genome of the sea lamprey (Petromyzon marinus) which revealed 23 intact globin genes and two hemoglobin pseudogenes. Analyses of the genome of the Arctic lamprey (Lethenteron camtschaticum) identified 18 full length and five partial globin gene sequences. The majority of the globin genes in both lamprey species correspond to the known agnathan hemoglobins. Both genomes harbor two copies of globin X, an ancient globin gene that has a broad phylogenetic distribution in the animal kingdom. Surprisingly, we found no evidence for an ortholog of neuroglobin in the lamprey genomes. Expression and phylogenetic analyses identified an ortholog of cytoglobin in the lampreys; in fact, our results indicate that cytoglobin is the only orthologous vertebrate-specific globin that has been retained in both gnathostomes and agnathans. Notably, we also found two globins that are highly expressed in the heart of P. marinus, thus representing functional myoglobins. Both genes have orthologs in L. camtschaticum. Phylogenetic analyses indicate that these heart-expressed globins are not orthologous to the myoglobins of jawed vertebrates (Gnathostomata), but originated independently within the agnathans. The agnathan myoglobin and hemoglobin proteins form a monophyletic group to the exclusion of functionally analogous myoglobins and hemoglobins of gnathostomes, indicating that specialized respiratory proteins for O2 transport in the blood and O2 storage in the striated muscles evolved independently in both lineages. This dual convergence of O2-transport and O2-storage proteins in

  17. Hb Wilde and Hb Patagonia: two novel elongated beta-globin variants causing dominant beta-thalassemia.

    Science.gov (United States)

    Scheps, Karen G; Hasenahuer, Marcia A; Parisi, Gustavo; Fornasari, María S; Pennesi, Sandra P; Erramouspe, Beatriz; Basack, Felisa N; Veber, Ernesto S; Aversa, Luis; Elena, Graciela; Varela, Viviana

    2015-06-01

    We describe here the molecular and hematological characteristics of novel frameshift mutations in exon 2 of the HBB gene (in heterozygous state) found in two Argentinean pediatric patients with dominant β-thalassemia-like features. In Hb Wilde, HBB:c.270_273delTGAG(p.Glu90Cysfs*67), we detected the deletion of the third base of the codon 89 (T) and the codon 90 (GAG), whereas in Hb Patagonia, HBB:c.296_297dupGT(p.Asp99Trpfs*59), the frameshift mutation was due to a duplication of a 'GT' dinucleotide after the second base of codon 98 (GTG). The Hb Patagonia and Hb Wilde mutations would result in elongated β-globin chains with modified C-terminal sequences and a total of 155 and 157 amino acids residues, respectively. Based on bioinformatics and structural analysis, as well as protein modeling, we predict that the elongated β-globins would affect the formation of the αβ dimers and their stability, which would further support the mechanism for the observed clinical features in both patients.

  18. Allele-specific enzymatic amplification of beta-globin genomic DNA for diagnosis of sickle cell anemia.

    OpenAIRE

    1989-01-01

    A rapid nonradioactive approach to the diagnosis of sickle cell anemia is described based on an allele-specific polymerase chain reaction (ASPCR). This method allows direct detection of the normal or the sickle cell beta-globin allele in genomic DNA without additional steps of probe hybridization, ligation, or restriction enzyme cleavage. Two allele-specific oligonucleotide primers, one specific for the sickle cell allele and one specific for the normal allele, together with another primer co...

  19. In silico analysis of single nucleotide polymorphism (SNPs in human β-globin gene.

    Directory of Open Access Journals (Sweden)

    Mohammed Alanazi

    Full Text Available Single amino acid substitutions in the globin chain are the most common forms of genetic variations that produce hemoglobinopathies--the most widespread inherited disorders worldwide. Several hemoglobinopathies result from homozygosity or compound heterozygosity to beta-globin (HBB gene mutations, such as that producing sickle cell hemoglobin (HbS, HbC, HbD and HbE. Several of these mutations are deleterious and result in moderate to severe hemolytic anemia, with associated complications, requiring lifelong care and management. Even though many hemoglobinopathies result from single amino acid changes producing similar structural abnormalities, there are functional differences in the generated variants. Using in silico methods, we examined the genetic variations that can alter the expression and function of the HBB gene. Using a sequence homology-based Sorting Intolerant from Tolerant (SIFT server we have searched for the SNPs, which showed that 200 (80% non-synonymous polymorphism were found to be deleterious. The structure-based method via PolyPhen server indicated that 135 (40% non-synonymous polymorphism may modify protein function and structure. The Pupa Suite software showed that the SNPs will have a phenotypic consequence on the structure and function of the altered protein. Structure analysis was performed on the key mutations that occur in the native protein coded by the HBB gene that causes hemoglobinopathies such as: HbC (E→K, HbD (E→Q, HbE (E→K and HbS (E→V. Atomic Non-Local Environment Assessment (ANOLEA, Yet Another Scientific Artificial Reality Application (YASARA, CHARMM-GUI webserver for macromolecular dynamics and mechanics, and Normal Mode Analysis, Deformation and Refinement (NOMAD-Ref of Gromacs server were used to perform molecular dynamics simulations and energy minimization calculations on β-Chain residue of the HBB gene before and after mutation. Furthermore, in the native and altered protein models, amino acid

  20. Erythroid cell-specific alpha-globin gene regulation by the CP2 transcription factor family.

    Science.gov (United States)

    Kang, Ho Chul; Chae, Ji Hyung; Lee, Yeon Ho; Park, Mi-Ae; Shin, June Ho; Kim, Sung-Hyun; Ye, Sang-Kyu; Cho, Yoon Shin; Fiering, Steven; Kim, Chul Geun

    2005-07-01

    We previously demonstrated that ubiquitously expressed CP2c exerts potent erythroid-specific transactivation of alpha-globin through an unknown mechanism. This mechanism is reported here to involve specific CP2 splice variants and protein inhibitor of activated STAT1 (PIAS1). We identify a novel murine splice isoform of CP2, CP2b, which is identical to CP2a except that it has an additional 36 amino acids encoded by an extra exon. CP2b has an erythroid cell-specific transcriptional activation domain, which requires the extra exon and can form heteromeric complexes with other CP2 isoforms, but lacks the DNA binding activity found in CP2a and CP2c. Transcriptional activation of alpha-globin occurred following dimerization between CP2b and CP2c in erythroid K562 and MEL cells, but this dimerization did not activate the alpha-globin promoter in nonerythroid 293T cells, indicating that an additional erythroid factor is missing in 293T cells. PIAS1 was confirmed as a CP2 binding protein by the yeast two-hybrid screen, and expression of CP2b, CP2c, and PIAS1 in 293T cell induced alpha-globin promoter activation. These results show that ubiquitously expressed CP2b exerts potent erythroid cell-specific alpha-globin gene expression by complexing with CP2c and PIAS1.

  1. Allele-specific enzymatic amplification of beta-globin genomic DNA for diagnosis of sickle cell anemia.

    Science.gov (United States)

    Wu, D Y; Ugozzoli, L; Pal, B K; Wallace, R B

    1989-04-01

    A rapid nonradioactive approach to the diagnosis of sickle cell anemia is described based on an allele-specific polymerase chain reaction (ASPCR). This method allows direct detection of the normal or the sickle cell beta-globin allele in genomic DNA without additional steps of probe hybridization, ligation, or restriction enzyme cleavage. Two allele-specific oligonucleotide primers, one specific for the sickle cell allele and one specific for the normal allele, together with another primer complementary to both alleles were used in the polymerase chain reaction with genomic DNA templates. The allele-specific primers differed from each other in their terminal 3' nucleotide. Under the proper annealing temperature and polymerase chain reaction conditions, these primers only directed amplification on their complementary allele. In a single blind study of DNA samples from 12 individuals, this method correctly and unambiguously allowed for the determination of the genotypes with no false negatives or positives. If ASPCR is able to discriminate all allelic variation (both transition and transversion mutations), this method has the potential to be a powerful approach for genetic disease diagnosis, carrier screening, HLA typing, human gene mapping, forensics, and paternity testing.

  2. A phylogenomic profile of globins

    Directory of Open Access Journals (Sweden)

    Dewilde Sylvia

    2006-04-01

    Full Text Available Abstract Background Globins occur in all three kingdoms of life: they can be classified into single-domain globins and chimeric globins. The latter comprise the flavohemoglobins with a C-terminal FAD-binding domain and the gene-regulating globin coupled sensors, with variable C-terminal domains. The single-domain globins encompass sequences related to chimeric globins and «truncated» hemoglobins with a 2-over-2 instead of the canonical 3-over-3 α-helical fold. Results A census of globins in 26 archaeal, 245 bacterial and 49 eukaryote genomes was carried out. Only ~25% of archaea have globins, including globin coupled sensors, related single domain globins and 2-over-2 globins. From one to seven globins per genome were found in ~65% of the bacterial genomes: the presence and number of globins are positively correlated with genome size. Globins appear to be mostly absent in Bacteroidetes/Chlorobi, Chlamydia, Lactobacillales, Mollicutes, Rickettsiales, Pastorellales and Spirochaetes. Single domain globins occur in metazoans and flavohemoglobins are found in fungi, diplomonads and mycetozoans. Although red algae have single domain globins, including 2-over-2 globins, the green algae and ciliates have only 2-over-2 globins. Plants have symbiotic and nonsymbiotic single domain hemoglobins and 2-over-2 hemoglobins. Over 90% of eukaryotes have globins: the nematode Caenorhabditis has the most putative globins, ~33. No globins occur in the parasitic, unicellular eukaryotes such as Encephalitozoon, Entamoeba, Plasmodium and Trypanosoma. Conclusion Although Bacteria have all three types of globins, Archaeado not have flavohemoglobins and Eukaryotes lack globin coupled sensors. Since the hemoglobins in organisms other than animals are enzymes or sensors, it is likely that the evolution of an oxygen transport function accompanied the emergence of multicellular animals.

  3. The structure of the human β-globin gene in β-thalassaemia

    NARCIS (Netherlands)

    Flavell, R.A.; Bernards, R.A.; Kooter, J.M.; Boer, E. de; Little, P.F.R.; Annison, G.; Williamson, R.

    1979-01-01

    Twenty-one cases of β⁰- and β⁺-thalassaemia have been analysed by restriction endonuclease mapping. In most cases no deletion in the regions surrounding the β- and δ-globin genes could be detected. However, in a single Asian case of β⁰-thalassaemia, homozygous clinically, one of the homologous chrom

  4. The effects of old and recent migration waves in the distribution of HBB*S globin gene haplotypes

    Directory of Open Access Journals (Sweden)

    Juliana D. Lindenau

    Full Text Available Abstract Sickle cell hemoglobin is the result of a mutation at the sixth amino acid position of the betaglobin chain. The HBB*S gene is in linkage disequilibrium with five main haplotypes in the β-globin-like gene cluster named according to their ethnic and geographic origins: Bantu (CAR, Benin (BEN, Senegal (SEN, Cameroon (CAM and Arabian-Indian (ARAB. These haplotypes demonstrated that the sickle cell mutation arose independently at least five times in human history. The distribution of βS haplotypes among Brazilian populations showed a predominance of the CAR haplotype. American populations were clustered in two groups defined by CAR or BEN haplotype frequencies. This scenario is compatible with historical records about the slave trade in the Americas. When all world populations where the sickle cell gene occurs were analyzed, three clusters were disclosed based on CAR, BEN or ARAB haplotype predominance. These patterns may change in the next decades due to recent migrations waves. Since these haplotypes show different clinical characteristics, these recent migrations events raise the necessity to develop optimized public health programs for sickle cell disease screening and management.

  5. The effects of old and recent migration waves in the distribution of HBB*S globin gene haplotypes.

    Science.gov (United States)

    Lindenau, Juliana D; Wagner, Sandrine C; Castro, Simone M de; Hutz, Mara H

    2016-01-01

    Sickle cell hemoglobin is the result of a mutation at the sixth amino acid position of the beta (β) globin chain. The HBB*S gene is in linkage disequilibrium with five main haplotypes in the β-globin-like gene cluster named according to their ethnic and geographic origins: Bantu (CAR), Benin (BEN), Senegal (SEN), Cameroon (CAM) and Arabian-Indian (ARAB). These haplotypes demonstrated that the sickle cell mutation arose independently at least five times in human history. The distribution of βS haplotypes among Brazilian populations showed a predominance of the CAR haplotype. American populations were clustered in two groups defined by CAR or BEN haplotype frequencies. This scenario is compatible with historical records about the slave trade in the Americas. When all world populations where the sickle cell gene occurs were analyzed, three clusters were disclosed based on CAR, BEN or ARAB haplotype predominance. These patterns may change in the next decades due to recent migrations waves. Since these haplotypes show different clinical characteristics, these recent migrations events raise the necessity to develop optimized public health programs for sickle cell disease screening and management.

  6. Promoter region sequence differences in the A and G gamma globin genes of Brazilian sickle cell anemia patients.

    Science.gov (United States)

    Barbosa, C G; Goncalves-Santos, N J; Souza-Ribeiro, S B; Moura-Neto, J P; Takahashi, D; Silva, D O; Hurtado-Guerrero, A F; Reis, M G; Goncalves, M S

    2010-08-01

    Fetal hemoglobin (HbF), encoded by the HBG2 and HBG1 genes, is the best-known genetic modulator of sickle cell anemia, varying dramatically in concentration in the blood of these patients. This variation is partially associated with polymorphisms located in the promoter region of the HBG2 and HBG1 genes. In order to explore known and unknown polymorphisms in these genes, the sequences of their promoter regions were screened in sickle cell anemia patients and correlated with both their HbF levels and their betaS-globin haplotypes. Additionally, the sequences were compared with genes from 2 healthy groups, a reference one (N = 104) and an Afro-descendant one (N = 98), to identify polymorphisms linked to the ethnic background.The reference group was composed by healthy individuals from the general population. Four polymorphisms were identified in the promoter region of HBG2 and 8 in the promoter region of HBG1 among the studied groups. Four novel single nucleotide polymorphisms (SNP) located at positions -324, -317, -309 and -307 were identified in the reference group. A deletion located between -396 and -391 in the HBG2 promoter region and the SNP -271 C-->T in the HBG1 promoter region were associated with the Central African Republic betaS-globin haplotype. In contrast, the -369 C-->G and 309 A-->G SNPs in the HBG2 promoter region were correlated to the Benin haplotype. The polymorphisms -396_-391 del HBG2, -369 SNP HBG2 and -271 SNP HBG1 correlated with HbF levels. Hence, we suggest an important role of HBG2 and HBG1 gene polymorphisms on the HbF synthesis.

  7. Sheep α-globin gene sequences: Implications for their concerted evolution and for the down-regulation of the 3' genes

    NARCIS (Netherlands)

    M.S. Ristaldi (Maria Serafina); S. Casula (S.); A. Rando (A.); R. Vestri (R.)

    1995-01-01

    textabstractIn sheep as in man and most other mammals, there are two α-globin genes (Iα and IIα), which are expressed at different levels, the upstream gene being the most efficient. In α-globin gene triplication and quadruplication, this trend is confirmed, i.e., the α-chain output of the downstrea

  8. Generation and Characterization of a Transgenic Mouse Carrying a Functional Human β -Globin Gene with the IVSI-6 Thalassemia Mutation.

    Science.gov (United States)

    Breveglieri, Giulia; Mancini, Irene; Bianchi, Nicoletta; Lampronti, Ilaria; Salvatori, Francesca; Fabbri, Enrica; Zuccato, Cristina; Cosenza, Lucia C; Montagner, Giulia; Borgatti, Monica; Altruda, Fiorella; Fagoonee, Sharmila; Carandina, Gianni; Rubini, Michele; Aiello, Vincenzo; Breda, Laura; Rivella, Stefano; Gambari, Roberto; Finotti, Alessia

    2015-01-01

    Mouse models that carry mutations causing thalassemia represent a suitable tool to test in vivo new mutation-specific therapeutic approaches. Transgenic mice carrying the β-globin IVSI-6 mutation (the most frequent in Middle-Eastern regions and recurrent in Italy and Greece) are, at present, not available. We report the production and characterization of a transgenic mouse line (TG-β-IVSI-6) carrying the IVSI-6 thalassemia point mutation within the human β-globin gene. In the TG-β-IVSI-6 mouse (a) the transgenic integration region is located in mouse chromosome 7; (b) the expression of the transgene is tissue specific; (c) as expected, normally spliced human β-globin mRNA is produced, giving rise to β-globin production and formation of a human-mouse tetrameric chimeric hemoglobin (mu) α-globin2/(hu) β-globin2 and, more importantly, (d) the aberrant β-globin-IVSI-6 RNAs are present in blood cells. The TG-β-IVSI-6 mouse reproduces the molecular features of IVSI-6 β-thalassemia and might be used as an in vivo model to characterize the effects of antisense oligodeoxynucleotides targeting the cryptic sites responsible for the generation of aberrantly spliced β-globin RNA sequences, caused by the IVSI-6 mutation. These experiments are expected to be crucial for the development of a personalized therapy for β-thalassemia.

  9. Versatile Cosmid Vectors for the Isolation, Expression, and Rescue of Gene Sequences: Studies with the Human α -globin Gene Cluster

    Science.gov (United States)

    Lau, Yun-Fai; Kan, Yuet Wai

    1983-09-01

    We have developed a series of cosmids that can be used as vectors for genomic recombinant DNA library preparations, as expression vectors in mammalian cells for both transient and stable transformations, and as shuttle vectors between bacteria and mammalian cells. These cosmids were constructed by inserting one of the SV2-derived selectable gene markers-SV2-gpt, SV2-DHFR, and SV2-neo-in cosmid pJB8. High efficiency of genomic cloning was obtained with these cosmids and the size of the inserts was 30-42 kilobases. We isolated recombinant cosmids containing the human α -globin gene cluster from these genomic libraries. The simian virus 40 DNA in these selectable gene markers provides the origin of replication and enhancer sequences necessary for replication in permissive cells such as COS 7 cells and thereby allows transient expression of α -globin genes in these cells. These cosmids and their recombinants could also be stably transformed into mammalian cells by using the respective selection systems. Both of the adult α -globin genes were more actively expressed than the embryonic zeta -globin genes in these transformed cell lines. Because of the presence of the cohesive ends of the Charon 4A phage in the cosmids, the transforming DNA sequences could readily be rescued from these stably transformed cells into bacteria by in vitro packaging of total cellular DNA. Thus, these cosmid vectors are potentially useful for direct isolation of structural genes.

  10. Regulation of the β-globin gene family expression, useful in the search for new therapeutic targets for hemoglobinopathies

    Directory of Open Access Journals (Sweden)

    Karen G. Scheps

    2016-12-01

    Full Text Available Different hemoglobin isoforms are expressed during the embryonic, fetal and postnatal stages. They are formed by combination of polypeptide chains synthesized from the α- and β-globin gene clusters. Based on the fact that the presence of high hemoglobin F levels is beneficial in both sickle cell disease and severe thalassemic syndromes, a revision of the regulation of the β-globin cluster expression is proposed, especially regarding the genes encoding the y-globin chains (HBG1 and HBG2. In this review we describe the current knowledge about transcription factors and epigenetic regulators involved in the switches of the β-globin cluster. It is expected that the consolidation of knowledge in this field will allow finding new therapeutic targets for the treatment of hemoglobinopathies.

  11. Total alpha-globin gene cluster deletion has high frequency in Filipinos

    Energy Technology Data Exchange (ETDEWEB)

    Hunt, J.A.; Haruyama, A.Z.; Chu, B.M. [Kapiolani Medical Center, Honolulu, HI (United States)] [and others

    1994-09-01

    Most {alpha}-thalassemias [Thal] are due to large deletions. In Southeast Asians, the (--{sup SEA}) double {alpha}-globin gene deletion is common, 3 (--{sup Tot}) total {alpha}-globin cluster deletions are known: Filipino (--{sup Fil}), Thai (--{sup Thai}), and Chinese (--{sup Chin}). In a Hawaii Thal project, provisional diagnosis of {alpha}-Thal-1 heterozygotes was based on microcytosis, normal isoelectric focusing, and no iron deficiency. One in 10 unselected Filipinos was an {alpha}-Thal-1 heterozygote, 2/3 of these had a (--{sup Tot}) deletion: a {var_sigma}-cDNA probe consistently showed fainter intensity of the constant 5.5 kb {var_sigma}{sub 2} BamHI band, with no heterzygosity for {var_sigma}-globin region polymorphisms; {alpha}-cDNA or {var_sigma}-cDNA probes showed no BamHI or BglII bands diagnostic of the (--{sup SEA}) deletion; bands for the (-{alpha}) {alpha}-Thal-2 single {alpha}-globin deletions were only seen in Hb H cases. A reliable monoclonal anti-{var_sigma}-peptide antibody test for the (--{sup SEA}) deletion was always negative in (--{sup Tot}) samples. Southern digests with the Lo probe, a gift from D. Higgs of Oxford Univ., confirmed that 49 of 50 (--{sup Tot}) chromosomes in Filipinos were (--{sup Fil}). Of 20 {alpha}-Thal-1 hydrops born to Filipinos, 11 were (--{sup Fil}/--{sup SEA}) compound heterozygotes; 9 were (--{sup SEA}/--{sup SEA}) homozygotes, but none was a (--{sup Fil}/--{sup Fil}).

  12. Functional Analysis of Multiple Transcription Factor Sites in a Regulatory Element of Human ε-Globin Gene

    Institute of Scientific and Technical Information of China (English)

    Chun-Hui HOU; Jian HUANG; Ruo-Lan QIAN

    2004-01-01

    The developmental control of the human ε-globin gene expression is mediated by transcriptional regulatory elements in the 5' flanking DNA of this gene. A previously identified negative regulatory element (-3028 to -2902 bp, termed ε-NRAII) was analyzed and one putative NF-κB site and two GATA sites locate at -3004 bp, -2975 bp and -2948 bp were characterized. Electrophoresis mobility shift assay (EMSA)showed that the putative NF-κB site was specifically bound by nuclear proteins of K562 cells. Data obtained from transient transfection showed that the expression of reporter gene could be upregulated about 50% or 100% respectively when ε-NRAII was inserted upstream of the SV40 promoter or ε-globin gene proximal promoter (-177 bp to +1 bp), suggesting that ε-NRAII might not be a classic silencer. Mutation in the putative NF-κB site or in the GATA site (at-2975 bp) slightly reduced the expression of reporter gene driven by SV40 promoter or ε-globin gene proximal promoter. However, the mutation of GATA site at -2948 bp remarkably reduced the reporter gene activity driven by SV40 promoter, but not by ε-globin gene proximal promoter. Further mutation analysis showed that the negative effect of mutation in GATA site at -2948 bp on SV40 promoter was not affected by the mutation of the putative NF-κB site, whereas it could be abolished by the mutation of GATA site at -2975 bp. Furthermore, the mutation of both GATA sites could synergistically reduce the reporter gene activity driven by ε-globin gene proximal promoter. Those results suggested that ε-NRAII might function differently on the SV40 promoter and ε-globin gene proximal promoter.

  13. Chromatin looping and eRNA transcription precede the transcriptional activation of gene in the β-globin locus.

    Science.gov (United States)

    Kim, Yea Woon; Lee, Sungkung; Yun, Jangmi; Kim, AeRi

    2015-03-18

    Enhancers are closely positioned with actively transcribed target genes by chromatin looping. Non-coding RNAs are often transcribed on active enhancers, referred to as eRNAs (enhancer RNAs). To explore the kinetics of enhancer-promoter looping and eRNA transcription during transcriptional activation, we induced the β-globin locus by chemical treatment and analysed cross-linking frequency between the β-globin gene and locus control region (LCR) and the amount of eRNAs transcribed on the LCR in a time course manner. The cross-linking frequency was increased after chemical induction but before the transcriptional activation of gene in the β-globin locus. Transcription of eRNAs was increased in concomitant with the increase in cross-linking frequency. These results show that chromatin looping and eRNA transcription precedes the transcriptional activation of gene. Concomitant occurrence of the two events suggests functional relationship between them.

  14. A novel erythroid differen tiation related gene EDRF2 inhibited α-globin gene expression in K562 cells

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    In previous studies, we found that EDRF2 was an erythroid differentiation related factor, whose expression was markedly upregulated during erythroid differentiation. It suggested that this factor played a role in erythropoiesis.By using rapid amplification of cDNA ends technology, we cloned EDRF2 gene 5 '- and 3 '-cDNA ends successfully.Transfection and Northern blot analysis demonstrated that EDRF2 inhibited mRNA expression of α-globin gene, while did not regulate γ-globin gene expression. Gel shift assay confirmed that DNA-binding activity of erythroid specific transcription factors GATA-1, NF-E2 and AP1 was not af fected by either forced overexpression or artificial down regulation of EDRF2 gene in K562 cells. However, we de tected that the negative regulator of expression of GATA-1 transcription factor was increased in EDRF2 overexpressed K562 cells. These results strongly suggested that EDRF2 was involved in α-globin gene expression and erythroid differen tiation and served as a negative regulator of PU.1 transcrip tion factor.

  15. A stage—specific protein factor binding to a CACCC motif in both human β—globin gene promoter and 5‘—HS2 region

    Institute of Scientific and Technical Information of China (English)

    SUNTONG; YADICHEN; 等

    1994-01-01

    The DNaseI hypersensitive site 2 (HS2) of human β-globin locus control region(LCR) is required for the high level expression of human β-globin genes.In the present study,a stage-specific protein factor (LPF-β) was identified in the nuclear extract prepared from mouse fetal liver at d 18 of gestation,which could bind to the HS2 region of human β-globin LCR.We also found that the shift band of LPF-β factor could be competed by human β-globin promoter.However,it couldn't be competed by human ε-globin promoter or by human Aγ-globin promoter.Furthermore,our data demonstrated that the binding-sequence of LPF-β factor is 5'CACACCCTA 3',which is located at the HS2 region of β-LCR(from-10845 to-10853 bp)and human β-globin promoter(from-92 to -84 bp).We speculated that these regions containing the CACCC box in both the human β-globin promoter and HS2 might function as stage selector elements in the regulation of human β-globin switching and the LPF-β factor might be a stage-specific protein factor involved in the regulation of human β-globin gene expression.

  16. Beta-Globin Gene Regulation and Nuclear Organisation

    NARCIS (Netherlands)

    J.A. Kooren (Jurgen)

    2007-01-01

    textabstractAll genetic information required for the development and functioning of an organism is stored in billions of base pairs of deoxyribonucleic acid (or DNA). In eukaryotes, DNA is organised in large units called chromosomes that are located inside the cells nucleus. On these chromosomes res

  17. Classical sickle beta-globin haplotypes exhibit a high degree of long-range haplotype similarity in African and Afro-Caribbean populations

    Directory of Open Access Journals (Sweden)

    Jallow Muminatou

    2007-08-01

    Full Text Available Abstract Background The sickle (βs mutation in the beta-globin gene (HBB occurs on five "classical" βs haplotype backgrounds in ethnic groups of African ancestry. Strong selection in favour of the βs allele – a consequence of protection from severe malarial infection afforded by heterozygotes – has been associated with a high degree of extended haplotype similarity. The relationship between classical βs haplotypes and long-range haplotype similarity may have both anthropological and clinical implications, but to date has not been explored. Here we evaluate the haplotype similarity of classical βs haplotypes over 400 kb in population samples from Jamaica, The Gambia, and among the Yoruba of Nigeria (Hapmap YRI. Results The most common βs sub-haplotype among Jamaicans and the Yoruba was the Benin haplotype, while in The Gambia the Senegal haplotype was observed most commonly. Both subtypes exhibited a high degree of long-range haplotype similarity extending across approximately 400 kb in all three populations. This long-range similarity was significantly greater than that seen for other haplotypes sampled in these populations (P s mutation. Conclusion Two different classical βs haplotypes, sampled from different populations, exhibit comparable and extensive long-range haplotype similarity and strong LD. This LD extends across the adjacent recombination hotspot, and is discernable at distances in excess of 400 kb. Although the multi-centric geographic distribution of βs haplotypes indicates strong subdivision among early Holocene sub-Saharan populations, we find no evidence that selective pressures imposed by falciparum malaria varied in intensity or timing between these subpopulations. Our observations also suggest that cis-acting loci, which may influence outcomes in sickle cell disease, could lie considerable distances away from β-globin.

  18. A novel in vivo transcription assay demonstrates the presence of globin-inducing trans-acting factors in uninduced Murine Erythroleukemia cells.

    NARCIS (Netherlands)

    N. Wrighton; F.G. Grosveld (Frank)

    1988-01-01

    textabstractWe report the development of a novel in vivo transcription assay for trans-acting factors regulating the human gamma- and beta-globin genes. A cDNA coding for the human tissue-type plasminogen activator (t-PA) was inserted into the globin genes. Simian virus 40 small T-antigen splice and

  19. Screening for mutations in human alpha-globin genes by nonradioactive single-strand conformation polymorphism

    Directory of Open Access Journals (Sweden)

    Jorge S.B.

    2003-01-01

    Full Text Available Point mutations and small insertions or deletions in the human alpha-globin genes may produce alpha-chain structural variants and alpha-thalassemia. Mutations can be detected either by direct DNA sequencing or by screening methods, which select the mutated exon for sequencing. Although small (about 1 kb, 3 exons and 2 introns, the alpha-globin genes are duplicate (alpha2 and alpha1 and highy G-C rich, which makes them difficult to denature, reducing sequencing efficiency and causing frequent artifacts. We modified some conditions for PCR and electrophoresis in order to detect mutations in these genes employing nonradioactive single-strand conformation polymorphism (SSCP. Primers previously described by other authors for radioactive SSCP and phast-SSCP plus denaturing gradient gel electrophoresis were here combined and the resultant fragments (6 new besides 6 original per alpha-gene submitted to silver staining SSCP. Nine structural and one thalassemic mutations were tested, under different conditions including two electrophoretic apparatus (PhastSystem(TM and GenePhor(TM, Amersham Biosciences, different polyacrylamide gel concentrations, run temperatures and denaturing agents, and entire and restriction enzyme cut fragments. One hundred percent of sensitivity was achieved with four of the new fragments formed, using the PhastSystem(TM and 20% gels at 15ºC, without the need of restriction enzymes. This nonradioactive PCR-SSCP approach showed to be simple, rapid and sensitive, reducing the costs involved in frequent sequencing repetitions and increasing the reliability of the results. It can be especially useful for laboratories which do not have an automated sequencer.

  20. Hb Bronte or alpha93(FG5)Val-->Gly: a new unstable variant of the alpha2-globin gene, associated with a mild alpha(+)-thalassemia phenotype.

    Science.gov (United States)

    Lacerra, Giuseppina; Testa, Rosario; De Angioletti, Maria; Schilirò, Gino; Carestia, Clementina

    2003-08-01

    We report a new unstable variant identified in three carriers of a family from East Sicily; it was named Hb Bronte after the place from which the family originated. DNA sequencing from nucleotides -181 to +894 (alpha1) and to +884 (alpha2) revealed a GTG-->GGG substitution at codon 93 of the alpha2-globin gene. The MCV and MCH values were at the lower end of the normal range in the carriers. On cation exchange high performance liquid chromatography (HPLC), the Hb A2 level was apparently increased to around 6%, and a small abnormal peak (0.3-0.4%) was detected after Hb A2. Two abnormal bands were detected by cellulose acetate electrophoresis: a major band (about 3-4%) migrated between Hb A and Hb F; a minor band (<1%) migrated between Hb A2 and carbonic anhydrase. Normal values of Hb A2 were detected by DEAE microchromatography. On reversed phase HPLC the variant chain was not detected, and most likely it was eluted with the alpha chain peak. The isopropanol stability test was very slightly positive in the carriers. Hemolytic symptoms were absent with the exception of indirect bilirubin, which was at high borderline in 2/3 carriers. In biosynthesis in vitro, the specific activity of the alpha chains was much higher than that of the beta-globin chains, and the alpha/beta biosynthetic ratio in the mother and proband was of the beta-thalassemia (thal) type (2.24 and 2.54, respectively). Time course experiments showed that the increase of the 3H-specific activity of the peak containing normal and variant alpha chains was not linear and was much higher than that of beta chains; moreover, the alpha/beta biosynthetic ratio varied during the 2 hours incubation.

  1. The First Report of a 290-bp Deletion in β-Globin Gene in the South of Iran

    Science.gov (United States)

    Hamid, Mohammad; Nejad, Ladan Dawoody; Shariati, Gholamreza; Galehdari, Hamid; Saberi, Alihossein; Mohammadi-Anaei, Marziye

    2017-01-01

    Background: β-thalassemia is one of the most widespread diseases in the world, including Iran. In this study, we reported, for the first time, a 290-bp β-globin gene deletion in the south of Iran. Methods: Four individuals from three unrelated families with Arabic ethnic background were studied in Khuzestan Province. Red blood cell indices and hemoglobin analysis were carried out according to the standard methods. Genomic DNA was obtained from peripheral blood cells by salting out procedures. β-globin gene amplification, multiplex ligation-dependent probe amplification (MLPA), and DNA sequencing were performed. Results: The PCR followed by sequencing and MLPA test of the β-globin gene confirmed the presence of a 290-bp deletion in the heterozygous form, along with -88C>A mutation. All the individuals had elevated hemoglobin A2 and normal fetal hemoglobin levels. Conclusions: This mutation causes β0-thalassemia and can be highly useful for prenatal diagnosis in compound heterozygous condition with different β-globin gene mutations. PMID:26948378

  2. Bergamot (Citrus bergamia Risso) fruit extracts as γ-globin gene expression inducers: phytochemical and functional perspectives.

    Science.gov (United States)

    Guerrini, Alessandra; Lampronti, Ilaria; Bianchi, Nicoletta; Zuccato, Cristina; Breveglieri, Giulia; Salvatori, Francesca; Mancini, Irene; Rossi, Damiano; Potenza, Rocco; Chiavilli, Francesco; Sacchetti, Gianni; Gambari, Roberto; Borgatti, Monica

    2009-05-27

    Epicarps of Citrus bergamia fruits from organic farming were extracted with the objective of obtaining derived products differently rich in coumarins and psoralens. The extracts were chemically characterized by (1)H nuclear magnetic resonance (NMR), gas chromatography-flame ionization detection (GC-FID), gas chromatography-mass spectrometry (GC-MS), and high-pressure liquid chromatography (HPLC) for detecting and quantifying the main constituents. Both bergamot extracts and chemical standards corresponding to the main constituents detected were then assayed for their capacity to increase erythroid differentiation of K562 cells and expression of γ-globin genes in human erythroid precursor cells. Three experimental cell systems were employed: (a) the human leukemic K562 cell line, (b) K562 cell clones stably transfected with a pCCL construct carrying green-enhanced green fluorescence protein (EGFP) under the γ-globin gene promoter, and (c) the two-phase liquid culture of human erythroid progenitors isolated from healthy donors. The results suggest that citropten and bergapten are powerful inducers of differentiation and γ-globin gene expression in human erythroid cells. These data could have practical relevance, because pharmacologically mediated regulation of human γ-globin gene expression, with the consequent induction of fetal hemoglobin, is considered to be a potential therapeutic approach in hematological disorders, including β-thalassemia and sickle cell anemia.

  3. Role of the duplicated CCAAT box region in γ-globin gene regulation and hereditary persistence of fetal haemoglobin.

    NARCIS (Netherlands)

    A. Ronchi (Antonella); M. Berry (Meera); S. Raguz (Selina); A.M.A. Imam (Ali); N. Yannoutsos (Nikos); S. Ottolenghi (Sergio); F.G. Grosveld (Frank); N.O. Dillon (Niall)

    1996-01-01

    textabstractHereditary persistence of fetal haemoglobin (HPFH) is a clinically important condition in which a change in the developmental specificity of the gamma-globin genes results in varying levels of expression of fetal haemoglobin in the adult. The condition is benign and can significantly all

  4. 1990 Mack Forster Prize lecture. The molecular genetics of the alpha globin gene family.

    Science.gov (United States)

    Higgs, D R

    1990-08-01

    The naturally occurring mutants described here provide an excellent opportunity for elucidating the relationship between structure and function of the alpha globin complex and the larger chromosomal region 16p13.3. From a practical point of view it is important to remember that millions of individuals throughout the world are carriers for alpha thalassaemia and every year many thousands of pregnancies are at risk of producing children with the severe alpha thalassaemia syndromes. The data summarized here provide the basis for accurately predicting the genotype in such cases and thus enabling appropriate prenatal testing. The less common larger rearrangements involving chromosomal band 16p13.3 may provide information on the nature of other genes that surround the alpha complex. Furthermore, the mechanism by which they have occurred provide some new and more general insights into the possible causes of other forms of unexplained mental handicap.

  5. Prevalence of β(S)-globin gene haplotypes, α-thalassemia (3.7 kb deletion) and redox status in patients with sickle cell anemia in the state of Paraná, Brazil.

    Science.gov (United States)

    Shimauti, Eliana LitsukoTomimatsu; Silva, Danilo Grunig Humberto; de Souza, Eniuce Menezes; de Almeida, Eduardo Alves; Leal, Francismar Prestes; Bonini-Domingos, Claudia Regina

    2015-01-01

    The aim of this study was to determine the frequency of beta S-globin gene (β(S) globin) haplotypes and alpha thalassemia with 3.7 kb deletion (-α(3.7kb) thalassemia) in the northwest region of Paraná state, and to investigate the oxidative and clinical-hematological profile of β(S) globin carriers in this population. Of the 77 samples analyzed, 17 were Hb SS, 30 were Hb AS and 30 were Hb AA. The β(S)globin haplotypes and -α(3.7kb) thalassemia were identified using polymerase chain reaction.Trolox equivalent antioxidant capacity (TEAC) and lipid peroxidation (LPO) were assessed spectophotometrically. Serum melatonin levels were determined using high-performance liquid chromatography coupled to coulometric electrochemical detection. The haplotype frequencies in the SS individuals were as follows: Bantu- 21 (62%), Benin - 11 (32%) and Atypical- 2 (6%). Bantu/Benin was the most frequent genotype. Of the 47 SS and AS individuals assessed, 17% (n = 8) had the -α(3.7kb) mutation. Clinical manifestations, as well as serum melatonin, TEAC and LPO levels did not differ between Bantu/Bantu and Bantu/Benin individuals (p > 0.05). Both genotypes were associated with high LPO and TEAC levels and decreased melatonin concentration. These data suggest that the level of oxidative stress in patients with Bantu/Bantu and Bantu/Benin genotypes may overload the antioxidant capacity.

  6. Generation and Characterization of a Transgenic Mouse Carrying a Functional Human β-Globin Gene with the IVSI-6 Thalassemia Mutation

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    Giulia Breveglieri

    2015-01-01

    Full Text Available Mouse models that carry mutations causing thalassemia represent a suitable tool to test in vivo new mutation-specific therapeutic approaches. Transgenic mice carrying the β-globin IVSI-6 mutation (the most frequent in Middle-Eastern regions and recurrent in Italy and Greece are, at present, not available. We report the production and characterization of a transgenic mouse line (TG-β-IVSI-6 carrying the IVSI-6 thalassemia point mutation within the human β-globin gene. In the TG-β-IVSI-6 mouse (a the transgenic integration region is located in mouse chromosome 7; (b the expression of the transgene is tissue specific; (c as expected, normally spliced human β-globin mRNA is produced, giving rise to β-globin production and formation of a human-mouse tetrameric chimeric hemoglobin αmu-globin2/βhu-globin2 and, more importantly, (d the aberrant β-globin-IVSI-6 RNAs are present in blood cells. The TG-β-IVSI-6 mouse reproduces the molecular features of IVSI-6 β-thalassemia and might be used as an in vivo model to characterize the effects of antisense oligodeoxynucleotides targeting the cryptic sites responsible for the generation of aberrantly spliced β-globin RNA sequences, caused by the IVSI-6 mutation. These experiments are expected to be crucial for the development of a personalized therapy for β-thalassemia.

  7. Accuracy of Reverse Dot-Blot PCR in Detection of Different β-Globin Gene Mutations.

    Science.gov (United States)

    El-Fadaly, N; Abd-Elhameed, A; Abd-Elbar, E; El-Shanshory, M

    2016-06-01

    Prevention programs for β-thalassemia based on molecular diagnosis of heterozygous carriers and/or patients require the use of reliable mutation screening methods. The aim of this study was to compare between direct DNA sequencing, and reverse dot-blot PCR in detection of different β-globin gene mutations in Egyptian children with β-thalassemia. Forty children with β-thalassemia were subjected to mutation analysis, performed by both direct DNA sequencing and β-globin Strip Assay MED™ (based on reverse dot-blot PCR). The most frequent mutant alleles detected by reverse dot-blot PCR were; IVSI-110 G>A (31.25 %), IVS I-6 T > C (21.25 %), and IVS I-1 G>A (20 %). Relatively less frequent mutant alleles detected by reverse dot-blot PCR were "IVSII-1 G>A (5 %), IVSII-745 C>G (5 %), IVSII-848 C>A (2.5 %), IVSI-5 G>C (2.5 %), -87 C>G(2.5 %), and cd39 C>T (2.5 %)", While the genotypes of three patients (6 alleles 7.5 %) were not detected by reverse dot-blot PCR. Mutant alleles detected by direct DNA sequencing were the same as reverse dot-blot PCR method except it revealed the genotypes of 3 undetected patients (one patient was homozygous IVSI-110 G>A, and two patients were homozygous IVS I-1 G>A. Sensitivity of the reverse dot-blot PCR was 92.5 % when compared to direct DNA sequencing for detecting β-thalassemia mutations. Our results therefore suggest that, direct DNA sequencing may be preferred over reverse dot-blot PCR in critical diagnostic situations like genetic counseling for prenatal diagnosis.

  8. Proteins binding to the 5‘—flanking regualtory elements of the human β—globin gene

    Institute of Scientific and Technical Information of China (English)

    CHENZHIGANG; YADICHEN; 等

    1993-01-01

    The binding of nuclear proteins prepared from mouse erythroid tissue in different developmental stages to the 5'-flanking regulatory elements of human β-globin gene,two negative control regions(NCR1,-610to-490 bp;NCR2,-338,to-233bp),was identified.Two stage specific protein factors corresponding to embryonic and fetal stages were found to be capable of binding to NCR2.These data provided evidence that the cis acting elements of the 5'-flanking region might be involved in the developmental control of β-globin gene and NCR2 might be responsible in art for the silence of β-glolbin gene in the embryonic and fetal stages.

  9. Acute chest syndrome is associated with single nucleotide polymorphism-defined beta globin cluster haplotype in children with sickle cell anaemia.

    Science.gov (United States)

    Bean, Christopher J; Boulet, Sheree L; Yang, Genyan; Payne, Amanda B; Ghaji, Nafisa; Pyle, Meredith E; Hooper, W Craig; Bhatnagar, Pallav; Keefer, Jeffrey; Barron-Casella, Emily A; Casella, James F; Debaun, Michael R

    2013-10-01

    Genetic diversity at the human β-globin locus has been implicated as a modifier of sickle cell anaemia (SCA) severity. However, haplotypes defined by restriction fragment length polymorphism sites across the β-globin locus have not been consistently associated with clinical phenotypes. To define the genetic structure at the β-globin locus more thoroughly, we performed high-density single nucleotide polymorphism (SNP) mapping in 820 children who were homozygous for the sickle cell mutation (HbSS). Genotyping results revealed very high linkage disequilibrium across a large region spanning the locus control region and the HBB (β-globin gene) cluster. We identified three predominant haplotypes accounting for 96% of the β(S) -carrying chromosomes in this population that could be distinguished using a minimal set of common SNPs. Consistent with previous studies, fetal haemoglobin level was significantly associated with β(S) -haplotypes. After controlling for covariates, an association was detected between haplotype and rate of hospitalization for acute chest syndrome (ACS) (incidence rate ratio 0·51, 95% confidence interval 0·29-0·89) but not incidence rate of vaso-occlusive pain or presence of silent cerebral infarct (SCI). Our results suggest that these SNP-defined β(S) -haplotypes may be associated with ACS, but not pain or SCI in a study population of children with SCA.

  10. Sequence change in the HS2-LCR and Gg-globin gene promoter region of sickle cell anemia patients

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    E.V. Adorno

    2008-02-01

    Full Text Available The fetal hemoglobin (HbF levels and ßS-globin gene haplotypes of 125 sickle cell anemia patients from Brazil were investigated. We sequenced the Gg- and Ag-globin gene promoters and the DNase I-2 hypersensitive sites in the locus control regions (HS2-LCR of patients with HbF level disparities as compared to their ßS haplotypes. Sixty-four (51.2% patients had CAR/Ben genotype; 36 (28.8% Ben/Ben; 18 (14.4% CAR/CAR; 2 (1.6% CAR/Atypical; 2 (1.6% Ben/Cam; 1 (0.8% CAR/Cam; 1 (0.8% CAR/Arab-Indian, and 1 (0.8% Sen/Atypical. The HS2-LCR sequence analyses demonstrated a c.-10.677G>A change in patients with the Ben haplotype and high HbF levels. The Gg gene promoter sequence analyses showed a c.-157T>C substitution shared by all patients, and a c.-222_-225del related to the Cam haplotype. These results identify new polymorphisms in the HS2-LCR and Gg-globin gene promoter. Further studies are required to determine the correlation between HbF synthesis and the clinical profile of sickle cell anemia patients.

  11. The Full Globin Repertoire of Turtles Provides Insights into Vertebrate Globin Evolution and Functions.

    Science.gov (United States)

    Schwarze, Kim; Singh, Abhilasha; Burmester, Thorsten

    2015-06-15

    Globins are small heme proteins that play an important role in oxygen supply, but may also have other functions. Globins offer a unique opportunity to study the functional evolution of genes and proteins. We have characterized the globin repertoire of two different turtle species: the Chinese softshell turtle (Pelodiscus sinensis) and the western painted turtle (Chrysemys picta bellii). In the genomes of both species, we have identified eight distinct globin types: hemoglobin (Hb), myoglobin, neuroglobin, cytoglobin, globin E, globin X, globin Y, and androglobin. Therefore, along with the coelacanth, turtles are so far the only known vertebrates with a full globin repertoire. This fact allows for the first time a comparative analysis of the expression of all eight globins in a single species. Phylogenetic analysis showed an early divergence of neuroglobin and globin X before the radiation of vertebrates. Among the other globins, cytoglobin diverged first, and there is a close relationship between myoglobin and globin E; the position of globin Y is not resolved. The globin E gene was selectively lost in the green anole, and the genes coding for globin X and globin Y were deleted in chicken. Quantitative real-time reverse transcription polymerase chain reaction experiments revealed that myoglobin, neuroglobin, and globin E are highly expressed with tissue-specific patterns, which are in line with their roles in the oxidative metabolism of the striated muscles, the brain, and the retina, respectively. Histochemical analyses showed high levels of globin E in the pigment epithelium of the eye. Globin E probably has a myoglobin-like role in transporting O2 across the pigment epithelium to supply in the metabolically highly active retina.

  12. Active expression of Gγ globin gene on chromosome 11 with Yunnanese (Ayγδβ)~0-thalasseinia deletion in MEL cells

    Institute of Scientific and Technical Information of China (English)

    张俊武; 乔军; 宋文风; 邱志明

    1996-01-01

    A permanent lymphocyte cell line of a heterozygote with Yunnanese (Aγδβ)0-thalassemia deletion, associated with an increased production of Cry globin in adult, was founded using Epstein-Barr virus transformation. The hybrids of the lymphocyte cell and mouse erythroleukemia cell (MEL) were achieved and the hybrids containing human chromosome 11 were selected with the monoclonal antibody 53/6. The subclones containing only either the normal or the abnormal human chromosome 11 were separated and the expression of the human globin genes was studied. Expression of the β-globin gene, but not the Cγ and Aγ, was observed in the hybrids containing only the normal human chromosome 11, while active expression of the Cγ globin gene was observed in the hybrids containing only the abnormal human chromosome 11. These results have confirmed that the DNA deletion in the β-globin gene cluster is the cause of persistent active expression of the Cγ globin gene in the Yunnanese mutant.

  13. Genetic heterogeneity of the β-globin gene in various geographic populations of Yunnan in southwestern China.

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    Jie Zhang

    Full Text Available The aim of this study was to investigate the geographic distribution of β-globin gene mutations in different ethnic groups in Yunnan province.From 2004 to 2014, 1,441 subjects with hemoglobin disorders, identified by PCR-reverse dot blot and DNA sequencing, were studied according to ethnicity and geographic origin. Haplotypes were examined among 41 unrelated thalassemia chromosomes.Eighteen β-thalassemia mutations and seven hemoglobin variants were identified for 1,616 alleles in 22 different ethnic groups from all 16 prefecture-level divisions of Yunnan. The prevalence of β-thalassemia was heterogeneous and regionally specific. CD 41-42 (-TCTT was the most prevalent mutation in the populations of northeastern Yunnan. CD 17 (A>T was the most common mutation in the populations of southeastern Yunnan, especially for the Zhuang minority, whereas Hb E (CD 26, G>A was the most prevalent mutation in populations of southwestern Yunnan, especially for the Dai minority. Among the seven types of haplotypes identified, CD 17 (A>T was mainly linked to haplotype VII (+ - - - - - + and IVS-II-654 (C>T was only linked to haplotype I (+ - - - - + +.Our data underline the heterogeneity of β-globin gene mutations in Yunnan. This distribution of β-globin mutations in the geographic regions and ethnic populations provided a detailed ethnic basis and evolutionary view of humans in southern China, which will be beneficial for genetic counseling and prevention strategies.

  14. Effect of ATRX and G-Quadruplex Formation by the VNTR Sequence on α-Globin Gene Expression.

    Science.gov (United States)

    Li, Yue; Syed, Junetha; Suzuki, Yuki; Asamitsu, Sefan; Shioda, Norifumi; Wada, Takahito; Sugiyama, Hiroshi

    2016-05-17

    ATR-X (α-thalassemia/mental retardation X-linked) syndrome is caused by mutations in chromatin remodeler ATRX. ATRX can bind the variable number of tandem repeats (VNTR) sequence in the promoter region of the α-globin gene cluster. The VNTR sequence, which contains the potential G-quadruplex-forming sequence CGC(GGGGCGGGG)n , is involved in the downregulation of α-globin expression. We investigated G-quadruplex and i-motif formation in single-stranded DNA and long double-stranded DNA. The promoter region without the VNTR sequence showed approximately twofold higher luciferase activity than the promoter region harboring the VNTR sequence. G-quadruplex stabilizers hemin and TMPyP4 reduced the luciferase activity, whereas expression of ATRX led to a recovery in reporter activity. Our results demonstrate that stable G-quadruplex formation by the VNTR sequence downregulates the expression of α-globin genes and that ATRX might bind to and resolve the G-quadruplex.

  15. Recent advances in globin research using genome-wide association studies and gene editing.

    Science.gov (United States)

    Orkin, Stuart H

    2016-03-01

    A long-sought goal in the hemoglobin field has been an improved understanding of the mechanisms that regulate the switch from fetal (HbF) to adult (HbA) hemoglobin during development. With such knowledge, the hope is that strategies for directed reactivation of HbF in adults could be devised as an approach to therapy for the β-hemoglobinopathies thalassemia and sickle cell disease. Recent genome-wide association studies (GWAS) led to identification of three loci (BCL11A, HBS1L-MYB, and the β-globin cluster itself) in which natural genetic variation is correlated with different HbF levels in populations. Here, the central role of BCL11A in control of HbF is reviewed from the perspective of how findings may be translated to gene therapy in the not-too-distant future. This summary traces the evolution of recent studies from the initial recognition of BCL11A through GWAS to identification of critical sequences in an enhancer required for its erythroid-specific expression, thereby highlighting an Achilles heel for genome editing.

  16. The sequence and phylogenesis of the ?-globin genes of Barbary sheep (Ammotragus lervia), goat (Capra hircus), European mouflon (Ovis aries musimon) and Cyprus mouflon (Ovis aries ophion).

    Science.gov (United States)

    Pirastru, Monica; Multineddu, Chiara; Mereu, Paolo; Sannai, Mara; El Sherbini, El Said; Hadjisterkotis, Eleftherios; Nàhlik, Andràs; Franceschi, Paul; Manca, Laura; Masala, Bruno

    2009-09-01

    In order to investigate the polymorphism of ?-globin chain of hemoglobin amongst caprines, the linked (I)? and (II)? globin genes of Barbary sheep (Ammotragus lervia), goat (Capra hircus), European mouflon (Ovis aries musimon), and Cyprus mouflon (Ovis aries ophion) were completely sequenced, including the 5? and 3? untranslated regions. European and Cyprus mouflons, which do not show polymorphic ? globin chains, had almost identical ? globin genes, whereas Barbary sheep exhibit two different chains encoded by two nonallelic genes. Four different ? genes were observed and sequenced in goat, validating previous observations of the existence of allelic and nonallelic polymorphism. As in other vertebrates, interchromosomal gene conversion appears to be responsible for such polymorphism. Evaluation of nucleotide sequences at the level of molecular evolution of the (I)?-globin gene family in the caprine taxa suggests a closer relationship between the genus Ammotragus and Capra. Molecular clock estimates suggest sheep-mouflon, goat-aoudad, and ancestor-caprine divergences of 2.8, 5.7, and 7.1 MYBP, respectively.

  17. Role of novel and rare nucleotide substitutions of the β-globin gene

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    Margherita Vinciguerra

    2012-11-01

    Full Text Available The Laboratory for Molecular Prenatal Diagnosis of Hemoglobinopathies at the Villa Sofia-Cervello Hospital in Palermo, Italy, carries out an intensive screening program aimed at identifying the healthy carriers of thalassemia and, consequently, the couples at risk of bearing an affected fetus. The diagnostic process is basically divided into two phases: i hematologic and hemoglobin data; ii molecular analysis of globin genes and, when possible, a genetic study of the family. Since 2003, we have been performing DNA sequence analysis on those cases in which classical molecular methods failed to give a complete diagnostic response, particularly in phenotypes with borderline values of HbA2 with mild or absent microcytosis. During ten years of screening activities (from 2003 to 2012, twenty- seven unknown or rare nucleotide changes of the β-globin gene have been identified; hematologic and hemoglobin data have been carefully evaluated and, wherever possible, we have conducted a family study to evaluate whether a phenotypic expression could be associated to these nucleotide changes. Because of the limited numbers of cases for each mutation, the significance of these nucleotide substitutions has still not been fully clarified, and this raises a number of questions that need to be answered when carrying out appropriate genetic counseling for couples presumed to be at risk. 意大利巴勒莫Villa Sofia-Cervello医院血红蛋白病分子产前诊断实验室进行密集的筛选程序,旨在识别健康的地中海贫血携带者和有怀上地中海贫血胎儿风险的夫妇。 诊断过程基本上分为两个阶段:1)血液及血红蛋白数据;2)珠蛋白基因分子分析以及家族遗传研究(如有可能)。 自2003年以来,我们已对这类病例进行DNA序列分析:传统的分子方法无法给出完整的诊断响应,尤其是有轻微小红细胞症或缺乏小红细胞症的HbA2临界值表型。

  18. Prevalence of βS-globin gene haplotypes, α-thalassemia (3.7 kb deletion and redox status in patients with sickle cell anemia in the state of Paraná, Brazil

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    Eliana LitsukoTomimatsu Shimauti

    2015-09-01

    Full Text Available The aim of this study was to determine the frequency of beta S-globin gene (βS globin haplotypes and alpha thalassemia with 3.7 kb deletion (−α3.7kb thalassemia in the northwest region of Paraná state, and to investigate the oxidative and clinical-hematological profile of βS globin carriers in this population. Of the 77 samples analyzed, 17 were Hb SS, 30 were Hb AS and 30 were Hb AA. The βSglobin haplotypes and −α3.7kb thalassemia were identified using polymerase chain reaction.Trolox equivalent antioxidant capacity (TEAC and lipid peroxidation (LPO were assessed spectophotometrically. Serum melatonin levels were determined using high-performance liquid chromatography coupled to coulometric electrochemical detection. The haplotype frequencies in the SS individuals were as follows: Bantu- 21 (62%, Benin - 11 (32% and Atypical- 2 (6%. Bantu/Benin was the most frequent genotype. Of the 47 SS and AS individuals assessed, 17% (n = 8 had the −α3.7kb mutation. Clinical manifestations, as well as serum melatonin, TEAC and LPO levels did not differ between Bantu/Bantu and Bantu/Benin individuals (p > 0.05. Both genotypes were associated with high LPO and TEAC levels and decreased melatonin concentration. These data suggest that the level of oxidative stress in patients with Bantu/Bantu and Bantu/Benin genotypes may overload the antioxidant capacity.

  19. mRNA expression of iron regulatory genes in beta-thalassemia intermedia and beta-thalassemia major mouse models.

    Science.gov (United States)

    Weizer-Stern, Orly; Adamsky, Konstantin; Amariglio, Ninette; Rachmilewitz, Eliezer; Breda, Laura; Rivella, Stefano; Rechavi, Gideon

    2006-07-01

    beta-Thalassemia is an inherited anemia in which synthesis of the hemoglobin beta-chain is decreased. The excess unmatched alpha-globin chains accumulate in the growing erythroid precursors, causing their premature death (ineffective erythropoiesis). Clinical features of beta-thalassemia include variably severe anemia and iron accumulation due to increased intestinal iron absorption. The most anemic patients require regular blood transfusions, which exacerbate their iron overload and result in damage to vital organs. The hepatic peptide hepcidin, a key regulator of iron metabolism in mammals, was recently found to be low in the urine of beta-thalassemia patients, compared with healthy controls, despite their iron overload. In our work, we measured by RQ-PCR the liver mRNA expression of hepcidin and other iron regulatory genes in beta-thalassemia major mouse model (C57Bl/6 Hbb(th3/th3)), and compared it with beta-thalassemia intermedia mouse model (C57Bl/6 Hbb(th3/+)) and control mice. We found decreased expression of hepcidin and TfR2 and increased expression of TfR1 and NGAL in the beta-thalassemia mouse models, compared with the control mice. Significant down-regulation of hepcidin expression in beta-thalassemia major, despite iron overload, might explain the increased iron absorption typically observed in thalassemia.

  20. Trans—acting factors from the human fetal liver binding to the human ε—globin gene silencer

    Institute of Scientific and Technical Information of China (English)

    YANZHIJIANG; CHUJIANG; 等

    1997-01-01

    The developmental stage-specific silencing of the human ε-globin gene during embryonic life is controlled,in part,by the silencer (-392bp- -177bp) upstream of this gene.In order to elucidate its role,the nuclear extract from the human fetal liver has been prepared and the interactions between trans-acting factors and this silencer element have been examined.By using DNaseI footprinting assay,a major protected region from -278bp to -235bp within this silencer element was identified.Furthermore,we found in gel mobility shift assay and Southwestern blotting assay that there were at least four trans-acting factors (MV≈32,28,26 and 22kD) in the nuclear extract isolated from the human fetal liver,which could specifically bind to this region.Our results suggested that these trans-acting factors might play an important role in silencing the human embryonic ε-globin gene expression at the fetal stage through the interactions with this silencer.

  1. The frequency of β-globin gene haplotypes, α-thalassemia and genetic polymorphisms of methylenetetrahydrofolate reductase, factor V Leiden and prothrombin genes in children with sickle cell disease in Rio de Janeiro, Brazil Frequência dos haplótipos da globina beta, da talassemia alfa e dos polimorfismos genéticos dos genes da metilenotetrahidrofolato redutase, do fator V Leiden e da protrombina em crianças com doença falciforme no Rio de Janeiro, Brasil

    Directory of Open Access Journals (Sweden)

    Isaac L. Silva Filho

    2010-02-01

    Full Text Available A freqüência dos haplótipos beta S e beta C do gene da globina e a prevalência de talassemia alfa e de mutações nos genes da metilenotetrahidrofolato redutase (MTHFR-C677T, do fator V de Leiden e da protrombina (G20210A foi estudada em crianças com doença falciforme do Rio de Janeiro. O haplótipo Bantu foi o mais freqüente (65,9%, 21,2% das crianças (18% heterozigotas e 3% homozigotas apresentam talassemia com mutação alfa 3.7kb, ao contrário da mutação alfa 4.2kb que não foi encontrada. Os alelos 677CT e 677TT da MTHFR foram observados em 20,2% e 4,8%, respectivamente. Os haplótipos Camarões, Árabe-Indiano e Senegal não foram detectados na amostra estudada, bem como mutações no gene do fator V de Leiden e da protrombina. Somente o haplótipo beta C CI foi observado. Esse é o primeiro estudo realizado em uma amostra proveniente do Programa de Triagem Neonatal para Hemoglobinopatias do estado do Rio de Janeiro. Apesar do Rio de Janeiro ser a segunda maior cidade brasileira e seus habitantes expressarem o elevado grau de miscigenação ocorrida no país, nossos resultados ainda coincidem com os registros históricos dos fluxos migratórios do gene beta S para o Brasil, bem como refletem a forte influência de indivíduos de origem africana na população do Rio de Janeiro.

  2. alpha-Globin genes: thalassemic and structural alterations in a Brazilian population

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    M.R.S.C. Wenning

    2000-09-01

    Full Text Available Seven unrelated patients with hemoglobin (Hb H disease and 27 individuals with alpha-chain structural alterations were studied to identify the alpha-globin gene mutations present in the population of Southeast Brazil. The -alpha3.7, --MED and -(alpha20.5 deletions were investigated by PCR, whereas non-deletional alpha-thalassemia (alphaHphalpha, alphaNcoIalpha, aaNcoI, alphaIcalpha and alphaTSaudialpha was screened with restriction enzymes and by nested PCR. Structural alterations were identified by direct DNA sequencing. Of the seven patients with Hb H disease, all of Italian descent, two had the -(alpha20.5/-alpha3.7 genotype, one had the --MED/-alpha3.7 genotype, one had the --MED/alphaHphalpha genotype and three showed interaction of the -alpha3.7 deletion with an unusual, unidentified form of non-deletional alpha-thalassemia [-alpha3.7/(aaT]. Among the 27 patients with structural alterations, 15 (of Italian descent had Hb Hasharon (alpha47Asp->His associated with the -alpha3.7 deletion, 4 (of Italian descent were heterozygous for Hb J-Rovigo (alpha53Ala->Asp, 4 (3 Blacks and 1 Caucasian were heterozygous for Hb Stanleyville-II (alpha78Asn->Lys associated with the alpha+-thalassemia, 1 (Black was heterozygous for Hb G-Pest (alpha74Asp->Asn, 1 (Caucasian was heterozygous for Hb Kurosaki (alpha7Lys->Glu, 1 (Caucasian was heterozygous for Hb Westmead (alpha122His->Gln, and 1 (Caucasian was the carrier of a novel silent variant (Hb Campinas, alpha26Ala->Val. Most of the mutations found reflected the Mediterranean and African origins of the population. Hbs G-Pest and Kurosaki, very rare, and Hb Westmead, common in southern China, were initially described in individuals of ethnic origin differing from those of the carriers reported in the present study and are the first cases to be reported in the Brazilian population.

  3. NF-Y recruits both transcription activator and repressor to modulate tissue- and developmental stage-specific expression of human γ-globin gene.

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    Xingguo Zhu

    Full Text Available The human embryonic, fetal and adult β-like globin genes provide a paradigm for tissue- and developmental stage-specific gene regulation. The fetal γ-globin gene is expressed in fetal erythroid cells but is repressed in adult erythroid cells. The molecular mechanism underlying this transcriptional switch during erythroid development is not completely understood. Here, we used a combination of in vitro and in vivo assays to dissect the molecular assemblies of the active and the repressed proximal γ-globin promoter complexes in K562 human erythroleukemia cell line and primary human fetal and adult erythroid cells. We found that the proximal γ-globin promoter complex is assembled by a developmentally regulated, general transcription activator NF-Y bound strongly at the tandem CCAAT motifs near the TATA box. NF-Y recruits to neighboring DNA motifs the developmentally regulated, erythroid transcription activator GATA-2 and general repressor BCL11A, which in turn recruit erythroid repressor GATA-1 and general repressor COUP-TFII to form respectively the NF-Y/GATA-2 transcription activator hub and the BCL11A/COUP-TFII/GATA-1 transcription repressor hub. Both the activator and the repressor hubs are present in both the active and the repressed γ-globin promoter complexes in fetal and adult erythroid cells. Through changes in their levels and respective interactions with the co-activators and co-repressors during erythroid development, the activator and the repressor hubs modulate erythroid- and developmental stage-specific transcription of γ-globin gene.

  4. Phylogenetic analysis reveals wide distribution of globin X

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    Makałowski Wojciech

    2011-10-01

    Full Text Available Abstract The vertebrate globin gene repertoire consists of seven members that differ in terms of structure, function and phyletic distribution. While hemoglobin, myoglobin, cytoglobin, and neuroglobin are present in almost all gnathostomes examined so far, other globin genes, like globin X, are much more restricted in their phyletic distribution. Till today, globin X has only been found in teleost fish and Xenopus. Here, we report that globin X is also present in the genomes of the sea lamprey, ghost shark and reptiles. Moreover, the identification of orthologs of globin X in crustacean, insects, platyhelminthes, and hemichordates confirms its ancient origin.

  5. Promoter region sequence differences in the A and G gamma globin genes of Brazilian sickle cell anemia patients

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    C.G. Barbosa

    2010-08-01

    Full Text Available Fetal hemoglobin (HbF, encoded by the HBG2 and HBG1 genes, is the best-known genetic modulator of sickle cell anemia, varying dramatically in concentration in the blood of these patients. This variation is partially associated with polymorphisms located in the promoter region of the HBG2 and HBG1 genes. In order to explore known and unknown polymorphisms in these genes, the sequences of their promoter regions were screened in sickle cell anemia patients and correlated with both their HbF levels and their βS-globin haplotypes. Additionally, the sequences were compared with genes from 2 healthy groups, a reference one (N = 104 and an Afro-descendant one (N = 98, to identify polymorphisms linked to the ethnic background.The reference group was composed by healthy individuals from the general population. Four polymorphisms were identified in the promoter region of HBG2 and 8 in the promoter region of HBG1 among the studied groups. Four novel single nucleotide polymorphisms (SNP located at positions -324, -317, -309 and -307 were identified in the reference group. A deletion located between -396 and -391 in the HBG2 promoter region and the SNP -271 C→T in the HBG1 promoter region were associated with the Central African Republic βS-globin haplotype. In contrast, the -369 C→G and 309 A→G SNPs in the HBG2 promoter region were correlated to the Benin haplotype. The polymorphisms -396_-391 del HBG2, -369 SNP HBG2 and -271 SNP HBG1 correlated with HbF levels. Hence, we suggest an important role of HBG2 and HBG1 gene polymorphisms on the HbF synthesis.

  6. A novel deletion/insertion caused by a replication error in the β-globin gene locus control region.

    Science.gov (United States)

    Joly, Philippe; Lacan, Philippe; Garcia, Caroline; Meley, Roland; Pondarré, Corinne; Francina, Alain

    2011-01-01

    Deletions in the β-globin locus control region (β-LCR) lead to (εγδβ)(0)-thalassemia [(εγδβ)(0)-thal]. In patients suffering from these rare deletions, a normal hemoglobin (Hb), phenotype is found, contrasting with a hematological thalassemic phenotype. Multiplex-ligation probe amplification (MLPA) is an efficient tool to detect β-LCR deletions combined with long-range polymerase chain reaction (PCR) and DNA sequencing to pinpoint deletion breakpoints. We present here a novel 11,155 bp β-LCR deletion found in a French Caucasian patient which removes DNase I hypersensitive site 2 (HS2) to HS4 of the β-LCR. Interestingly, a 197 bp insertion of two inverted sequences issued from the HS2-HS3 inter-region is present and suggests a complex rearrangement during replication. Carriers of this type of thalassemia can be misdiagnosed as an α-thal trait. Consequently, a complete α- and β-globin gene cluster analysis is required to prevent a potentially damaging misdiagnosis in genetic counselling.

  7. Hb Filottrano [codon 120 (-A)]: a novel frameshift mutation in exon 3 of the β-globin gene causing dominantly inherited β-thalassemia intermedia.

    Science.gov (United States)

    Amato, Antonio; Cappabianca, Maria Pia; Perri, Maria; Zaghis, Ivo; Mastropietro, Fabrizio; Ponzini, Donatella; Di Biagio, Paola; Piscitelli, Roberta

    2012-01-01

    We report a novel frameshift mutation in exon 3 of the β-globin gene, that, in the heterozygous state, leads to a β-thalassemia intermedia (β-TI) phenotype (marked anemia, splenomegaly, hyperbilirubinemia, jaundice, unbalanced synthesis of α/non-α chains in a 34-year-old Italian woman. This frameshift mutation, due to the deletion of the first nucleotide (-A) at codon 120, results in a β-globin chain that is elongated to 156 amino acid residues. These highly unstable abnormal chains precipitate in the erythroblasts as inclusion bodies, thus causing inefficient erythropoiesis and ultimately resulting in the observed dominant clinical phenotype.

  8. Identification of the development stage—specific factors in mouse fetal liver binding to the human β—globin gene promoter

    Institute of Scientific and Technical Information of China (English)

    CHENYADI; YULONGHU; 等

    1994-01-01

    In order to elucidate the molecular mechanisms of globin gene expression during embryonic development,the nuclear extracts from mouse hematopoietic tissue at different stages of development have been prepared.By using DNase I footprinting and gel mobility shift assays,the binding of protein factors in these extracts to the human β-globin promoter was analyzed.The differences in the binding patterns of protein factors during development were observed.An erythroid-specific and stage-specific nuclear protein in the nuclear extrace from d 18 mouse fetal liver was identified,which can bind to the sequence(from-66bp to-90bp) of human β-globin promoter.We therefore speculate that the function of this cis-acting element may be similar to stage selector element(SSE) in chicken βA-promoter.

  9. A new Frameshift mutation on the α2-globin gene causing α⁺-thalassemia: codon 43 (TTC>-TC or TTC>T-C).

    Science.gov (United States)

    Joly, Philippe; Lacan, Philippe; Garcia, Caroline; Barro, Claire; Francina, Alain

    2012-01-01

    We report a new mutation on the α2-globin gene causing α(+)-thalassemia (α(+)-thal) with a deletion of a single nucleotide (T) at amino acid residue 43 [HBA2:c.130delT or HBA2:c.131delT]. This frameshift deletion gives rise to a premature termination codon at codon 47.

  10. Mi2β is required for γ-globin gene silencing: temporal assembly of a GATA-1-FOG-1-Mi2 repressor complex in β-YAC transgenic mice.

    Directory of Open Access Journals (Sweden)

    Flávia C Costa

    Full Text Available Activation of γ-globin gene expression in adults is known to be therapeutic for sickle cell disease. Thus, it follows that the converse, alleviation of repression, would be equally effective, since the net result would be the same: an increase in fetal hemoglobin. A GATA-1-FOG-1-Mi2 repressor complex was recently demonstrated to be recruited to the -566 GATA motif of the (Aγ-globin gene. We show that Mi2β is essential for γ-globin gene silencing using Mi2β conditional knockout β-YAC transgenic mice. In addition, increased expression of (Aγ-globin was detected in adult blood from β-YAC transgenic mice containing a T>G HPFH point mutation at the -566 GATA silencer site. ChIP experiments demonstrated that GATA-1 is recruited to this silencer at day E16, followed by recruitment of FOG-1 and Mi2 at day E17 in wild-type β-YAC transgenic mice. Recruitment of the GATA-1-mediated repressor complex was disrupted by the -566 HPFH mutation at developmental stages when it normally binds. Our data suggest that a temporal repression mechanism is operative in the silencing of γ-globin gene expression and that either a trans-acting Mi2β knockout deletion mutation or the cis-acting -566 (Aγ-globin HPFH point mutation disrupts establishment of repression, resulting in continued γ-globin gene transcription during adult definitive erythropoiesis.

  11. α(+)-Thalassemia Due to a Frameshift Mutation of the α2-Globin Gene [codons 55/56 (+T) or HBA2: c.168dup].

    Science.gov (United States)

    Waye, John S; Eng, Barry; Hanna, Meredith; Hohenadel, Betty-Ann; Nakamura, Lisa M; Walker, Lynda

    2015-01-01

    We report a case of α(+)-thalassemia (α(+)-thal) trait in a Chinese-Canadian family caused by a novel frameshift mutation of the α2-globin gene, specifically the duplication of a single nucleotide at amino acid codon 56 [HBA2: c.168dup]. The mutation results in substitution of a termination codon (TAA) for lysine (AAG) at amino acid position 56.

  12. Novel Inducers of Fetal Globin Identified through High Throughput Screening (HTS Are Active In Vivo in Anemic Baboons and Transgenic Mice.

    Directory of Open Access Journals (Sweden)

    Michael S Boosalis

    Full Text Available High-level fetal (γ globin expression ameliorates clinical severity of the beta (β hemoglobinopathies, and safe, orally-bioavailable γ-globin inducing agents would benefit many patients. We adapted a LCR-γ-globin promoter-GFP reporter assay to a high-throughput robotic system to evaluate five diverse chemical libraries for this activity. Multiple structurally- and functionally-diverse compounds were identified which activate the γ-globin gene promoter at nanomolar concentrations, including some therapeutics approved for other conditions. Three candidates with established safety profiles were further evaluated in erythroid progenitors, anemic baboons and transgenic mice, with significant induction of γ-globin expression observed in vivo. A lead candidate, Benserazide, emerged which demonstrated > 20-fold induction of γ-globin mRNA expression in anemic baboons and increased F-cell proportions by 3.5-fold in transgenic mice. Benserazide has been used chronically to inhibit amino acid decarboxylase to enhance plasma levels of L-dopa. These studies confirm the utility of high-throughput screening and identify previously unrecognized fetal globin inducing candidates which can be developed expediently for treatment of hemoglobinopathies.

  13. Two new beta-chain variants: Hb Tripoli [beta26(B8)Glu-->Ala] and Hb Tizi-Ouzou [beta29(B11)Gly-->Ser].

    Science.gov (United States)

    Lacan, Philippe; Becchi, Michel; Zanella-Cleon, Isabelle; Aubry, Martine; Ffrench, Martine; Couprie, Nicole; Francina, Alain

    2004-08-01

    Two new beta-globin chain variants: Hb Tripoli: codon 26, GAG-->GCG [beta26(B8)Glu-->Ala] and Hb Tizi-Ouzou: codon 29, GGC-->AGC [beta29(B11)Gly-->Ser] are described on the first exon of the beta-globin gene. The two variants are characterized by DNA sequencing and mass spectrometry (MS). Hematological abnormalities were found in the two carriers. The presence of microcytosis and hypochromia is explained by an additional homozygous 3.7 kb alpha(+) thalassemic deletion for the carrier of Hb Tizi-Ouzou. Hb Tizi-Ouzou showed a slight instability in vitro. The same hematological abnormalities associated with anemia are difficult to explain for Hb Tripoli's carrier in the absence of an alpha-globin genes abnormality and could suggest a possible abnormal splicing.

  14. Isolation of β-globin related genes from a human cosmid library.

    NARCIS (Netherlands)

    F.G. Grosveld (Frank); H.H.M. Dahl; R.A. Flavell (Richard); E. de Boer (Ernie)

    1981-01-01

    textabstractA human gene library was constructed using an improved cloning technique for cosmid vectors. Human placental DNA was partially digested with restriction endonuclease MboI; size-fractionated and ligated to BamHI-cut and phosphatase-treated cosmid vector pJB8. After packaging in lambda pha

  15. Erythroid-Specific Expression of LIN28A Is Sufficient for Robust Gamma-Globin Gene and Protein Expression in Adult Erythroblasts.

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    Y Terry Lee

    Full Text Available Increasing fetal hemoglobin (HbF levels in adult humans remains an active area in hematologic research. Here we explored erythroid-specific LIN28A expression for its effect in regulating gamma-globin gene expression and HbF levels in cultured adult erythroblasts. For this purpose, lentiviral transduction vectors were produced with LIN28A expression driven by erythroid-specific gene promoter regions of the human KLF1 or SPTA1 genes. Transgene expression of LIN28A with a linked puromycin resistance marker was restricted to the erythroid lineage as demonstrated by selective survival of erythroid colonies (greater than 95% of all colonies. Erythroblast LIN28A over-expression (LIN28A-OE did not significantly affect proliferation or inhibit differentiation. Greater than 70% suppression of total let-7 microRNA levels was confirmed in LIN28A-OE cells. Increases in gamma-globin mRNA and protein expression with HbF levels reaching 30-40% were achieved. These data suggest that erythroblast targeting of LIN28A expression is sufficient for increasing fetal hemoglobin expression in adult human erythroblasts.

  16. In silico mutation analysis of human beta globin gene in sickle cell disease patients

    Directory of Open Access Journals (Sweden)

    Hira Mubeen

    2016-05-01

    Conclusion: Studies suggested that there is need to maintain a primary prevention program to detect sickle cell disease at earlier stages despite having a large high risk. Preventive diagnosis and follow-up would reduce infant mortality by preventing the development of severe anemia as well as dangerous complications. In short, sickle cell disease surveillance would avert loss of life, measured as the number of years lost due to ill-health, disability or early death. [Int J Res Med Sci 2016; 4(5.000: 1673-1677

  17. The Role of Crowding Forces in Juxtaposing β-Globin Gene Domain Remote Regulatory Elements in Mouse Erythroid Cells.

    Directory of Open Access Journals (Sweden)

    Arkadiy K Golov

    Full Text Available The extremely high concentration of macromolecules in a eukaryotic cell nucleus indicates that the nucleoplasm is a crowded macromolecular solution in which large objects tend to gather together due to crowding forces. It has been shown experimentally that crowding forces support the integrity of various nuclear compartments. However, little is known about their role in control of chromatin dynamics in vivo. Here, we experimentally addressed the possible role of crowding forces in spatial organization of the eukaryotic genome. Using the mouse β-globin domain as a model, we demonstrated that spatial juxtaposition of the remote regulatory elements of this domain in globin-expressing cells may be lost and restored by manipulation of the level of macromolecular crowding. In addition to proving the role of crowding forces in shaping interphase chromatin, our results suggest that the folding of the chromatin fiber is a major determinant in juxtaposing remote genomic elements.

  18. Interactions between HMG proteins (HMG1/2 and HMG14/17) and humanε-globin gene promoter(ε-promoter)

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    High mobility group (HMG) proteins are abundant non-histone proteins in the nuclei of eukaryocytes. It has been shown that HMG proteins may play important roles in the structure and function of chromatin. In the present study, thebinding of HMG proteins (HMG1/2 and HMG14/17) to the human ε-globin gene promoter (ε-promo- ter, -177-+1 bp) has been examined by using both the in vitro nucleosome reconstitution and the electrophoresis mobility shift assay (EMSA). We found that HMG1/2 proteins could bind to the naked ε-promoter DNA, however, HMG14/17 could not. Using the in vitro nucleosome recons- titution, we revealed that HMG14/17 could bind to the mononucleosome reconstituted in vitro with ε-promoter,whi- le HMG1/2 could not. Those results indicate that the binding of HMG proteins to ε-promoter is dynamic as the nucleo- some assembling and disassembling. Wespeculated that this selective binding of HMG proteins to ε-promoter might playa critical role in the regulation of ε-globin gene expression.

  19. The prevalence and molecular spectrum of α- and β-globin gene mutations in 14,332 families of Guangdong Province, China.

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    Aihua Yin

    Full Text Available OBJECTIVE: To reveal the familial prevalence and molecular variation of α- and β-globin gene mutations in Guangdong Province. METHODS: A total of 40,808 blood samples from 14,332 families were obtained and analyzed for both hematological and molecular parameters. RESULTS: A high prevalence of α- and β-globin gene mutations was found. Overall, 17.70% of pregnant women, 15.94% of their husbands, 16.03% of neonates, and 16.83% of couples (pregnant women and their husbands were heterozygous carriers of α- or β-thalassemia. The regions with the highest prevalence were the mountainous and western regions, followed by the Pearl River Delta; the region with the lowest prevalence was Chaoshan. The total familial carrier rate (both spouses were α- or β-thalassemia carriers was 1.87%, and the individual carrier rates of α- and β-thalassemia were 1.68% and 0.20%, respectively. The total rate of moderate-to-severe fetal thalassemia was 12.78% among couples in which both parents were carriers. CONCLUSIONS: There was a high prevalence of α- and β-thalassemia in Guangdong Province. This study will contribute to the development of thalassemia prevention and control strategies in Guangdong Province.

  20. Analysis of betaS and betaA genes in a Mexican population with African roots.

    Science.gov (United States)

    Magaña, María Teresa; Ongay, Zoyla; Tagle, Juan; Bentura, Gilberto; Cobián, José G; Perea, F Javier; Casas-Castañeda, Maricela; Sánchez-López, Yoaly J; Ibarra, Bertha

    2002-01-01

    To investigate the origin of the beta(A) and beta(S) genes in a Mexican population with African roots and a high frequency of hemoglobin S, we analyzed 467 individuals (288 unrelated) from different towns in the states of Guerrero and Oaxaca in the Costa Chica region. The frequency of the sickle-cell trait was 12.8%, which may represent a public health problem. The frequencies of the beta-haplotypes were determined from 350 nonrelated chromosomes (313 beta(A) and 37 beta(S)). We observed 15 different beta(A) haplotypes, the most common of which were haplotypes 1 (48.9%), 2 (13.4%), and 3 (13.4%). The calculation of pairwise distributions and Nei's genetic distance analysis using 32 worldwide populations showed that the beta(A) genes are more closely related to those of Mexican Mestizos and North Africans. Bantu and Benin haplotypes and haplotype 9 were related to the beta(S) genes, with frequencies of 78.8, 18.2, and 3.0%, respectively. Comparison of these haplotypes with 17 other populations revealed a high similitude with the population of the Central African Republic. These data suggest distinct origins for the beta(A) and beta(S) genes in Mexican individuals from the Costa Chica region.

  1. Progress toward the genetic treatment of the beta-thalassemias.

    Science.gov (United States)

    Sadelain, Michel; Lisowski, Leszek; Samakoglu, Selda; Rivella, Stefano; May, Chad; Riviere, Isabelle

    2005-01-01

    The beta-thalassemias are congenital anemias that are caused by mutations that reduce or abolish expression of the beta-globin gene. They can be cured by allogeneic hematopoietic stem cell (HSC) transplantation, but this therapeutic option is not available to most patients. The transfer of a regulated beta-globin gene in autologous HSCs is a highly attractive alternative treatment. This strategy, which is simple in principle, raises major challenges in terms of controlling expression of the globin transgene, which ideally should be erythroid specific, differentiation- and stage-restricted, elevated, position independent, and sustained over time. Using lentiviral vectors, May et al. demonstrated in 2000 that an optimized combination of proximal and distal transcriptional control elements permits lineage-specific and elevated beta-globin expression, resulting in therapeutic hemoglobin production and correction of anemia in beta-thalassemic mice. Several groups have by now replicated and extended these findings to various mouse models of severe hemoglobinopathies, thus fueling enthusiasm for a potential treatment of beta-thalassemia based on globin gene transfer. Current investigation focuses on safety issues and the need for improved vector production methodologies. The safe implementation of stem cell-based gene therapy requires the prevention of the formation of replication-competent viral genomes and minimization of the risk of insertional oncogenesis. Importantly, globin vectors, in which transcriptional activity is highly restricted, have a lesser risk of activating oncogenes in hematopoietic progenitors than non-tissue-specific vectors, by virtue of their late-stage erythroid specificity. As such, they provide a general paradigm for improving vector safety in stem cell-based gene therapy.

  2. Molecular basis of asymptomatic beta-thalassemia major in an African American individual.

    Science.gov (United States)

    Ballas, S K; Cai, S P; Gabuzda, T; Chehab, F F

    1997-03-17

    The beta-thalassemia syndromes are a heterogeneous group of genetic disorders characterized by reduced or absent expression of the beta-globin gene. To date, over 300 beta-thalassemia alleles have been characterized in or around the beta-globin region. Thalassemia major is severe anemia necessitating chronic blood transfusions, splenectomy, iron chelation therapy, and bone marrow transplantation. Usually thalassemia major results from homozygosity or compound heterozygosity for severe betaO- and/or beta+-thalassemia mutations. Thalassemia intermedia is a clinical diagnosis that describes a symptomatic but less severe condition than beta-thalassemia major. beta-thalassemia intermedia may arise from several different combinations of alpha- and/or beta-thalassemia mutations. Heterozygous beta-thalassemia is typically characterized by a mild microcytic hypochromic anemia without any significant clinical implications. In this report, we describe a 63-year-old Africian American woman with asymptomatic homozygous beta-thalassemia, who seems to carry 2 copies of the -29 mutation in the promoter region of the beta-globin gene. Her elevated hemoglobin F level of 83% was associated with heterozygosity for the Xmn I polymorphism upstream of the Ggamma-globin gene. Southern blot analysis at the alpha-globin locus did not show any deletion that would account for the mildness of her phenotype. Therefore, homozygosity for the -29 mutation along with the Xmn I polymorphism appears to confer an extremely mild beta-thalassemia phenotype. This observation has important implications in the prenatal diagnosis and genetic counseling of families segregating this type of genetic defect.

  3. Wide diversity in structure and expression profiles among members of the Caenorhabditis elegans globin protein family

    Directory of Open Access Journals (Sweden)

    Vinogradov Serge

    2007-10-01

    Full Text Available Abstract Background The emergence of high throughput genome sequencing facilities and powerful high performance bioinformatic tools has highlighted hitherto unexpected wide occurrence of globins in the three kingdoms of life. In silico analysis of the genome of C. elegans identified 33 putative globin genes. It remains a mystery why this tiny animal might need so many globins. As an inroad to understanding this complexity we initiated a structural and functional analysis of the globin family in C. elegans. Results All 33 C. elegans putative globin genes are transcribed. The translated sequences have the essential signatures of single domain bona fide globins, or they contain a distinct globin domain that is part of a larger protein. All globin domains can be aligned so as to fit the globin fold, but internal interhelical and N- and C-terminal extensions and a variety of amino acid substitutions generate much structural diversity among the globins of C. elegans. Likewise, the encoding genes lack a conserved pattern of intron insertion positioning. We analyze the expression profiles of the globins during the progression of the life cycle, and we find that distinct subsets of globins are induced, or repressed, in wild-type dauers and in daf-2(e1370/insulin-receptor mutant adults, although these animals share several physiological features including resistance to elevated temperature, oxidative stress and hypoxic death. Several globin genes are upregulated following oxygen deprivation and we find that HIF-1 and DAF-2 each are required for this response. Our data indicate that the DAF-2 regulated transcription factor DAF-16/FOXO positively modulates hif-1 transcription under anoxia but opposes expression of the HIF-1 responsive globin genes itself. In contrast, the canonical globin of C. elegans, ZK637.13, is not responsive to anoxia. Reduced DAF-2 signaling leads to enhanced transcription of this globin and DAF-16 is required for this effect

  4. DNA polymorphisms at the BCL11A, HBS1L-MYB, and beta-globin loci associate with fetal hemoglobin levels and pain crises in sickle cell disease.

    Science.gov (United States)

    Lettre, Guillaume; Sankaran, Vijay G; Bezerra, Marcos André C; Araújo, Aderson S; Uda, Manuela; Sanna, Serena; Cao, Antonio; Schlessinger, David; Costa, Fernando F; Hirschhorn, Joel N; Orkin, Stuart H

    2008-08-19

    Sickle cell disease (SCD) is a debilitating monogenic blood disorder with a highly variable phenotype characterized by severe pain crises, acute clinical events, and early mortality. Interindividual variation in fetal hemoglobin (HbF) expression is a known and potentially heritable modifier of SCD severity. High HbF levels are correlated with reduced morbidity and mortality. Common single nucleotide polymorphisms (SNPs) at the BCL11A and HBS1L-MYB loci have been implicated previously in HbF level variation in nonanemic European populations. We recently demonstrated an association between a BCL11A SNP and HbF levels in one SCD cohort [Uda M, et al. (2008) Proc Natl Acad Sci USA 105:1620-1625]. Here, we genotyped additional BCL11A SNPs, HBS1L-MYB SNPs, and an SNP upstream of (G)gamma-globin (HBG2; the XmnI polymorphism), in two independent SCD cohorts: the African American Cooperative Study of Sickle Cell Disease (CSSCD) and an SCD cohort from Brazil. We studied the effect of these SNPs on HbF levels and on a measure of SCD-related morbidity (pain crisis rate). We strongly replicated the association between these SNPs and HbF level variation (in the CSSCD, P values range from 0.04 to 2 x 10(-42)). Together, common SNPs at the BCL11A, HBS1L-MYB, and beta-globin (HBB) loci account for >20% of the variation in HbF levels in SCD patients. We also have shown that HbF-associated SNPs associate with pain crisis rate in SCD patients. These results provide a clear example of inherited common sequence variants modifying the severity of a monogenic disease.

  5. The human thyrotropin beta-subunit gene differs in 5' structure from murine TSH-beta genes.

    Science.gov (United States)

    Guidon, P T; Whitfield, G K; Porti, D; Kourides, I A

    1988-12-01

    The gene encoding the beta-subunit of human thyrotropin (hTSH-beta) was isolated, and its nucleotide sequence was determined. The gene is 4.3 kb in length, consists of three exons and two introns, and is present as a single copy as determined by Southern blot analysis of total genomic DNA. The protein coding portion of the gene, which includes exons 2 and 3, was isolated from a human genomic phage library, while exon 1, which encodes only 5' untranslated mRNA sequence, was isolated from a plasmid library of size-selected genomic DNA fragments. Here we describe the isolation of the 5' untranslated exon of the hTSH-beta subunit and 5'-flanking region. The structure of the hTSH-beta gene is very similar to the previously characterized TSH-beta genes from mouse and rat. The genes from all three species have two distinct promoter regions, but while both promoters are utilized by the murine TSH-beta genes, the human TSH-beta gene apparently utilizes only the proximal promoter for transcription initiation. A striking difference in hTSH-beta gene structure compared to the murine genes is that exon 1 of the human gene is 36 nucleotides. An analysis of the mouse, rat, and human exon 1 and 5'-flanking region shows a high percentage of sequence homology, with the exception of a 9-nucleotide insertion 13 bases 3' from the proximal TATA box found in the human gene but not found in the other two species. We propose that this insertion results in the additional length of human exon 1 compared to the mouse and rat genes. By isolating the promoter region of the hTSH-beta gene, we can begin to identify specific sequences involved in the regulation of hTSH gene expression.

  6. The 5‘—flanking cis—acting elements of the human ε—globin gene associates with the nuclear matrix and binds to the nuclear matrix proteins

    Institute of Scientific and Technical Information of China (English)

    YANZHIJIANG; RUOLANQIAN

    1998-01-01

    The nuclear matrix attachment regions(MARs) and the binding nuclear matrix proteins in the 5'-flanking cisacting elements of the human ε-globin gene have been examined.Using in vitro DNA-matrix binding assay,it has been shown that the positive stage-specific regulatory element (ε-PREII,-446bp- -419bp) upstream of this gene could specifically associate with the nuclear matrix from K562 cells,indicating that ε-PREII may be an erythroidspecific facultative MAR.In gel mobility shift assay and Southwestern blotting assay,an erythroid-specific nuclear matrix protein (ε-NMPk) in K562 cells has been revealed to bind to this positive regulatory element (ε-PREII).Furthermore,we demonstrated that the silencer (-392bp- -177bp) upstream of the human ε-globin gene could associate with the nuclear matrices from K562,HEL and Raji cells.In addition,the nuclear matrix proteins prepared from these three cell lines could also bind to this silencer,suggesting that this silencer element might be a constitutive nuclear matrix attachment region(constitutive MAR).Our results demonstrated that the nuclear matrix and nuclear matrix proteins might play an important role in the regulation of the human ε-globin gene expression.

  7. A bioinformatics insight to rhizobial globins: gene identification and mapping, polypeptide sequence and phenetic analysis, and protein modeling. [v1; ref status: indexed, http://f1000r.es/5ai

    Directory of Open Access Journals (Sweden)

    Reinier Gesto-Borroto

    2015-05-01

    Full Text Available Globins (Glbs are proteins widely distributed in organisms. Three evolutionary families have been identified in Glbs: the M, S and T Glb families. The M Glbs include flavohemoglobins (fHbs and single-domain Glbs (SDgbs; the S Glbs include globin-coupled sensors (GCSs, protoglobins and sensor single domain globins, and the T Glbs include truncated Glbs (tHbs. Structurally, the M and S Glbs exhibit 3/3-folding whereas the T Glbs exhibit 2/2-folding. Glbs are widespread in bacteria, including several rhizobial genomes. However, only few rhizobial Glbs have been characterized. Hence, we characterized Glbs from 62 rhizobial genomes using bioinformatics methods such as data mining in databases, sequence alignment, phenogram construction and protein modeling. Also, we analyzed soluble extracts from Bradyrhizobium japonicum USDA38 and USDA58 by (reduced + carbon monoxide (CO minus reduced differential spectroscopy. Database searching showed that only fhb, sdgb, gcs and thb genes exist in the rhizobia analyzed in this work. Promoter analysis revealed that apparently several rhizobial glb genes are not regulated by a -10 promoter but might be regulated by -35 and Fnr (fumarate-nitrate reduction regulator-like promoters. Mapping analysis revealed that rhizobial fhbs and thbs are flanked by a variety of genes whereas several rhizobial sdgbs and gcss are flanked by genes coding for proteins involved in the metabolism of nitrates and nitrites and chemotaxis, respectively. Phenetic analysis showed that rhizobial Glbs segregate into the M, S and T Glb families, while structural analysis showed that predicted rhizobial SDgbs and fHbs and GCSs globin domain and tHbs fold into the 3/3- and 2/2-folding, respectively. Spectra from B. japonicum USDA38 and USDA58 soluble extracts exhibited peaks and troughs characteristic of bacterial and vertebrate Glbs thus indicating that putative Glbs are synthesized in B. japonicum USDA38 and USDA58.

  8. Clusters of conserved beta cell marker genes for assessment of beta cell phenotype

    DEFF Research Database (Denmark)

    Martens, Geert A; Jiang, Lei; Hellemans, Karine H;

    2011-01-01

    The aim of this study was to establish a gene expression blueprint of pancreatic beta cells conserved from rodents to humans and to evaluate its applicability to assess shifts in the beta cell differentiated state. Genome-wide mRNA expression profiles of isolated beta cells were compared to those...... of a large panel of other tissue and cell types, and transcripts with beta cell-abundant and -selective expression were identified. Iteration of this analysis in mouse, rat and human tissues generated a panel of conserved beta cell biomarkers. This panel was then used to compare isolated versus laser capture...... microdissected beta cells, monitor adaptations of the beta cell phenotype to fasting, and retrieve possible conserved transcriptional regulators....

  9. Splice-site mutations cause Rrp6-mediated nuclear retention of the unspliced RNAs and transcriptional down-regulation of the splicing-defective genes.

    Directory of Open Access Journals (Sweden)

    Andrea B Eberle

    Full Text Available BACKGROUND: Eukaryotic cells have developed surveillance mechanisms to prevent the expression of aberrant transcripts. An early surveillance checkpoint acts at the transcription site and prevents the release of mRNAs that carry processing defects. The exosome subunit Rrp6 is required for this checkpoint in Saccharomyces cerevisiae, but it is not known whether Rrp6 also plays a role in mRNA surveillance in higher eukaryotes. METHODOLOGY/PRINCIPAL FINDINGS: We have developed an in vivo system to study nuclear mRNA surveillance in Drosophila melanogaster. We have produced S2 cells that express a human beta-globin gene with mutated splice sites in intron 2 (mut beta-globin. The transcripts encoded by the mut beta-globin gene are normally spliced at intron 1 but retain intron 2. The levels of the mut beta-globin transcripts are much lower than those of wild type (wt ss-globin mRNAs transcribed from the same promoter. We have compared the expression of the mut and wt beta-globin genes to investigate the mechanisms that down-regulate the production of defective mRNAs. Both wt and mut beta-globin transcripts are processed at the 3', but the mut beta-globin transcripts are less efficiently cleaved than the wt transcripts. Moreover, the mut beta-globin transcripts are less efficiently released from the transcription site, as shown by FISH, and this defect is restored by depletion of Rrp6 by RNAi. Furthermore, transcription of the mut beta-globin gene is significantly impaired as revealed by ChIP experiments that measure the association of the RNA polymerase II with the transcribed genes. We have also shown that the mut beta-globin gene shows reduced levels of H3K4me3. CONCLUSIONS/SIGNIFICANCE: Our results show that there are at least two surveillance responses that operate cotranscriptionally in insect cells and probably in all metazoans. One response requires Rrp6 and results in the inefficient release of defective mRNAs from the transcription site. The

  10. Clusters of conserved beta cell marker genes for assessment of beta cell phenotype

    DEFF Research Database (Denmark)

    Martens, Geert A; Jiang, Lei; Hellemans, Karine H;

    2011-01-01

    The aim of this study was to establish a gene expression blueprint of pancreatic beta cells conserved from rodents to humans and to evaluate its applicability to assess shifts in the beta cell differentiated state. Genome-wide mRNA expression profiles of isolated beta cells were compared to those...... microdissected beta cells, monitor adaptations of the beta cell phenotype to fasting, and retrieve possible conserved transcriptional regulators.......The aim of this study was to establish a gene expression blueprint of pancreatic beta cells conserved from rodents to humans and to evaluate its applicability to assess shifts in the beta cell differentiated state. Genome-wide mRNA expression profiles of isolated beta cells were compared to those...... of a large panel of other tissue and cell types, and transcripts with beta cell-abundant and -selective expression were identified. Iteration of this analysis in mouse, rat and human tissues generated a panel of conserved beta cell biomarkers. This panel was then used to compare isolated versus laser capture...

  11. Investigating alpha-globin structural variants: a retrospective review of 135,000 Brazilian individuals

    Directory of Open Access Journals (Sweden)

    Elza Miyuki Kimura

    2015-04-01

    Full Text Available Background: Brazil has a multiethnic population with a high diversity of hemoglobinopathies. While screenings for beta-globin mutations are far more common, alterations affecting alpha-globin genes are usually more silent and less well known. The aim of this study was to describe the results of a screening program for alpha-globin gene mutations in a representative sample of the Southeastern Brazilian population. Methods: A total of 135,000 individuals, including patients with clinical suspicion of hemoglobinopathies and their family members, randomly chosen individuals submitted to blood tests and blood donors who were abnormal hemoglobin carriers were analyzed. The variants were screened by alkaline and acid electrophoreses, isoelectric focusing and cation-exchange high performance liquid chromatography (HPLC and the abnormal chains were investigated by reverse-phase high performance liquid chromatography (RP-HPLC. Mutations were identified by molecular analyses, and the oxygen affinity, heme-heme cooperativity and Bohr effect of the variants were evaluated by functional tests. Results: Four new and 22 rare variants were detected in 98 families. Some of these variants were found in co-inheritance with other hemoglobinopathies. Of the rare hemoglobins, Hasharon, Stanleyville II and J-Rovigo were the most common, the first two being S-like and associated with alpha-thalassemia. Conclusion: The variability of alpha-globin alterations reflects the high degree of racial miscegenation and an intense internal migratory flow between different Brazilian regions. This diversity highlights the importance of programs for diagnosing hemoglobinopathies and preventing combinations that may lead to important clinical manifestations in multiethnic populations.

  12. An erythroid chaperone that facilitates folding of α-globin subunits for hemoglobin synthesis

    Science.gov (United States)

    Yu, Xiang; Kong, Yi; Dore, Louis C.; Abdulmalik, Osheiza; Katein, Anne M.; Zhou, Suiping; Choi, John K.; Gell, David; Mackay, Joel P.; Gow, Andrew J.; Weiss, Mitchell J.

    2007-01-01

    Erythrocyte precursors produce abundant α- and β-globin proteins, which assemble with each other to form hemoglobin A (HbA), the major blood oxygen carrier. αHb-stabilizing protein (AHSP) binds free α subunits reversibly to maintain their structure and limit their ability to generate reactive oxygen species. Accordingly, loss of AHSP aggravates the toxicity of excessive free α-globin caused by β-globin gene disruption in mice. Surprisingly, we found that AHSP also has important functions when free α-globin is limited. Thus, compound mutants lacking both Ahsp and 1 of 4 α-globin genes (genotype Ahsp–/–α-globin*α/αα) exhibited more severe anemia and Hb instability than mice with either mutation alone. In vitro, recombinant AHSP promoted folding of newly translated α-globin, enhanced its refolding after denaturation, and facilitated its incorporation into HbA. Moreover, in erythroid precursors, newly formed free α-globin was destabilized by loss of AHSP. Therefore, in addition to its previously defined role in detoxification of excess α-globin, AHSP also acts as a molecular chaperone to stabilize nascent α-globin for HbA assembly. Our findings illustrate what we believe to be a novel adaptive mechanism by which a specialized cell coordinates high-level production of a multisubunit protein and protects against various synthetic imbalances. PMID:17607360

  13. Identification of a NF-kB site in the negative regulatory element (εNRAII) of human ε-globin gene and its binding protein NF-κB p50 in the nuclei of K562 cells

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The developmental control of the human ε-globin gene expression is mediated by transcription regulatory elements in the 5' flanking DNA of this gene. Sequence analysis has revealed a DNA motif (GGGGAATTTGCT) similar to NF-κB consensus sequence resides in the negative regulatory element (-3028bp ~ -2902bp, termed ε-NRAII) 5' to the cap site of this gene. NRF DNA fragment (-3010bp ~-2986bp) containing the NF-κB motif similar sequence was synthesized and used in electrophoresis mobility shift assay (EMSA) and competitive analysis. Data showed that a protein factor from nuclear extracts of K562 cells specifically interacted with NRF DNA fragment. The synthetic NF DNA fragment (containing NF-κB consensus sequence) could competed for the protein binding, but MNF DNA fragment (mutated NF-κB motif) could not, suggesting that the binding protein is a member of NF-κB/Rel family. Western blot assay demonstrated that the molecular weight of NF-κB protein in the nuclei of K562 cells is 50ku. We suggested that NF-κB p50 may play an important role in the regulation of human ε-globin gene expression.

  14. HbF增高合并β地中海贫血患者的α-珠蛋白基因拷贝数变异分析%Analysis of copy number variations in α-globin gene in the cases with combined increased levels of HbF with β thalassemia

    Institute of Scientific and Technical Information of China (English)

    刘思平; 宋兰林; 熊丽; 王克; 申洪; 钟梅

    2015-01-01

    目的 阐述α珠蛋白基因拷贝数丢失与增加对胎儿血红蛋白(fetal hemoglobin,HbF)水平的影响.方法 收集15例HbF增高合并β地中海贫血的患者,首先采用Gap-PCR检测常见α-地中海贫血的3种缺失型,之后应用多重探针连接扩增技术对α-珠蛋白基因簇行片段缺失与重复分析.结果 15例患者中,检出-SEA缺失杂合子3例,-α3.7缺失杂合子1例,-α4.2缺失纯合子1例,-α3.7与-SEA双重缺失杂合子1例,α珠蛋白基因簇大片段重复1例,类α-珠蛋白基因杂合缺失1例,7例样本未见α拷贝数的丢失与增加.结论 α拷贝数的增多会生成过多的α链,加重α与β链的不平衡性,同时过量的α链与γ链组成过多的HbF,导致HbF水平的升高;α拷贝数的减少会修正α与β链比例的失衡,减轻β0/β0或β0/β+的贫血症状,这类病例归为中间型β地中海贫血,一般具有较高的HbF水平.%Objective To detect copy number changes of α-globin gene,and analyze molecular mechanism of the impacts of fetal hemoglobin (HbF) levels for α-globin gene copy numbers loss or increase.Methods A total of 15 cases with combined increased levels of fetal hemoglobin with β thalassemia were collected.Firstly,three common α-thalassemia deletions were validated by Gap-PCR.Secondly,the largest deletions of the ββ-globin gene cluster were detected by multiplex ligation-dependent probe amplification (MLPA).Result Among the 15 cases,there was 1 case with duplication of the α-globin gene cluster,3 cases of SEA heterozygote deletion of the α-globin gene,1 cases of alpha 3.7 deletion heterozygote of the α-globin gene,1 case of alpha 4.2 deletion homozygote of the α-globin gene,1 case of deletion homozygote in the like α-globin gene.A compound heterozygous for SEA and alpha 3.7 of the α-globin gene was also detected.However,7 cases showed no copy numbers loss and increase of the the α-globin gene cluster.Conclusion Additional α-globin gene can produce

  15. Atypical beta(s) haplotypes are generated by diverse genetic mechanisms.

    Science.gov (United States)

    Zago, M A; Silva, W A; Dalle, B; Gualandro, S; Hutz, M H; Lapoumeroulie, C; Tavella, M H; Araujo, A G; Krieger, J E; Elion, J; Krishnamoorthy, R

    2000-02-01

    The majority of the chromosomes with the beta(S) gene have one of the five common haplotypes, designated as Benin, Bantu, Senegal, Cameroon, and Arab-Indian haplotypes. However, in every large series of sickle cell patients, 5-10% of the chromosomes have less common haplotypes, usually referred to as "atypical" haplotypes. In order to explore the genetic mechanisms that could generate these atypical haplotypes, we extended our analysis to other rarely studied polymorphic markers of the beta(S)-gene cluster, in a total of 40 chromosomes with uncommon haplotypes from Brazil and Cameroon. The following polymorphisms were examined: seven restriction site polymorphisms of the epsilongammadeltabeta-cluster, the pre-(G)gamma framework sequence including the 6-bp deletion/insertion pattern, HS-2 LCR (AT)xR(AT)y and pre-beta (AT)xTy repeat motifs, the GC/TT polymorphism at -1105-1106 of (G)gamma-globin gene, the C/T polymorphism at -551 of the beta-globin gene, and the intragenic beta-globin gene framework. Among the Brazilian subjects, the most common atypical structure (7/16) was a Bantu 3'-subhaplotype associated with different 5'-sequences, while in two chromosomes a Benin 3'-subhaplotype was associated with two different 5'-subhaplotypes. A hybrid Benin/Bantu configuration was also observed. In three chromosomes, the atypical haplotype differed from the typical one by the change of a single restriction site. In 2/134 chromosomes identified as having a typical Bantu RFLP-haplotype, a discrepant LCR repeat sequence was observed, probably owing to a crossover 5' to the epsilon-gene. Among 80 beta(S) chromosomes from Cameroon, 22 were associated with an atypical haplotype. The most common structure was represented by a Benin haplotype (from the LCR to the beta-gene) with a non-Benin segment 3' to the beta-globin gene. In two cases a Bantu LCR was associated with a Benin haplotype and a non-Benin segment 3' to the beta-globin gene. In three other cases, a more complex

  16. Fetal stromal niches enhance human embryonic stem cell-derived hematopoietic differentiation and globin switch.

    Science.gov (United States)

    Lee, King Yiu; Fong, Benny Shu Pan; Tsang, Kam Sze; Lau, Tze Kin; Ng, Pak Cheung; Lam, Audrey Carmen; Chan, Kathy Yuen Yee; Wang, Chi Chiu; Kung, Hsiang Fu; Li, Chi Kong; Li, Karen

    2011-01-01

    Hematopoiesis during mammalian embryonic development has been perceived as a migratory phenomenon, from the yolk sac blood island to the aorta-gonad-mesonephros (AGM) region, fetal liver (FL), and subsequently, the fetal bone marrow. In this study, we investigated the effects of primary stromal cells from fetal hematopoietic niches and their conditioned media (CM), applied singly or in sequential orders, on induction of human embryonic stem cells, H1, H9, and H14 lines, to hematopoietic cells. Our results demonstrated that stromal support of FL, AGM + FL, and AGM + FL + fetal bone marrow significantly increased the proliferation of embryoid bodies (EB) at day 18 of hematopoietic induction in the presence of thrombopoietin, stem cell factor, and Flt-3 ligand. AGM + FL also increased hematopoietic colony-forming unit (CFU) formation. CM did not enhance EB proliferation but CM of FL and AGM + FL significantly increased the density of total CFU and early erythroid (burst-forming unit) progenitors. Increased commitment to the hematopoietic lineage was demonstrated by enhanced expressions of CD45, alpha-, beta-, and gamma-globins in CFU at day 32, compared with EB at day 18. CM of FL significantly increased these globin expressions, indicating enhanced switches from embryonic to fetal and adult erythropoiesis. Over 50% and 10% of cells derived from CFU expressed CD45 and beta-globin proteins, respectively. Expressions of hematopoietic regulatory genes (Bmi-1, β-Catenin, Hox B4, GATA-1) were increased in EB or CFU cultures supported by FL or sequential CM. Our study has provided a strategy for derivation of hematopoietic cells from embryonic stem cells under the influence of primary hematopoietic niches and CM, particularly the FL.

  17. Repair of Thalassemic Human β -globin mRNA in Mammalian Cells by Antisense Oligonucleotides

    Science.gov (United States)

    Sierakowska, Halina; Sambade, Maria J.; Agrawal, Sudhir; Kole, Ryszard

    1996-11-01

    In one form of β -thalassemia, a genetic blood disorder, a mutation in intron 2 of the β -globin gene (IVS2-654) causes aberrant splicing of β -globin pre-mRNA and, consequently, β -globin deficiency. Treatment of mammalian cells stably expressing the IVS2-654 human β -globin gene with antisense oligonucleotides targeted at the aberrant splice sites restored correct splicing in a dose-dependent fashion, generating correct human β -globin mRNA and polypeptide. Both products persisted for up to 72 hr posttreatment. The oligonucleotides modified splicing by a true antisense mechanism without overt unspecific effects on cell growth and splicing of other pre-mRNAs. This novel approach in which antisense oligonucleotides are used to restore rather than to down-regulate the activity of the target gene is applicable to other splicing mutants and is of potential clinical interest.

  18. The globins of Campylobacter jejuni.

    Science.gov (United States)

    Tinajero-Trejo, Mariana; Shepherd, Mark

    2013-01-01

    Campylobacter jejuni is a zoonotic Gram-negative bacterial pathogen that is exposed to reactive nitrogen species, such as nitric oxide, from a variety of sources. To combat the toxic effects of this nitrosative stress, C. jejuni upregulates a small regulon under the control of the transcriptional activator NssR, which positively regulates the expression of a single-domain globin protein (Cgb) and a truncated globin protein (Ctb). Cgb has previously been shown to detoxify nitric oxide, but the role of Ctb remains contentious. As C. jejuni is amenable to genetic manipulation, and its globin proteins are easily expressed and purified, a combination of mutagenesis, complementation, transcriptomics, spectroscopic characterisation and structural analyses has been used to probe the regulation, function and structure of Cgb and Ctb. This ability to study Cgb and Ctb with such a multi-pronged approach is a valuable asset, especially since only a small fraction of known globin proteins have been functionally characterised.

  19. A DNA-binding protein factor in K562 nuclear extract interacts with positive control region (PCR) in the 5'-flanking sequence of human β-globin gene

    Institute of Scientific and Technical Information of China (English)

    HUYULONG; YADICHEN; TONGSUN; RUOLANQIAN

    1993-01-01

    It has been known that there are at least three regulatory regions (NCR1. NCR2 and PCR) in the 5'-flanking sequence (from -610 bp to +1 bp) of human β-glohin geneand that the function of PCR is unique to the human erythroleukemia (Ksfi2) ceils. Here we have detected a DNA-binding protein factor (termed NFEa) in K562 ceils. which can bind specifically to the PCR of human β-globin gene. The sequence of the binding site is 5'ACTGATG3' (between -222 bp and -216 bp). The NFEa is erythroidspecific and perhaps specific for K562 cells. It seemed that this factor differed from the erythroid-specific transcriptional factor (NFE-1) ,nsing competition assay. The presence of the NFEa further supported that the funciton of the cis-acting element PCR was specitic for K562 cells. and helps us to understand the mechauism of the regulation of the expression of lmman β-globin gene in the human K562 cells.

  20. Interactions between HMG proteins and the core sequence of DNaseI hypersensitive site 2 in the locus control region (LCR) of the human β-like globin gene cluster

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    HMG proteins are abundant chromosomal non-histone proteins. It has been suggested that the HMG proteins may play an important role in the structure and function of chromatin. In the present study, the binding of HMG proteins (HMG1/2 and HMG14/17) to the core DNA sequence of DNaseI hypersensitive site 2 (HS2core DNA sequence, -10681--10970 bp) in the locus control region (LCR) of the human b-like globin gene cluster has been examined by using both the in vitro nucleosome reconstitution and the gel mobility shift assays. Here we show that HMG1/2 can bind to the naked HS2core DNA sequence, however, HMG14/17 cannot. Using the in vitro nucleosome reconstitution we demonstrate that HMG14/17 can bind to the HS2core DNA sequence which is assembled into nucleosomes with the core histone octamer transferred from chicken erythrocytes. In contrast, HMG1/2 cannot bind to the nucleosomes reconstituted in vitro with the HS2core DNA sequence. These results indicate that the binding patterns between HMG proteins and the HS2core DNA sequence which exists in different states (the naked DNA or the in vitro reconstituted nucleosomal DNA) are quite different. We speculate that HMG proteins might play a critical role in the regulation of the human β-like globin gene's expression.

  1. Interactions between HMG proteins and the core sequence of DNaseI hypersensitive site 2 in the locus control region (LCR) of the human b-like globin gene cluster

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    HMG proteins are abundant chromosomal non-histone proteins. It has been suggested that the HMG proteins may play an important role in the structure and function of chromatin. In the present study, the binding of HMG proteins (HMG1/2 and HMG14/17) to the core DNA sequence of DNaseI hypersensitive site 2 (HS2core DNA sequence, -10681--10970 bp) in the locus control region (LCR) of the human b-like globin gene cluster has been examined by using both the in vitro nucleosome reconstitution and the gel mobility shift assays. Here we show that HMG1/2 can bind to the naked HS2core DNA sequence, however, HMG14/17 cannot. Using the in vitro nucleosome reconstitution we demonstrate that HMG14/17 can bind to the HS2core DNA sequence which is assembled into nucleosomes with the core histone octamer transferred from chicken erythrocytes. In contrast, HMG1/2 cannot bind to the nucleosomes reconstituted in vitro with the HS2core DNA sequence. These results indicate that the binding patterns between HMG proteins and the HS2core DNA sequence which exists in different states (the naked DNA or the in vitro reconstituted nucleosomal DNA) are quite different. We speculate that HMG proteins might play a critical role in the regulation of the human b-like globin gene's expression.

  2. Characterization of beta-R1, a gene that is selectively induced by interferon beta (IFN-beta) compared with IFN-alpha.

    Science.gov (United States)

    Rani, M R; Foster, G R; Leung, S; Leaman, D; Stark, G R; Ransohoff, R M

    1996-09-13

    We report preliminary characterization of a gene designated beta-R1, which is selectively expressed in response to interferon beta (IFN-beta) compared with IFN-alpha. In human astrocytoma cells, beta-R1 was induced to an equivalent extent by 10 IU/mL IFN-beta or 2500 IU/mL IFN-alpha2. To address the mechanism of this differential response, we analyzed induction of the beta-R1 gene in fibrosarcoma cells and derivative mutant cells lacking components required for signaling by type I IFNs. beta-R1 was readily induced by IFN-beta in the parental 2fTGH cell line, but not by recombinant IFN-alpha2, IFN-alpha Con1, or a mixture of IFN-alpha subtypes. IFN-alpha8 induced beta-R1 weakly. beta-R1 was not induced by IFN-beta in mutant cell lines U2A, U3A, U4A, and U6A, which lack, respectively, p48, STAT1, JAK1, and STAT2. U5A cells, which lack the Ifnar 2.2 component of the IFN-alpha and -beta receptor, also failed to express beta-R1. U1A cells are partially responsive to IFN-beta and IFN-alpha8 but lacked beta-R1 expression, indicating that TYK2 protein is essential for induction of this gene. Taken together, these results suggest that the expression of beta-R1 in response to type I IFN requires IFN-stimulated gene factor 3 plus an additional component, which is more efficiently formed on induction by IFN-beta compared with IFN-alpha.

  3. The gene for familial Mediterranean fever in both Armenians and non-Ashkenzai Jews is linked to the [alpha]-globin complex on 16p: Evidence for locus homogeneity

    Energy Technology Data Exchange (ETDEWEB)

    Shohat, M.; Shohat, T.; Magal, N.; Danon, Y. (Tel Aviv Univ. (Israel)); Xiangdong Bu; Fischel-Ghodsian, N.; Schwabe, A.D.; Rotter, J.I. (Univ. of California, Los Angeles (United States)); Nakamura, Yusuke (Cancer Inst., Tokyo (Japan)); Schlezinger, M. (Barzilai Medical Center, Ashkelon (Israel))

    1992-12-01

    Familial Mediterranean fever (FMF) is a recurrent inflammatory disorder characterized by short episodes of fever, peritonitis, pleuritis, and arthritis. While FMF has been shown to be inherited in an autosomal recessive fashion in both non-Ashkenazi Jews and Armenian families, clinical differences have raised the possibility of genetic heterogeneity. As its pathogenesis is unknown, mapping of the gene for FMF may provide the first objective method for early and accurate diagnosis of this disease. After excluding 45% of the entire human genome, the authors studied 14 Armenian and 9 non-Ashkenazi Jewish families with FMF and tested linkage with the [alpha]-globin locus on chromosome 16. Analysis of the PvuII length polymorphism of the 3[prime] HVR (hypervariable region) probe showed significant linkage with the FMF gene (maximum lod score [lod[sub max

  4. Two new δ-globin gene variants: Hb A(2)-Saint-Etienne [δ14(A11)Leu→Pro (HBD: c.44T>C)] and Hb A(2)-Marseille [δ22(B4) Ala→Lys (HBD: c.67G>A;68C>A)].

    Science.gov (United States)

    Joly, Philippe; Lacan, Philippe; Garcia, Caroline; Desbrée, Aurélie; Couprie, Nicole; Francina, Alain

    2013-01-01

    We report two new variants of the δ-globin gene: Hb A(2)-Saint-Etienne [δ14(A11)Leu→Pro] and Hb A(2)-Marseille [δ22(B4)Ala→Lys]. The first variant has a low rate of expression, the second results from a double nucleotide mutation on the same codon.

  5. Gene expression of beta carotene genes in transgenic biofortified cassava

    OpenAIRE

    Telengech, P. K.; Maling’a, J. N.; Nyende, A. B.; Gichuki, S. T.; Wanjala, B. W.

    2014-01-01

    Cassava is an important food for millions of people around the world. However, cassava is deficient in protein, iron, zinc, pro-vitamin A and vitamin E. Cassava biofortified with pro-vitamin A can help reduce Vitamin A Deficiency among the undernourished communities that rely upon it for sustenance. BioCassava Plus project has developed transgenic cassava that expresses beta carotene in roots using root specific patatin promoter. This study aimed at confirming expression of nptII, crtB and DX...

  6. Reversal of hemochromatosis by apotransferrin in non-transfused and transfused Hbbth3/+ (heterozygous B1/B2 globin gene deletion) mice.

    Science.gov (United States)

    Gelderman, Monique P; Baek, Jin Hyen; Yalamanoglu, Ayla; Puglia, Michele; Vallelian, Florence; Burla, Bo; Vostal, Jaroslav; Schaer, Dominik J; Buehler, Paul W

    2015-05-01

    Intermediate beta-thalassemia has a broad spectrum of sequelae and affected subjects may require occasional blood transfusions over their lifetime to correct anemia. Iron overload in intermediate beta-thalassemia results from a paradoxical intestinal absorption, iron release from macrophages and hepatocytes, and sporadic transfusions. Pathological iron accumulation in parenchyma is caused by chronic exposure to non-transferrin bound iron in plasma. The iron scavenger and transport protein transferrin is a potential treatment being studied for correction of anemia. However, transferrin may also function to prevent or reduce iron loading of tissues when exposure to non-transferrin bound iron increases. Here we evaluate the effects of apotransferrin administration on tissue iron loading and early tissue pathology in non-transfused and transfused Hbb(th3/+) mice. Mice with the Hbb(th3/+) phenotype have mild to moderate anemia and consistent tissue iron accumulation in the spleen, liver, kidneys and myocardium. Chronic apotransferrin administration resulted in normalization of the anemia. Furthermore, it normalized tissue iron content in the liver, kidney and heart and attenuated early tissue changes in non-transfused Hbb(th3/+) mice. Apotransferrin treatment was also found to attenuate transfusion-mediated increases in plasma non-transferrin bound iron and associated excess tissue iron loading. These therapeutic effects were associated with normalization of transferrin saturation and suppressed plasma non-transferrin bound iron. Apotransferrin treatment modulated a fundamental iron regulatory pathway, as evidenced by decreased erythroid Fam132b gene (erythroferrone) expression, increased liver hepcidin gene expression and plasma hepcidin-25 levels and consequently reduced intestinal ferroportin-1 in apotransferrin-treated thalassemic mice.

  7. Preliminary identification of hemoglobin q-iran in an Iranian family from central province of Iran by globin chain analysis on HPLC.

    Science.gov (United States)

    Khatami, Shohreh; Najmabadi, Hossein; Rouhi, Soghra; Mirzazadeh, Roghieh; Bayat, Parastoo; Sadeghi, Sedigheh

    2013-12-01

    Many abnormal α-chain hemoglobins (Hbs) are caused by single nucleotide mutations in α1- or α2-goblin genes. One of these Hbs is Hb Q-Iran which is resulted from a point mutation at codon 75 of the α1-globin gene (Asp→His). The identification of Hb Q-Iran was observed in two members of a family from the Central Province of Iran. In this study, Globin chain analysis on high performance liquid chromatography (HPLC) and DNA sequencing were applied. An unusual Hb variant, like HbS on alkaline pH electrophoresis was identified from samples of a father and his son from Arak city in the Central Province of Iran. The variant was further characterized by globin chain analysis and DNA sequencing methods. Globin chain analysis revealed an unknown globin chain peak after α-globin chain peak with a different retention time from βs-globin chain, as the control in both samples. Genetic analysis led to the identification of an unknown Hb variant, Hb Q-Iran. Globin chain analysis showed the presence of an unknown globin chain, and likewise DNA sequencing revealed HbQ-Iran. In other words, Globin chain analysis procedure could preliminarily detect an unknown globin chain.

  8. Molecular mechanisms associated with increased fetal hemoglobin G gamma-type in part-aboriginal family with beta thalassemia.

    Science.gov (United States)

    Motum, P I; Lammi, A; Trent, R J

    1989-07-01

    A part-Aboriginal family with beta thalassemia and raised hemoglobin F (HbF) was studied at the molecular level to determine if there were identifiable gene changes associated with increased production of HbF. Two beta thalassemia heterozygotes aged eight years and 18 months had raised HbF levels of 2.9% and 22% respectively. HbF was predominantly G gamma in composition. Five family members were typed for restriction fragment length polymorphisms (RFLPs) using nine restriction enzymes and five DNA probes specific for the beta globin cluster on chromosome 11. RFLPs were combined to construct haplotypes for the beta thalassemia and the high HbF defects. A beta globin subhaplotype comprising only 5' RFLP markers (-(+)-(+) +) co-segregated with the high HbF determinant. This has previously been associated with increased G gamma expression in beta thalassemia and sickle cell anemia. An additional Xmnl RFLP 5' to the G gamma gene, which has been described in individuals with elevated G gamma expression, was also demonstrated in those family members with increased G gamma levels. In this study both the 5' beta globin subhaplotype (-(+)-(+) +) and the Xmnl/gamma RFLP are present in the one family but the relative contributions of each cannot be determined.

  9. Genetic therapy for beta-thalassemia: from the bench to the bedside.

    Science.gov (United States)

    Arumugam, Paritha; Malik, Punam

    2010-01-01

    Beta-thalassemia is a genetic disorder with mutations in the β-globin gene that reduce or abolish β-globin protein production. Patients with β-thalassemia major (Cooley's anemia) become severely anemic by 6 to 18 months of age, and are transfusion dependent for life, while those with thalassemia intermedia, a less-severe form of thalassemia, are intermittently or rarely transfused. An allogeneically matched bone marrow transplant is curative, although it is restricted to those with matched donors. Gene therapy holds the promise of "fixing" one's own bone marrow cells by transferring the normal β-globin or γ-globin gene into hematopoietic stem cells (HSCs) to permanently produce normal red blood cells. Requirements for effective gene transfer for the treatment of β-thalassemia are regulated, erythroid-specific, consistent, and high-level β-globin or γ-globin expression. Gamma retroviral vectors have had great success with immune-deficiency disorders, but due to vector-associated limitations, they have limited utility in hemoglobinopathies. Lentivirus vectors, on the other hand, have now been shown in several studies to correct mouse and animal models of thalassemia. The immediate challenges of the field as it moves toward clinical trials are to optimize gene transfer and engraftment of a high proportion of genetically modified HSCs and to minimize the adverse consequences that can result from random integration of vectors into the genome by improving current vector design or developing novel vectors. This article discusses the current state of the art in gene therapy for β-thalassemia and some of the challenges it faces in human trials.

  10. Gene expression analysis of interferon-beta treatment in multiple sclerosis

    DEFF Research Database (Denmark)

    Sellebjerg, F.; Datta, P.; Larsen, J.;

    2008-01-01

    by treatment with IFN-beta. We use DNA microarrays to study gene expression in 10 multiple sclerosis (MS) patients who began de novo treatment with IFN-beta. After the first injection of IFN-beta, the expression of 74 out of 3428 genes changed at least two-fold and statistically significantly (after Bonferroni...... correction). In contrast, we observed no persisting effects of IFN-beta on gene expression. Among the most strongly induced genes was MXA, which has been used in previous biomarker studies in MS. In addition, the study identified the induction of LGALS9 and TCIR1G, involved in negative regulation of T helper......Treatment with interferon-beta (IFN-beta) induces the expression of hundreds of genes in blood mononuclear cells, and the expression of several genes has been proposed as a marker of the effect of treatment with IFN-beta. However, to date no molecules have been identified that are stably induced...

  11. Evidence for the multicentric origin of the sickle cell hemoglobin gene in Africa.

    OpenAIRE

    Pagnier, J.; Mears, J G; Dunda-Belkhodja, O; Schaefer-Rego, K E; Beldjord, C; Nagel, R L; Labie, D

    1984-01-01

    Previous studies of the Hpa I cleavage site-sickle cell hemoglobin gene linkage in various African populations suggested that the sickle gene arose independently more than once. In the present study we have performed restriction endonuclease haplotype analysis for the beta-globin-like gene cluster from four separate geographic areas in Africa, all of which possess the sickle gene. In Benin (Central West Africa) and Algeria (Arab North Africa) all chromosomes carrying the sickle gene possess a...

  12. Regulation of laminin beta2 chain gene expression in human cancer cell lines

    DEFF Research Database (Denmark)

    Durkin, M E; Nielsen, F C; Loechel, F;

    2001-01-01

    The laminin beta2 chain is a basement membrane component expressed in a tissue- and developmental stage-specific manner. In this report we have examined the transcriptional and post-transcriptional regulation of the human laminin beta2 chain in human tumor cell lines. Both the A204 rhabdomyosarcoma...... and clone A colon carcinoma cells express the laminin beta2 chain mRNA, but only the A204 cells secrete laminin heterotrimers containing the beta2 chain. Segments of the beta2 chain gene promoter region were cloned into luciferase reporter vectors, and their ability to stimulate transcription was tested...... of the human laminin beta2 chain gene generates two isoforms of the 5' untranslated region of the beta2 chain mRNA. The translational efficiencies of the two laminin beta2 chain leaders did not differ significantly, when assayed by polysome profile analysis of endogenous clone A cell beta2 chain m...

  13. Variation in RNA-Seq transcriptome profiles of peripheral whole blood from healthy individuals with and without globin depletion.

    Directory of Open Access Journals (Sweden)

    Heesun Shin

    Full Text Available BACKGROUND: The molecular profile of circulating blood can reflect physiological and pathological events occurring in other tissues and organs of the body and delivers a comprehensive view of the status of the immune system. Blood has been useful in studying the pathobiology of many diseases. It is accessible and easily collected making it ideally suited to the development of diagnostic biomarker tests. The blood transcriptome has a high complement of globin RNA that could potentially saturate next-generation sequencing platforms, masking lower abundance transcripts. Methods to deplete globin mRNA are available, but their effect has not been comprehensively studied in peripheral whole blood RNA-Seq data. In this study we aimed to assess technical variability associated with globin depletion in addition to assessing general technical variability in RNA-Seq from whole blood derived samples. RESULTS: We compared technical and biological replicates having undergone globin depletion or not and found that the experimental globin depletion protocol employed removed approximately 80% of globin transcripts, improved the correlation of technical replicates, allowed for reliable detection of thousands of additional transcripts and generally increased transcript abundance measures. Differential expression analysis revealed thousands of genes significantly up-regulated as a result of globin depletion. In addition, globin depletion resulted in the down-regulation of genes involved in both iron and zinc metal ion bonding. CONCLUSIONS: Globin depletion appears to meaningfully improve the quality of peripheral whole blood RNA-Seq data, and may improve our ability to detect true biological variation. Some concerns remain, however. Key amongst them the significant reduction in RNA yields following globin depletion. More generally, our investigation of technical and biological variation with and without globin depletion finds that high-throughput sequencing by RNA

  14. 人胎儿γ-珠蛋白基因的再活化及在β血红蛋白病治疗中的%Reactivation of Humanγ-globin Gene in Adult and its Significance for β Hemoglobinopathies Treatment

    Institute of Scientific and Technical Information of China (English)

    于海川; 张俊武

    2011-01-01

    During the development of an individual, expression of human β-like globin genes displays two switches: from embryonic ( ε- ) to fetal ( Gγand Aγ) globin and from the fetal to adult ( β- ) globin. β-thalassemia and sickle cell disease ( SCD ) are two of the most common genetic disorders in the world. Reactivation of fetal γ-globin gene in adulthood had been considered as an effetive strategy for the treatment of the diseases. Several transcriptional factors and cofactors that can promote γ-globin gene expression have been identified, and some pharmaceuticals that can induce reactivation of γ-globin gene in the adult have also been reported. Some studies have partially explored the mechanisms by which these factors and pharmaceuticals function. These studies have provided important clues and preliminary experimental evidence for effective treatment of SCD and β-thalassemia by increasing fetal hemoglobin production in the adult.%在个体发育过程中,人β类珠蛋白基因的表达存在从胎儿(γ)到成人(β)珠蛋白基因的表达转换(或称开关).β-地中海贫血和镰刀型贫血症是两种最为常见的严重危害人类健康的单基因遗传病,通过诱导胎儿期血红蛋白(HbF,α2γ2)在成人期表达对该病的治疗是一种有效的策略.一些活化γ珠蛋白基因表达的转录因子和辅助因子已经被鉴定,一些可以增加胎儿血红蛋白在成人红细胞中表达的药物也已被鉴别和实验,它们的作用机制被部分揭示,这些研究为发展通过活化γ-珠蛋白基因治疗镰刀形细胞贫血和重型β-地中海贫血的方法提供了重要线索和实验依据.

  15. A membrane-bound vertebrate globin.

    Directory of Open Access Journals (Sweden)

    Miriam Blank

    Full Text Available The family of vertebrate globins includes hemoglobin, myoglobin, and other O(2-binding proteins of yet unclear functions. Among these, globin X is restricted to fish and amphibians. Zebrafish (Danio rerio globin X is expressed at low levels in neurons of the central nervous system and appears to be associated with the sensory system. The protein harbors a unique N-terminal extension with putative N-myristoylation and S-palmitoylation sites, suggesting membrane-association. Intracellular localization and transport of globin X was studied in 3T3 cells employing green fluorescence protein fusion constructs. Both myristoylation and palmitoylation sites are required for correct targeting and membrane localization of globin X. To the best of our knowledge, this is the first time that a vertebrate globin has been identified as component of the cell membrane. Globin X has a hexacoordinate binding scheme and displays cooperative O(2 binding with a variable affinity (P(50∼1.3-12.5 torr, depending on buffer conditions. A respiratory function of globin X is unlikely, but analogous to some prokaryotic membrane-globins it may either protect the lipids in cell membrane from oxidation or may act as a redox-sensing or signaling protein.

  16. Understanding the mechanisms of ATPase beta family genes for cellular thermotolerance in crossbred bulls

    Science.gov (United States)

    Deb, Rajib; Sajjanar, Basavaraj; Singh, Umesh; Alex, Rani; Raja, T. V.; Alyethodi, Rafeeque R.; Kumar, Sushil; Sengar, Gyanendra; Sharma, Sheetal; Singh, Rani; Prakash, B.

    2015-12-01

    Na+/K+-ATPase is an integral membrane protein composed of a large catalytic subunit (alpha), a smaller glycoprotein subunit (beta), and gamma subunit. The beta subunit is essential for ion recognition as well as maintenance of the membrane integrity. Present study was aimed to analyze the expression pattern of ATPase beta subunit genes (ATPase B1, ATPase B2, and ATPase B3) among the crossbred bulls under different ambient temperatures (20-44 °C). The present study was also aimed to look into the relationship of HSP70 with the ATPase beta family genes. Our results demonstrated that among beta family genes, transcript abundance of ATPase B1 and ATPase B2 is significantly ( P P < 0.01) with HSP70, representing that the change in the expression pattern of these genes is positive and synergistic. These may provide a foundation for understanding the mechanisms of ATPase beta family genes for cellular thermotolerance in cattle.

  17. The globins of cyanobacteria and algae.

    Science.gov (United States)

    Johnson, Eric A; Lecomte, Juliette T J

    2013-01-01

    Approximately, 20 years ago, a haemoglobin gene was identified within the genome of the cyanobacterium Nostoc commune. Haemoglobins have now been confirmed in multiple species of photosynthetic microbes beyond N. commune, and the diversity of these proteins has recently come under increased scrutiny. This chapter summarizes the state of knowledge concerning the phylogeny, physiology and chemistry of globins in cyanobacteria and green algae. Sequence information is by far the best developed and the most rapidly expanding aspect of the field. Structural and ligand-binding properties have been described for just a few proteins. Physiological data are available for even fewer. Although activities such as nitric oxide dioxygenation and oxygen scavenging are strong candidates for cellular function, dedicated studies will be required to complete the story on this intriguing and ancient group of proteins.

  18. Developmental regulation of a complete 70kb human β-globin locus in transgenic mice.

    NARCIS (Netherlands)

    J. Strouboulis (John); N.O. Dillon (Niall); F.G. Grosveld (Frank)

    1992-01-01

    textabstractWe have used a linker-based ligation strategy to combine two 35-kb cosmid inserts from the human beta-globin locus into one linear fragment containing the entire locus. This 70-kb fragment was introduced into transgenic mice by microinjection of fertilized eggs. Southern blot analysis sh

  19. Synthetic Human β-Globin 5'HS2 Constructs Function as Partially Active Locus Control Regions.

    NARCIS (Netherlands)

    J. Ellis (James); D. Talbot; N.O. Dillon (Niall); F.G. Grosveld (Frank)

    1993-01-01

    textabstractTransgenes linked to the beta-globin locus control region (LCR) are transcribed in a copy-dependent manner that is independent of the integration site. It has previously been shown that the LCR 5'HS2 region does not require its NF-E2 dimer binding site for LCR activity. In this paper we

  20. Induction of Fetal Hemoglobin In Vivo Mediated by a Synthetic γ-Globin Zinc Finger Activator

    Directory of Open Access Journals (Sweden)

    Flávia C. Costa

    2012-01-01

    Full Text Available Sickle cell disease (SCD and β-thalassemia patients are phenotypically normal if they carry compensatory hereditary persistence of fetal hemoglobin (HPFH mutations that result in increased levels of fetal hemoglobin (HbF, γ-globin chains in adulthood. Thus, research has focused on manipulating the reactivation of γ-globin gene expression during adult definitive erythropoiesis as the most promising therapy to treat these hemoglobinopathies. Artificial transcription factors (ATFs are synthetic proteins designed to bind at a specific DNA sequence and modulate gene expression. The artificial zinc finger gg1-VP64 was designed to target the −117 region of the Aγ-globin gene proximal promoter and activate expression of this gene. Previous studies demonstrated that HbF levels were increased in murine chemical inducer of dimerization (CID-dependent bone marrow cells carrying a human β-globin locus yeast artificial chromosome (β-YAC transgene and in CD34+ erythroid progenitor cells from normal donors and β-thalassemia patients. Herein, we report that gg1-VP64 increased γ-globin gene expression in vivo, in peripheral blood samples from gg1-VP64 β-YAC double-transgenic (bigenic mice. Our results demonstrate that ATFs function in an animal model to increase gene expression. Thus, this class of reagent may be an effective gene therapy for treatment of some inherited diseases.

  1. Genetic origin of Behçet's disease population in Denizli, Turkey; population genetics data analysis; historical demography and geographical perspectives based on β-globin gene cluster haplotype variation.

    Science.gov (United States)

    Ozturk, O; Arikan, S; Bahadir, A; Atalay, A; Atalay, E O

    2017-01-01

    In our study, we aimed to investigate the possible genetic drift, relationships, expansion and historical origin based on haplotype frequencies of the β-globin gene cluster of normal and Behçet's disease (BD) population in Denizli, Turkey. We examined blood DNA samples obtained from our DNA bank. The association of population genetic parameters such as haplotypes, diversity, differentiation, Hardy-Weinberg equilibrium and demographic analysis for two populations was performed by Arlequin ver. 3.5. Our results show that both populations have high similarity in genetic parameters in terms of development and expansion based on haplotype diversity through the history. We found that historical levels of gene flow were significantly higher between the two populations. According to historical population, growth parameter of τ values for normal and BD populations dated approximately 42 000 to 38 000 ybp, respectively. In conclusion, historically, two populations show similar genetic parameters and unimodal growth distribution. Our results are consistent with the view that the BD may have occurred in area, independent from Silk Road.

  2. The biosynthetic gene cluster for the beta-lactam carbapenem thienamycin in Streptomyces cattleya.

    Science.gov (United States)

    Núñez, Luz Elena; Méndez, Carmen; Braña, Alfredo F; Blanco, Gloria; Salas, José A

    2003-04-01

    beta-lactam ring formation in carbapenem and clavam biosynthesis proceeds through an alternative mechanism to the biosynthetic pathway of classic beta-lactam antibiotics. This involves the participation of a beta-lactam synthetase. Using available information from beta-lactam synthetases, we generated a probe for the isolation of the thienamycin cluster from Streptomyces cattleya. Genes homologous to carbapenem and clavulanic acid biosynthetic genes have been identified. They would participate in early steps of thienamycin biosynthesis leading to the formation of the beta-lactam ring. Other genes necessary for the biosynthesis of thienamycin have also been identified in the cluster (methyltransferases, cysteinyl transferases, oxidoreductases, hydroxylase, etc.) together with two regulatory genes, genes involved in exportation and/or resistance, and a quorum sensing system. Involvement of the cluster in thienamycin biosynthesis was demonstrated by insertional inactivation of several genes generating thienamycin nonproducing mutants.

  3. Intestinal lactase as an autologous beta-galactosidase reporter gene for in vivo gene expression studies.

    Science.gov (United States)

    Salehi, Siamak; Eckley, Lorna; Sawyer, Greta J; Zhang, Xiaohong; Dong, Xuebin; Freund, Jean-Noel; Fabre, John W

    2009-01-01

    Intestinal lactase has potential as an autologous beta-galactosidase reporter gene for long-term gene expression studies in vivo, using chromogenic, luminescent, and fluorogenic substrates developed for Escherichia coli beta-galactosidase. In normal rat tissues, reactivity with a chromogenic fucopyranoside (X-Fuc, the preferred substrate of lactase) was present only at the lumenal surface of small intestine epithelial cells. Full-length lactase (domains I-IV), mature lactase (domains III and IV), and a cytosolic form of mature lactase (domains III and IV, without the signal sequence or transmembrane region) were evaluated. Transfection of HuH-7 cells in vitro, and hydrodynamic gene delivery to the liver in vivo, resulted in excellent gene expression. The full-length and mature (homodimeric, membrane-bound) forms reacted strongly with X-Fuc but not with the corresponding galactopyranoside (X-Gal). However, the presumptively monomeric cytosolic lactase unexpectedly reacted equally well with both substrates. The fluorogenic substrate fluorescein-di-beta-D-galactopyranoside was cleaved by cytosolic lactase, but not by full-length or mature lactase. Full-length lactase, when expressed ectopically in hepatocytes in vivo, localized exclusively to the bile canalicular membrane. Intestinal lactase is highly homologous in mice, rats, and humans and has considerable potential for evaluating long-term gene expression in experimental animals and the clinic.

  4. Interactions between HMG proteins and the core sequence of DNaseI hypersensitive site 2 in the locus control region (LCR) of the human β-Mike globin gene cluster

    Institute of Scientific and Technical Information of China (English)

    赵晖; 张树冰; 蒋俶; 钱若兰

    2000-01-01

    HMG proteins are abundant chromosomal non-histone proteins. It has been suggested that the HMG proteins may play an important role in the structure and function of chromatin. In the present study, the binding of HMG proteins (HMG1/2 and HMG14/17) to the core DNA sequence of DNasel hypersensitive site 2 (HS2core DNA sequence, -10681-10970 bp) in the locus control region (LCR) of the human β-like globin gene cluster has been examined by using both the in vitro nucleosome reconstitution and the gel mobility shift assays. Here we show that HMG1/2 can bind to the naked HS2core DNA sequence, however, HMG 14/17 cannot. Using the in vitro nucleosome reconstitution we demonstrate that HMG14/17 can bind to the HS2core DNA sequence which is assembled into nucleosomes with the core histone octamer transferred from chicken erythrocytes. In contrast, HMG 1/2 cannot bind to the nucleosomes reconstituted in vitro with the HS2core DNA sequence. These results indicate that the binding patterns between HMG proteins and t

  5. Expression profiling of genes regulated by TGF-beta: Differential regulation in normal and tumour cells

    Directory of Open Access Journals (Sweden)

    Takahashi Takashi

    2007-04-01

    Full Text Available Abstract Background TGF-beta is one of the key cytokines implicated in various disease processes including cancer. TGF-beta inhibits growth and promotes apoptosis in normal epithelial cells and in contrast, acts as a pro-tumour cytokine by promoting tumour angiogenesis, immune-escape and metastasis. It is not clear if various actions of TGF-beta on normal and tumour cells are due to differential gene regulations. Hence we studied the regulation of gene expression by TGF-beta in normal and cancer cells. Results Using human 19 K cDNA microarrays, we show that 1757 genes are exclusively regulated by TGF-beta in A549 cells in contrast to 733 genes exclusively regulated in HPL1D cells. In addition, 267 genes are commonly regulated in both the cell-lines. Semi-quantitative and real-time qRT-PCR analysis of some genes agrees with the microarray data. In order to identify the signalling pathways that influence TGF-beta mediated gene regulation, we used specific inhibitors of p38 MAP kinase, ERK kinase, JNK kinase and integrin signalling pathways. The data suggest that regulation of majority of the selected genes is dependent on at least one of these pathways and this dependence is cell-type specific. Interestingly, an integrin pathway inhibitor, RGD peptide, significantly affected TGF-beta regulation of Thrombospondin 1 in A549 cells. Conclusion These data suggest major differences with respect to TGF-beta mediated gene regulation in normal and transformed cells and significant role of non-canonical TGF-beta pathways in the regulation of many genes by TGF-beta.

  6. Globin-like proteins in Caenorhabditis elegans: in vivo localization, ligand binding and structural properties

    Directory of Open Access Journals (Sweden)

    Van Doorslaer Sabine

    2010-04-01

    Full Text Available Abstract Background The genome of the nematode Caenorhabditis elegans contains more than 30 putative globin genes that all are transcribed. Although their translated amino acid sequences fit the globin fold, a variety of amino-acid substitutions and extensions generate a wide structural diversity among the putative globins. No information is available on the physicochemical properties and the in vivo expression. Results We expressed the globins in a bacterial system, characterized the purified proteins by optical and resonance Raman spectroscopy, measured the kinetics and equilibria of O2 binding and determined the crystal structure of GLB-1* (CysGH2 → Ser mutant. Furthermore, we studied the expression patterns of glb-1 (ZK637.13 and glb-26 (T22C1.2 in the worms using green fluorescent protein technology and measured alterations of their transcript abundances under hypoxic conditions.GLB-1* displays the classical three-over-three α-helical sandwich of vertebrate globins, assembled in a homodimer associated through facing E- and F-helices. Within the heme pocket the dioxygen molecule is stabilized by a hydrogen bonded network including TyrB10 and GlnE7.GLB-1 exhibits high ligand affinity, which is, however, lower than in other globins with the same distal TyrB10-GlnE7 amino-acid pair. In the absence of external ligands, the heme ferrous iron of GLB-26 is strongly hexacoordinated with HisE7, which could explain its extremely low affinity for CO. This globin oxidizes instantly to the ferric form in the presence of oxygen and is therefore incapable of reversible oxygen binding. Conclusion The presented data indicate that GLB-1 and GLB-26 belong to two functionally-different globin classes.

  7. Effect of β‑globin MAR characteristic elements on transgene expression.

    Science.gov (United States)

    Li, Qin; Dong, Weihua; Wang, Tianyun; Liu, Zhonghe; Wang, Fang; Wang, Xiaoyin; Zhao, Chunpeng; Zhang, Junhe; Wang, Li

    2013-06-01

    The aim of the present study was to investigate the effect of the characteristic elements of matrix attachment region (MAR) on transgene expression. Human β‑globin MAR was obtained by PCR amplification. A splicing MAR fragment containing all the characteristic elements of β‑globin MAR was artificially synthesized and then cloned into the eukaryotic expression vector. Following digestion and sequence identification, we transfected Chinese hamster ovary (CHO) cells with the two vectors, and then screened for the transformation of stable cells. The transgene expression level was analyzed by ELISA, and the copy numbers of the CAT gene were analyzed by real‑time fluorescent quantitative PCR. β‑globin MAR enhanced CAT reporter gene expression by 2.1489‑fold, whereas the β‑globin MAR characteristic elements did not enhance this expression. The real‑time fluorescent quantitative PCR analysis demonstrated that the relative copy numbers of the CAT gene of the β‑globin MAR expression vector were 1.2‑fold higher compared with those of the non‑MAR expression vector. MAR was able to improve the transgene expression level to a certain extent. The MAR characteristic elements did not improve the transgene expression alone. The transgenic expression levels were not linear with the transgene copy number; however, the enhancement of transgenic expression was relative to the increase in the gene copy number.

  8. A novel erythroid differentiation related gene EDRF1 upregulating globin gene expression in HEL cells%新的红细胞分化相关基因EDRF1增强HEL细胞的珠蛋白表达

    Institute of Scientific and Technical Information of China (English)

    王敦成; 黎燕; 沈倍奋

    2002-01-01

    目的观察EDRF1基因在HEL细胞中对红细胞终末分化和珠蛋白表达的扮演的角色.方法构建EDRF1真核正义和反义表达载体,转染HEL细胞并筛选稳定表达细胞克隆.然后利用Northern blot和反转录PCR (reverse transcription-polymerase chain reaction, RT-PCR)的方法观察红系标志基因的表达水平改变.凝胶阻滞电泳(electrophoresis mobility shift assay, EMSA)的方法观察红系转录因子的DNA结合活性的改变.结果与对照相比,EDRF1过表达的HEL细胞中α-珠蛋白合成明显增加,EDRF1反义表达载体转染的HEL细胞中的α、γ-珠蛋白合成下调,而红细胞生成素受体(erythropoietin receptor, ER)表达水平没有明显改变,这提示EDRF1并非通过调控ER信号通路而影响HEL细胞的红细胞分化.红细胞特异的转录因子GATA-1和NF-E2 mRNA的表达在转染前后没有明显改变.另外,GATA-1转录因子的DNA结合活性在反义表达载体转染的HEL细胞中受到明显的抑制,而NF-E2的转录活性则没有明显的改变.结论 EDRF1下调珠蛋白的合成是通过抑制红系特异的转录因子GATA-1的DNA结合活性实现的.%Objective To further characterize the differentiation inducing properties of EDRF1 and demonstrate its functional pathway involved in regulation of globin gene expression. Methods By transfecting EDRF1 sense and antisense constructs into HEL cells, we identified the expression of globin and erythropoietin receptor genes by Northern blot analysis. RT-PCR and EMSA (electrophoresis mobility shift assay) were performed to monitor the expression and DNA-binding activity of erythroid specific transcription factors GATA-1 and NF-E2. Results It was shown that when EDRF1 was overexpressed, production of α-globin increased. In antisense EDRF1, overexpression of HEL cells, significant loss of α-, γ-globin mRNA synthesis was observed. The transcription of endogenous GATA-1 and NF-E2 mRNA expression were maintained at the same levels

  9. Molecular Identification Of Sicilian (deltabeta) Degrees-thalassemia Associated With Beta-thalassemia And Hemoglobin S In Brazil.

    OpenAIRE

    2015-01-01

    We describe the clinical and molecular characteristics of two unrelated Brazilian families with an association of the Sicilian form of (deltabeta) degrees -thalassemia with hemoglobin S and beta-thalassemia. Direct sequencing of the beta-globin gene showed only the hemoglobin S mutation in patient 1 and the beta-thalassemia IVS1-110 in patient 2. The other allele was deleted in both patients and PCR of DNA samples of the breakpoint region of both patients showed a band of approximately 1,150 ...

  10. Tumor-produced, active Interleukin-1 {beta} regulates gene expression in carcinoma-associated fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Dudas, Jozsef, E-mail: Jozsef.Dudas@i-med.ac.at [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Fullar, Alexandra, E-mail: fullarsz@gmail.com [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); 1st Institute of Pathology and Experimental Cancer Research, Semmelweis University, Ulloei ut 26, H-1085 Budapest (Hungary); Bitsche, Mario, E-mail: Mario.Bitsche@i-med.ac.at [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Schartinger, Volker, E-mail: Volker.Schartinger@i-med.ac.at [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Kovalszky, Ilona, E-mail: koval@korb1.sote.hu [1st Institute of Pathology and Experimental Cancer Research, Semmelweis University, Ulloei ut 26, H-1085 Budapest (Hungary); Sprinzl, Georg Mathias, E-mail: Georg.Sprinzl@i-med.ac.at [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria); Riechelmann, Herbert, E-mail: Herbert.Riechelmann@i-med.ac.at [Department of Otorhinolaryngology, Medical University Innsbruck, Anichstrasse 35, A-6020 Innsbruck (Austria)

    2011-09-10

    Recently we described a co-culture model of periodontal ligament (PDL) fibroblasts and SCC-25 lingual squamous carcinoma cells, which resulted in conversion of normal fibroblasts into carcinoma-associated fibroblasts (CAFs), and in epithelial-mesenchymal transition (EMT) of SCC-25 cells. We have found a constitutive high interleukin-1{beta} (IL1-{beta}) expression in SCC-25 cells in normal and in co-cultured conditions. In our hypothesis a constitutive IL1-{beta} expression in SCC-25 regulates gene expression in fibroblasts during co-culture. Co-cultures were performed between PDL fibroblasts and SCC-25 cells with and without dexamethasone (DEX) treatment; IL1-{beta} processing was investigated in SCC-25 cells, tumor cells and PDL fibroblasts were treated with IL1-{beta}. IL1-{beta} signaling was investigated by western blot and immunocytochemistry. IL1-{beta}-regulated genes were analyzed by real-time qPCR. SCC-25 cells produced 16 kD active IL1-{beta}, its receptor was upregulated in PDL fibroblasts during co-culture, which induced phosphorylation of interleukin-1 receptor-associated kinase-1 (IRAK-1), and nuclear translocalization of NF{kappa}B{alpha}. Several genes, including interferon regulatory factor 1 (IRF1) interleukin-6 (IL-6) and prostaglandin-endoperoxide synthase 2 (COX-2) were induced in CAFs during co-culture. The most enhanced induction was found for IL-6 and COX-2. Treatment of PDL fibroblasts with IL1-{beta} reproduced a time- and dose-dependent upregulation of IL1-receptor, IL-6 and COX-2. A further proof was achieved by DEX inhibition for IL1-{beta}-stimulated IL-6 and COX-2 gene expression. Constitutive expression of IL1-{beta} in the tumor cells leads to IL1-{beta}-stimulated gene expression changes in tumor-associated fibroblasts, which are involved in tumor progression. -- Graphical abstract: SCC-25 cells produce active, processed IL1-{beta}. PDL fibroblasts possess receptor for IL1-{beta}, and its expression is increased 4.56-times in the

  11. Activation of the human beta interferon gene by the adenovirus type 12 E1B gene

    Energy Technology Data Exchange (ETDEWEB)

    Shiroki, K.; Toth, M.

    1988-01-01

    The transcription of endogenous beta interferon mRNA was activated in human embryo kidney (HEK) cells infected with adenovirus 12 (Ad12) but was activated only inefficiently or not at all in HEK cells infected with Ad5 and rc-1 (Ad5 dl312 containing the Ad12 E1A region). The analysis with Ad12 mutants showed that Ad12 E1B products, especially the 19K protein, were important for the expression of the endogenous beta interferon gene and Ad12 E1A products were not involved in the expression. The expression of exogeneously transfected pIFN-CAT (a hybrid plasmid having the human beta interferon promoter fused with the CAT gene) was activated in HEK and chicken embryo fibroblast (CEF) cells infected with either Ad12 or Ad5. The analysis of cotransfection of CEF cells with pIFN-CAT and plasmids containing fragments of Ad12 or Ad5 DNA showed that Ad12 or Ad5 E1B (possibly the 19K protein) was and E1A was not involved in the expression of the exogenous pIFN-CAT.

  12. Structure and expression of the mouse beta-hexosaminidase genes, Hexa and Hexb.

    Science.gov (United States)

    Yamanaka, S; Johnson, O N; Norflus, F; Boles, D J; Proia, R L

    1994-06-01

    Two genes, HEXA and HEXB, encode the alpha- and beta-subunits, respectively, of human beta-hexosaminidase. In the mouse, the corresponding genes are termed Hexa and Hexb. The subunits dimerize to yield three isozymes, beta-hexosaminidase A (alpha beta), B (beta beta), and S (alpha alpha), that have the capacity to degrade a variety of substrates containing beta-linked N-acetylglucosamine and N-acetylgalactosamine residues. Mutations in the HEXA or HEXB gene resulting in a beta-hexosaminidase deficiency cause Tay-Sachs or Sandhoff disease, respectively. As a prelude to the creation of mouse models of these lysosomal storage diseases, we have characterized the molecular biology of the mouse beta-hexosaminidase system. Protein sequences derived from the cloned Hexa and Hexb cDNAs were 55% identical to each other and were also very similar to the cognate human sequences: 84% sequence identity with human HEXA and 75% with HEXB. The mouse hexosaminidase subunits, when expressed in HeLa cells from the cDNAs, displayed specificity toward synthetic substrates similar to the human subunits. The Hexa and Hexb genes were 25 and 22 kb in length, respectively. Each gene was divided into 14 exons, with the positions of introns precisely matching those of the corresponding human genes. The 5' flanking regions of the mouse genes demonstrated promoter activity as ascertained by their ability to drive chloramphenicol acetyltransferase gene expression in transfected NIH 3T3 cells. The sequences of these regulatory regions were G+C-rich in the 200 bp upstream of the respective initiator ATGs. Several putative promoter elements were present, including Sp1, AP2, CAAT, and TATA motifs.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. [Hemoglobin beta S haplotype in the Kebili region (southern Tunisia)].

    Science.gov (United States)

    Frikha, M; Fakhfakh, F; Mseddi, S; Gargouri, J; Ghali, L; Labiadh, Z; Harrabi, M; Souissi, T; Ayadi, H

    1998-04-01

    Sickle cell anemia is a monogenic hereditary disease characterized by a mutation in the beta globin gene. Five major haplotypes associated with the beta S mutation have been defined: Benin, Bantu, Senegalian, Camerounian, and Arabo-Indian. Previous studies in northern Tunisia showed that sickle cell anemia was of Benin origin in this region. Patients from the south of Tunisia, mainly from the Kebili region, were not previously concerned. In this study, we have determined the beta S haplotype and evaluated phenotypical expression of the disease in 14 patients from this latter region. The use of four restriction endonucleases having polymorphic sites in the beta globin gene showed that all patients had the Benin haplotype, confirming the Benin origin of sickle cell anemia in Tunisia. This haplotype is associated with an heterogeneous expression of fetal hemoglobin (HbF) with extremes varying from 2.4 to 16.3% and a mean expression rate of 8.16%, which is in accordance with literature data. In spite of the haplotype homogeneity in our patients, clinical heterogeneity was noted. A unique case of alpha-thalassemia could not explain this heterogeneity. In contrast, we found a certain correlation between fetal hemoglobin expression and clinical severity.

  14. The transforming growth factor-beta receptor genes and the risk of intracranial aneurysms

    NARCIS (Netherlands)

    Ruigrok, Ynte M.; Baas, Annette F.; Medic, Jelena; Wijmenga, Cisca; Rinkel, Gabriel J. E.

    2012-01-01

    Background Mutations in the receptor genes of the transforming growth factor beta pathway, TGFBR1 and TGFBR2, cause syndromes with thoracic aortic aneurysms, while genetic variants in TGFBR1 and TGFBR2 are associated with abdominal aortic aneurysms. The transforming growth factor-beta pathway may be

  15. Molecular cloning and characterization of mutant and wild-type human. beta. -actin genes

    Energy Technology Data Exchange (ETDEWEB)

    Leavitt, J.; Gunning, P.; Porreca, P.; Ng, S.Y.; Lin, C.H.; Kedes, L.

    1984-10-01

    There are more than 20 ..beta..-actin-specific sequences in the human genome, many of which are pseudogenes. To facilitate the isolation of potentially functional ..beta..-actin genes, they used the new method of B. Seed for selecting genomic clones by homologous recombination. A derivative of the ..pi..VX miniplasmid, ..pi..AN7..beta..1, was constructed by insertion of the 600-base-pair 3' untranslated region of the ..beta..-actin mRNA expressed in human fibroblasts. Five clones containing ..beta..-actin sequences were selected from an amplified human fetal gene library by homologous recombination between library phage and the miniplasmid. One of these clones contained a complete ..beta..-actin gene with a coding sequence identical to that determined for the mRNA of human fibroblasts. A DNA fragment consisting of mostly intervening sequences from this gene was then use to identify 13 independent recombinant copies of the analogous gene from two specially constructed gene libraries, each containing one of the two types of mutant ..beta..-actin genes found in a line of neoplastic human fibroblasts. The amino acid and nucleotide sequences encoded by the unmutated gene predict that a guanine-to-adenine transition is responsible for the glycine-to-aspartic acid mutation at codon 244 and would also result in the loss of a HaeIII site. Detection of this HaeIII polymorphism among the fibroblast-derived closed verified the identity of the ..beta..-actin gene expressed in human fibroblasts.

  16. Analysis of β-globin chromatin micro-environment using a novel 3C variant, 4Cv.

    Directory of Open Access Journals (Sweden)

    Ryan C Pink

    Full Text Available Higher order chromatin folding is critical to a number of developmental processes, including the regulation of gene expression. Recently developed biochemical techniques such as RNA TRAP and chromosome conformation capture (3C have provided us with the tools to probe chromosomal structures. These techniques have been applied to the β-globin locus, revealing a complex pattern of interactions with regions along the chromosome that the gene resides on. However, biochemical and microscopy data on the nature of β-globin interactions with other chromosomes is contradictory. Therefore we developed a novel 4C variant, Complete-genome 3C by vectorette amplification (4Cv, which allows an unbiased and quantitative method to examine chromosomal structure. We have used 4Cv to study the microenvironment of the β-globin locus in mice and show that a significant proportion of the interactions of β-globin are inter-chromosomal. Furthermore, our data show that in the liver, where the gene is active, β-globin is more likely to interact with other chromosomes, compared to the brain where the gene is silent and is more likely to interact with other regions along the same chromosome. Our data suggest that transcriptional activation of the β-globin locus leads to a change in nuclear position relative to the chromosome territory.

  17. 地西他滨对K562细胞γ-珠蛋白基因表达及细胞增殖的影响%Effects of Decitabine on γ-globin Gene Expression and Proliferation in K562 Cells

    Institute of Scientific and Technical Information of China (English)

    谢冬梅; 赖永榕; 刘容容; 谭滨彬; 杨高晖

    2012-01-01

    目的 研究地西他滨对K562细胞γ-珠蛋白基因表达及细胞增殖的影响,探讨药物治疗β-地中海贫血的新思路.方法 分别用不同浓度(0 nmol/L、50 nmol/L、100 nmol/L、200 nmol/L、300 nmol/L)地西他滨作用于K562细胞24 h、48 h、72 h,用CCK-8检测药物对K562细胞存活率的影响,采用流式细胞术检测细胞凋亡率,采用实时荧光定量RT-PCR检测γ-珠蛋白基因表达.结果 地西他滨对K562细胞生长有抑制作用,作用后细胞凋亡率增加,γ-珠蛋白基因表达显著提高(P<0.05).结论 地西他滨可以提高K562细胞γ-珠蛋白基因表达.%Objective To investigate the effects of decitabine on γ-globin gene expression( HBG ) and proliferation in K562 cells,and provide a new way for β-thalassemia gene therapy. Methods K562 cells were treated with different concentrations of decitabine( 0 nmol/L,50 nmol/L,100 nmol/L,200 nmol/L,300 nmol/L ) for 24 h,48 h,72 h,respectively. The survival of K562 cells was examined by cell counting kit-8( CCK8 ),the Annexin V +/PI- cells were detected by flow cytometry( FCM ). Real-time quantitative reverse transcription polymerase chain reaction( RT-PCR ) was applied to detect the expression of γ-globin gene. Results K562 cells were inhibited by decitabine,and the significant increase in apoptosis and expression of γ-globin gene was found( P < 0.05 ). Conclusion Decitabine can improve γ-globin gene expression in K562cell.

  18. Two new hemoglobin variants: Hb Brem-sur-Mer [beta9(A6)Ser-->Tyr] and Hb Passy [alpha81(F2)Ser-->Pro (alpha2)].

    Science.gov (United States)

    Lacan, Philippe; Moreau, Mathieu; Becchi, Michel; Zanella-Cleon, Isabelle; Aubry, Martine; Louis, Jean-Jacques; Couprie, Nicole; Francina, Alain

    2005-01-01

    Two new hemoglobin (Hb) variants: Hb Brem-sur-Mer [codon 9 (TCT-->TAT); beta9(A6)Ser-->Tyr] on the first exon of the beta-globin gene and Hb Passy [codon 81 (TCC-->CCC); alpha81(F2)Ser-->Pro (alpha2)] on the second exon of the alpha2-globin gene, are described. The two variants were characterized by DNA sequencing and mass spectrometry (MS). Hematological abnormalities: microcytosis and hypochromia were found only in the carrier of Hb Passy. In the absence of an association with an alpha-thalassemic deletion or mutation, the mutation 81(F2)Pro could induce a possible alpha-thalassemia (thal).

  19. Effects of ultrasound on Transforming Growth Factor-beta genes in bone cells

    Directory of Open Access Journals (Sweden)

    J Harle

    2005-12-01

    Full Text Available Therapeutic ultrasound (US is a widely used form of biophysical stimulation that is increasingly applied to promote fracture healing. Transforming growth factor-beta (TGF-beta, which is encoded by three related but different genes, is known to play a major part in bone growth and repair. However, the effects of US on the expression of the TGF-beta genes and the physical acoustic mechanisms involved in initiating changes in gene expression in vitro, are not yet known. The present study demonstrates that US had a differential effect on these TGF-beta isoforms in a human osteoblast cell line, with the highest dose eliciting the most pronounced up-regulation of both TGF-beta1 and TGF-beta3 at 1 hour after treatment and thereafter declining. In contrast, US had no effect on TGF-beta2 expression. Fluid streaming rather than thermal effects or cavitation was found to be the most likely explanation for the gene responses observed in vitro.

  20. Cloning and sequencing of the beta-glucosidase gene from Acetobacter xylinum ATCC 23769.

    Science.gov (United States)

    Tajima, K; Nakajima, K; Yamashita, H; Shiba, T; Munekata, M; Takai, M

    2001-12-31

    The beta-glucosidase gene (bglxA) was cloned from the genomic DNA of Acetobacter xylinum ATCC 23769 and its nucleotide sequence (2200 bp) was determined. This bglxA gene was present downstream of the cellulose synthase operon and coded for a polypeptide of molecular mass 79 kDa. The overexpression of the beta-glucosidase in A. xylinum caused a tenfold increase in activity compared to the wild-type strain. In addition, the action pattern of the enzyme was identified as G3ase activity. The deduced amino acid sequence of the bglxA gene showed 72.3%, 49.6%, and 45.1% identity with the beta-glucosidases from A. xylinum subsp. sucrofermentans, Cellvibrio gilvus, and Mycobacterium tuberculosis, respectively. Based on amino acid sequence similarities, the beta-glucosidase (BglxA) was assigned to family 3 of the glycosyl hydrolases.

  1. Bradykinin stimulation of nitric oxide production is not sufficient for gamma-globin induction

    Directory of Open Access Journals (Sweden)

    Čokić Vladan P.

    2014-01-01

    Full Text Available Introduction. Hydroxycarbamide, used in therapy of hemoglobinopathies, enhances nitric oxide (NO production both in primary human umbilical vein endothelial cells (HUVECs and human bone marrow endothelial cell line (TrHBMEC. Moreover, NO increases γ-globin and fetal hemoglobin levels in human erythroid progenitors. Objective. In order to find out whether simple physiologic stimulation of NO production by components of hematopoietic microenvironment can increase γ-globin gene expression, the effects of NO-inducer bradykinin were examined in endothelial cells. Methods. The study was performed in co-cultures of human erythroid progenitors, TrHBMEC and HUVECs by ozone-based chemiluminescent determination of NO and real-time quantitative RT-PCR. Results. In accordance with previous reports, the endogenous factor bradykinin increased endothelial cell production of NO in a dose- and time-dependent manner (0.1-0.6 μM up to 30 minutes. This induction of NO in HUVECs and TrHBMEC by bradykinin was blocked by competitive inhibitors of NO synthase (NOS, demonstrating NOS-dependence. It has been shown that bradykinin significantly reduced endothelial NOS (eNOS mRNA level and eNOS/Я-actin ratio in HUVEC (by twofold. In addition, bradykinin failed to increase γ-globin mRNA expression in erythroid progenitors only, as well as in co-culture studies of erythroid progenitors with TrHBMEC and HUVEC after 24 hours of treatment. Furthermore, bradykinin did not induce γ/β globin ratio in erythroid progenitors in co-cultures with HUVEC. Conclusion. Bradykinin mediated eNOS activation leads to short time and low NO production in endothelial cells, insufficient to induce γ-globin gene expression. These results emphasized the significance of elevated and extended NO production in augmentation of γ-globin gene expression. [Projekat Ministarstva nauke Republike Srbije, br. 175053

  2. A dominant chromatin opening activity in 5' hypersensitive site 3 of the human β-globin locus control region.

    NARCIS (Netherlands)

    J. Ellis (James); K.C. Tan-Un; A. Harper; D. Michalovich (David); P.J. Fraser (Peter); N. Yannoutsos (Nikos); F.G. Grosveld (Frank)

    1996-01-01

    textabstractSingle-copy human beta-globin transgenes are very susceptible to suppression by position effects of surrounding closed chromatin. However, these position effects are overcome by a 20 kbp DNA fragment containing the locus control region (LCR). Here we show that the 6.5 kbp microlocus LCR

  3. Elucidation of IL-1/TGF-beta interactions in mouse chondrocyte cell line by genome-wide gene expression

    DEFF Research Database (Denmark)

    Takahashi, N; Rieneck, K; van der Kraan, P M;

    2005-01-01

    To elucidate the antagonism between interleukin-1 (IL-1) and transforming growth factor-beta (TGF-beta) at the gene expression level, as IL-1 and TGF-beta are postulated to be critical mediators of cartilage degeneration/protection in rheumatic diseases.......To elucidate the antagonism between interleukin-1 (IL-1) and transforming growth factor-beta (TGF-beta) at the gene expression level, as IL-1 and TGF-beta are postulated to be critical mediators of cartilage degeneration/protection in rheumatic diseases....

  4. Arrested rearrangement of TCR V[beta] genes in thymocytes from children with x-linked severe combined immunodeficiency disease

    Energy Technology Data Exchange (ETDEWEB)

    Sleasman, J.W.; Harville, T.O.; White, G.B.; Barrett, D.J. (Univ. of Florida College of Medicine, Gainsville, FL (United States)); George, J.F. (Univ. of Alabama, Birmingham, AL (United States)); Goodenow, M.M. (Univ. of Florida College of Medicine, Gainsville, FL (United States) Univ. of Alabama, Birmingham, AL (United States))

    1994-07-01

    Human X-linked severe combined immunodeficiency disease (SCID) is an immunodeficiency disorder in which T cell development is arrested in the thymic cortex. B lymphocytes in children with X-linked SCID seem to differentiate normally. X-linked SCID is associated with a mutation in the gene that encodes the IL-2R [gamma]-chain. Because TCR-[beta] gene recombination is a pivotal initial event in T lymphocyte onteogeny within the thymus, the authors hypothesized that a failure to express normal IL-2R[gamma] could lead to impaired TCR-[beta] gene recombination in early thymic development. PCR was used to determine the status of TCR-[beta] gene-segment rearrangements in thymic DNA that had been obtained from children with X-linked SCID. The initial step in TCR-[beta] gene rearrangement, that of D[beta] to J[beta] recombination, was readily detected in all thymus samples from children with X-linked SCID; in contrast, V[beta] to DJ[beta] gene rearrangements were undetectable in the same samples. Both D[beta] to J[beta] and V[beta] to DJ[beta] TCR genes were rearranged in the thymic tissues obtained from immunologically normal children. The authors conclude that TCR[beta]-chain gene rearrangement is arrested in children with X-linked SCID. The results suggest a causative relationship between the failure of TCR [beta]-chain gene arrangements to proceed beyond DJ[beta] rearrangements and the production of a nonfunctional IL-2R [gamma]-chain. 45 refs., 3 figs.

  5. Structure of the gene for human. beta. /sub 2/-adrenergic receptor: expression and promoter characterization

    Energy Technology Data Exchange (ETDEWEB)

    Emorine, L.J.; Marullo, S.; Delavier-Klutchko, C.; Kaveri, S.V.; Durieu-Trautmann, O.; Strosberg, A.D.

    1987-10-01

    The genomic gene coding for the human ..beta../sub 2/-adrenergic receptor (..beta../sub 2/AR) from A431 epidermoid cells has been isolated. Transfection of the gene into eukaryotic cells restores a fully active receptor/GTP-binding protein/adenylate cyclase complex with ..beta../sub 2/AR properties. Southern blot analyses with ..beta../sub 2/AR-specific probes show that a single ..beta../sub 2/AR gene is common to various human tissues and that its flanking sequences are highly conserved among humans and between man and rabbit, mouse, and hamster. Functional significance of these regions is supported by the presence of a promoter region (including mRNA cap sites, two TATA boxes, a CAAT box, and three G + C-rich regions that resemble binding sites for transcription factor Sp1) 200-300 base pairs 5' to the translation initiation codon. In the 3' flanking region, sequences homologous to glucocorticoid-response elements might be responsible for the increased expression of the ..beta../sub 2/AR gene observed after treatment of the transfected cells with hydrocortisone. In addition, 5' to the promoter region, an open reading frame encodes a 251-residue polypeptide that displays striking homologies with protein kinases and other nucleotide-binding proteins.

  6. Usage of U7 small nuclear ribonucleic acid in gene therapy of hemoglobin D Punjab disorder: Rationale?

    Directory of Open Access Journals (Sweden)

    Viroj Wiwanitkit

    2013-01-01

    Full Text Available Background: Hemoglobin (Hb D Punjab disorder is a congenital hemoglobinopathy described in India. It is a disorder due to defect in beta-globin gene. Materials and Methods: Here, the author assesses the possibility of U7.623 gene therapy for Hb D Punjab disorder. A standard bioinformatic analysis to study the effect of co-expression between nucleic acid sequence for human Hb D Punjab beta-globin chain and U7.623 was performed. Result: It can be seen that fully recovery of Hb function and biological process can be derived via gene ontology study. Conclusion: Here, there is a rationale to use U7 small nuclear ribonucleic acid as a possible tool for gene therapy in Hb D Punjab disorder.

  7. IFN-beta gene deletion leads to augmented and chronic demyelinating experimental autoimmune encephalomyelitis

    DEFF Research Database (Denmark)

    Teige, Ingrid; Treschow, Alexandra; Teige, Anna;

    2003-01-01

    Since the basic mechanisms behind the beneficial effects of IFN-beta in multiple sclerosis (MS) patients are still obscure, here we have investigated the effects of IFN-beta gene disruption on the commonly used animal model for MS, experimental autoimmune encephalomyelitis (EAE). We show that IFN......-beta knockout (KO) mice are more susceptible to EAE than their wild-type (wt) littermates; they develop more severe and chronic neurological symptoms with more extensive CNS inflammation and demyelination. However, there was no discrepancy observed between wt and KO mice regarding the capacity of T cells...... to proliferate or produce IFN-gamma in response to recall Ag. Consequently, we addressed the effect of IFN-beta on encephalitogenic T cell development and the disease initiation phase by passive transfer of autoreactive T cells from KO or wt littermates to both groups of mice. Interestingly, IFN-beta KO mice...

  8. Genes and mechanisms involved in beta-amyloid generation and Alzheimer's disease.

    Science.gov (United States)

    Steiner, H; Capell, A; Leimer, U; Haass, C

    1999-01-01

    Alzheimer's disease is characterized by the invariable accumulation of senile plaques that are predominantly composed of amyloid beta-peptide (Abeta). Abeta is generated by proteolytic processing of the beta-amyloid precursor protein (betaAPP) involving the combined action of beta- and gamma-secretase. Cleavage within the Abeta domain by alpha-secretase prevents Abeta generation. In some very rare cases of familial AD (FAD), mutations have been identified within the betaAPP gene. These mutations are located close to or at the cleavage sites of the secretases and pathologically effect betaAPP processing by increasing Abeta production, specifically its highly amyloidogenic 42 amino acid variant (Abeta42). Most of the mutations associated with FAD have been identified in the two presenilin (PS) genes, particularly the PS1 gene. Like the mutations identified within the betaAPP gene, mutations in PS1 and PS2 cause the increased generation of Abeta42. PS1 has been shown to be functionally involved in Notch signaling, a key process in cellular differentation, and in betaAPP processing. A gene knock out of PS1 in mice leads to an embryonic lethal phenotype similar to that of mice lacking Notch. In addition, absence of PS1 results in reduced gamma-secretase cleavage and leads to an accumulation of betaAPP C-terminal fragments and decreased amounts of Abeta. Recent work may suggest that PS1 could be the gamma-secretase itself, exhibiting the properties of a novel aspartyl protease. Mutagenesis of either of two highly conserved intramembraneous aspartate residues of PS1 leads to reduced Abeta production as observed in the PS1 knockout. A corresponding mutation in PS2 interfered with betaAPP processing and Notch signaling suggesting a functional redundancy of both presenilins. In this issue, some of the recent work on the molecular mechanisms involved in Alzheimer's disease (AD) as well as novel diagnostic approaches and risk factors for AD will be discussed. In the first

  9. Isolation and characterization of BetaM protein encoded by ATP1B4 - a unique member of the Na,K-ATPase {beta}-subunit gene family

    Energy Technology Data Exchange (ETDEWEB)

    Pestov, Nikolay B. [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States); Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow 117997 (Russian Federation); Zhao, Hao [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States); Basrur, Venkatesha [Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109 (United States); Modyanov, Nikolai N., E-mail: nikolai.modyanov@utoledo.edu [Department of Physiology and Pharmacology, University of Toledo College of Medicine, 3000 Arlington Ave., Toledo, OH 43614 (United States)

    2011-09-09

    Highlights: {yields} Structural properties of BetaM and Na,K-ATPase {beta}-subunits are sharply different. {yields} BetaM protein is concentrated in nuclear membrane of skeletal myocytes. {yields} BetaM does not associate with a Na,K-ATPase {alpha}-subunit in skeletal muscle. {yields} Polypeptide chain of the native BetaM is highly sensitive to endogenous proteases. {yields} BetaM in neonatal muscle is a product of alternative splice mRNA variant B. -- Abstract: ATP1B4 genes represent a rare instance of the orthologous gene co-option that radically changed functions of encoded BetaM proteins during vertebrate evolution. In lower vertebrates, this protein is a {beta}-subunit of Na,K-ATPase located in the cell membrane. In placental mammals, BetaM completely lost its ancestral role and through acquisition of two extended Glu-rich clusters into the N-terminal domain gained entirely new properties as a muscle-specific protein of the inner nuclear membrane possessing the ability to regulate gene expression. Strict temporal regulation of BetaM expression, which is the highest in late fetal and early postnatal myocytes, indicates that it plays an essential role in perinatal development. Here we report the first structural characterization of the native eutherian BetaM protein. It should be noted that, in contrast to structurally related Na,K-ATPase {beta}-subunits, the polypeptide chain of BetaM is highly sensitive to endogenous proteases that greatly complicated its isolation. Nevertheless, using a complex of protease inhibitors, a sample of authentic BetaM was isolated from pig neonatal skeletal muscle by a combination of ion-exchange and lectin-affinity chromatography followed by SDS-PAGE. Results of the analysis of the BetaM tryptic digest using MALDI-TOF and ESI-MS/MS mass spectrometry have demonstrated that native BetaM in neonatal skeletal muscle is a product of alternative splice mRNA variant B and comprised of 351 amino acid residues. Isolated BetaM protein was

  10. A new sickle cell disease phenotype associating Hb S trait, severe pyruvate kinase deficiency (PK Conakry), and an alpha2 globin gene variant (Hb Conakry).

    Science.gov (United States)

    Cohen-Solal, M; Préhu, C; Wajcman, H; Poyart, C; Bardakdjian-Michau, J; Kister, J; Promé, D; Valentin, C; Bachir, D; Galactéros, F

    1998-12-01

    A Guinean woman, heterozygous for haemoglobin (Hb) S, was studied because of episodes of marked anaemia, repeated typical metaphyseal painful crises and haemosiderosis. Her sickling syndrome resulted from the association of Hb S trait with a severe pyruvate kinase deficiency leading to a 2,3-DPG concentration of twice normal levels. Sequence of the PK-R gene revealed an undescribed mutation in the homozygous or hemizygous state within exon 5 (nucleotide 2670 C-->A), leading to the interchange of Ser 130 into Tyr (PK Conakry). In addition, the patient carried a new haemoglobin variant, Hb Conakry [alpha80(F1) Leu-->Val], which seemed to have a mild effect. The high intraerythrocytic 2,3-DPG concentration induced by the PK deficiency resulted in a decreased oxygen affinity which favoured sickling to a level almost similar to that of Hb S/C compound heterozygous patients. This was confirmed by oxygen binding measurements of Hb A/Hb S erythrocytes in which 2,3-DPG content was modified in vitro. Hysteresis between deoxy- and reoxygenation curves, as well as increase in the n(max) value, demonstrated that the extent of HbS polymerization in the propositus was almost the same as that of RBCs from a homozygous sickle cell patient or those of an A/S heterozygous patient with an artificial in vitro increase of 2,3-DPG concentration.

  11. Highly efficient expression of interleukin-2 under the control of rabbit β-globin intron II gene enhances protective immune responses of porcine reproductive and respiratory syndrome (PRRS DNA vaccine in pigs.

    Directory of Open Access Journals (Sweden)

    Yijun Du

    Full Text Available Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV had caused catastrophic losses in swine industry in China. The current inactivated vaccine provided only limited protection, and the attenuated live vaccine could protect piglets against the HP-PRRSV but there was a possibility that the attenuated virus returned to high virulence. In this study, the eukaryotic expression vector pVAX1© was modified under the control of rabbit β-globin intron II gene and the modified vector pMVAX1© was constructed. Porcine interleukin-2 (IL-2 and GP3-GP5 fusion protein of HP-PRRSV strain SD-JN were highly expressed by pMVAX1©. Mice inoculated with pMVAX1©-GP35 developed significantly higher PRRSV-specific antibody responses and T cell proliferation than those vaccinated with pVAX1©-GP35. pMVAX1©-GP35 was selected as PRRS DNA vaccine candidate and co-administrated with pVAX1©-IL-2 or pMVAX1©-IL-2 in pigs. pMVAX1©-IL-2+pMVAX1©-GP35 could provide enhanced PRRSV-specific antibody responses, T cell proliferation, Th1-type and Th2-type cytokine responses and CTL responses than pMVAX1©-GP35 and pVAX1©-IL-2+pMVAX1©-GP35. Following homologous challenge with HP-PRRSV strain SD-JN, similar with attenuated PRRS vaccine group, pigs inoculated with pMVAX1©-IL-2+pMVAX1©-GP35 showed no clinical signs, almost no lung lesions and no viremia, as compared to those in pMVAX1©-GP35 and pVAX1©-IL-2+pMVAX1©-GP35 groups. It indicated that pMVAX1©-IL-2 effectively increases humoral and cell mediated immune responses of pMVAX1©-GP35. Co-administration of pMVAX1©-IL-2 and pMVAX1©-GP35 might be attractive candidate vaccines for preventing HP-PRRSV infections.

  12. A New Intergenic α-Globin Deletion (α-αΔ125) Found in a Kabyle Population.

    Science.gov (United States)

    Singh, Amrathlal Rabbind; Lacan, Philippe; Cadet, Estelle; Bignet, Patricia; Dumesnil, Cécile; Vannier, Jean-Pierre; Joly, Philippe; Rochette, Jacques

    2016-01-01

    We have identified a deletion of 125 bp (α-α(Δ125)) (NG_000006.1: g.37040_37164del) in the α-globin gene cluster in a Kabyle population. A combination of singlex and multiplex polymerase chain reaction (PCR)-based assays have been used to identify the molecular defect. Sequencing of the abnormal PCR amplification product revealed a novel α1-globin promoter deletion. The endpoints of the deletion were characterized by sequencing the deletion junctions of the mutated allele. The observed deletion was located 378 bp upstream of the α1-globin gene transcription initiation site and leaves the α2 gene intact. In some patients, the α-α(Δ125) deletion was shown to segregate with Hb S (HBB: c.20A>T) and/or Hb C (HBB: c.19G>A) or a β-thalassemic allele. The α-α(Δ125) deletion has no discernible effect on red cell indices when inherited with no other abnormal globin genes. The family study demonstrated that the deletion is heritable. This is the only example of an intergenic α2-α1 non coding DNA deletion, leaving the α2-globin gene and the α1 coding part intact.

  13. Description of two new alpha variants: Hb Canuts [alpha85(F6)Asp-->His (alpha1)] and Hb Ambroise Pare [alpha117(GH5)Phe-->Ile (alpha2)]; two new beta variants: Hb Beaujolais [beta84(EF8)Thr-->Asn] and Hb Monplaisir [beta147 (Tyr-Lys-Leu-Ala-Phe-Phe-Leu-Leu-Ser-Asn-Phe-Tyr-158-COOH)] and one new delta variant: Hb (A2)North Africa [delta59(E3)Lys-->Met].

    Science.gov (United States)

    Joly, Philippe; Lacan, Philippe; Bererd, Martine; Garcia, Caroline; Zanella-Cleon, Isabelle; Becchi, Michel; Aubry, Martine; Couprie, Nicole; Francina, Alain

    2009-01-01

    We present here five new hemoglobin (Hb) variants which have been identified during routine Hb analysis before their genotypic characterization. Four of these result from a classical missense mutation: Hb Canuts [alpha85(F6)Asp-->His (alpha1)], Hb Ambroise Pare [alpha117(GH5)Phe-->Ile (alpha2)], Hb Beaujolais [beta84(EF8)Thr-->Asn] and HbA(2)-North Africa [delta59(E3)Lys-->Met]. The last one, Hb Monplaisir [beta147 (Tyr-Lys-Leu-Ala-Phe-Phe-Leu-Leu-Ser-Asn-Phe-Tyr-158-COOH)], results from a frameshift mutation at the stop codon of the beta-globin gene which leads to a modified C-terminal sequence in the beta-globin chain. None of these variants seem to have a particular clinical expression in the heterozygous state. The circumstances of the discovery of these five new Hb variants emphasize the fact that an association of techniques is necessary for a complete screening of Hb variants during routine Hb analysis. Globin chain separation by reversed phase liquid chromatography (RP-LC) appears to be the most relevant method.

  14. The Survey of Genes Encoding Beta-Lactamases, in Escherichia Coli Resistant to Beta-Lactam and Non-Beta-Lactam Antibiotics

    Directory of Open Access Journals (Sweden)

    Fereshteh Shahcheraghi

    2010-01-01

    Full Text Available Resistance to the new generation of cephalosporins which is mediated by Extended-Spectrum beta-lactamases (ESBLs has been found amongEscherichia coli isolates throughout the world. These resistance genes and their producers, the micro-organisms carrying beta-lactamases, are responsible for serious clinical and therapeutic problems among inpatients and it is necessary to pay more attention to detection of ESBLs producing organisms.Materiasl and MethodsCollectively 260 isolates of E. coli were obtained from 6 hospitals in Tehran (Iran during April-2006 to April-2007. The antibiotic susceptibility patterns of isolates were determined by disk diffusion method. phenotypic confirmatory test (PCT was carried out for screening of ESBLs. Microbroth dilution assay was used to determine the minimum inhibitory concentration (MIC of ceftazidime. Isolates showing MIC≥2 μg/ml were subjected to polymerase chain reaction (PCR targeting blaTEM, blaSHV, blaCTX and blaPER genes. ResultsThe PCT showed that 48.08% of isolates are ESBL producers (125 of 260. The majority of cefotaxime resistant (90.8% and ceftazidime resistant (92.5% isolates were ESBL producers. The obtained results by PCR revealed that 5.77% (n=15 of 260 and 24.23 (n=63 of isolates can produce SHV and TEM type enzymes respectively. blaCTX was detected in 20.38% of isolates (n=53 and none of them could produce blaPER type beta-lactamases. ConclusionThe results of our study showed that the ESBL genes have high prevalence among clinical isolates of E. coli. Such high dissemination of ESBLs is a serious problem for public health and therefore, it's necessary to seek a program for monitoring ESBLs in hospitals.

  15. Promoters for the human beta-hexosaminidase genes, HEXA and HEXB.

    Science.gov (United States)

    Norflus, F; Yamanaka, S; Proia, R L

    1996-02-01

    Human lysosomal beta-hexosaminidases are encoded by two genes, HEXA and HEXB, specifying an alpha- and a beta-subunit, respectively. The subunits dimerize to form beta-hexosaminidase A (alpha beta), beta-hexosaminidase B (beta beta), and beta-hexosaminidase S (alpha alpha). This enzyme system has the capacity to degrade a variety of cellular substrates: oligosaccharides, glycosaminoglycans, and glycolipids containing beta-linked N-acetylglucosaminyl or N-galactosaminyl residues. Mutations in either the HEXA gene or HEXB gene lead to an accumulation of GM2 ganglioside in neurons, resulting in the severe neurodegenerative disorders termed the GM2 gangliosidoses. To identify the DNA elements responsible for hexosaminidase expression, we ligated the 5'-flanking sequences of both the human and mouse hexosaminidase genes to a chloramphenicol acetyltransferase (CAT) gene. The resulting plasmids were transfected into NIH-3T3 cells and CAT activity was determined as a measure of promoter strength. By 5' deletion analysis, it was found that essential sequences for HEXA expression resided within a 40-bp region between 100 bp and 60 bp upstream of the ATG initiation codon. This area contained two potential estrogen response element half-sites as well as potential binding sites for transcription factors NF-E1 and AP-2. Similarly, important HEXB promoter sequences were localized to a 60-bp region between 150 bp and 90 bp upstream of the ATG codon. By performing scanning mutagenesis on a 60-bp region within the 150-bp HEXB construct, we defined an essential promoter element of 12 bp that contained two potential AP-1 sites. The mouse Hexa and Hexb 5'-flanking sequences were found to contain regions similar in sequence, location, and activity to the essential promoter elements defined in the cognate human genes. No sequence similarity was found, however, between 5'-flanking regions of the HEXA and HEXB genes. These essential promoter elements represent potential sites for HEXA and

  16. Elucidation of IL-1/TGF-beta interactions in mouse chondrocyte cell line by genome-wide gene expression

    DEFF Research Database (Denmark)

    Takahashi, N; Rieneck, K; van der Kraan, P M;

    2005-01-01

    To elucidate the antagonism between interleukin-1 (IL-1) and transforming growth factor-beta (TGF-beta) at the gene expression level, as IL-1 and TGF-beta are postulated to be critical mediators of cartilage degeneration/protection in rheumatic diseases....

  17. SIRT1 deacetylates SATB1 to facilitate MAR HS2-MAR ε interaction and promote ε-globin expression.

    Science.gov (United States)

    Xue, Zheng; Lv, Xiang; Song, Wei; Wang, Xing; Zhao, Guang-Nian; Wang, Wen-Tian; Xiong, Jian; Mao, Bei-Bei; Yu, Wei; Yang, Ben; Wu, Jie; Zhou, Li-Quan; Hao, De-Long; Dong, Wen-Ji; Liu, De-Pei; Liang, Chih-Chuan

    2012-06-01

    The higher order chromatin structure has recently been revealed as a critical new layer of gene transcriptional control. Changes in higher order chromatin structures were shown to correlate with the availability of transcriptional factors and/or MAR (matrix attachment region) binding proteins, which tether genomic DNA to the nuclear matrix. How posttranslational modification to these protein organizers may affect higher order chromatin structure still pending experimental investigation. The type III histone deacetylase silent mating type information regulator 2, S. cerevisiae, homolog 1 (SIRT1) participates in many physiological processes through targeting both histone and transcriptional factors. We show that MAR binding protein SATB1, which mediates chromatin looping in cytokine, MHC-I and β-globin gene loci, as a new type of SIRT1 substrate. SIRT1 expression increased accompanying erythroid differentiation and the strengthening of β-globin cluster higher order chromatin structure, while knockdown of SIRT1 in erythroid k562 cells weakened the long-range interaction between two SATB1 binding sites in the β-globin locus, MAR(HS2) and MAR(ε). We also show that SIRT1 activity significantly affects ε-globin gene expression in a SATB1-dependent manner and that knockdown of SIRT1 largely blocks ε-globin gene activation during erythroid differentiation. Our work proposes that SIRT1 orchestrates changes in higher order chromatin structure during erythropoiesis, and reveals the dynamic higher order chromatin structure regulation at posttranslational modification level.

  18. Utilization of denaturing gradient gel electrophoresis for diagnosis of {beta}-thalassemia and ascertainment of new mutations

    Energy Technology Data Exchange (ETDEWEB)

    Ngo, K.Y.; Liu, D.; Lee, J. [Univ. of California, San Diego (United States)] [and others

    1994-09-01

    During the past two years we have tested 2,300 Southeast Asians for alpha- and beta-thaleassemia mutations. We found the incidence of hemoglobin E ({beta}{sup 26}) to be 47% among Laotians and 38% among Cambodians. The incidence of beta thalassemia trait is 9% for Laotians and 6% for Cambodians. Thus, the risk for hemoglobin E/{beta}{sup 26} thalassemia, a transfusion-dependent disorder, is increased in these two population groups. Denaturing gradient gel electrophoresis (DGGE) has proven to be useful in testing for beta-thalassemia carriers and identifying new mutations in the beta globin gene. DNA was extracted from venous blood obtained from patients with elevated Hgb A2 (>4%). Five DNA fragments, encompassing the beta globin gene cluster, were amplified by PCR and analyzed, along with known beta gene mutations as controls, by DGGE using different denaturing gradient concentrations. Different mutations at the same nucleotide position can be distinguished by migration pattern on the DGGE (e.g., in IVS-I-1, G{r_arrow}A and T). Compound heterozygotes for {beta}-thalassemia can be detected on the same gel (e.g., HbE/mutation codon 17). New mutations are identified by their migration pattern compared with controls and determined by subsequent sequencing. We have identified three new mutations: codon 82 CAA{r_arrow}AAA in one Cambodian patient; IVS-II-667, T{r_arrow}C and IVS-II-672, A{r_arrow}C in two Laotian patients. When the parent`s genotypes are known, prenatal diagnosis can be obtained within 24 hours. Thus, PCR/DGGE combination is a rapid and reliable diagnostic approach to clinically significant {beta}-thalassemia. The most important steps are carefully designed primers and predetermined gradient concentrations for DGGE.

  19. Alpha beta T-cell development is not affected by inversion of TCR beta gene enhancer sequences: polar enhancement of gene expression regardless of enhancer orientation.

    Science.gov (United States)

    Huang, Fang; Cabaud, Olivier; Verthuy, Christophe; Hueber, Anne-Odile; Ferrier, Pierre

    2003-08-01

    V(D)J recombination and expression of the T-cell receptor beta (TCRbeta) gene are required for the development of the alphabeta T lymphocyte lineage. These processes depend on a transcriptional enhancer (Ebeta) which acts preferentially on adjacent upstream sequences, and has little impact on the 5' distal and 3' proximal regions of the TCRbeta locus. Using knock-in mice, we show that alphabeta T-cell differentiation and TCRbeta gene recombination and expression are not sensitive to the orientation of Ebeta sequences. We discuss the implication of these results regarding the mode of enhancer function at this locus during T lymphocyte development.

  20. Estrogen Receptor beta 2 Induces Hypoxia Signature of Gene Expression by Stabilizing HIF-1 alpha in Prostate Cancer

    OpenAIRE

    Prasenjit Dey; Velazquez-Villegas, Laura A.; Michelle Faria; Anthony Turner; Philp Jonsson; Paul Webb; Cecilia Williams; Jan-Åke Gustafsson; Ström, Anders M.

    2015-01-01

    The estrogen receptor (ER) beta variant ER beta 2 is expressed in aggressive castration-resistant prostate cancer and has been shown to correlate with decreased overall survival. Genome-wide expression analysis after ER beta 2 expression in prostate cancer cells revealed that hypoxia was an overrepresented theme. Here we show that ER beta 2 interacts with and stabilizes HIF-1 alpha protein in normoxia, thereby inducing a hypoxic gene expression signature. HIF-1 alpha is known to stimulate met...

  1. Mapping of the Mouse Actin Capping Protein Beta Subunit Gene

    Directory of Open Access Journals (Sweden)

    Cooper John A

    2000-07-01

    Full Text Available Abstract Background Capping protein (CP, a heterodimer of α and β subunits, is found in all eukaryotes. CP binds to the barbed ends of actin filaments in vitro and controls actin assembly and cell motility in vivo. Vertebrates have three isoforms of CPβ produced by alternatively splicing from one gene; lower organisms have one gene and one isoform. Results We isolated genomic clones corresponding to the β subunit of mouse CP and identified its chromosomal location by interspecies backcross mapping. Conclusions The CPβ gene (Cappb1 mapped to Chromosome 4 between Cdc42 and D4Mit312. Three mouse mutations, snubnose, curly tail, and cribriform degeneration, map in the vicinity of the β gene.

  2. Pseudomonas aeruginosa biofilms exposed to imipenem exhibit changes in global gene expression and beta-lactamase and alginate production

    DEFF Research Database (Denmark)

    Bagge, Niels; Schuster, Martin; Hentzer, Morten

    2004-01-01

    . The treatment often includes beta-lactam antibiotics. How these antibiotics influence gene expression in the surviving biofilm population of P. aeruginosa is not clear. Thus, we used the microarray technology to study the effects of subinhibitory concentrations of a beta-lactam antibiotic, imipenem, on gene...

  3. Nonblack patients with sickle cell disease have African. beta. sup s gene cluster haplotypes

    Energy Technology Data Exchange (ETDEWEB)

    Rogers, Z.R.; Powars, D.R.; Williams, W.D. (Univ. of Southern California School of Medicine, Los Angeles (USA)); Kinney, T.R. (Duke Univ., Durham, NC (USA)); Schroeder, W.A. (California Institute of Technology, Pasadena (USA))

    1989-05-26

    Of 18 nonblack patients with sickle cell disease, 14 had sickle cell anemia, 2 had hemoglobin SC disease, and 2 had hemoglobin S-{beta}{sup o}-thalassemia. The {beta}{sup s} gene cluster haplotypes that were determined in 7 patients were of African origin and were identified as Central African Republic, Central African Republic minor II, Benin, and Senegal. The haplotype Central African Republic minor II was present on the {beta}{sup o}-thalassemia chromosome in 2 patients. None of 10 patients whose {alpha}-gene status was determined had {alpha}-thalassemia-2. These data strongly support the concept that the {beta}{sup s} gene on chromosome 11 of these individuals is of African origin and that the {alpha}-gene locus on chromosome 16 is of white or native American origin. The clinical severity of the disease in these nonblack patients is appropriate to their haplotype without {alpha}-thalassemia-2 and is comparable with that of black patients. All persons with congenital hemolytic anemia should be examined for the presence of sickle cell disease regardless of physical appearance or ethnic background.

  4. A gene duplication led to specialized gamma-aminobutyrate and beta-alanine aminotransferase in yeast

    DEFF Research Database (Denmark)

    Andersen, Gorm; Andersen, Birgit; Dobritzsch, D.

    2007-01-01

    In humans, beta-alanine (BAL) and the neurotransmitter gamma-aminobutyrate (GABA) are transaminated by a single aminotransferase enzyme. Apparently, yeast originally also had a single enzyme, but the corresponding gene was duplicated in the Saccharomyces kluyveri lineage. SkUGA1 encodes a homologue...

  5. Structure of the gene encoding the murine protein kinase CK2 beta subunit

    DEFF Research Database (Denmark)

    Boldyreff, B; Issinger, O G

    1995-01-01

    The mouse protein kinase CK2 beta subunit gene (Csnk2b) is composed of seven exons contained within 7874 bp. The exon and intron lengths extend from 76 to 321 and 111 to 1272 bp, respectively. The lengths of the murine coding exons correspond exactly to the lengths of the exons in the human CK2...

  6. Organ specificity of beta-carotene induced lung gene-expression changes in Bcmo 1-/- mice

    NARCIS (Netherlands)

    Helden, Y.G.J.; Godschalk, R.W.L.; Schooten, van F.J.; Keijer, J.

    2013-01-01

    Scope - Whole genome transcriptome analysis of male and female beta-carotene 15,15'-monooxygenase knockout (Bcmo1-/-) and Bcmo1+/+ (wild-type) mice with or without 14 wk of BC supplementation was done. We previously showed that only 1.8% of the genes regulated by BC in lung were also regulated in li

  7. Using the NCBI Genome Databases to Compare the Genes for Human & Chimpanzee Beta Hemoglobin

    Science.gov (United States)

    Offner, Susan

    2010-01-01

    The beta hemoglobin protein is identical in humans and chimpanzees. In this tutorial, students see that even though the proteins are identical, the genes that code for them are not. There are many more differences in the introns than in the exons, which indicates that coding regions of DNA are more highly conserved than non-coding regions.

  8. 中国汉族人群β-珠蛋白基因BP1蛋白结合区多态性分析%Polymorphism in BP1 Binding Site Upstream of β-Globin Gene in Chinese Han Population

    Institute of Scientific and Technical Information of China (English)

    孙顺昌; 周指明; 彭运生; 谢春英; 陈群蓉; 王燮衡

    2011-01-01

    本研究旨在对中国汉族人群β-珠蛋白基因BP1蛋白结合区序列进行分析,获取β-珠蛋白基因BP1蛋白结合区的多态性信息,为探讨BP1蛋白结合区多态性与β-珠蛋白表达的相关性奠定基础.收集110例健康中国汉族人群的外周血,并抽提基因组DNA,通过聚合酶链反应扩增B-珠蛋白基因BPI蛋白结合区序列,经DNA测序确定BP1蛋白结合区的多态性序列.结果显示:在中国汉族人群的β-珠蛋白基因BP1蛋白结合区共发现2个多态性位点,它们分别是- 551位点C/T、- 530位点(AC)n(AT)xTy.在-551位点存在C和T碱基,其频率分别为60.4%和39.6%,而- 530位点(AC)n(AT)xTy多态性序列存在(AC)2(AT) 9T5、(AC)2 (AT) 8T5、(AC)2(AT)7T7、(AC)3(AT)7T5、(AC)2(AT)8T9、(AC)3(AT)8T5、(AC)2 (AT)10 T3、( AC)2 (AT)11 T3和(AC)2(AT)7T5共9种单倍型,它们在人群中的频率分别为33.2%、29.1%、24.1%、5.4%、3.2%、1.8%、1.4%、0.9%和0.9%.结论:在中国汉族人群B-珠蛋白基因BP1蛋白结合区内,- 530位点(AC)n(AT)xTy多态性信息丰富,其中( AC)2 (AT) 9T5、( AC)2 (AT) 8T5和(AC)2 (AT) 7T7是三种常见单倍型.(AC)3(AT)8T5是一种新类型多态性序列.这些(AC)n(AT)xTy多态性与β-珠蛋白表达的相关性有待深入研究.%This study was aimed to analyze the BP1 binding site sequence upstream of B-globin gene in Chinese Han population, and to investigate polymorphism in the BP1 binding site upstream of p-globin gene, so as to provide the basis for exploration of relation between polymorphisms in the BP1 binding site and p-globin expression. Genomic DNA was extracted from peripheral leukocytes of 110 healthy individuals in Chinese Han population. Sequence of the BP1 binding site upstream of p-globin gene was amplified by polymerase chain reaction, the polymorphic variation in the BP1 binding site was determined by DNA sequencing. The results indicated that 2 polymorphism loci were found in the BP1 binding site

  9. Isolation and functional characterization of a lycopene beta-cyclase gene that controls fruit colour of papaya (Carica papaya L.).

    Science.gov (United States)

    Devitt, Luke C; Fanning, Kent; Dietzgen, Ralf G; Holton, Timothy A

    2010-01-01

    The colour of papaya fruit flesh is determined largely by the presence of carotenoid pigments. Red-fleshed papaya fruit contain lycopene, whilst this pigment is absent from yellow-fleshed fruit. The conversion of lycopene (red) to beta-carotene (yellow) is catalysed by lycopene beta-cyclase. This present study describes the cloning and functional characterization of two different genes encoding lycopene beta-cyclases (lcy-beta1 and lcy-beta2) from red (Tainung) and yellow (Hybrid 1B) papaya cultivars. A mutation in the lcy-beta2 gene, which inactivates enzyme activity, controls lycopene production in fruit and is responsible for the difference in carotenoid production between red and yellow-fleshed papaya fruit. The expression level of both lcy-beta1 and lcy-beta2 genes is similar and low in leaves, but lcy-beta2 expression increases markedly in ripe fruit. Isolation of the lcy-beta2 gene from papaya, that is preferentially expressed in fruit and is correlated with fruit colour, will facilitate marker-assisted breeding for fruit colour in papaya and should create possibilities for metabolic engineering of carotenoid production in papaya fruit to alter both colour and nutritional properties.

  10. Beta-Adrenergic gene therapy for cardiovascular disease

    Directory of Open Access Journals (Sweden)

    Koch Walter J

    2000-10-01

    Full Text Available Abstract Gene therapy using in vivo recombinant adenovirus-mediated gene transfer is an effective technique that offers great potential to improve existing drug treatments for the complex cardiovascular diseases of heart failure and vascular smooth muscle intimal hyperplasia. Cardiac-specific adenovirus-mediated transfer of the carboxyl-terminus of the β-adrenergic receptor kinase (βARKct, acting as a Gβγ-β-adrenergic receptor kinase (βARK1 inhibitor, improves basal and agonist-induced cardiac performance in both normal and failing rabbit hearts. In addition, βARKct adenovirus infection of vascular smooth muscle is capable of significantly diminishing neointimal proliferation after angioplasty. Therefore, further investigation is warranted to determine whether inhibition of βARK1 activity and sequestration of Gβγ via an adenovirus that encodes the βARKct transgene might be a useful clinical tool for the treatment of cardiovascular pathologies.

  11. Effects of 1-beta-D-arabinofuranosylcytosine and phorbol ester on differentiation of human K562 erythroleukemia cells.

    Science.gov (United States)

    Watanabe, T; Mitchell, T; Sariban, E; Sabbath, K; Griffin, J; Kufe, D

    1985-06-01

    We have previously demonstrated that 1-beta-D-arabinofuranosylcytosine (ara-C) induces hemoglobin synthesis in human K562 erythroleukemia cells. The present study extends these findings by demonstrating that ara-C treatment of K562 cells results in both increased heme synthesis and accumulation of alpha-, gamma-, epsilon-, and zeta-globin RNA. The results also demonstrate that ara-C enhances K562 cell surface expression of glycophorin. Furthermore, we demonstrate that phorbol ester (12-O-tetradecanoylphorbol-13-acetate; TPA) inhibits the effects of ara-C on heme production, accumulation of globin RNA, and glycophorin expression. The inhibitory effect occurs maximally when K562 cells are treated with TPA before undergoing ara-C-induced commitment to erythroid differentiation. These findings suggest that TPA inhibits an early step in the process required for ara-C to enhance expression of genes involved in the erythroid program.

  12. Class II genes of miniature swine. II. Molecular identification and characterization of B (beta) genes from the SLAc haplotype.

    Science.gov (United States)

    Pratt, K; Sachs, D H; Germana, S; el-Gamil, M; Hirsch, F; Gustafsson, K; LeGuern, C

    1990-01-01

    Genomic clones corresponding to class II beta genes of the SLAc haplotype of miniature swine have been isolated and characterized. These genes have been grouped into seven non-overlapping clusters on the basis of restriction mapping. Ordering of exons within each cluster was accomplished by hybridization of Southern blots of restriction fragments with exon-specific probes. The two clusters (clusters 2 and 3) encoding the DRB and DQB genes were identified on the basis of hybridization with locus-specific 3' untranslated cDNA probes. Cluster 4 contained exons of both DOB and DQB genes, the basis for which remains to be determined. The remaining four clusters (1, 5, 6, 7) were identified as containing DP, DR, and DO coding sequences, respectively, on the basis of sequence analysis. The porcine class II region appears very similar to that of man in number and nature of the class II genes identified and in the intron/exon organization of corresponding genes.

  13. Advances in stem cell transplantation and gene therapy in the β-hemoglobinopathies.

    Science.gov (United States)

    Payen, Emmanuel; Leboulch, Philippe

    2012-01-01

    High-level production of β-globin, γ-globin, or therapeutic mutant globins in the RBC lineage by hematopoietic stem cell gene therapy ameliorates or cures the hemoglobinopathies sickle cell disease and beta thalassemia, which are major causes of morbidity and mortality worldwide. Considerable efforts have been made in the last 2 decades in devising suitable gene-transfer vectors and protocols to achieve this goal. Five years ago, the first β(E)/β(0)-thalassemia major (transfusion-dependent) patient was treated by globin lentiviral gene therapy without injection of backup cells. This patient has become completely transfusion independent for the past 4 years and has global amelioration of the thalassemic phenotype. Partial clonal dominance for an intragenic site (HMGA2) of chromosomal integration of the vector was observed in this patient without a loss of hematopoietic homeostasis. Other patients are now receiving transplantations while researchers are carefully weighing the benefit/risk ratio and continuing the development of further modified vectors and protocols to improve outcomes further with respect to safety and efficacy.

  14. Mx1 and IP-10: biomarkers to measure IFN-beta activity in mice following gene-based delivery.

    Science.gov (United States)

    Petry, Harald; Cashion, Linda; Szymanski, Paul; Ast, Oliver; Orme, Ann; Gross, Cynthia; Bauzon, Maxine; Brooks, Alan; Schaefer, Caralee; Gibson, Heather; Qian, Husheng; Rubanyi, Gabor M; Harkins, Richard N

    2006-10-01

    Recombinant interferon-beta (IFN-beta) protein is used successfully for the treatment of multiple sclerosis (MS). Gene therapy might be an alternative approach to overcome drawbacks occurring with IFN-beta protein therapy. A critical issue in developing a new approach is detection of biologically active IFN-beta in preclinical models. The goal of the present study was to determine if Mx1 and IP-10, which are known to be activated after IFN-beta treatment in humans, can be used as biomarkers in mice. In three in vivo experiments, the correlation between different methods of murine IFN-beta (MuIFN-beta) delivery and biomarker induction was studied: (1) bolus protein delivery by intravenous (i.v.) or intramuscular (i.m.) injection, (2) gene-based delivery of IFN- beta by i.m. injection of plasmid DNA, followed by electroporation, and (3) gene-based delivery of IFN-beta by i.m. injection of adenovirus-associated type 1 (AAV1). Short-term induction of Mx1 mRNA and IP-10 was observed after treatment with bolus MuIFN-beta protein. Long-term induction of both biomarkers was observed after IFN-beta plasmid DNA delivery or when AAV1 was used as the vector. The experiments demonstrate that gene-based delivery provides sustained levels of IFN-beta compared with bolus protein injection and that Mx1 RNA and IP-10 can be used to monitor biologically active circulating plasma MuIFN-beta protein in mice.

  15. Transforming growth factor-beta inhibits aromatase gene transcription in human trophoblast cells via the Smad2 signaling pathway

    Directory of Open Access Journals (Sweden)

    Fu Guodong

    2009-12-01

    Full Text Available Abstract Background Transforming growth factor-beta (TGF-beta is known to exert multiple regulatory functions in the human placenta, including inhibition of estrodial production. We have previously reported that TGF-beta1 decreased aromatase mRNA levels in human trophoblast cells. The objective of this study was to investigate the molecular mechanisms underlying the regulatory effect of TGF-beta1 on aromatase expression. Methods To determine if TGF-beta regulates aromatase gene transcription, several reporter constructs containing different lengths of the placental specific promoter of the human aromatase gene were generated. JEG-3 cells were transiently transfected with a promoter construct and treated with or without TGF-beta1. The promoter activity was measured by luciferase assays. To examine the downstream signaling molecule mediating the effect of TGF-beta on aromatase transcription, cells were transiently transfected with dominant negative mutants of TGF-beta type II (TbetaRII and type I receptor (ALK5 receptors before TGF-beta treatment. Smad2 activation was assessed by measuring phophorylated Smad2 protein levels in cytosolic and nuclear fractions. Smad2 expression was silenced using a siRNA expression construct. Finally, aromatase mRNA half-life was determined by treating cells with actinomycin D together with TGF-beta1 and measuring aromatase mRNA levels at various time points after treatment. Results and Discussion TGF-beta1 inhibited the aromatase promoter activity in a time- and dose-dependent manner. Deletion analysis suggests that the TGF-β1 response element resides between -422 and -117 nucleotides upstream from the transcription start site where a Smad binding element was found. The inhibitory effect of TGF-beta1 was blocked by dominant negative mutants of TbetaRII and ALK5. TGF-beta1 treatment induced Smad2 phosphorylation and translocation into the nucleus. On the other hand, knockdown of Smad2 expression reversed the

  16. O-Linked N-Acetylglucosamine (O-GlcNAc) Transferase and O-GlcNAcase Interact with Mi2β Protein at the Aγ-Globin Promoter.

    Science.gov (United States)

    Zhang, Zhen; Costa, Flávia C; Tan, Ee Phie; Bushue, Nathan; DiTacchio, Luciano; Costello, Catherine E; McComb, Mark E; Whelan, Stephen A; Peterson, Kenneth R; Slawson, Chad

    2016-07-22

    One mode of γ-globin gene silencing involves a GATA-1·FOG-1·Mi2β repressor complex that binds to the -566 GATA site relative to the (A)γ-globin gene cap site. However, the mechanism of how this repressor complex is assembled at the -566 GATA site is unknown. In this study, we demonstrate that the O-linked N-acetylglucosamine (O-GlcNAc) processing enzymes, O-GlcNAc-transferase (OGT) and O-GlcNAcase (OGA), interact with the (A)γ-globin promoter at the -566 GATA repressor site; however, mutation of the GATA site to GAGA significantly reduces OGT and OGA promoter interactions in β-globin locus yeast artificial chromosome (β-YAC) bone marrow cells. When WT β-YAC bone marrow cells are treated with the OGA inhibitor Thiamet-G, the occupancy of OGT, OGA, and Mi2β at the (A)γ-globin promoter is increased. In addition, OGT and Mi2β recruitment is increased at the (A)γ-globin promoter when γ-globin becomes repressed in postconception day E18 human β-YAC transgenic mouse fetal liver. Furthermore, we show that Mi2β is modified with O-GlcNAc, and both OGT and OGA interact with Mi2β, GATA-1, and FOG-1. Taken together, our data suggest that O-GlcNAcylation is a novel mechanism of γ-globin gene regulation mediated by modulating the assembly of the GATA-1·FOG-1·Mi2β repressor complex at the -566 GATA motif within the promoter.

  17. Analysis and expression of the alpha-expansin and beta-expansin gene families in maize

    Science.gov (United States)

    Wu, Y.; Meeley, R. B.; Cosgrove, D. J.

    2001-01-01

    Expansins comprise a multigene family of proteins in maize (Zea mays). We isolated and characterized 13 different maize expansin cDNAs, five of which are alpha-expansins and eight of which are beta-expansins. This paper presents an analysis of these 13 expansins, as well as an expression analysis by northern blotting with materials from young and mature maize plants. Some expansins were expressed in restricted regions, such as the beta-expansins ExpB1 (specifically expressed in maize pollen) and ExpB4 (expressed principally in young husks). Other expansins such as alpha-expansin Exp1 and beta-expansin ExpB2 were expressed in several organs. The expression of yet a third group was not detected in the selected organs and tissues. An analysis of expansin sequences from the maize expressed sequence tag collection is also presented. Our results indicate that expansin genes may have general, overlapping expression in some instances, whereas in other cases the expression may be highly specific and limited to a single organ or cell type. In contrast to the situation in Arabidopsis, beta-expansins in maize seem to be more numerous and more highly expressed than are alpha-expansins. The results support the concept that beta-expansins multiplied and evolved special functions in the grasses.

  18. Molecular cloning and functional characterization of beta-N-acetylglucosaminidase genes from Sf9 cells.

    Science.gov (United States)

    Aumiller, Jared J; Hollister, Jason R; Jarvis, Donald L

    2006-06-01

    Sf9, a cell line derived from the lepidopteran insect, Spodoptera frugiperda, is widely used as a host for recombinant glycoprotein expression and purification by baculovirus vectors. Previous studies have shown that this cell line has one or more beta-N-acetylglucosaminidase activities that may be involved in the degradation and/or processing of N-glycoprotein glycans. However, these enzymes and their functions remain poorly characterized. Therefore, the goal of this study was to isolate beta-N-acetylglucosaminidase genes from Sf9 cells, over-express the gene products, and characterize their enzymatic activities. A degenerate PCR approach yielded three Sf9 cDNAs, which appeared to encode two distinct beta-N-acetylglucosaminidases, according to bioinformatic analyses. Baculovirus-mediated expression of these two cDNA products induced membrane-associated beta-N-acetylglucosaminidase activities in Sf9 cells, which cleaved terminal N-acetylglucosamine residues from the alpha-3 and -6 branches of a biantennary N-glycan substrate with acidic pH optima and completely hydrolyzed chitotriose to its constituent N-acetylglucosamine monomers. GFP-tagged forms of both enzymes exhibited punctate cytoplasmic fluorescence, which did not overlap with either lysosomal or Golgi-specific dyes. Together, these results indicated that the two new Sf9 genes identified in this study encode broad-spectrum beta-N-acetylglucosaminidases that appear to have unusual intracellular distributions. Their relative lack of substrate specificity and acidic pH optima are consistent with a functional role for these enzymes in glycoprotein glycan and chitin degradation, but not with a role in N-glycoprotein glycan processing.

  19. Evidence for the association of the S100beta gene with low cognitive performance and dementia in the elderly

    DEFF Research Database (Denmark)

    Lambert, J-C; Ferreira, S; Gussekloo, J

    2007-01-01

    Variations in the S100beta gene may be instrumental in producing a continuum from mild cognitive decline to overt dementia. After screening 25 single nucleotide polymorphisms (SNPs) in S100beta, we observed association of the rs2300403 intron 2 SNP with poorer cognitive function in three...... corresponding mRNA isoform was called S100beta2). S100beta2 expression was increased in AD brain compared with controls, and the rs2300403 SNP was associated with elevated levels of S100beta2 mRNA in AD brains, especially in women. Therefore, this genetic variant in S100beta increases the risk of low cognitive...... performance and dementia, possibly by favouring a splicing event increasing S100beta2 isoform expression in the brain....

  20. Altered DNA-binding specificity mutants of EKLF and Sp1 show that EKLF is an activator of the β-globin locus control region in vivo.

    NARCIS (Netherlands)

    N. Gillemans (Nynke); R. Tewari (Rita); F.A. Lindeboom (Fokke); R.J. Rottier (Robbert); M.G.J.M. Wijgerde (Mark); F.G. Grosveld (Frank); J.N.J. Philipsen (Sjaak); T.P.M. de Wit (Ton)

    1998-01-01

    textabstractThe locus control region of the beta-globin cluster contains five DNase I hypersensitive sites (5'HS1-5) required for locus activation. 5'HS3 contains six G-rich motifs that are essential for its activity. Members of a protein family, characterized by three zinc fingers highly homologous

  1. Spectrum of Common α-Globin Deletion Mutations in the Southern Region of Vietnam.

    Science.gov (United States)

    Bui Thi Kim, Ly; Phu Chi, Dung; Hoang Thanh, Chi

    2016-06-01

    The common deletion mutations of α-globin genes in the Vietnamese population is not well known. Here we report the presence of five deletional mutations of Southeast Asia in the southern region of Vietnam. The - -(SEA) (NG_000006.1: g.26264_45564del19301) mutation is the most common type of deletion (87.35%), followed by the -α(3.7) (rightward) (NG_000006.1: g.34164_37967del3804) deletion (9.64%), -α(4.2) (leftward) (AF221717) deletion (2.41%) and - -(THAI) (NG_000006.1: g.10664_44164del33501) (0.6%) mutation in this region. The - -(FIL) (NG_000006.1: g.11684_43534del31581) mutation was not detected in this study. This result provided a view of the distribution of common α-globin gene mutations in Vietnam and could serve as a baseline for further investigations into these genetic defects.

  2. Haplotypes of beta S chromosomes among patients with sickle cell anemia from Georgia.

    Science.gov (United States)

    Hattori, Y; Kutlar, F; Kutlar, A; McKie, V C; Huisman, T H

    1986-01-01

    Fetal hemoglobin and G gamma levels have been correlated with the presence or absence of eight restriction sites within the beta globin gene cluster (haplotypes) for numerous sickle cell anemia patients from Georgia. The most common haplotypes were #19 (Benin) and #20 (CAR); all patients with haplotype combinations 19/19, 20/20, and 19/20 were severely affected with low Hb F and low G gamma levels. A modified #19 beta S chromosome with a -G gamma-G gamma- globin gene arrangement, instead of -G gamma-A gamma-, was present in SS and SC newborn babies with G gamma values above 80%. Haplotype #3 (Senegal) was present among 15% of the beta S chromosomes; the two adult patients with the 3/3 combination were mildly affected with high Hb F and G gamma values. The haplotype AT with the variant A gamma T chain was a rarity. A new haplotype was found in one 17-year-old SS patient and five of his Hb S heterozygous relatives. This haplotype is associated with an increased production of Hb F in heterozygous and homozygous Hb S individuals; this Hb F contained primarily A gamma chains. A comparison was made between the different haplotypes among SS patients and normal Black individuals, and a remarkable similarity was noted in the fetal hemoglobin data for subjects with these different chromosomes.

  3. Genetic structures at the origin of acquisition of the beta-lactamase bla KPC gene.

    Science.gov (United States)

    Naas, Thierry; Cuzon, Gaelle; Villegas, Maria-Virginia; Lartigue, Marie-Frédérique; Quinn, John P; Nordmann, Patrice

    2008-04-01

    Genetic structures surrounding the carbapenem-hydrolyzing Ambler class A bla KPC gene were characterized in several KPC-positive Klebsiella pneumoniae and Pseudomonas aeruginosa strains isolated from the United States, Colombia, and Greece. The bla KPC genes were associated in all cases with transposon-related structures. In the K. pneumoniae YC isolate from the United States, the beta-lactamase bla KPC-2 gene was located on a novel Tn3-based transposon, Tn4401. Tn4401 was 10 kb in size, was delimited by two 39-bp imperfect inverted repeat sequences, and harbored, in addition to the beta-lactamase bla KPC-2 gene, a transposase gene, a resolvase gene, and two novel insertion sequences, ISKpn6 and ISKpn7. Tn4401 has been identified in all isolates. However, two isoforms of this transposon were found: Tn4401a was found in K. pneumoniae YC and K. pneumoniae GR from the United States and Greece, respectively, and differed by a 100-bp deletion, located just upstream of the bla KPC-2 gene, compared to the sequence of Tn4401b, which was found in the Colombian isolates. In all isolates tested, Tn4401 was flanked by a 5-bp target site duplication, the signature of a recent transposition event, and was inserted in different open reading frames located on plasmids that varied in size and nature. Tn4401 is likely at the origin of carbapenem-hydrolyzing beta-lactamase KPC mobilization to plasmids and its further insertion into various-sized plasmids identified in nonclonally related K. pneumoniae and P. aeruginosa isolates.

  4. Enantioselective Degradation Mechanism of Beta-Cypermethrin in Soil From the Perspective of Functional Genes.

    Science.gov (United States)

    Yang, Zhong-Hua; Ji, Guo-Dong

    2015-12-01

    The behavior and mechanisms of the enantioselective degradation of beta-cypermethrin were studied in soil. The four isomers were degraded at different rates, and the enantiomer fractions of alpha-cypermethrin and theta-cypermethrin exceeded 0.5. Moreover, 3-phenoxybenzoic acid, phenol, and protocatechuic acid were detected; based on the presence of these metabolites, we predicted the degradation pathway and identified the functional genes that are related to this degradation process. We established quantitative relationships between the data on degradation kinetics and functional genes; we found that the quantitative relationships between different enantiomers differed even under the same conditions, and the genes pobA and pytH played key roles in limiting the degradation rate. Data obtained using path analysis revealed that the same gene had different direct and indirect effects on the degradation of different isomers. A mechanism was successfully proposed to explain the selective degradation of chiral compounds based on the perspective of functional genes.

  5. Production of β-globin and adult hemoglobin following G418 treatment of erythroid precursor cells from homozygous β039 thalassemia patients

    Science.gov (United States)

    Salvatori, Francesca; Breveglieri, Giulia; Zuccato, Cristina; Finotti, Alessia; Bianchi, Nicoletta; Borgatti, Monica; Feriotto, Giordana; Destro, Federica; Canella, Alessandro; Brognara, Eleonora; Lampronti, Ilaria; Breda, Laura; Rivella, Stefano; Gambari, Roberto

    2013-01-01

    In several types of thalassemia (including β039-thalassemia), stop codon mutations lead to premature translation termination and to mRNA destabilization through nonsense-mediated decay. Drugs (for instance aminoglycosides) can be designed to suppress premature termination, inducing a ribosomal readthrough. These findings have introduced new hopes for the development of a pharmacologic approach to the cure of this disease. However, the effects of aminoglycosides on globin mRNA carrying β-thalassemia stop mutations have not yet been investigated. In this study, we have used a lentiviral construct containing the β039- thalassemia globin gene under control of the β-globin promoter and a LCR cassette. We demonstrated by fluorescence-activated cell sorting (FACS) analysis the production of β-globin by K562 cell clones expressing the β039-thalassemia globin gene and treated with G418. More importantly, after FACS and high-performance liquid chromatography (HPLC) analyses, erythroid precursor cells from β039-thalassemia patients were demonstrated to be able to produce β-globin and adult hemoglobin after treatment with G418. This study strongly suggests that ribosomal readthrough should be considered a strategy for developing experimental strategies for the treatment of β0-thalassemia caused by stop codon mutations. PMID:19810011

  6. HisE11 and HisF8 provide bis-histidyl heme hexa-coordination in the globin domain of Geobacter sulfurreducens globin-coupled sensor.

    Science.gov (United States)

    Pesce, Alessandra; Thijs, Liesbet; Nardini, Marco; Desmet, Filip; Sisinni, Lorenza; Gourlay, Louise; Bolli, Alessandro; Coletta, Massimiliano; Van Doorslaer, Sabine; Wan, Xuehua; Alam, Maqsudul; Ascenzi, Paolo; Moens, Luc; Bolognesi, Martino; Dewilde, Sylvia

    2009-02-13

    Among heme-based sensors, recent phylogenomic and sequence analyses have identified 34 globin coupled sensors (GCS), to which an aerotactic or gene-regulating function has been tentatively ascribed. Here, the structural and biochemical characterization of the globin domain of the GCS from Geobacter sulfurreducens (GsGCS(162)) is reported. A combination of X-ray crystallography (crystal structure at 1.5 A resolution), UV-vis and resonance Raman spectroscopy reveals the ferric GsGCS(162) as an example of bis-histidyl hexa-coordinated GCS. In contrast to the known hexa-coordinated globins, the distal heme-coordination in ferric GsGCS(162) is provided by a His residue unexpectedly located at the E11 topological site. Furthermore, UV-vis and resonance Raman spectroscopy indicated that ferrous deoxygenated GsGCS(162) is a penta-/hexa-coordinated mixture, and the heme hexa-to-penta-coordination transition does not represent a rate-limiting step for carbonylation kinetics. Lastly, electron paramagnetic resonance indicates that ferrous nitrosylated GsGCS(162) is a penta-coordinated species, where the proximal HisF8-Fe bond is severed.

  7. Conditional beta1-integrin gene deletion in neural crest cells causes severe developmental alterations of the peripheral nervous system

    DEFF Research Database (Denmark)

    Pietri, Thomas; Eder, Olivier; Breau, Marie Anne;

    2004-01-01

    Integrins are transmembrane receptors that are known to interact with the extracellular matrix and to be required for migration, proliferation, differentiation and apoptosis. We have generated mice with a neural crest cell-specific deletion of the beta1-integrin gene to analyse the role of beta1-...

  8. Decreased gene expression of human beta-defensin-1 in the development of squamous cell carcinoma of the oral cavity.

    NARCIS (Netherlands)

    Wenghoefer, M.H.; Pantelis, A.; Dommisch, H.; Reich, R.; Martini, M.; Allam, J.P.; Novak, N.; Berge, S.; Jepsen, S.; Winter, J.

    2008-01-01

    The aim of this study was to investigate the gene expression of human beta-defensin-1, -2, -3 (hBD-1, -2, -3), interleukin-1beta, tumour necrosis factor-alpha and cyclooxygenase-2 in oral squamous cell carcinoma (OSCC) compared to benign and premalignant lesions as well as healthy controls. Biopsies

  9. Defective haematopoiesis in fetal liver resulting from inactivation of the EKLF gene.

    Science.gov (United States)

    Nuez, B; Michalovich, D; Bygrave, A; Ploemacher, R; Grosveld, F

    1995-05-25

    Erythroid Krüppel-like factor (EKLF) was originally isolated from erythroid cell RNA by differential screening and shown to be erythroid-specific, although a low level of EKLF was found in mast cell lines. EKLF contains three zinc-fingers homologous to those found in the Krüppel family of transcription factors. Because it binds the sequence CCACACCCT, EKLF may affect erythroid development as a result of its ability to bind to the CAC box in the promoter of the beta-globin gene. Mutation of this element leads to reduced beta-globin expression and it appears to mediate the effect of the globin locus control region on the promoter. Here we inactivate the EKLF gene through insertion of a lacZ reporter gene by homologous recombination in embryonic stem (ES) cells. Heterozygous EKLF+/- mice show that the reporter gene is expressed in a developmentally specific manner in all types of erythroblasts in the fetal liver and adult bone marrow. Homozygous EKLF-/- mice appear normal during the embryonic stage of haematopoiesis in the yolk sac, but develop a fatal anaemia during early fetal life when haematopoiesis has switched to the fetal liver. Enucleated erythrocytes are formed but these do not contain the proper amount of haemoglobin. We conclude that the transcription factor EKLF is essential for the final steps of definitive erythropoiesis in fetal liver.

  10. Isolation of novel human cDNA (hGMF-gamma) homologous to Glia Maturation Factor-beta gene.

    Science.gov (United States)

    Asai, K; Fujita, K; Yamamoto, M; Hotta, T; Morikawa, M; Kokubo, M; Moriyama, A; Kato, T

    1998-03-13

    A novel full-length human cDNA homologous to Glia Maturation Factor-beta (GMF-beta) gene was isolated. Sequence analysis of the entire cDNA revealed an open reading frame of 426 nucleotides with a deduced protein sequence of 142 amino acid residues. The deduced amino acid sequences of its putative product is highly homologous to human GMF-beta (82% identity) and named for GMF-gamma. Northern blot analysis indicated that a message of 0.9 kb long, but not 4.1 kb of GMF-beta, is predominantly expressed in human lung, heart, and placenta.

  11. Effect of polymorphic variants of GH, Pit-1, and beta-LG genes on milk production of Holstein cows.

    Science.gov (United States)

    Heidari, M; Azari, M A; Hasani, S; Khanahmadi, A; Zerehdaran, S

    2012-04-01

    Effect of polymorphic variants of growth hormone (GH), beta-lactoglobulin (beta-LG), and Pit-1 genes on milk yield was analyzed in a Holstein herd. Genotypes of the cows for these genes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Allele frequencies were 0.884 and 0.116 for L and V variants of GH, 0.170 and 0.830 for A and B variants of Pit-1, and 0.529 and 0.471 for A and B variants of beta-LG, respectively. GLM procedure of SAS software was used to test the effects of these genes on milk yield. Results indicated significant effects of these genes on milk yield (P LG gene, milk yield of animals with AA genotype was more than BB genotype (P LG (AA) were superior compared to heterozygote genotypes, whereas, the heterozygote genotype of Pit-1 gene (AB) was desirable.

  12. Características fenotípicas dos pacientes com anemia falciforme de acordo com os haplótipos do gene da βS-globina em Fortaleza, Ceará Phenotypic characteristics of patients with sickle cell anemia related to βS-Globin gene haplotypes in Fortaleza, Ceara

    Directory of Open Access Journals (Sweden)

    Lilianne B. Silva

    2010-02-01

    Full Text Available Foram analisados 47 pacientes com diagnóstico clínico, laboratorial e molecular de anemia falciforme, residentes em Fortaleza, Ceará, com a finalidade de fornecer informações sobre a influência dos haplótipos do gene da βS- globina nas características fenotípicas desta doença. A determinação dos valores hematológicos foi realizada em contador automático de células sanguíneas, e os níveis de HbF foram determinados pela técnica da desnaturação alcalina. O DNA foi isolado de leucócitos, a partir de amostras de sangue total. A análise dos haplótipos da mutação βS foi realizada por PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Lenght Polymorphism, sendo analisados seis sítios polimórficos de restrição. Os pacientes foram divididos em cinco grupos, de acordo com o tipo de haplótipo: Bantu/Bantu, Benin/Benin, Bantu/Benin, Bantu/Atípico e Benin/Atípico. O nível de significância considerado nas análises foi pWe analyzed 47 patients living in Fortaleza, Ceará with clinical, laboratory and molecular diagnosis of sickle cell anemia, in order to provide information on the influence of the βS-globin gene haplotypes on the phenotypic characteristics of this disease. The evaluation of hematological values was performed using an automated blood cell counter and the levels of HbF were determined by the alkali denaturation technique. The DNA was isolated from leukocytes from a whole blood sample. The analysis of the haplotypes of the βS mutation was achieved by PCR-RFLP, with an assessment of six polymorphic restriction sites. The patients were divided in 5 groups according to the type of haplotype: Bantu/Bantu, Benin/Benin, Bantu/Benin, Bantu/Atypical and Benin/Atypical. The level of significance was set for a p-value < 0.05. In the comparison between the haplotypes and the hematological characteristics, statistically significant differences were seen only for the values of HbF and Ht. The levels of HbF were

  13. Nuclear orphan receptor TLX affects gene expression, proliferation and cell apoptosis in beta cells.

    Science.gov (United States)

    Shi, Xiaoli; Xiong, Xiaokan; Dai, Zhe; Deng, Haohua; Sun, Li; Hu, Xuemei; Zhou, Feng; Xu, Yancheng

    Nuclear orphan receptor TLX is an essential regulator of the growth of neural stem cells. However, its exact function in pancreatic islet cells is still unknown. In the present study, gene expression profiling analysis revealed that overexpression of TLX in beta cell line MIN6 causes suppression of 176 genes and upregulation of 49 genes, including a cadre of cell cycle, cell proliferation and cell death control genes, such as Btg2, Ddit3 and Gadd45a. We next examined the effects of TLX overexpression on proliferation, apoptosis and insulin secretion in MIN6 cells. Proliferation analysis using EdU assay showed that overexpression of TLX increased percentage of EdU-positive cells. Cell cycle and apoptosis analysis revealed that overexpression of TLX in MIN6 cells resulted in higher percentage of cells exiting G1 into S-phase, and a 58.8% decrease of cell apoptosis induced by 0.5 mM palmitate. Moreover, TLX overexpression did not cause impairment of insulin secretion. Together, we conclude that TLX is among factors capable of controlling beta cell proliferation and survival, which may serve as a target for the development of novel therapies for diabetes.

  14. INS-gene mutations: from genetics and beta cell biology to clinical disease.

    Science.gov (United States)

    Liu, Ming; Sun, Jinhong; Cui, Jinqiu; Chen, Wei; Guo, Huan; Barbetti, Fabrizio; Arvan, Peter

    2015-04-01

    A growing list of insulin gene mutations causing a new form of monogenic diabetes has drawn increasing attention over the past seven years. The mutations have been identified in the untranslated regions of the insulin gene as well as the coding sequence of preproinsulin including within the signal peptide, insulin B-chain, C-peptide, insulin A-chain, and the proteolytic cleavage sites both for signal peptidase and the prohormone convertases. These mutations affect a variety of different steps of insulin biosynthesis in pancreatic beta cells. Importantly, although many of these mutations cause proinsulin misfolding with early onset autosomal dominant diabetes, some of the mutant alleles appear to engage different cellular and molecular mechanisms that underlie beta cell failure and diabetes. In this article, we review the most recent advances in the field and discuss challenges as well as potential strategies to prevent/delay the development and progression of autosomal dominant diabetes caused by INS-gene mutations. It is worth noting that although diabetes caused by INS gene mutations is rare, increasing evidence suggests that defects in the pathway of insulin biosynthesis may also be involved in the progression of more common types of diabetes. Collectively, the (pre)proinsulin mutants provide insightful molecular models to better understand the pathogenesis of all forms of diabetes in which preproinsulin processing defects, proinsulin misfolding, and ER stress are involved.

  15. Unexpected expression of α- and β-globin in mesencephalic dopaminergic neurons and glial cells

    Science.gov (United States)

    Biagioli, Marta; Pinto, Milena; Cesselli, Daniela; Zaninello, Marta; Lazarevic, Dejan; Roncaglia, Paola; Simone, Roberto; Vlachouli, Christina; Plessy, Charles; Bertin, Nicolas; Beltrami, Antonio; Kobayashi, Kazuto; Gallo, Vittorio; Santoro, Claudio; Ferrer, Isidro; Rivella, Stefano; Beltrami, Carlo Alberto; Carninci, Piero; Raviola, Elio; Gustincich, Stefano

    2009-01-01

    The mesencephalic dopaminergic (mDA) cell system is composed of two major groups of projecting cells in the substantia nigra (SN) (A9 neurons) and the ventral tegmental area (VTA) (A10 cells). A9 neurons form the nigrostriatal pathway and are involved in regulating voluntary movements and postural reflexes. Their selective degeneration leads to Parkinson's disease. Here, we report that gene expression analysis of A9 dopaminergic neurons (DA) identifies transcripts for α- and β-chains of hemoglobin (Hb). Globin immunoreactivity decorates the majority of A9 DA, a subpopulation of cortical and hippocampal astrocytes and mature oligodendrocytes. This pattern of expression was confirmed in different mouse strains and in rat and human. We show that Hb is expressed in the SN of human postmortem brain. By microarray analysis of dopaminergic cell lines overexpressing α- and β-globin chains, changes in genes involved in O2 homeostasis and oxidative phopshorylation were observed, linking Hb expression to mitochondrial function. Our data suggest that the most famed oxygen-carrying globin is not exclusively restricted to the blood, but it may play a role in the normal physiology of the brain and neurodegenerative diseases. PMID:19717439

  16. Comprehensive identification of single nucleotide polymorphisms associated with beta-lactam resistance within pneumococcal mosaic genes.

    Directory of Open Access Journals (Sweden)

    Claire Chewapreecha

    2014-08-01

    Full Text Available Traditional genetic association studies are very difficult in bacteria, as the generally limited recombination leads to large linked haplotype blocks, confounding the identification of causative variants. Beta-lactam antibiotic resistance in Streptococcus pneumoniae arises readily as the bacteria can quickly incorporate DNA fragments encompassing variants that make the transformed strains resistant. However, the causative mutations themselves are embedded within larger recombined blocks, and previous studies have only analysed a limited number of isolates, leading to the description of "mosaic genes" as being responsible for resistance. By comparing a large number of genomes of beta-lactam susceptible and non-susceptible strains, the high frequency of recombination should break up these haplotype blocks and allow the use of genetic association approaches to identify individual causative variants. Here, we performed a genome-wide association study to identify single nucleotide polymorphisms (SNPs and indels that could confer beta-lactam non-susceptibility using 3,085 Thai and 616 USA pneumococcal isolates as independent datasets for the variant discovery. The large sample sizes allowed us to narrow the source of beta-lactam non-susceptibility from long recombinant fragments down to much smaller loci comprised of discrete or linked SNPs. While some loci appear to be universal resistance determinants, contributing equally to non-susceptibility for at least two classes of beta-lactam antibiotics, some play a larger role in resistance to particular antibiotics. All of the identified loci have a highly non-uniform distribution in the populations. They are enriched not only in vaccine-targeted, but also non-vaccine-targeted lineages, which may raise clinical concerns. Identification of single nucleotide polymorphisms underlying resistance will be essential for future use of genome sequencing to predict antibiotic sensitivity in clinical microbiology.

  17. Insect and wound induced GUS gene expression from a Beta vulgaris proteinase inhibitor gene promoter

    Science.gov (United States)

    Inducible gene promoters that are specifically activated by pathogen invasion or insect pest attack are needed for effective expression of resistance genes to control plant diseases. In the present study, a promoter from a serine proteinase inhibitor gene (BvSTI) shown to be up-regulated in resist...

  18. Transcriptome analysis of sugar beet root maggot (Tetanops myopaeformis) genes modulated by the Beta vulgaris host.

    Science.gov (United States)

    Li, Haiyan; Smigocki, Ann C

    2016-10-03

    Sugar beet root maggot (SBRM, Tetanops myopaeformis von Röder) is a major but poorly understood insect pest of sugar beet (Beta vulgaris L.). The molecular mechanisms underlying plant defense responses are well documented, however, little information is available about complementary mechanisms for insect adaptive responses to overcome host resistance. To date, no studies have been published on SBRM gene expression profiling. Suppressive subtractive hybridization (SSH) generated more than 300 SBRM ESTs differentially expressed in the interaction of the pest with a moderately resistant (F1016) and a susceptible (F1010) sugar beet line. Blast2GO v. 3.2 search indicated that over 40% of the differentially expressed genes had known functions, primarily driven by fruit fly D. melanogaster genes. Expression patterns of 18 selected EST clones were confirmed by RT-PCR analysis. Gene Ontology (GO) analysis predicted a dominance of metabolic and catalytic genes involved in the interaction of SBRM with its host. SBRM genes functioning during development, regulation, cellular process, signaling and under stress conditions were annotated. SBRM genes that were common or unique in response to resistant or susceptible interactions with the host were identified and their possible roles in insect responses to the host are discussed.

  19. Effect of casein genes - beta-LGB, DGAT1, GH, and LHR - on milk production and milk composition traits in crossbred Holsteins.

    Science.gov (United States)

    Molee, A; Poompramun, C; Mernkrathoke, P

    2015-03-30

    The objectives of this study were to determine the effects of a single gene and composite genotype of the casein gene family, including the beta-lactoglobulin gene (beta-LGB), acyl-CoA: diacylglycerol acyltransferase 1 gene (DGAT1), growth hormone gene (GH), and luteinizing hormone receptor gene (LHR) on milk yield, milk composition, the percentage of fat, protein, solids-not-fat, and total solid in crossbred Holsteins. A total of 231 crossbred Holstein cows were examined for the study. The genotype of the beta-casein gene was analyzed by allele-specific polymerase chain reaction, while the alpha-S1, alpha-S2, kappa-casein, DGAT1, beta-LGB, and GH genes were analyzed using a polymerase chain reaction-restriction fragment length polymorphism assay. The association between genes and milk yield and milk composition was analyzed. Three pairs of genes, for which significant associations were detected, were beta + kappa-casein, DGAT1 + beta-casein, and GH + beta-LGB. In the single-gene model, most loci are significantly associated with traits. A significant association between the composite genotype and the traits was detected in all composite genotypes. GH + beta-LGB appears to be the most suitable variants for improving milk production and percentage of milk protein. Overall, the effects of the composite genotype and single gene were different. A physical or functional relationship between genes is necessary for investigating gene markers.

  20. Molecular cloning and analysis of zebrafish voltage-gated sodium channel beta subunit genes: implications for the evolution of electrical signaling in vertebrates

    Directory of Open Access Journals (Sweden)

    Zhong Tao P

    2007-07-01

    Full Text Available Abstract Background Action potential generation in excitable cells such as myocytes and neurons critically depends on voltage-gated sodium channels. In mammals, sodium channels exist as macromolecular complexes that include a pore-forming alpha subunit and 1 or more modulatory beta subunits. Although alpha subunit genes have been cloned from diverse metazoans including flies, jellyfish, and humans, beta subunits have not previously been identified in any non-mammalian species. To gain further insight into the evolution of electrical signaling in vertebrates, we investigated beta subunit genes in the teleost Danio rerio (zebrafish. Results We identified and cloned single zebrafish gene homologs for beta1-beta3 (zbeta1-zbeta3 and duplicate genes for beta4 (zbeta4.1, zbeta4.2. Sodium channel beta subunit loci are similarly organized in fish and mammalian genomes. Unlike their mammalian counterparts, zbeta1 and zbeta2 subunit genes display extensive alternative splicing. Zebrafish beta subunit genes and their splice variants are differentially-expressed in excitable tissues, indicating tissue-specific regulation of zbeta1-4 expression and splicing. Co-expression of the genes encoding zbeta1 and the zebrafish sodium channel alpha subunit Nav1.5 in Chinese Hamster Ovary cells increased sodium current and altered channel gating, demonstrating functional interactions between zebrafish alpha and beta subunits. Analysis of the synteny and phylogeny of mammalian, teleost, amphibian, and avian beta subunit and related genes indicated that all extant vertebrate beta subunits are orthologous, that beta2/beta4 and beta1/beta3 share common ancestry, and that beta subunits are closely related to other proteins sharing the V-type immunoglobulin domain structure. Vertebrate sodium channel beta subunit genes were not identified in the genomes of invertebrate chordates and are unrelated to known subunits of the para sodium channel in Drosophila. Conclusion The

  1. CK2(beta)tes gene encodes a testis-specific isoform of the regulatory subunit of casein kinase 2 in Drosophila melanogaster

    DEFF Research Database (Denmark)

    Kalmykova, Alla I; Shevelyov, Yuri Y; Polesskaya, Oksana O

    2002-01-01

    An earlier described CK2(beta)tes gene of Drosophila melanogaster is shown to encode a male germline specific isoform of regulatory beta subunit of casein kinase 2. Western-analysis using anti-CK2(beta)tes Ig revealed CK2(beta)tes protein in Drosophila testes extract. Expression of a CK2(beta...... and coimmunoprecipitation analysis of protein extract from Drosophila testes, we demonstrated an association between CK2(beta)tes and CK2alpha. Northern-analysis has shown that another regulatory (beta') subunit found recently in D. melanogaster genome is also testis-specific. Thus, we describe the first example of two...

  2. Gene expression profile of amyloid beta protein-injected mouse model for Alzheimer disease

    Institute of Scientific and Technical Information of China (English)

    Ling-na KONG; Ping-ping ZUO; Liang MU; Yan-yong LIU; Nan YANG

    2005-01-01

    Aim: To investigate the gene expression profile changes in the cerebral cortex of mice injected icv with amyloid beta-protein (Aβ) fragment 25-35 using cDNA microarray. Methods: Balb/c mice were randomly divided into a control group and Aβ-treated group. The Morris water maze test was performed to detect the effect of Aβ-injection on the learning and memory of mice. Atlas Mouse 1.2 Expression Arrays containing 1176 genes were used to investigate the gene expression pattern of each group. Results: The gene expression profiles showed that 19 genes including TBX1, NF-κB, AP-1/c-Jun, cadherin, integrin, erb-B2, and FGFR1 were up-regulated after 2 weeks oficv administration of Aβ; while 12 genes were downregulated, including NGF, glucose phosphate isomerase 1, AT motif binding factor 1, Na+/K+-ATPase, and Akt. Conclusions: The results provide important leads for pursuing a more complete understanding of the molecular events of Aβ-injection into mice with Alzheimer disease.

  3. Beta s gene in Central Iran is in linkage disequilibrium with the Indian-Arab haplotype.

    Science.gov (United States)

    Rahgozar, S; Poorfathollah, A A; Moafi, A R; Old, J M

    2000-11-01

    Sickle cell anemia is not considered to be a significant disease in Iran, although the sickle cell trait is estimated to have a high incidence in the Southern provinces. Since 1977, when the presence of a mild sickle cell anemia was reported in this country, there have been no further investigations published giving precise data on the incidence and origins of the sickle cell mutation in Iran. We report here the finding of patients with the sickle cell trait, sickle cell anemia, and sickle-beta thalassemia in Central Iran. A survey of 300 individuals from a village in Southeast Esfahan revealed an incidence of the sickle cell trait of 8.33%. "Cascade screening" enabled 96 relatives in four surrounding villages to be tested, and the at-risk couples were offered counseling as a "sickle cell control program." The hematological indices and HbF levels of the affected patients were determined. The HbF levels were unusually high, ranging from 18% to 41.4%, and SS patients with the highest levels were asymptomatic. Linkage analysis revealed the betaS gene haplotype in this population to be the Indian-Arab haplotype.

  4. Effect of interleukin-1beta gene functional polymorphism on dorsolateral prefrontal cortex activity in schizophrenic patients.

    Science.gov (United States)

    Papiol, Sergi; Molina, Vicente; Rosa, Araceli; Sanz, Javier; Palomo, Tomás; Fañanás, Lourdes

    2007-12-05

    Hypoactivity of the dorsolateral prefrontal cortex (DLPFC) during cognitive tasks is among the most consistent findings in schizophrenia. The biological factors contributing to this hypofrontality are only partially known. Previous reports have shown the influence of genes mapped to IL-1 cluster (i) in the risk to develop schizophrenia and (ii) on brain morphological abnormalities in these patients. Moreover, Interleukin-1beta (IL-1beta), encoded by IL-1B gene (IL-1 cluster, chromosome 2q13) has a key role in dopaminergic differentiation and dendrite growth in developing cortical neurons. The authors explored the role of a genetic functional polymorphism at IL-1B gene in relation to DLPFC activity. DLPFC (left and right) metabolic activity was measured in a sample of 19 DSM-IV diagnosed schizophrenic patients of Spanish origin using a procedure based on MRI/PET image fusion. During PET studies, subjects performed a contingent Continuous Performance Test aiming to activate DLPFC. Functional promoter polymorphism -511 C/T (rs16944) of IL-1B gene was genotyped in these patients. Those patients who were allele 2 (-511 T) carriers showed a lower metabolic activity in the left DLPFC with respect to patients homozygous for allele 1 (-511 C) (U = 16, z = -2.32, P = 0.02). Our results suggest that hypofrontality reported in some schizophrenic patients might be explained, at least in part, by this functional polymorphism at IL-1B gene. Genetic variants with influence on brain functionality may account for the neurocognitive heterogeneity observed in schizophrenic patients.

  5. Globin X is a six-coordinate globin that reduces nitrite to nitric oxide in fish red blood cells

    Science.gov (United States)

    Corti, Paola; Xue, Jianmin; Tejero, Jesús; Wajih, Nadeem; Sun, Ming; Stolz, Donna B.; Tsang, Michael; Kim-Shapiro, Daniel B.; Gladwin, Mark T.

    2016-01-01

    The discovery of novel globins in diverse organisms has stimulated intense interest in their evolved function, beyond oxygen binding. Globin X (GbX) is a protein found in fish, amphibians, and reptiles that diverged from a common ancestor of mammalian hemoglobins and myoglobins. Like mammalian neuroglobin, GbX was first designated as a neuronal globin in fish and exhibits six-coordinate heme geometry, suggesting a role in intracellular electron transfer reactions rather than oxygen binding. Here, we report that GbX to our knowledge is the first six-coordinate globin and the first globin protein apart from hemoglobin, found in vertebrate RBCs. GbX is present in fish erythrocytes and exhibits a nitrite reduction rate up to 200-fold faster than human hemoglobin and up to 50-fold higher than neuroglobin or cytoglobin. Deoxygenated GbX reduces nitrite to form nitric oxide (NO) and potently inhibits platelet activation in vitro, to a greater extent than hemoglobin. Fish RBCs also reduce nitrite to NO and inhibit platelet activation to a greater extent than human RBCs, whereas GbX knockdown inhibits this nitrite-dependent NO signaling. The description of a novel, six-coordinate globin in RBCs with dominant electron transfer and nitrite reduction functionality provides new insights into the evolved signaling properties of ancestral heme-globins. PMID:27407144

  6. Pseudomonas aeruginosa biofilms exposed to imipenem exhibit changes in global gene expression and beta-lactamase and alginate production

    DEFF Research Database (Denmark)

    Bagge, N.; Schuster, M.; Hentzer, Morten

    2004-01-01

    . As expected, the most strongly induced gene was ampC, which codes for chromosomal beta-lactamase. We also found that genes coding for alginate biosynthesis were induced by exposure to imipenem. Alginate production is correlated to the development of impaired lung function, and P. aeruginosa strains isolated...

  7. Characterization of the molecularly cloned murine alpha-globin transcription factor CP2.

    Science.gov (United States)

    Lim, L C; Fang, L; Swendeman, S L; Sheffery, M

    1993-08-25

    We recently cloned human and murine cDNAs that encode CP2, a transcription factor that interacts with the murine alpha-globin promoter. In this report, we exploited our ability to express CP2 in bacteria and eukaryotic cells to further investigate factor activities in vitro and in vivo. CP2 expressed in bacteria was significantly enriched and used in a series of DNase I footprinting and electrophoretic gel shift assays. The results suggest that CP2 binds a hyphenated recognition sequence motif that spans one DNA helix turn. In addition, the enriched bacterial protein activated transcription of alpha-globin promoter templates approximately 3- to 4-fold in vitro. We then tested the effect of elevating CP2 levels 2.5- to 5.5-fold in vivo using both transient and stable transformation assays. When a reporter construct comprised of the intact murine alpha-globin promoter driving the bacterial chloramphenicol acetyltransferase (CAT) gene was introduced into these overexpressing cells, we observed a 3- to 6-fold increase in CAT activity when compared to cells expressing normal levels of CP2. These results define the CP2 factor binding site in more detail and help characterize the activities of the factor in vivo.

  8. Detection of genes mediating beta-lactamase production in isolates of enterobacteria recovered from wild pets in Saudi Arabia

    Directory of Open Access Journals (Sweden)

    Sabry A. Hassan

    2015-12-01

    Full Text Available Aim: To determine the genetic basis and types of beta-lactamase encountered among enterobacterial isolates of wild pets from the animal exhibit. Materials and Methods: A total of 17 beta-lactamase-producing enterobacteria recovered from fecal samples of wild pet animals were analyzed for a selected beta-lactamase gene by polymerase chain reaction. Results: Molecular analysis identified one or more β-lactamase-encoding genes in 14 enterobacterial isolates as a single or gene combination. The most frequent extended-spectrum β-lactamases types were transmission electron microscopy and CTX-M, and the most common AmpC enzymes were CMY-2 and DHA types. Conclusions: The study is the first in Saudi Arabia, have established the presence of β-lactamase-encoding genes in the fecal isolates of wild pets.

  9. Inefficiency in GM2 ganglioside elimination by human lysosomal beta-hexosaminidase beta-subunit gene transfer to fibroblastic cell line derived from Sandhoff disease model mice.

    Science.gov (United States)

    Itakura, Tomohiro; Kuroki, Aya; Ishibashi, Yasuhiro; Tsuji, Daisuke; Kawashita, Eri; Higashine, Yukari; Sakuraba, Hitoshi; Yamanaka, Shoji; Itoh, Kohji

    2006-08-01

    Sandhoff disease (SD) is an autosomal recessive GM2 gangliosidosis caused by the defect of lysosomal beta-hexosaminidase (Hex) beta-subunit gene associated with neurosomatic manifestations. Therapeutic effects of Hex subunit gene transduction have been examined on Sandhoff disease model mice (SD mice) produced by the allelic disruption of Hexb gene encoding the murine beta-subunit. We demonstrate here that elimination of GM2 ganglioside (GM2) accumulated in the fibroblastic cell line derived from SD mice (FSD) did not occur when the HEXB gene only was transfected. In contrast, a significant increase in the HexB (betabeta homodimer) activity toward neutral substrates, including GA2 (asialo-GM2) and oligosaccharides carrying the terminal N-acetylglucosamine residues at their non-reducing ends (GlcNAc-oligosaccharides) was observed. Immunoblotting with anti-human HexA (alphabeta heterodimer) serum after native polyacrylamide gel electrophoresis (Native-PAGE) revealed that the human HEXB gene product could hardly form the chimeric HexA through associating with the murine alpha-subunit. However, co-introduction of the HEXA encoding the human alpha-subunit and HEXB genes caused significant corrective effect on the GM2 degradation by producing the human HexA. These results indicate that the recombinant human HexA could interspeciesly associate with the murine GM2 activator protein to degrade GM2 accumulated in the FSD cells. Thus, therapeutic effects of the recombinant human HexA isozyme but not human HEXB gene product could be evaluated by using the SD mice.

  10. Structural and functional properties of designed globins

    Indian Academy of Sciences (India)

    Yasuhiro Isogai; Anna Ishii; Manabu Ishida; Masahiro Mukai; Motonori Ota; Ken Nishikawa; Tetsutaro Iizuka

    2000-06-01

    De novo design of artificial proteins is an essential approach to elucidate the principles of protein architecture and to understand specific functions of natural proteins and also to yield novel molecules for medical and industrial aims. We have designed artificial sequences of 153 amino acids to fit the main-chain framework of the sperm whale myoglobin structure based on the knowledge-based energy functions to evaluate the compatibility between protein tertiary structures and amino acid sequences. The synthesized artificial globins bind a single heme per protein molecule as designed, which show well-defined electrochemical and spectroscopic features characteristic of proteins with a low-spin heme. Redox and ligand binding reactions of the artificial heme proteins were investigated and these heme-related functions were found to vary with their structural uniqueness. Relationships between the structural and functional properties are discussed.

  11. Genomic organization of the human {beta}-catenin gene (CTNNB1)

    Energy Technology Data Exchange (ETDEWEB)

    Nollet, F.; Berx, G.; Molemans, F.; Roy, F. van [Univ. of Ghent (Belgium)

    1996-03-05

    The cytoplasmic {beta}-catenin protein is implicated in signal transduction and associates with both the cell-cell adhesion protein E-cadherin and the tumor suppressor gene product APC. We determined the primary structure of the human {beta}-catenin gene (CTNNB1) by analysis cDNA and genomic clones. The size of the complete gene was determined to be 23.2 kb. Restriction mapping and partial sequence analysis revealed 16 exons. All splice donor and acceptor sites were conformable to the GT/AG rule. The exon size ranged from 61 to 790 bp. Half of the introns were smaller than 550 bp, with the smallest being 84 pb and the longest being 6700 bp. The intron-exon boundaries did not coincide either with conserved sites in the 12 armadillo repeat sequences of {beta}-catenin or with intron-exon boundaries in the armadillo gene of Drosophila. A major site for transcription initiation was identified as an A residue 214 nucleotides upstream of the ATG initiation codon. The resulting transcript is 3362 nucleotides long. Compared to the previously published mRNA sequence, additional residues were identified, 16 at the 5{prime} end and 766 at the 3{prime} end of the mRNA. An alternative splice acceptor site within exon 16 reduced the 3{prime} UTR sequence by 159 bp. Polymerase chain reaction on cDNA from 14 human cell lines demonstrated the general occurrence of both splice variants. The 5{prime}-flanking region is highly GC-rich and lacks a CCAAT box, but contains a TATA box and potential binding sites for several transcription factors, such as NFkB, SP1, AP2, and EGR1. Both a 437-bp fragment and a 6-kb fragment, containing about 4.7 kb of the 5{prime}-flanking region in addition to the noncoding exon 1 and 1 kb of intron 1, showed clear promoter activity when these fragments were linked to a secreted alkaline phosphatase reporter gene and transfected into a mouse epithelial cell line. 53 refs., 5 figs., 2 tabs.

  12. Detection of Amp C genes encoding for beta-lactamases in Escherichia coli and Klebsiella pneumoniae

    Directory of Open Access Journals (Sweden)

    M Shanthi

    2012-01-01

    Full Text Available Purpose : Amp C beta-lactamase are Ambler class C enzymes that confer resistance to extended spectrum cephalosporins and are not inhibited by beta-lactamase inhibitors. Their detection is crucial, since the phenotypic tests are not standardised leading to ambiguity in interpretation of results. This study was done to detect the types of Amp C prevalent in Escherichia coli and Klebsiella pneumoniae by multiplex polymerase chain reaction (PCR. Materials and Methods : Seventy-seven consecutive cefoxitin resistant clinical isolates of E. coli (n = 25 and K. pneumoniae (n = 52 were included in the study. Antibiotic susceptibility testing to various classes of antibiotics was performed by disc diffusion using Clinical Laboratory Standards Institute (CLSI guidelines. Minimum inhibitory concentration (MIC to cefoxitin, imipenem and meropenem were determined by broth microdilution method. Isolates were screened for production of Extended Spectrum Beta-Lactamase (ESBL. Multiplex PCR was performed for the detection of Amp C genes after phenotypic testing (Hodge test and inhibitor based test. Results : Cefoxitin Hodge test was positive in 40 isolates which included 20 E. coli and 20 K. pneumoniae. There was zone enhancement with boronic acid in 55 isolates, of which 36 were K. pneumoniae and 19 were E. coli. Multiplex PCR detected Amp C in 11/25 E. coli and 12/52 K. pneumoniae isolates. The Amp C genes detected were CIT (Amp C origin - Citrobacter freundii, DHA (Dhahran Hospital, Saudi Arabia, ACC (Ambler class C, EBC (Amp C origin - Enterobacter cloacae groups. ESBL was co-produced in 54 isolates. Conclusions : Amp C was detected in 29.87% of the study isolates. Majority of them co-produced ESBL. The most common Amp C was the CIT family. Screen tests for cefoxitin resistance may be falsely positive due to production of carbapenamases.

  13. Expression of herpes simplex virus. beta. and. gamma. genes integrated in mammalian cells and their induction by an. cap alpha. gene product

    Energy Technology Data Exchange (ETDEWEB)

    Sandri-Goldin, R.M.; Goldin, A.L.; Holland, L.E.

    1983-11-01

    The proteins of herpes simplex virus type 1 (HSV-1) form three kinetic groups termed ..cap alpha..,..beta..,and ..gamma.., whose synthesis is regulated in a cascade fashion, ..cap alpha.. products are synthesized first during infection, and they are required for synthesis of ..beta.. and ..gamma.. proteins. To examine the expression of several HSV-1 ..beta.. and ..gamma.. genes in the absence of ..cap alpha.. functions, we transferred into mammalian cells a plasmid containing a region of the HSV-1 genome that codes for only ..beta.. and ..gamma.. genes (0.315 to 0.421 map units). The authors found stable integration of at least one copy of the intact plasmid in each cell line. Four HSV-1 transcripts of the ..beta.. and ..gamma.. classes were transcribed constitutively in the cells, including the genes for glycoprotein B and DNA-binding protein. No constitutive synthesis of these two proteins could be demonstrated, however. The integrated HSV-1 genes responded to viral regulatory signals in that they could be induced by infection with HSV-1 mutants resulting in a high level of synthesis of both glycoprotein B and DNA-binding protein. The HSV-1 ..cap alpha.. gene product ICP4 was necessary for this induction, and it was found to be most efficient at a low multiplicity of infection. Functional expression of four genes was demonstrated in that the cell lines complemented infecting HSV-1 temperature-sensitive mutants. The same genes were not available for homologous recombination with infecting virus, however, since no recombinant wild-type virus could be detected. These data demonstrate that HSV-1 ..beta.. and ..gamma.. genes can be transcribed in the absence of ..cap alpha.. functions in mammalian cells, but that they still respond to HSV-1 regulatory signals such as the ..cap alpha.. gene product ICP4.

  14. Pancreatic beta-cell overexpression of the glucagon receptor gene results in enhanced beta-cell function and mass

    DEFF Research Database (Denmark)

    Gelling, Richard W; Vuguin, Patricia M; Du, Xiu Quan

    2009-01-01

    in response to glucagon and glucose, the glucose excursion resulting from both a glucagon challenge and intraperitoneal glucose tolerance test (IPGTT) was significantly reduced in RIP-Gcgr mice compared with controls. However, RIP-Gcgr mice display similar glucose responses to an insulin challenge. beta...... in vivo, we generated mice overexpressing the Gcgr specifically on pancreatic beta-cells (RIP-Gcgr). In vivo and in vitro insulin secretion in response to glucagon and glucose was increased 1.7- to 3.9-fold in RIP-Gcgr mice compared with controls. Consistent with the observed increase in insulin release...... and impaired glucose tolerance (IGT) were reduced in RIP-Gcgr mice compared with controls. Furthermore, the insulin response of RIP-Gcgr mice to an IPGTT was twice that of controls when fed the HFD. These data indicate that increased pancreatic beta-cell expression of the Gcgr increased insulin secretion...

  15. Beta-thalassemia

    Directory of Open Access Journals (Sweden)

    Origa Raffaella

    2010-05-01

    , deletions in the beta globin gene on chromosome 11, leading to reduced (beta+ or absent (beta0 synthesis of the beta chains of hemoglobin (Hb. Transmission is autosomal recessive; however, dominant mutations have also been reported. Diagnosis of thalassemia is based on hematologic and molecular genetic testing. Differential diagnosis is usually straightforward but may include genetic sideroblastic anemias, congenital dyserythropoietic anemias, and other conditions with high levels of HbF (such as juvenile myelomonocytic leukemia and aplastic anemia. Genetic counseling is recommended and prenatal diagnosis may be offered. Treatment of thalassemia major includes regular RBC transfusions, iron chelation and management of secondary complications of iron overload. In some circumstances, spleen removal may be required. Bone marrow transplantation remains the only definitive cure currently available. Individuals with thalassemia intermedia may require splenectomy, folic acid supplementation, treatment of extramedullary erythropoietic masses and leg ulcers, prevention and therapy of thromboembolic events. Prognosis for individuals with beta-thalassemia has improved substantially in the last 20 years following recent medical advances in transfusion, iron chelation and bone marrow transplantation therapy. However, cardiac disease remains the main cause of death in patients with iron overload.

  16. Beta-thalassemia.

    Science.gov (United States)

    Galanello, Renzo; Origa, Raffaella

    2010-05-21

    globin gene on chromosome 11, leading to reduced (beta+) or absent (beta0) synthesis of the beta chains of hemoglobin (Hb). Transmission is autosomal recessive; however, dominant mutations have also been reported. Diagnosis of thalassemia is based on hematologic and molecular genetic testing. Differential diagnosis is usually straightforward but may include genetic sideroblastic anemias, congenital dyserythropoietic anemias, and other conditions with high levels of HbF (such as juvenile myelomonocytic leukemia and aplastic anemia). Genetic counseling is recommended and prenatal diagnosis may be offered. Treatment of thalassemia major includes regular RBC transfusions, iron chelation and management of secondary complications of iron overload. In some circumstances, spleen removal may be required. Bone marrow transplantation remains the only definitive cure currently available. Individuals with thalassemia intermedia may require splenectomy, folic acid supplementation, treatment of extramedullary erythropoietic masses and leg ulcers, prevention and therapy of thromboembolic events. Prognosis for individuals with beta-thalassemia has improved substantially in the last 20 years following recent medical advances in transfusion, iron chelation and bone marrow transplantation therapy. However, cardiac disease remains the main cause of death in patients with iron overload.

  17. Cochlear implantation effect on deaf children with gap junction protein beta 2 gene mutation

    Institute of Scientific and Technical Information of China (English)

    KONG Ying; LIU Sha; WANG Su-ju; Li Shu-jing; LIANG Shuang

    2013-01-01

    Background The popularization and promotion of gene diagnosis technology makes it possible to detect deafness genes for children with congenital hearing impairment,and the proportion of gap junction protein beta 2 (GJB2) gene mutations in cochlear implant patients is 26.5% We did follow-up evaluation on auditory rehabilitation effect for all 31 deaf children with GJB2 gene mutation after cochlear implantation to provide a reference for such patients.Methods Application of “the genetic deafness gene chip detection kit” and “gene complete sequence analysis” were applied to conduct detection on common genetic deafness gene mutation hotspots of the hearing impaired children with cochlear implantation.To conduct auditory rehabilitation effect evaluation on all 31 cases of patients with GJB2 genetic deafness after 3,6 and 12 months of the operation respectively.The single factor repeated measure analysis of variance (ANOVA) was applied to analysis whether there were significant difference among the results of initial consonant of a Chinese syllable recognition at 3 different stages after the operation,the results of vowel of a Chinese syllable recognition at 3 different stages after the operation,and the results of two-syllable recognition at 3 different stages after the operation.Results The 235delC is the high-incidence mutational site in 31 cases of patients with GJB2 genetic deafness,and the total detection rate is up to 90.3% (28/31).There were significant differences in the initial consonant and the vowel of a Chinese syllable recognition rate,and the two-syllable recognition rates at 3,6,and 12 months after the operation (P<0.01).Conclusion Cochlear implantation is a safe and effective measure for auditory reconstruction,enabling patients with GJB2 hereditary severe sensorineural deafness to achieve auditory speech recognition effectively.

  18. Massive parallel gene expression profiling of RINm5F pancreatic islet beta-cells stimulated with interleukin-1beta

    DEFF Research Database (Denmark)

    Rieneck, K; Bovin, L F; Josefsen, K

    2000-01-01

    Interleukin 1 (IL-1) is a pleiotropic cytokine with the potential to kill pancreatic beta-cells, and this unique property is thought to be involved in the pathogenesis of type I diabetes mellitus. We therefore determined the quantitative expression of 24,000 mRNAs of RINm5F, an insulinoma cell line...

  19. Candidate Gene Study of TRAIL and TRAIL Receptors: Association with Response to Interferon Beta Therapy in Multiple Sclerosis Patients

    Science.gov (United States)

    Órpez-Zafra, Teresa; Pinto-Medel, María Jesús; Oliver-Martos, Begoña; Ortega-Pinazo, Jesús; Arnáiz, Carlos; Guijarro-Castro, Cristina; Varadé, Jezabel; Álvarez-Lafuente, Roberto; Urcelay, Elena; Sánchez-Jiménez, Francisca

    2013-01-01

    TRAIL and TRAIL Receptor genes have been implicated in Multiple Sclerosis pathology as well as in the response to IFN beta therapy. The objective of our study was to evaluate the association of these genes in relation to the age at disease onset (AAO) and to the clinical response upon IFN beta treatment in Spanish MS patients. We carried out a candidate gene study of TRAIL, TRAILR-1, TRAILR-2, TRAILR-3 and TRAILR-4 genes. A total of 54 SNPs were analysed in 509 MS patients under IFN beta treatment, and an additional cohort of 226 MS patients was used to validate the results. Associations of rs1047275 in TRAILR-2 and rs7011559 in TRAILR-4 genes with AAO under an additive model did not withstand Bonferroni correction. In contrast, patients with the TRAILR-1 rs20576-CC genotype showed a better clinical response to IFN beta therapy compared with patients carrying the A-allele (recessive model: p = 8.88×10−4, pc = 0.048, OR = 0.30). This SNP resulted in a non synonymous substitution of Glutamic acid to Alanine in position 228 (E228A), a change previously associated with susceptibility to different cancer types and risk of metastases, suggesting a lack of functionality of TRAILR-1. In order to unravel how this amino acid change in TRAILR-1 would affect to death signal, we performed a molecular modelling with both alleles. Neither TRAIL binding sites in the receptor nor the expression levels of TRAILR-1 in peripheral blood mononuclear cell subsets (monocytes, CD4+ and CD8+ T cells) were modified, suggesting that this SNP may be altering the death signal by some other mechanism. These findings show a role for TRAILR-1 gene variations in the clinical outcome of IFN beta therapy that might have relevance as a biomarker to predict the response to IFN beta in MS. PMID:23658636

  20. Candidate gene study of TRAIL and TRAIL receptors: association with response to interferon beta therapy in multiple sclerosis patients.

    Directory of Open Access Journals (Sweden)

    Carlos López-Gómez

    Full Text Available TRAIL and TRAIL Receptor genes have been implicated in Multiple Sclerosis pathology as well as in the response to IFN beta therapy. The objective of our study was to evaluate the association of these genes in relation to the age at disease onset (AAO and to the clinical response upon IFN beta treatment in Spanish MS patients. We carried out a candidate gene study of TRAIL, TRAILR-1, TRAILR-2, TRAILR-3 and TRAILR-4 genes. A total of 54 SNPs were analysed in 509 MS patients under IFN beta treatment, and an additional cohort of 226 MS patients was used to validate the results. Associations of rs1047275 in TRAILR-2 and rs7011559 in TRAILR-4 genes with AAO under an additive model did not withstand Bonferroni correction. In contrast, patients with the TRAILR-1 rs20576-CC genotype showed a better clinical response to IFN beta therapy compared with patients carrying the A-allele (recessive model: p = 8.88×10(-4, pc = 0.048, OR = 0.30. This SNP resulted in a non synonymous substitution of Glutamic acid to Alanine in position 228 (E228A, a change previously associated with susceptibility to different cancer types and risk of metastases, suggesting a lack of functionality of TRAILR-1. In order to unravel how this amino acid change in TRAILR-1 would affect to death signal, we performed a molecular modelling with both alleles. Neither TRAIL binding sites in the receptor nor the expression levels of TRAILR-1 in peripheral blood mononuclear cell subsets (monocytes, CD4+ and CD8+ T cells were modified, suggesting that this SNP may be altering the death signal by some other mechanism. These findings show a role for TRAILR-1 gene variations in the clinical outcome of IFN beta therapy that might have relevance as a biomarker to predict the response to IFN beta in MS.

  1. beta-catenin siRNA regulation of apoptosis- and angiogenesis-related gene expression in hepatocellular carcinoma cells: potential uses for gene therapy.

    Science.gov (United States)

    Wang, Xin-Hong; Sun, Xun; Meng, Xiang-Wei; Lv, Zhi-Wu; Du, Ya-Ju; Zhu, Yan; Chen, Jing; Kong, De-Xia; Jin, Shi-Zhu

    2010-10-01

    The molecular mechanism responsible for hepatocellular carcinoma (HCC) development remains to be defined although a number of gene pathways have been shown to play an active role, such as Wnt/beta-catenin signaling. In this study, beta-catenin small interfering RNA (siRNA) was designed, synthesized, and transfected into HCC HepG2 cells. RT-PCR and western blot assays were performed to detect expression of altered genes and proteins, and the MTT assay was used to detect cell viability. Our data showed that beta-catenin mRNA and protein expression levels were effectively knocked down by beta-catenin siRNA and subsequently, tumor cell proliferation was significantly suppressed. Flow cytometry assay showed that tumor cells were arrested at the G0/G1 phase of the cell cycles. Molecularly, expression of Smad3, p-caspase-3, and Grp78 protein were upregulated after 72 h of beta-catenin siRNA transfection, whereas expression of TERT, caspase-3, XIAP, MMP-2, MMP-9, VEGF-A, VEGF-c, and bFGF protein were reduced. However, there was no change between the expression of STAT3 and the HSP27 protein following transfection. The results from the current study demonstrated the importance of the Wnt/beta-catenin signaling pathway in regulation of gene expression in HCC. Further studies are required to investigate the role of this pathway in HCC development and targeting of this pathway to control HCC.

  2. Detection of a major gene for heterocellular hereditary persistence of fetal hemoglobin after accounting for genetic modifiers

    Energy Technology Data Exchange (ETDEWEB)

    Thein, S.L.; Weatherall, D.J. (Institute of Molecular Medicine, Oxford (United Kingdom)); Sampietro, M.; Rohde, K.; Rochette, J.; Lathrop, G.M.; Demenais, F.

    1994-02-01

    [open quotes]Heterocellular hereditary persistence of fetal hemoglobin[close quotes] (HPFH) is the term used to describe the genetically determined persistence of fetal hemoglobin (Hb F) production into adult life, in the absence of any related hematological disorder. Whereas some forms are caused by mutations in the [beta]-globin gene cluster on chromosome 11, others segregate independently. While the latter are of particular interest with respect to the regulation of globin gene switching, it has not been possible to determine their chromosomal location, mainly because their mode of inheritance is not clear, but also because several other factors are known to modify Hb F production. The authors have examined a large Asian Indian pedigree which includes individuals with heterocellular HPFH associated with [beta]-thalassemia and/or [alpha]-thalassemia. Segregation analysis was conducted on the HPFH trait FC, defined to be the percentage of Hb F-containing cells (F-cells), using the class D regressive model. The results provide evidence for the presence of a major gene, dominant or codominant, which controls the FC values with residual familial correlations. The major gene was detected when the effects of genetic modifiers, notably [beta]-thalassemia and the XmnI-[sup G][gamma] polymorphism, are accounted for in this analysis. Linkage with the [beta]-globin gene cluster is excluded. The transmission of the FC values in this pedigree is informative enough to allow detection of linkage with an appropriate marker(s). The analytical approach outlined in this study, using simple regression to allow for genetic modifiers and thus allowing the mode of inheritance of a trait to be dissected out, may be useful as a model for segregation and linkage analyses of other complex phenotypes. 39 refs., 4 figs., 6 tabs.

  3. DNA methylation of retrotransposons, DNA transposons and genes in sugar beet (Beta vulgaris L.).

    Science.gov (United States)

    Zakrzewski, Falk; Schmidt, Martin; Van Lijsebettens, Mieke; Schmidt, Thomas

    2017-03-03

    The methylation of cytosines shapes the epigenetic landscape of plant genomes, coordinates transgenerational epigenetic inheritance, represses activity of transposable elements (TEs), affects gene expression, and, hence, can influence the phenotype. Sugar beet (Beta vulgaris ssp. vulgaris), an important crop that accounts for 30% of the worldwide sugar needs, has a relatively small genome size (758 Mbp) consisting of approximately 485 Mbp repetitive DNA (64%) in particular, satellite DNA, retrotransposons, and DNA transposons. Genome-wide cytosine methylation in the sugar beet genome was studied in leaves and leaf-derived callus with a focus on repetitive sequences, including retrotransposons and DNA transposons, the major groups of repetitive DNA sequences and compared with gene methylation. Genes showed a specific methylation pattern for CG, CHG (H=A, C, and T), and CHH sites, whereas the TE pattern differed, depending on the classes 1 (retrotransposons) and 2 (DNA transposons), respectively. Along genes and TEs, the CG and CHG methylation was higher than that of adjacent genomic regions. In contrast to the relatively low CHH methylation in retrotransposons and genes, the level of CHH methylation in DNA transposons was strongly increased, pointing toward a functional role of asymmetric methylation in DNA transposon silencing. Comparison of genome-wide DNA methylation between sugar beet leaves and callus revealed a differential methylation upon tissue culture. Potential epialleles were hypomethylated (lower methylation) at CG and CHG sites in retrotransposons and genes and hypermethylated (higher methylation) at CHH sites in DNA transposons of callus when compared to leaves. This article is protected by copyright. All rights reserved.

  4. Mutation analysis of beta thalassemia gene in Guangxi Zhuang Autonomous Region%广西壮族自治区β-地中海贫血基因突变分析

    Institute of Scientific and Technical Information of China (English)

    张强; 范歆; 何升; 唐燕青; 陈秋丽; 郑陈光

    2014-01-01

    目的 分析广西壮族自治区(简称广西)常见、罕见类型β-地中海贫血(简称地贫)基因突变的发生情况,为减少漏诊、误诊提供临床帮助.方法 2010年1月至2013年12月,选取在广西妇幼保健院就诊及转诊的可疑地贫患者42 770例,其中男性20 740例,女性22 030例.年龄范围在1个月~ 54岁.所有患者进行血常规,血红蛋白电泳,血清铁、铁蛋白筛查;筛查阳性患者通过反向点杂交(RDB)进行基因诊断;筛查阳性但基因诊断常见基因未发现突变位点的患者进行β珠蛋白基因扩增并测序.结果 42 770名可疑地贫患者中,共确诊地贫患者28 101例,其中β-地贫患者10 891例,包括β-地贫纯合子49例,杂合子10 718例,双重 杂合子124例;患者进行β珠蛋白基因扩增并测序后,发现14种南方常见类型地贫,7种罕见β-地贫突变类型,罕见地贫检出率为17.949%(7/39).结论 广西地区β-地贫突变类型复杂多样;常规地贫基因检测正常但有地贫表型的患者,应对其进行罕见地贫的检测,减少漏诊,以便更好地为临床开展地贫诊断提供帮助.%Objective To investigate the occurrence of regular and rare types of beta thalassemia in Guangxi,and to reduce the misdiagnosis and missed diagnosis.Methods Between Jan 2010 and Dec 2013,42 770 patients (20 740 males and 22 030 females,one month to fifty-four years old) from Maternal and Child Healthy Hospital of Guangxi,who were suspected with thalassemia were involved in this study.All these patients were went through the following screening tests:routine blood cell count,hemoglobin electrophoresis test,and serum iron and ferritin tests.Positive patients in the screening test would be taken gene diagnosis with regular reverse dot blot (RDB) method; negative patients in gene diagnosis but positive in the screening test would be under the test of beta globin gene sequencing.Results Totally 28 101 patients were confirmed with thalassemia from 42

  5. Molecular analysis of T-cell receptor beta genes in cutaneous T-cell lymphoma reveals Jbeta1 bias.

    Science.gov (United States)

    Morgan, Suzanne M; Hodges, Elizabeth; Mitchell, Tracey J; Harris, Susan; Whittaker, Sean J; Smith, John L

    2006-08-01

    Molecular characterization of T-cell receptor junctional region sequences in cutaneous T-cell lymphoma had not been previously reported. We have examined in detail the features of the T-cell receptor beta (TCRB) gene rearrangements in 20 individuals with well-defined stages of cutaneous T-cell lymphoma (CTCL) comprising 10 cases with early-stage mycosis fungoides (MF) and 10 cases with late-stage MF or Sezary syndrome. Using BIOMED-2 PCR primers, we detected a high frequency of clonally rearranged TCR gamma and TCRB genes (17/20 and 15/20 cases, respectively). We carried out sequencing analysis of each complete clonal variable (V)beta-diversity (D)beta-joining(J)beta fingerprint generated by PCR amplification, and determined the primary structure of the Vbeta-Dbeta-Jbeta junctional regions. We observed considerable diversity in the T-cell receptor Vbeta gene usage and complementarity-determining region 3 loops. Although we found that TCRB gene usage in CTCL and normal individuals share common features, our analysis also revealed preferential usage of Jbeta1 genes in all cases with advanced stages of disease.

  6. TGF-beta 1 Gene-Activated Matrices Regulated the Osteogenic Differentiation of BMSCs

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Poly (lactic acid/glycolic acid/asparagic acid-co-polyethylene glycol)(PLGA-[ASP-PEG]) scaffold materials were linked with a novel nonviral vector (K)16GRGDSPC through cross linker Sulfo-LC-SPDP to construct a new type of nonviral gene transfer system. Eukaryotic expressing vector containing transforming growth factor beta 1 (pcDNA3-TGFβ1) was encapsulated by the system. Bone marrow stromal cells (BMSCs) obtained from rabbit were cultured on PLGA-[ASP-PEG] modified by (K)16GRGDSPC and TGF-β1 gene and PLGA-[ASP-PEG] modified by (K)16GRGDSPC and empty vector pcDNA3 as control.The expressions of osteogenic makers of the BMSCs cultured on the TGF-β1 gene-activated scaffold materials were found significantly higher than those of the control group (P<0.05). A brand-new way was provided for regulating seed cells to directionally differentiate into osteoblasts for bone defect restoration in bone tissue engineering.

  7. Relationship Between Polymorphism of Cystathionine beta Synthase Gene and Congenital Heart Disease in Chinese Nuclear Families

    Institute of Scientific and Technical Information of China (English)

    XIAO-MING SONG; XIAO-YING ZHENG; WEN-LI ZHU; LEI HUANG; YONG LI

    2006-01-01

    Objective To study the relationship between polymorphism of cystathionine beta synthase (CBS) gene and development of congenital heart disease (CHD). Methods One hundred and twenty-seven CHD case-parent triads were recruited from Liaoning Province as patient group, and 129 healthy subjects without family history of birth defect were simultaneously recruited as control group together with their biological parents. For all subjects the polymorphism of CBS gene G919A locus was examined by PCR-ARMS method. Results The frequencies of three genotypes (w/w, w/m, and m/m) in control group were 27.2%, 58.4%, and 14.4%, respectively, with no significant difference in gender. A significant difference in the allele frequency was found between CHD patients and controls, the wild allele frequency was 67.9% in patients and 55.7% in controls.CHD parents' genotype distribution was significantly different from that in controls. Further comparison of each type of CHD showed that genotype frequencies in several CHD subtypes were significantly different from those in their corresponding controls. The results of TDT analysis showed that no allele transmission disequilibrium existed in CHD nuclear families.Conclusions CBS gene G919A mutation is associated with the development of CHD, and the mutated allele may decrease the risk of CHD.

  8. The GATA1-HS2 enhancer allows persistent and position-independent expression of a β-globin transgene.

    Directory of Open Access Journals (Sweden)

    Annarita Miccio

    Full Text Available Gene therapy of genetic diseases requires persistent and position-independent expression of a therapeutic transgene. Transcriptional enhancers binding chromatin-remodeling and modifying complexes may play a role in shielding transgenes from repressive chromatin effects. We tested the activity of the HS2 enhancer of the GATA1 gene in protecting the expression of a β-globin minigene delivered by a lentiviral vector in hematopoietic stem/progenitor cells. Gene expression from proviruses carrying GATA1-HS2 in both LTRs was persistent and resistant to silencing at most integration sites in the in vivo progeny of human hematopoietic progenitors and murine long-term repopulating stem cells. The GATA1-HS2-modified vector allowed correction of murine β-thalassemia at low copy number without inducing clonal selection of erythroblastic progenitors. Chromatin immunoprecipitation studies showed that GATA1 and the CBP acetyltransferase bind to GATA1-HS2, significantly increasing CBP-specific histone acetylations at the LTRs and β-globin promoter. Recruitment of CBP by the LTRs thus establishes an open chromatin domain encompassing the entire provirus, and increases the therapeutic efficacy of β-globin gene transfer by reducing expression variegation and epigenetic silencing.

  9. Live Borrelia burgdorferi preferentially activate interleukin-1 beta gene expression and protein synthesis over the interleukin-1 receptor antagonist.

    Science.gov (United States)

    Miller, L C; Isa, S; Vannier, E; Georgilis, K; Steere, A C; Dinarello, C A

    1992-01-01

    Lyme arthritis is one of the few forms of chronic arthritis in which the cause is known with certainty. Because cytokines are thought to contribute to the pathogenesis of chronic arthritis, we investigated the effect of the Lyme disease spirochete, Borrelia burgdorferi, on the gene expression and synthesis of IL-1 beta and the IL-1 receptor antagonist (IL-1ra) in human peripheral blood mononuclear cells. Live B. burgdorferi induced fivefold more IL-1 beta than IL-1 alpha and sevenfold more IL-1 beta than IL-1ra; LPS or sonicated B. burgdorferi induced similar amounts of all three cytokines. This preferential induction of IL-1 beta was most dramatic in response to a low passage, virulent preparation of B. burgdorferi vs. three high passage avirulent strains. No difference in induction of IL-1ra was seen between these strains. The marked induction of IL-1 beta was partially diminished by heat-treatment and abrogated by sonication; IL-1ra was not affected. This suggested that a membrane component(s) accounted for the preferential induction of IL-1 beta. However, recombinant outer surface protein beta induced little IL-1 beta. By 4 h after stimulation, B. burgdorferi induced sixfold more IL-1 beta protein than LPS. In contrast to LPS-induced IL-1 beta mRNA which reached maximal accumulation after 3 h, B. burgdorferi-induced IL-1 beta mRNA showed biphasic elevations at 3 and 18 h. B. burgdorferi-induced IL-1ra mRNA peaked at 12 h, whereas LPS-induced IL-1ra mRNA peaked at 9 h. IL-1 beta synthesis increased in response to increasing numbers of spirochetes, whereas IL-1ra synthesis did not. The preferential induction by B. burgdorferi of IL-1 beta over IL-1ra is an example of excess agonist over antagonist synthesis induced by a microbial pathogen, and may contribute to the destructive lesion of Lyme arthritis. Images PMID:1387885

  10. Early peroxisome proliferator-activated receptor gamma regulated genes involved in expansion of pancreatic beta cell mass

    Directory of Open Access Journals (Sweden)

    Vivas Yurena

    2011-12-01

    Full Text Available Abstract Background The progression towards type 2 diabetes depends on the allostatic response of pancreatic beta cells to synthesise and secrete enough insulin to compensate for insulin resistance. The endocrine pancreas is a plastic tissue able to expand or regress in response to the requirements imposed by physiological and pathophysiological states associated to insulin resistance such as pregnancy, obesity or ageing, but the mechanisms mediating beta cell mass expansion in these scenarios are not well defined. We have recently shown that ob/ob mice with genetic ablation of PPARγ2, a mouse model known as the POKO mouse failed to expand its beta cell mass. This phenotype contrasted with the appropriate expansion of the beta cell mass observed in their obese littermate ob/ob mice. Thus, comparison of these models islets particularly at early ages could provide some new insights on early PPARγ dependent transcriptional responses involved in the process of beta cell mass expansion Results Here we have investigated PPARγ dependent transcriptional responses occurring during the early stages of beta cell adaptation to insulin resistance in wild type, ob/ob, PPARγ2 KO and POKO mice. We have identified genes known to regulate both the rate of proliferation and the survival signals of beta cells. Moreover we have also identified new pathways induced in ob/ob islets that remained unchanged in POKO islets, suggesting an important role for PPARγ in maintenance/activation of mechanisms essential for the continued function of the beta cell. Conclusions Our data suggest that the expansion of beta cell mass observed in ob/ob islets is associated with the activation of an immune response that fails to occur in POKO islets. We have also indentified other PPARγ dependent differentially regulated pathways including cholesterol biosynthesis, apoptosis through TGF-β signaling and decreased oxidative phosphorylation.

  11. Massive parallel gene expression profiling of RINm5F pancreatic islet beta-cells stimulated with interleukin-1beta

    DEFF Research Database (Denmark)

    Rieneck, K; Bovin, L F; Josefsen, K;

    2000-01-01

    the genes into groups according to functional relations on the basis of knowledge of the structure or function ascribed to the individual genes. Many of the differentially regulated genes are known to play a role in immune- and stress-related pathways as well as in insulin secretion and vesicle trafficking......, e.g. alpha-endosulfine and K+ channel Kir6.2 are differentially regulated. A number of transcripts in the biosynthesis pathway for cholesterol are also differentially regulated....

  12. PIAS1 regulates CP2c localization and active promoter complex formation in erythroid cell-specific α-globin expression

    OpenAIRE

    Chul Kang, Ho; Hyung Chae, Ji; Jeon, Jinseon; Kim, Won; Hyun Ha, Dae; Ho Shin, June; Gil Kim, Chan; Geun Kim, Chul

    2010-01-01

    Data presented here extends our previous observations on α-globin transcriptional regulation by the CP2 and PIAS1 proteins. Using RNAi knockdown, we have now shown that CP2b, CP2c and PIAS1 are each necessary for synergistic activation of endogenous α-globin gene expression in differentiating MEL cells. In this system, truncated PIAS1 mutants lacking the ring finger domain recruited CP2c to the nucleus, as did wild-type PIAS1, demonstrating that this is a sumoylation-independent process. In v...

  13. Association of polymorphisms in the beta-2 adrenergic receptor gene with fracture risk and bone mineral density

    NARCIS (Netherlands)

    Veldhuis-Vlug, A G; Oei, L; Souverein, P C; Tanck, M W T; Rivadeneira, F; Zillikens, M C; Kamphuisen, P W; Maitland-van der Zee, A H; de Groot, M C H; Hofman, A; Uitterlinden, A G; Fliers, E; de Boer, A; Bisschop, P H

    2015-01-01

    Signaling through the beta-2 adrenergic receptor (B2AR) on the osteoblast influences bone remodeling in rodents. In the B2AR gene, three polymorphisms influence receptor function. We show that these polymorphisms are not associated with fracture risk or bone mineral density in the UCP, Rotterdam Stu

  14. Association of polymorphisms in the beta-2 adrenergic receptor gene with fracture risk and bone mineral density

    NARCIS (Netherlands)

    A.G. Veldhuis-Vlug; L. Oei (Ling); P. Souverein (Patrick); M.W.T. Tanck (Michael); F. Rivadeneira Ramirez (Fernando); M.C. Zillikens (Carola); P.W. Kamphuisen; A-H. Maitland-van der Zee (Anke-Hilse); M.C.H. de Groot; A. Hofman (Albert); A.G. Uitterlinden (André); E. Fliers (Eric); A.C. de Boer (Anthonius); P.H. Bisschop

    2015-01-01

    textabstractSummary: Signaling through the beta-2 adrenergic receptor (B2AR) on the osteoblast influences bone remodeling in rodents. In the B2AR gene, three polymorphisms influence receptor function. We show that these polymorphisms are not associated with fracture risk or bone mineral density in t

  15. Association of polymorphisms in the beta-2 adrenergic receptor gene with fracture risk and bone mineral density

    NARCIS (Netherlands)

    Veldhuis-Vlug, A. G.; Oei, L.; Souverein, P. C.; Tanck, M. W T; Rivadeneira, F.; Zillikens, M. C.; Kamphuisen, P. W.; Maitland - van der Zee, A. H.; de Groot, M. C H; Hofman, A.; Uitterlinden, A. G.; Fliers, E.; de Boer, A.; Bisschop, P. H.

    2015-01-01

    Summary: Signaling through the beta-2 adrenergic receptor (B2AR) on the osteoblast influences bone remodeling in rodents. In the B2AR gene, three polymorphisms influence receptor function. We show that these polymorphisms are not associated with fracture risk or bone mineral density in the UCP, Rott

  16. The diversity and abundance of phytase genes (beta-propeller phytases) in bacterial communities of the maize rhizosphere

    NARCIS (Netherlands)

    Cotta, S.R.; Cavalcante Franco Dias, A.; Seldin, L.; Andreote, F. D.; van Elsas, J. D.

    2016-01-01

    The ecology of microbial communities associated with organic phosphorus (P) mineralization in soils is still understudied. Here, we assessed the abundance and diversity of bacteria harbouring genes encoding beta-propeller phytases (BPP) in the rhizosphere of traditional and transgenic maize cultivat

  17. The dnaN gene codes for the beta subunit of DNA polymerase III holoenzyme of escherichia coli.

    Science.gov (United States)

    Burgers, P M; Kornberg, A; Sakakibara, Y

    1981-09-01

    An Escherichia coli mutant, dnaN59, stops DNA synthesis promptly upon a shift to a high temperature; the wild-type dnaN gene carried in a transducing phage encodes a polypeptide of about 41,000 daltons [Sakakibara, Y. & Mizukami, T. (1980) Mol. Gen. Genet. 178, 541-553; Yuasa, S. & Sakakibara, Y. (1980) Mol. Gen. Genet. 180, 267-273]. We now find that the product of dnaN gene is the beta subunit of DNA polymerase III holoenzyme, the principal DNA synthetic multipolypeptide complex in E. coli. The conclusion is based on the following observations: (i) Extracts from dnaN59 cells were defective in phage phi X174 and G4 DNA synthesis after the mutant cells had been exposed to the increased temperature. (ii) The enzymatic defect was overcome by addition of purified beta subunit but not by other subunits of DNA polymerase III holoenzyme or by other replication proteins required for phi X174 DNA synthesis. (iii) Partially purified beta subunit from the dnaN mutant, unlike that from the wild type, was inactive in reconstituting the holoenzyme when mixed with the other purified subunits. (iv) Increased dosage of the dnaN gene provided by a plasmid carrying the gene raised cellular levels of the beta subunit 5- to 6-fold.

  18. Identification of a Novel Single Nucleotide Polymorphism in Porcine Beta-Defensin-1 Gene.

    Science.gov (United States)

    Pruthviraj, D R; Usha, A P; Venkatachalapathy, R T

    2016-03-01

    Porcine beta-defensin-1 (PBD-1) gene plays an important role in the innate immunity of pigs. The peptide encoded by this gene is an antimicrobial peptide that has direct activity against a wide range of microbes. This peptide is involved in the co-creation of an antimicrobial barrier in the oral cavity of pigs. The objective of the present study was to detect polymorphisms, if any, in exon-1 and exon-2 regions of PBD-1 gene in Large White Yorkshire (LWY) and native Ankamali pigs of Kerala, India. Blood samples were collected from 100 pigs and genomic DNA was isolated using phenol chloroform method. The quantity of DNA was assessed in a spectrophotometer and quality by gel electrophoresis. Exon-1 and exon-2 regions of PBD-1 gene were amplified by polymerase chain reaction (PCR) and the products were subjected to single strand conformation polymorphism (SSCP) analysis. Subsequent silver staining of the polyacrylamide gels revealed three unique SSCP banding patterns in each of the two exons. The presence of single nucleotide polymorphisms (SNPs) was confirmed by nucleotide sequencing of the PCR products. A novel SNP was found in the 5'-UTR region of exon-1 and a SNP was detected in the mature peptide coding region of exon-2. In exon-1, the pooled population frequencies of GG, GT, and TT genotypes were 0.67, 0.30, and 0.03, respectively. GG genotype was predominant in both the breeds whereas TT genotype was not detected in LWY breed. Similarly, in exon-2, the pooled population frequencies of AA, AG, and GG genotypes were 0.50, 0.27, and 0.23, respectively. AA genotype was predominant in LWY pigs whereas GG genotype was predominant in native pigs. These results suggest that there exists a considerable genetic variation at PBD-1 locus and further association studies may help in development of a PCR based genotyping test to select pigs with better immunity.

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

    Science.gov (United States)

    Foltz, David W

    2007-11-01

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

  20. The expression of pregnancy-specific {beta}1-glycoprotein genes in Meckel-Gruber syndrome fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Shao-Ming; Cham, Wai-Yee [Georgetown Univ. Medical Center, Washington, DC (United States)

    1994-09-01

    Meckel-Gruber syndrome (MS) is an autosomal recessive disorder with multiple congenital malformations. The only available prenatal diagnostic marker for this disorder is the amniotic fluid level of pregnancy-specific {beta}1-glycoprotein (PSG). PSG is a family of proteins which are expressed at high levels during pregnancy. Increasing maternal serum PSG levels correlate with the progression of pregnancy and can be used as indicators for pregnancy outcome and fetal well-being. The amniotic fluid PSG level is about one-tenth of that of the maternal serum level in normal pregnancy, but are elevated in all cases of MS examined so far. On the other hand, the maternal serum PSG level and third trimester placental PSG content are normal in most cases of MS. This study aims at comparing the expression of PSG in fibroblasts derived from a fetus afflicted with MS. Total cellular RNA was extracted from two MS cultured fibroblast lines (M3206 and GM7817) and four age- and sex-matched control fibroblast lines obtained from the Human Genetic Mutant Cell Repository, Camden, NJ. The expression of eight PSG genes namely, PSG1, PSG2, PSG3, PSG4, PSG5, PSG6, PSG9 and PSG11, were examined with reverse transcription-polymerase chain reaction (RT-PCR). All PSG transcripts present in the cell were first amplified using universal primers in a 28-cycle PCR. Specific PSG gene products were then amplified with PSG gene-specific primers. Results showed that there is no significant difference in PSG expression between control and disease fibroblasts. In both cases, the most abundant transcript was the type II transcript of PSG5 followed by the type I transcripts of PSG1 and PG4. PSG9, PSG11 and PSG 3 were expressed at very low levels or not expressed at all in MS as well as in normal control fibroblasts. These results showed that PSG gene expression was not altered in MS fibroblasts.

  1. Transcriptional profiling of type 1 diabetes genes on chromosome 21 in a rat beta-cell line and human pancreatic islets

    DEFF Research Database (Denmark)

    Bergholdt, R.; Karlsen, A.E.; Hagedorn, Peter;

    2007-01-01

    We recently finemapped a type 1 diabetes (T1D)-linked region on chromosome 21, indicating that one or more T1D-linked genes exist in this region with 33 annotated genes. In the current study, we have taken a novel approach using transcriptional profiling in predicting and prioritizing the most...... likely candidate genes influencing beta-cell function in this region. Two array-based approaches were used, a rat insulinoma cell line (INS-1alphabeta) overexpressing pancreatic duodenum homeobox 1 (pdx-1) and treated with interleukin 1beta (IL-1beta) as well as human pancreatic islets stimulated...... with a mixture of cytokines. Several candidate genes with likely functional significance in T1D were identified. Genes showing differential expression in the two approaches were highly similar, supporting the role of these specific gene products in cytokine-induced beta-cell damage. These were genes involved...

  2. Structural alterations of transforming growth factor-beta receptor genes in human cervical carcinoma

    NARCIS (Netherlands)

    Chen, TP; De Vries, EGE; Hollema, H; Yegen, HA; Vellucci, VF; Strickler, HD; Hildesheim, A; Reiss, M

    1999-01-01

    The development and progression of invasive uterine cervical carcinomas appear to be associated with the progressive loss of sensitivity to transforming growth factor-beta (TGF beta)-mediated cell cycle arrest. In order to identify possible molecular mechanisms responsible for TGF beta resistance, w

  3. Gene expression profiles of Beta-cell enriched tissue obtained by laser capture microdissection from subjects with type 2 diabetes.

    Directory of Open Access Journals (Sweden)

    Lorella Marselli

    Full Text Available BACKGROUND: Changes in gene expression in pancreatic beta-cells from type 2 diabetes (T2D should provide insights into their abnormal insulin secretion and turnover. METHODOLOGY/PRINCIPAL FINDINGS: Frozen sections were obtained from cadaver pancreases of 10 control and 10 T2D human subjects. Beta-cell enriched samples were obtained by laser capture microdissection (LCM. RNA was extracted, amplified and subjected to microarray analysis. Further analysis was performed with DNA-Chip Analyzer (dChip and Gene Set Enrichment Analysis (GSEA software. There were changes in expression of genes linked to glucotoxicity. Evidence of oxidative stress was provided by upregulation of several metallothionein genes. There were few changes in the major genes associated with cell cycle, apoptosis or endoplasmic reticulum stress. There was differential expression of genes associated with pancreatic regeneration, most notably upregulation of members of the regenerating islet gene (REG family and metalloproteinase 7 (MMP7. Some of the genes found in GWAS studies to be related to T2D were also found to be differentially expressed. IGF2BP2, TSPAN8, and HNF1B (TCF2 were upregulated while JAZF1 and SLC30A8 were downregulated. CONCLUSIONS/SIGNIFICANCE: This study made possible by LCM has identified many novel changes in gene expression that enhance understanding of the pathogenesis of T2D.

  4. Assessment of AmpC Beta-Lactamase Genes among Clinical Escherichia coli Isolates

    Directory of Open Access Journals (Sweden)

    HedrooshaMolla Agha-Mirzaeie

    2015-11-01

    Full Text Available Background: AmpC bta lactamases play a significant role in creating resistance to third generation cephalosporins worldwide. They mostly express on chromosome of Enterobacteriaceae especially Escherichia coli and cause consequential problem inclinical treatment and lead to failure in diagnosis and phenotypic test recommended byClinical and Laboratory Standards Institute.Methods:Totally 200 E. coli isolates from different hospitals of Tehran were collected. The isolates were screened by disk diffusion method according to the CLSI guidelines. The profiles and prevalence surveys of AmpC (Dha, CITM, Mox and FOX-type β-lactamase genes in clinical isolates of E. coli by phenotypic and molecular methods.  Results:Out of 200 Ecoli isolated, 115 (89.8% and 13 (10.2% isolates were identified as ESBL- and AmpC- beta-lactamase producers, respectively. Among mpC producers, 13 (100% and 5 (38.5% isolates was reported by PCR assay as bla-CITM and Dha respectively. Mox and FOX genes were not detected in any sample.Conclusions:Our results highlight the importance of using molecular detection methods to identify β-lactamase-producer that have resistance to antibiotics. 

  5. Analysis of beta-casein gene (CSN2 polymorphism in different breeds of cattle

    Directory of Open Access Journals (Sweden)

    Martina Miluchová

    2014-11-01

    Full Text Available Normal 0 21 false false false SK X-NONE X-NONE MicrosoftInternetExplorer4 The goal of work was identification of b - casein gene polymorphism in different breeds of cow. The beta - casein constitutes up to 45 % of the casein of bovine milk. The most common forms of beta-casein in dairy cattle breeds are A1 and A2, while B is less common. The b-casein A1 variant was associated with the incidence of diabetes mellitus 1st type, coronary heart disease and autism. The A2 variant reduces serum cholesterol. The material involved 287 cows (Simmental breed – 111 cows, Pinzgau breed – 89 cows, Holstein breed – 87 cows. Bovine genomic DNA was extracted from whole blood by using commercial kit and used in order to estimate b - casein genotypes by means of PCR-RFLP method. In the populations included in the study were detected all three genotypes – homozygote genotype A1A1, heterozygote genotype A1A2 and homozygote genotype A2A2 with frequencies 0.1261, 0.3333 and 0.5405 in Simmental breed; 0.1379, 0.4598 and 0.4023 in Holstein breed, 0.3034, 0.5168 and 0.1798 in Pinzgau breed. In population of Simmental breed and Holstein breed was higher frequency of allele A2 (0.7072 and 0.6322. In opposite, in population of Pinzgau breed was present higher frequency of the allele A1 (0.5618.

  6. Quantitative response relationships between degradation rates and functional genes during the degradation of beta-cypermethrin in soil.

    Science.gov (United States)

    Yang, Zhong-Hua; Ji, Guo-Dong

    2015-12-15

    In the present study, the degradation mechanisms of beta-cypermethrin and its metabolites in soil were explored through the quantitative response relationships between the degradation rates and related functional genes. We found that the degradation rate of beta-cypermethrin was rapid in unsterilized soil but not in sterilized soil, which indicated that the degradation process is microbially based. Moreover, three metabolites (3-phenoxybenzoic acid, phenol and protocatechuic acid) were detected during the degradation process and used to identify the degradation pathway and functional genes related to the degradation process. The key rate-limiting functional genes were pytH and pobA, and the relative contributions of these genes to the degradation process were examined with a path analysis. The path analysis revealed that the genes pobA and pytH had the greatest direct effects on the degradation of beta-cypermethrin (pobA), alpha-cypermethrin (pobA), theta-cypermethrin (pytH) and 3-phenoxybenzoic acid (pytH).

  7. Baseline Gene Expression Signatures in Monocytes from Multiple Sclerosis Patients Treated with Interferon-beta

    Science.gov (United States)

    Bustamante, Marta F.; Nurtdinov, Ramil N.; Río, Jordi; Montalban, Xavier; Comabella, Manuel

    2013-01-01

    Background A relatively large proportion of relapsing-remitting multiple sclerosis (RRMS) patients do not respond to interferon-beta (IFNb) treatment. In previous studies with peripheral blood mononuclear cells (PBMC), we identified a subgroup of IFNb non-responders that was characterized by a baseline over-expression of type I IFN inducible genes. Additional mechanistic experiments carried out in IFNb non-responders suggested a selective alteration of the type I IFN signaling pathway in the population of blood monocytes. Here, we aimed (i) to investigate whether the type I IFN signaling pathway is up-regulated in isolated monocytes from IFNb non-responders at baseline; and (ii) to search for additional biological pathways in this cell population that may be implicated in the response to IFNb treatment. Methods Twenty RRMS patients classified according to their clinical response to IFNb treatment and 10 healthy controls were included in the study. Monocytes were purified from PBMC obtained before treatment by cell sorting and the gene expression profiling was determined with oligonucleotide microarrays. Results and discussion Purified monocytes from IFNb non-responders were characterized by an over-expression of type I IFN responsive genes, which confirms the type I IFN signature in monocytes suggested from previous studies. Other relevant signaling pathways that were up-regulated in IFNb non-responders were related with the mitochondrial function and processes such as protein synthesis and antigen presentation, and together with the type I IFN signaling pathway, may also be playing roles in the response to IFNb. PMID:23637780

  8. Pest protection conferred by a Beta vulgaris serine proteinase inhibitor gene.

    Directory of Open Access Journals (Sweden)

    Ann C Smigocki

    Full Text Available Proteinase inhibitors provide a means of engineering plant resistance to insect pests. A Beta vulgaris serine proteinase inhibitor gene (BvSTI was fused to the constitutive CaMV35S promoter for over-expression in Nicotiana benthamiana plants to study its effect on lepidopteran insect pests. Independently derived BvSTI transgenic tobacco T2 homozygous progeny were shown to have relatively high BvSTI gene transcript levels. BvSTI-specific polyclonal antibodies cross-reacted with the expected 30 kDA recombinant BvSTI protein on Western blots. In gel trypsin inhibitor activity assays revealed a major clear zone that corresponded to the BvSTI proteinase inhibitor that was not detected in the untransformed control plants. BvSTI-transgenic plants were bioassayed for resistance to five lepidopteran insect pests. Spodoptera frugiperda, S. exigua and Manduca sexta larvae fed BvSTI leaves had significant reductions in larval weights as compared to larvae fed on untransformed leaves. In contrast, larval weights increased relative to the controls when Heliothis virescens and Agrotis ipsilon larvae were fed on BvSTI leaves. As the larvae entered the pupal stage, pupal sizes reflected the overall larval weights. Some developmental abnormalities of the pupae and emerging moths were noted. These findings suggest that the sugar beet BvSTI gene may prove useful for effective control of several different lepidopteran insect pests in genetically modified tobacco and other plants. The sugar beet serine proteinase inhibitor may be more effective for insect control because sugar beet is cropped in restricted geographical areas thus limiting the exposure of the insects to sugar beet proteinase inhibitors and build up of non-sensitive midgut proteases.

  9. Pest protection conferred by a Beta vulgaris serine proteinase inhibitor gene.

    Science.gov (United States)

    Smigocki, Ann C; Ivic-Haymes, Snezana; Li, Haiyan; Savić, Jelena

    2013-01-01

    Proteinase inhibitors provide a means of engineering plant resistance to insect pests. A Beta vulgaris serine proteinase inhibitor gene (BvSTI) was fused to the constitutive CaMV35S promoter for over-expression in Nicotiana benthamiana plants to study its effect on lepidopteran insect pests. Independently derived BvSTI transgenic tobacco T2 homozygous progeny were shown to have relatively high BvSTI gene transcript levels. BvSTI-specific polyclonal antibodies cross-reacted with the expected 30 kDA recombinant BvSTI protein on Western blots. In gel trypsin inhibitor activity assays revealed a major clear zone that corresponded to the BvSTI proteinase inhibitor that was not detected in the untransformed control plants. BvSTI-transgenic plants were bioassayed for resistance to five lepidopteran insect pests. Spodoptera frugiperda, S. exigua and Manduca sexta larvae fed BvSTI leaves had significant reductions in larval weights as compared to larvae fed on untransformed leaves. In contrast, larval weights increased relative to the controls when Heliothis virescens and Agrotis ipsilon larvae were fed on BvSTI leaves. As the larvae entered the pupal stage, pupal sizes reflected the overall larval weights. Some developmental abnormalities of the pupae and emerging moths were noted. These findings suggest that the sugar beet BvSTI gene may prove useful for effective control of several different lepidopteran insect pests in genetically modified tobacco and other plants. The sugar beet serine proteinase inhibitor may be more effective for insect control because sugar beet is cropped in restricted geographical areas thus limiting the exposure of the insects to sugar beet proteinase inhibitors and build up of non-sensitive midgut proteases.

  10. Studies of the variability of the hepatocyte nuclear factor-1beta (HNF-1beta / TCF2) and the dimerization cofactor of HNF-1 (DcoH / PCBD) genes in relation to type 2 diabetes mellitus and beta-cell function

    DEFF Research Database (Denmark)

    Ek, J; Grarup, N; Urhammer, S A

    2001-01-01

    , and 46 type 2 diabetic patients with an impaired beta-cell function by combined single-strand conformation polymorphism (SSCP) and heteroduplex analysis. Analysis of the promoter and nine exons including intron-exon boundaries of the HNF-1beta gene revealed one novel silent polymorphism and three...... previously reported intronic variants. The silent polymorphism (I91I) was found in one patient with late-onset type 2 diabetes. One of the intronic variant (IVS6+26T-->C) was examined further. Among 584 type 2 diabetic patients the allelic frequency was 13.1% (11.2-15.0%) compared to 11.6% (8.6-14.5%) in 229...... comprising the DCoH gene revealed a previously described A-->G polymorphism located in the 3' untranslated region, which was not investigated further. In conclusion, mutations in HNF-1beta and DCoH are not a major cause of MODY or late onset type 2 diabetes in Danish Caucasian subjects....

  11. Construction of a fusion gene comprising the Taka-amylase A promoter and the Escherichia coli beta-glucuronidase gene and analysis of its expression in Aspergillus oryzae.

    Science.gov (United States)

    Tada, S; Gomi, K; Kitamoto, K; Takahashi, K; Tamura, G; Hara, S

    1991-10-01

    Northern blot analysis of glucose-grown and starch-grown mycelia of Aspergillus oryzae RIB40 was conducted using the cloned Taka-amylase A (TAA) gene as a probe. The amount of mRNA homologous to the TAA gene was increased when this fungus was grown with starch as a sole carbon source. In order to analyze the induction mechanism, we inserted the Escherichia coli uidA gene encoding beta-glucuronidase (GUS) down-stream of the TAA promoter and introduced the resultant fusion gene into the A. oryzae genome. Production of a functional GUS protein was induced by starch, but not by glucose. When the effects of various sugars on expression of the fusion gene were examined, the results suggested that the expression of the fusion gene was under control of the TAA gene promoter.

  12. Localization of the human {beta}-catenin gene (CTNNB1) to 3p21: A region implicated in tumor development

    Energy Technology Data Exchange (ETDEWEB)

    Kraus, C.; Liehr, T.; Ballhausen, G. [Institut fuer Humangenetik der Universitaet, Erlangen (Germany)] [and others

    1994-09-01

    The human {beta}-catenin locus (CTNNB1) was mapped by in situ fluorescence analysis to band p21 on the short arm of chromosome 3, a region frequently affected by somatic alterations in a variety of tumors. PCR primers for the genomic amplification of {beta}-catenin sequences were selected on the basis of homology to exon 4 of the Drosophila armadillo gene. Analysis of a panel of somatic cell hybrids confirmed the localization of {beta}-catenin on human chromosome 3. Furthermore, exclusion mapping of three hybrids carrying defined fragments of the short arm of human chromosome 3 allowed us to determine the position of the CTNNB1 locus close to the marker D3S2 in 3p21. 22 refs., 3 figs.

  13. Role of transcription factor KLF11 and its diabetes-associated gene variants in pancreatic beta cell function

    DEFF Research Database (Denmark)

    Neve, Bernadette; Fernandez-Zapico, Martin E; Ashkenazi-Katalan, Vered

    2005-01-01

    a role in free radical clearance that may render beta cells more sensitive to oxidative stress. Thus, both functional and genetic analyses reveal that KLF11 plays a role in the regulation of pancreatic beta cell physiology, and its variants may contribute to the development of diabetes.......KLF11 (TIEG2) is a pancreas-enriched transcription factor that has elicited significant attention because of its role as negative regulator of exocrine cell growth in vitro and in vivo. However, its functional role in the endocrine pancreas remains to be established. Here, we report, for the first...... in beta cells. Genetic analysis of the KLF11 gene revealed two rare variants (Ala347Ser and Thr220Met) that segregate with diabetes in families with early-onset type 2 diabetes, and significantly impair its transcriptional activity. In addition, analysis of 1,696 type 2 diabetes mellitus and 1...

  14. The Beta-Binomial Distribution for Estimating the Number of False Rejections in Microarray Gene Expression Studies.

    Science.gov (United States)

    Hunt, Daniel L; Cheng, Cheng; Pounds, Stanley

    2009-03-15

    In differential expression analysis of microarray data, it is common to assume independence among null hypotheses (and thus gene expression levels). The independence assumption implies that the number of false rejections V follows a binomial distribution and leads to an estimator of the empirical false discovery rate (eFDR). The number of false rejections V is modeled with the beta-binomial distribution. An estimator of the beta-binomial false discovery rate (bbFDR) is then derived. This approach accounts for how the correlation among non-differentially expressed genes influences the distribution of V. Permutations are used to generate the observed values for V under the null hypotheses and a beta-binomial distribution is fit to the values of V. The bbFDR estimator is compared to the eFDR estimator in simulation studies of correlated non-differentially expressed genes and is found to outperform the eFDR for certain scenarios. As an example, this method is also used to perform an analysis that compares the gene expression of soft tissue sarcoma samples to normal tissue samples.

  15. High-level production of beta-carotene in Saccharomyces cerevisiae by successive transformation with carotenogenic genes from Xanthophyllomyces dendrorhous.

    Science.gov (United States)

    Verwaal, René; Wang, Jing; Meijnen, Jean-Paul; Visser, Hans; Sandmann, Gerhard; van den Berg, Johan A; van Ooyen, Albert J J

    2007-07-01

    To determine whether Saccharomyces cerevisiae can serve as a host for efficient carotenoid and especially beta-carotene production, carotenogenic genes from the carotenoid-producing yeast Xanthophyllomyces dendrorhous were introduced and overexpressed in S. cerevisiae. Because overexpression of these genes from an episomal expression vector resulted in unstable strains, the genes were integrated into genomic DNA to yield stable, carotenoid-producing S. cerevisiae cells. Furthermore, carotenoid production levels were higher in strains containing integrated carotenogenic genes. Overexpression of crtYB (which encodes a bifunctional phytoene synthase and lycopene cyclase) and crtI (phytoene desaturase) from X. dendrorhous was sufficient to enable carotenoid production. Carotenoid production levels were increased by additional overexpression of a homologous geranylgeranyl diphosphate (GGPP) synthase from S. cerevisiae that is encoded by BTS1. Combined overexpression of crtE (heterologous GGPP synthase) from X. dendrorhous with crtYB and crtI and introduction of an additional copy of a truncated 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene (tHMG1) into carotenoid-producing cells resulted in a successive increase in carotenoid production levels. The strains mentioned produced high levels of intermediates of the carotenogenic pathway and comparable low levels of the preferred end product beta-carotene, as determined by high-performance liquid chromatography. We finally succeeded in constructing an S. cerevisiae strain capable of producing high levels of beta-carotene, up to 5.9 mg/g (dry weight), which was accomplished by the introduction of an additional copy of crtI and tHMG1 into carotenoid-producing yeast cells. This transformant is promising for further development toward the biotechnological production of beta-carotene by S. cerevisiae.

  16. Association of beta 2 -adrenergic receptor gene polymorphisms and nocturnal asthma in Saudi patients

    Directory of Open Access Journals (Sweden)

    Al-Rubaish Abdullah

    2011-01-01

    Full Text Available Background and Objectives : Two polymorphisms of beta 2 -adrenergic receptor (β2 -AR gene, namely the substitution from arginine (Arg to glycine (Gly at codon 16 and from glutamine (Gln to glutamic (Glu at codon 27, are linked with functional changes in the β2 -AR in the respiratory system even though they are not deemed to be susceptibility genes for asthma per se. The objective of this study was to investigate this association in a subset of asthmatic patients, namely those with nocturnal asthma. Methods : The β2 -AR gene polymorphisms at codon 16 and 27 were assessed in 40 patients clinically diagnosed with nocturnal asthma and 96 normal controls. Genomic DNA was obtained from whole blood and genotyping was carried out by a PCR based restriction fragment length polymorphism technique. Results : There was a statistically significant difference in genotype frequencies at codon 16 (Arg/Gly between nocturnal asthmatic patients and normal control subjects (P < 0.05. However, there was no statistically significant difference in allele frequencies between the two groups. In addition, there was a significant association between Arg16-Gly genotype with nocturnal asthma compared to homozygous Gly16 (codominant model P = 0.0033, OR = 3.69: 95% CI: 1.49-9.12. However, there were no statistically significant differences in genotype and allele frequencies at codon 27 (Gln/Glu between the normal control and nocturnal asthmatic groups (χ2 = 1.81, P = 0.41. The results also indicate that linkage disequilibrium existed between the β2 -AR codon 16 and β2 -AR codon 27 polymorphism (/ D΄/ = 0.577. The data for all haplotypes did not show a statistically significant association. Conclusion : We present the genotype and allele frequencies of β2 -AR gene polymorphisms in normal Saudi subjects and nocturnal asthmatic patients. There was a significant difference in genotype frequencies at codon 16 (Arg/Gly. However, our study indicates a poor association of

  17. Mutations in the dopamine beta-hydroxylase gene are associated with human norepinephrine deficiency

    Science.gov (United States)

    Kim, Chun-Hyung; Zabetian, Cyrus P.; Cubells, Joseph F.; Cho, Sonhae; Biaggioni, Italo; Cohen, Bruce M.; Robertson, David; Kim, Kwang-Soo

    2002-01-01

    Norepinephrine (NE), a key neurotransmitter of the central and peripheral nervous systems, is synthesized by dopamine beta-hydroxylase (DBH) that catalyzes oxidation of dopamine (DA) to NE. NE deficiency is a congenital disorder of unknown etiology, in which affected patients suffer profound autonomic failure. Biochemical features of the syndrome include undetectable tissue and circulating levels of NE and epinephrine, elevated levels of DA, and undetectable levels of DBH. Here, we report identification of seven novel variants including four potentially pathogenic mutations in the human DBH gene (OMIM 223360) from analysis of two unrelated patients and their families. Both patients are compound heterozygotes for variants affecting expression of DBH protein. Each carries one copy of a T-->C transversion in the splice donor site of DBH intron 1, creating a premature stop codon. In patient 1, there is a missense mutation in DBH exon 2. Patient 2 carries missense mutations in exons 1 and 6 residing in cis. We propose that NE deficiency is an autosomal recessive disorder resulting from heterogeneous molecular lesions at DBH. Copyright 2002 Wiley-Liss, Inc.

  18. Effects of intra-gastric beta-casomorphin-7 on somatostatin and gastrin gene expression in rat gastric mucosa

    Institute of Scientific and Technical Information of China (English)

    Ya-Feng Zong; Wei-Hua Chen; Yuan-Shu Zhang; Si-Xiang Zou

    2007-01-01

    AIM: To investigate the in vivo effect of beta-casomorphin-7on the regulation of gastric somatostatin and gastrin messenger RNA in rat gastric mucosa.METHODS: Somatostatin and gastrin mRNA were quantified by RT-PCR and in situ hybridization (ISH)in 24 rats. The rats were divided into three treatment groups: basal diet + physiological saline (n = 8), basal diet + beta-casomorphin-7 (7.5 × 10-7 mol) (n = 8),and basal diet + poly-Gly-7 (containing equal mol of N with 7.5 x 10-7 mol beta-casomorphin-7) (n = 8).After oral administration for 30 days, rats were killed by exsanguinations.RESULTS: After intra-gastric administration of betacasomorphin-7 for 30 d, gastrin mRNA increased by 52.8% (P < 0.05, n = 8), and somatostatin mRNA levels decreased by 30.7% compared with the controls (P <0.01, n = 8). No significant differences in the expression of the two genes were observed in the poly-Gly-treated group, although gastrin mRNA expression was elevated by 35.6% as against the control group (P = 0.15, n =8). The long-term oral administration of a casomorphin solution significantly decreased the even gray of D-cells,but did not lower the number of D-cells both in the antrum and fundus. Interestingly, the number of G-cells increased in the antrum and fundus, but its average density was augmented only in the antrum.CONCLUSION: Beta-casomorphin-7 is capable of modulating gene expression of the regulatory peptides from G and D cells. Data from in situ hybridization studies indicate that beta-casomorphin-7 affects gastrin gene expression indirectly by means of the paracrine action of somatostatin, and depends on its intrinsic molecular function.

  19. Human-specific SNP in obesity genes, adrenergic receptor beta2 (ADRB2, Beta3 (ADRB3, and PPAR γ2 (PPARG, during primate evolution.

    Directory of Open Access Journals (Sweden)

    Akiko Takenaka

    Full Text Available UNLABELLED: Adrenergic-receptor beta2 (ADRB2 and beta3 (ADRB3 are obesity genes that play a key role in the regulation of energy balance by increasing lipolysis and thermogenesis. The Glu27 allele in ADRB2 and the Arg64 allele in ADRB3 are associated with abdominal obesity and early onset of non-insulin-dependent diabetes mellitus (NIDDM in many ethnic groups. Peroxisome proliferator-activated receptor γ (PPARG is required for adipocyte differentiation. Pro12Ala mutation decreases PPARG activity and resistance to NIDDM. In humans, energy-expense alleles, Gln27 in ADRB2 and Trp64 in ADRB3, are at higher frequencies than Glu27 and Arg64, respectively, but Ala12 in PPARG is at lower frequency than Pro12. Adaptation of humans for lipolysis, thermogenesis, and reduction of fat accumulation could be considered by examining which alleles in these genes are dominant in non-human primates (NHP. All NHP (P. troglodytes, G. gorilla, P. pygmaeus, H. agilis and macaques had energy-thrifty alleles, Gly16 and Glu27 in ADRB2, and Arg64 in ADRB3, but did not have energy-expense alleles, Arg16, Gln27 and Trp64 alleles. In PPARG gene, all NHP had large adipocyte accumulating type, the Pro12 allele. CONCLUSIONS: These results indicate that a tendency to produce much more heat through the energy-expense alleles developed only in humans, who left tropical rainforests for savanna and developed new features in their heat-regulation systems, such as reduction of body hair and increased evaporation of water, and might have helped the protection of entrails from cold at night, especially in glacial periods.

  20. Human-Specific SNP in Obesity Genes, Adrenergic Receptor Beta2 (ADRB2), Beta3 (ADRB3), and PPAR γ2 (PPARG), during Primate Evolution

    Science.gov (United States)

    Takenaka, Akiko; Nakamura, Shin; Mitsunaga, Fusako; Inoue-Murayama, Miho; Udono, Toshifumi; Suryobroto, Bambang

    2012-01-01

    Adrenergic-receptor beta2 (ADRB2) and beta3 (ADRB3) are obesity genes that play a key role in the regulation of energy balance by increasing lipolysis and thermogenesis. The Glu27 allele in ADRB2 and the Arg64 allele in ADRB3 are associated with abdominal obesity and early onset of non-insulin-dependent diabetes mellitus (NIDDM) in many ethnic groups. Peroxisome proliferator-activated receptor γ (PPARG) is required for adipocyte differentiation. Pro12Ala mutation decreases PPARG activity and resistance to NIDDM. In humans, energy-expense alleles, Gln27 in ADRB2 and Trp64 in ADRB3, are at higher frequencies than Glu27 and Arg64, respectively, but Ala12 in PPARG is at lower frequency than Pro12. Adaptation of humans for lipolysis, thermogenesis, and reduction of fat accumulation could be considered by examining which alleles in these genes are dominant in non-human primates (NHP). All NHP (P. troglodytes, G. gorilla, P. pygmaeus, H. agilis and macaques) had energy-thrifty alleles, Gly16 and Glu27 in ADRB2, and Arg64 in ADRB3, but did not have energy-expense alleles, Arg16, Gln27 and Trp64 alleles. In PPARG gene, all NHP had large adipocyte accumulating type, the Pro12 allele. Conclusions These results indicate that a tendency to produce much more heat through the energy-expense alleles developed only in humans, who left tropical rainforests for savanna and developed new features in their heat-regulation systems, such as reduction of body hair and increased evaporation of water, and might have helped the protection of entrails from cold at night, especially in glacial periods. PMID:22937051

  1. Interleukin (IL)1beta, IL-1alpha, and IL-1 receptor antagonist gene polymorphisms in patients with temporal lobe epilepsy.

    Science.gov (United States)

    Kanemoto, K; Kawasaki, J; Miyamoto, T; Obayashi, H; Nishimura, M

    2000-05-01

    Proinflammatory cytokines, including interleukin (IL)-1beta, are known to modulate effects of neurotoxic neurotransmitters discharged during excitation or inflammation in the central nervous system (CNS). They also regulate development of glial scars at sites of CNS injury. To elucidate a genetic predisposition of temporal lobe epilepsy with hippocampal sclerosis (TLE-HS+), we studied polymorphisms in the IL-1beta, IL-1alpha, and IL-1 receptor antagonist (IL-1RA) genes in 50 patients with TLE-HS+ and in 112 controls. Fifty-three patients who had TLE without HS were also examined (TLE-HS-) as disease controls. The distribution of the biallelic polymorphism in the promoter region at position -511 of the IL-1beta gene (IL-1B-511) was significantly different both between TLE-HS+ patients and controls and between TLE-HS+ and TLE-HS- patients. The differences were due to overrepresentation of the homozygotes for IL-1B-511*2, which is suggested to be a high producer of IL-1beta, in TLE-HS+ patients compared with both controls and TLE-HS- patients. In contrast, there was no difference between TLE-HS- patients and controls. Our data suggest that, in the homozygotes for IL-IB-511*2, minor events in development such as febrile convulsions could set up a cascade leading to HS.

  2. Occurrence of bacteria producing broad-spectrum beta-lactamases and qnr genes in hospital and urban wastewater samples.

    Science.gov (United States)

    Röderová, Magdaléna; Sedláková, Miroslava Htoutou; Pudová, Vendula; Hricová, Kristýna; Silová, Romana; Imwensi, Peter Eghonghon Odion; Bardoň, Jan; Kolář, Milan

    2016-04-01

    The aims were to investigate the level of antibiotic-resistant bacteria in hospital and urban wastewater and to determine the similarity of isolates obtained from wastewater and hospitalized patients. Wastewater samples were collected in September 2013 and 2014. After identification using MALDI-TOF MS, beta-lactamase production was determined by relevant phenotypic tests. Genes responsible for the production of single beta-lactamase groups and Qnr proteins were established. The epidemiological relationship of the isolates from wastewater and hospitalized patients was determined by PFGE. A total of 51 isolates of enterobacteria were obtained. Overall, 45.1% of them produced broad-spectrum beta-lactamases. Genes encoding TEM, SHV, CTX-M, CIT, DHA and EBC types of enzymes and Qnr proteins were detected. No broad-spectrum beta-lactamase production was confirmed in the urban wastewater treatment plant. The most important finding was the detection of two identical isolates of K. pneumoniae in 2013, one from a patient's urinary catheter and the other from a wastewater sample.

  3. Sequential mutations in the interleukin-3 (IL3)/granulocyte-macrophage colony-stimulating factor/IL5 receptor beta-subunit genes are necessary for the complete conversion to growth autonomy mediated by a truncated beta C subunit.

    Science.gov (United States)

    Hannemann, J; Hara, T; Kawai, M; Miyajima, A; Ostertag, W; Stocking, C

    1995-05-01

    An amino-terminally truncated beta C receptor (beta C-R) subunit of the interleukin-3 (IL3)/granulocyte-macrophage colony-stimulating factor/IL5 receptor complex mediates factor-independent and tumorigenic growth in two spontaneous mutants of a promyelocytic cell line. The constitutive activation of the JAK2 protein kinase in these mutants confirms that signaling occurs through the truncated receptor protein. Noteworthily, in addition to a 10-kb deletion in the beta C-R subunit gene encoding the truncated receptor, several secondary and independent mutations that result in the deletion or functional inactivation of the allelic beta C-R subunit and the closely related beta IL3-R subunit genes were observed in both mutants, suggesting that such mutations are necessary for the full oncogenic penetrance of the truncated beta C-R subunit. Reversion of these mutations by the expression of the wild-type beta C-R in the two mutants resulted in a fivefold decrease in cloning efficiency of the mutants in the absence of IL3, confirming a functional interaction between the wild-type and truncated proteins. Furthermore, expression of the truncated beta C-R subunit in factor-dependent myeloid cells did not immediately render the cells autonomous but increased the spontaneous frequency to factor-independent growth by 4 orders of magnitude. Implications for both leukemogenic progression and receptor-subunit interaction and signaling are discussed.

  4. Missense mutations in the MEFV gene are associated with fibromyalgia syndrome and correlate with elevated IL-1beta plasma levels.

    Directory of Open Access Journals (Sweden)

    Jinong Feng

    Full Text Available BACKGROUND: Fibromyalgia syndrome (FMS, a common, chronic, widespread musculoskeletal pain disorder found in 2% of the general population and with a preponderance of 85% in females, has both genetic and environmental contributions. Patients and their parents have high plasma levels of the chemokines MCP-1 and eotaxin, providing evidence for both a genetic and an immunological/inflammatory origin for the syndrome (Zhang et al., 2008, Exp. Biol. Med. 233: 1171-1180. METHODS AND FINDINGS: In a search for a candidate gene affecting inflammatory pathways, among five screened in our patient samples (100 probands with FMS and their parents, we found 10 rare and one common alleles for MEFV, a gene in which various compound heterozygous mutations lead to Familial Mediterranean Fever (FMF. A total of 2.63 megabases of genomic sequence of the MEFV gene were scanned by direct sequencing. The collection of rare missense mutations (all heterozygotes and tested in the aggregate had a significant elevated frequency of transmission to affecteds (p = 0.0085, one-sided, exact binomial test. Our data provide evidence that rare missense variants of the MEFV gene are, collectively, associated with risk of FMS and are present in a subset of 15% of FMS patients. This subset had, on average, high levels of plasma IL-1beta (p = 0.019 compared to FMS patients without rare variants, unaffected family members with or without rare variants, and unrelated controls of unknown genotype. IL-1beta is a cytokine associated with the function of the MEFV gene and thought to be responsible for its symptoms of fever and muscle aches. CONCLUSIONS: Since misregulation of IL-1beta expression has been predicted for patients with mutations in the MEFV gene, we conclude that patients heterozygous for rare missense variants of this gene may be predisposed to FMS, possibly triggered by environmental factors.

  5. Cloning and expression in Escherichia coli of a new gene of Schistosoma japonicum encoding casein kinase Ⅱ beta subunit

    Institute of Scientific and Technical Information of China (English)

    彭寨玉; 余新炳; 吴忠道; 徐劲; 吴德; 李孜

    2004-01-01

    Background Nowadays it is now a focus topic in schistosomiasis research to find ideal vaccine candidates and new drug targets for developing anti-schistosomiasis vaccine. We cloned a new gene, casein kinase Ⅱ beta subunit, of Schistosoma japonicum (S. japonicum) and express it in Escherichia coli (E.coli).Methods The ESTs obtained in our laboratory were analyzed by homologous searching, and a new gene was recognized. The full-length cDNA of the new gene was obtained by joining the 3'RACE PCR fragment and the EST clone. To express the new gene, the cDNA was cloned into pGEX-4T-1 vector and then transformed into E.coli JM109. The recombinant protein was analyzed by SDS-PAGE and Western-blot. Results A 908 bp cDNA was isolated from S. japonicum and identified to be casein kinase Ⅱ beta subunit gene by sequence analysis. The open reading frame of the gene encodes a protein of 217 amino acids exhibiting 75.8%, 75.8%, 73.9%, 68.2%, 51.6% identity to the amino acids sequence of the corresponding genes of Homo sapiens (H. sapiens), Xenopus laevi (X. laevi), Drosophila melanogaster (D. melanogaster), Caenorhabditis elegan (C. elegan), and Schizosaccharomyces pombe (S. promber) respectively. The predicted molecular weight of the protein was 24.921 kDa. The new cDNA sequence had been submitted to GenBank, and its accession number is AY241391. This cDNA was subcloned into the pGEX-4T-1 vector and expressed in E.coli JM109.The recombinant protein could be recognized by the S. japonicum infected rabbit serum. Conclusion The full-length cDNA sequences encoding S. japonicum casein kinase Ⅱ beta subunit were firstly sequenced, cloned, and expressed in E.coli.

  6. Fetal globin expression is regulated by Friend of Prmt1

    NARCIS (Netherlands)

    T.B. van Dijk (Thamar); N. Gillemans (Nynke); F. Pourfarzad, F. (Farzin); K. van Lom (Kirsten); M.M. von Lindern (Marieke); F.G. Grosveld (Frank); J.N.J. Philipsen (Sjaak)

    2010-01-01

    textabstractAnestimated 6% to 7% of the earth's population carries a mutation affecting red blood cell function. The β-thalassemias and sickle cell disease are the most common monogenic disorders caused by these mutations. Increased levels of γ-globin ameliorate the severity of these diseases becaus

  7. The gene for familial Mediterranean fever in both Armenians and non-Ashkenazi Jews is linked to the alpha-globin complex on 16p: evidence for locus homogeneity.

    OpenAIRE

    1992-01-01

    Familial Mediterranean fever (FMF) is a recurrent inflammatory disorder characterized by short episodes of fever, peritonitis, pleuritis, and arthritis. While FMF has been shown to be inherited in an autosomal recessive fashion in both non-Ashkenazi Jews and Armenian families, clinical differences have raised the possibility of genetic heterogeneity. As its pathogenesis is unknown, mapping of the gene for FMF may provide the first objective method for early and accurate diagnosis of this dise...

  8. A second glutamine synthetase gene with expression in the gills of the gulf toadfish (opsanus beta)

    Energy Technology Data Exchange (ETDEWEB)

    Walsh, Patrick J.; Mayer, Gregory D.; Medina, Monica; Bernstein, Matthew L.; Barimo, John F.; Mommsen, Thomas P.

    2003-05-08

    Enzyme and molecular biology approaches were used to more completely characterize the expression of the nitrogen metabolism enzyme glutamine synthetase [GSase; L-glutamate: ammonia ligase (ADP-forming), E.C. 6.3.1.2] in a variety of tissues of the gulf toadfish (Opsanus beta) subjected to unconfined (ammonotelic) and confined (ureotelic) conditions. Enzymological results demonstrate that while weight-specific GSase activities rank in the order of brain > liver > stomach {approx} kidney > intestine > gill> heart/spleen > muscle, when tissue mass is used to calculate a glutamine synthetic potential, the liver has the greatest, followed by muscle > stomach and intestine with minor contributions from the remaining tissues. Additionally, during confinement stress, GSase activity only increases significantly in liver (5-fold) and muscle (2-fold), tissues which previously showed significant expression of the other enzymes of urea synthesis. RT PCR and RACE PCR revealed the presence of a second GSas e cDNA from gill tissue that appears to share relatively low nucleotide and amino acid sequence similarity ({approx}73 percent) with the original GSase cloned from liver, and furthermore lacks a mitochondrial leader targeting sequence. RT PCR and restriction digestion experiments demonstrated that mRNA from the original ''liver'' GSase is expressed in all tissues examined (liver, gill, stomach, intestine, kidney, brain and muscle), whereas the new ''gill'' form shows expression primarily in the gill. Enzyme activities of gill GSase also exhibit a different subcellular compartmentation with apparent exclusive expression in the soluble compartment, whereas other tissues expressing the ''liver'' form show both cytoplasmic and mitochondrial activities. Finally, phylogenetic analysis of a number of GSases demonstrates that the toadfish gill GSase has a greater affinity for a clade that includes the Xenopus GSase genes and

  9. Genes involved in the transforming growth factor beta signalling pathway and the risk of intracranial aneurysms

    NARCIS (Netherlands)

    Ruigrok, Y. M.; Tan, S.; Medic, J.; Rinkel, G. J. E.; Wijmenga, C.

    2008-01-01

    Background and purpose: The 19q13.3 locus for intracranial aneurysms (IA) partly overlaps with the 19q13 locus for abdominal aortic aneurysms (AAA). A common genetic risk factor located in this locus for the two aneurysm types seems plausible. The transforming growth factor beta (TGF-beta) signallin

  10. 164Ile allele in the beta2-Adrenergic receptor gene is associated with risk of elevated blood pressure in women. The Copenhagen City Heart Study

    DEFF Research Database (Denmark)

    Sethi, Amar A; Tybjaerg-Hansen, Anne; Jensen, Gorm B;

    2005-01-01

    Since beta2-adrenergic receptors are important regulators of blood pressure, genetic variation in this receptor could explain risk of elevated blood pressure in selected individuals. We tested the hypothesis that Gly16Arg, Gln27Glu, and Thr164Ile in the beta2-adrenergic receptor gene associated w...

  11. Analysis of beta, gamma, and delta T-cell receptor genes in mycosis fungoides and Sezary syndrome.

    Science.gov (United States)

    Whittaker, S J; Smith, N P; Jones, R R; Luzzatto, L

    1991-10-01

    The authors have analyzed the configuration of immunoglobulin (Ig) and beta, gamma and delta T-cell receptor (TCR) genes in DNA extracted from skin, lymph nodes, and peripheral blood mononuclear cells obtained from 41 patients with mycosis fungoides (MF), 14 patients with Sezary syndrome, and 13 patients with benign inflammatory dermatoses. No discrete rearranged bands (DRB) were detected in patients with inflammatory dermatoses. In tissue DNA from 19 patients with MF DRB were detected with beta and gamma, but not delta TCR probes. Only one patient with MF had a rearrangement of gamma and delta with germ line beta TCR genes. In 13 patients multiple biopsies were analyzed and DRB, when present, were identical in different lesions from individual patients. In three patients analysis of DNA from dermatopathic lymph nodes did not reveal DRB. Analysis of peripheral blood DNA from 24 patients revealed a discrete rearrangement of the gamma TCR gene in four patients and both beta and gamma genes in four additional patients. In MF DRB were detected more frequently with advancing stage of disease in tissues (P less than 0.01) but not in peripheral blood (P equals 0.36). Of 14 patients with Sezary syndrome, eight had DRB in peripheral blood DNA with both beta and gamma probes and in three of these patients identical DRB were also detected in DNA from skin biopsy samples. In contrast, DRB were not detected in the peripheral blood of the other six patients. In both MF and Sezary syndrome there was no restricted usage of particular V gamma genes. These results indicate that in MF (1) T-cell clones can be detected in skin biopsy specimens from the majority of patients with early stage disease, (2) gamma delta T-cell clones are only rarely found, and (3) TCR gene analysis can detect T-cell clones in the peripheral blood with a greater degree of specificity than conventional light microscopic study. In Sezary syndrome these studies also suggest that a subset of patients have a

  12. PARP-1 and YY1 are important novel regulators of CXCL12 gene transcription in rat pancreatic beta cells.

    Directory of Open Access Journals (Sweden)

    Jelena Marković

    Full Text Available Despite significant progress, the molecular mechanisms responsible for pancreatic beta cell depletion and development of diabetes remain poorly defined. At present, there is no preventive measure against diabetes. The positive impact of CXCL12 expression on the pancreatic beta cell prosurvival phenotype initiated this study. Our aim was to provide novel insight into the regulation of rat CXCL12 gene (Cxcl12 transcription. The roles of poly(ADP-ribose polymerase-1 (PARP-1 and transcription factor Yin Yang 1 (YY1 in Cxcl12 transcription were studied by examining their in vitro and in vivo binding affinities for the Cxcl12 promoter in a pancreatic beta cell line by the electrophoretic mobility shift assay and chromatin immunoprecipitation. The regulatory activities of PARP-1 and YY1 were assessed in transfection experiments using a reporter vector with a Cxcl12 promoter sequence driving luciferase gene expression. Experimental evidence for PARP-1 and YY1 revealed their trans-acting potential, wherein PARP-1 displayed an inhibitory, and YY1 a strong activating effect on Cxcl12 transcription. Streptozotocin (STZ-induced general toxicity in pancreatic beta cells was followed by changes in Cxcl12 promoter regulation. PARP-1 binding to the Cxcl12 promoter during basal and in STZ-compromised conditions led us to conclude that PARP-1 regulates constitutive Cxcl12 expression. During the early stage of oxidative stress, YY1 exhibited less affinity toward the Cxcl12 promoter while PARP-1 displayed strong binding. These interactions were accompanied by Cxcl12 downregulation. In the later stages of oxidative stress and intensive pancreatic beta cell injury, YY1 was highly expressed and firmly bound to Cxcl12 promoter in contrast to PARP-1. These interactions resulted in higher Cxcl12 expression. The observed ability of PARP-1 to downregulate, and of YY1 to upregulate Cxcl12 promoter activity anticipates corresponding effects in the natural context where the

  13. The chicken beta 2-microglobulin gene is located on a non-major histocompatibility complex microchromosome: a small, G+C-rich gene with X and Y boxes in the promoter

    DEFF Research Database (Denmark)

    Riegert, P; Andersen, R; Bumstead, N

    1996-01-01

    beta 2-Microglobulin is an essential subunit of major histocompatibility complex (Mhc) class I molecules, which present antigenic peptides to T lymphocytes. We sequenced a number of cDNAs and two genomic clones corresponding to chicken beta 2-microglobulin. The chicken beta 2-microglobulin gene has...... a similar genomic organization but smaller introns and higher G+C content than mammalian beta 2-microglobulin genes. The promoter region is particularly G+C-rich and contains, in addition to interferon regulatory elements, potential S/W, X, and Y boxes that were originally described for mammalian class II...

  14. Mutations in the lysosomal [beta]-galactosidase gene that cause the adult form of GMI gangliosidosis

    Energy Technology Data Exchange (ETDEWEB)

    Chakraborty, S.; Rafi, M.A.; Wenger, D.A. (Thomas Jefferson Univ., Philadelphia, PA (United States))

    1994-06-01

    Three adult patients with acid-galactosidase deficiency/GM1 gangliosidosis who were from two unrelated families of Scandinavian descent were found to share a common point mutation in the coding region of the corresponding gene. The patients share common clinical features, including early dysarthria, mild ataxia, and bone abnormalities. When cDNA from the two patients in family 1 was PCR amplified and sequenced, most (39/41) of the clones showed a C-to-T transition (C[yields]T) at nucleotide 245 (counting from the initiation codon). This mutation changes the codon for the Thr(ACG) to Met(ATG). Mutant and normal sequences were also found in that position in genomic DNA, indicating the presence of another mutant allele. Genomic DNA from the patient in family 2 revealed the same point mutation in one allele. It was determined that in each family only the father carried the C[yields]T mutation. Expression studies showed that this mutation produced 3%-4% of [beta]-galactosidase activity, confirming its deleterious effects. The cDNA clones from the patients in family 1 that did not contain the C[yields]T revealed a 20-bp insertion of intronic sequence between nucleotides 75 and 76, the location of the first intron. Further analysis showed the insertion of a T near the 5[prime] splice donor site which led to the use of a cryptic splice site. It appears that the C[yields]T mutation results in enough functional enzyme to produce a mild adult form of the disease, even in the presence of a second mutation that likely produces nonfunctional enzyme. 31 refs., 7 figs., 1 tab.

  15. Exclusion of the phosphatidylinositol-specific phospholipase C beta 3 (PLC beta 3) gene as candidate for the multiple endocrine neoplasia type 1 (MEN 1) gene

    NARCIS (Netherlands)

    de Wit, M J; Landsvater, R M; Sinke, R J; Geurts van Kessel, A; Lips, C J; Höppener, J W

    1997-01-01

    Multiple endocrine neoplasia type 1 (MEN 1) is inherited as an autosomal dominant disorder, characterized by hyperplasia and neoplasia in several endocrine organs. The MEN 1 gene, which is most probably a tumor suppressor gene, has been localized to a 900-kb region on chromosome 11q13. The human pho

  16. Cloning and characterization of genes encoding alpha and beta subunits of glutamate-gated chloride channel protein in Cylicocyclus nassatus.

    Science.gov (United States)

    Tandon, Ritesh; LePage, Keith T; Kaplan, Ray M

    2006-11-01

    The invertebrate glutamate-gated chloride channels (GluCls) are receptor molecules and targets for the avermectin-milbemycin (AM) group of anthelmintics. Mutations in GluCls are associated with ivermectin resistance in the soil dwelling nematode Caenorhabditis elegans and the parasitic nematode Cooperia oncophora. In this study, full-length cDNAs encoding alpha and beta subunits of GluCl were cloned and sequenced in Cylicocyclus nassatus, a common and important cyathostomin nematode parasite of horses. Both genes possess the sequence characteristics typical of GluCls, and phylogenetic analysis confirms that these genes are evolutionarily closely related to GluCls of other nematodes and flies. Complete coding sequences of C. nassatus GluCl-alpha and GluCl-beta were subcloned into pTL1 mammalian expression vector, and proteins were expressed in COS-7 cells. Ivermectin-binding characteristics were determined by incubating COS-7 cell membranes expressing C. nassatus GluCl-alpha and GluCl-beta proteins with [(3)H]ivermectin. In competitive binding experiments, fitting the data to a one site competition model, C. nassatus GluCl-alpha was found to bind [(3)H]ivermectin with a high amount of displaceable binding (IC(50)=208 pM). Compared to the mock-transfected COS-7 cells, the means of [(3)H]ivermectin binding were significantly different for C. nassatus GluCl-alpha and the Haemonchus contortus GluCl (HcGluCla) (p=0.018 and 0.023, respectively) but not for C. nassatus GluCl-beta (p=0.370). This is the first report of orthologs of GluCl genes and in vitro expression of an ivermectin-binding protein in a cyathostomin species. These data suggest the likelihood of a similar mechanism of action of AM drugs in these parasites, and suggest that mechanisms of resistance may also be similar.

  17. Prevalence PER and VEB beta-lactamase Genes among Acinetobacter baumannii Isolated from Patients in Tehran by PCR

    Directory of Open Access Journals (Sweden)

    Abbas Nazari Monazam

    2014-12-01

    Full Text Available Background and Aim: According to numerous reports of infections caused by spectrum beta lactamases (ESBLs producing Acinetobacter baumannii in our country in recent years, this study was performed to define the antibiotic susceptibility patterns and detect the prevalence PER  and VEB beta-lactamase genes among  A.baumannii isolated from patients in Tehran by PCR. Materials and Methods: 100 A.baumannii clinical isolates collected from various hospitals in Tehran during a year, using special culture media and biochemical tests were identified. The antibiograms of the isolates against 11 antibiotics by disk diffusion method (Disk diffusion according to Clinical and Laboratory Standards Institute (CLSI guidelines was performed. Then minimum inhibitory concentrations (MIC was determined for cefepime and ceftazidime and for the identification of ESBL-producing strains was used in combination disk method, and finally to assess the prevalence of PER and VEB beta-lactamase genes using specific primers PCR was performed. Results: Antibiograms results showed that the greatest resistance to the antibiotics amikacin, cefepime, and less resistance to polymyxin B were obtained. Rates of multi-drug resistant strains of about 70% was achieved. Of the isolates studied, the MIC of ceftazidime in 84% and for cefepime in 91% were ≥ 128 μg/ml. The results of the combined-disk test demonstrated that 20% of samples were ESBL positive. The PCR results showed that 47% and 32% of our isolates had PER and VEB genes respectively. Conclusions: Regarding to existence of PER and VEB genes in this bacterium and possibility of transformation of these genes to the other bacteria, reconsideration in antibiotics consumption patterns and more attention to nosocomial infections control criteria are inevitable.

  18. Mechanism of fiber carcinogenesis: from reactive radical species to silencing of the beta igH3 gene.

    Science.gov (United States)

    Hei, Tom K; Xu, An; Huang, Sarah X; Zhao, Yongliang

    2006-11-01

    Although the U.S. Environmental Protection Agency has restricted the industrial use of regulated forms of asbestos in the United States since the early 1970s, environmental exposure to asbestos remains a health concern in the United States and is a significant health issue among developing countries. Exposure to asbestos is associated with chronic pulmonary diseases and cancer of the lung, pleura, and peritoneum. The mechanism of fiber carcinogenesis is far from clear and is likely to be complex, depending on fiber dimensions, surface properties, and physical durability. The induction of reactive oxygen and nitrogen species upon phagocytosis of fibers plays an important role in fiber genotoxicity. The beta igH3, a secreted protein induced by the transforming growth factor-beta and essential for cell adhesion, is downregulated in asbestos-induced tumorigenic human bronchial epithelial cells. Ectopic expression of the beta igH3 gene abrogates the tumorigenic phenotype and suggests that the gene plays a causal role in fiber carcinogenesis. A better understanding of the carcinogenic mechanism of asbestos and other mineral fibers will provide useful information on interventional and preventive measures for asbestos-mediated diseases such as human pleural and peritoneal mesotheliomas.

  19. Localization of eight additional genes in the human major histocompatibility complex, including the gene encoding the casein kinase II {beta} subunit (CSNK2B)

    Energy Technology Data Exchange (ETDEWEB)

    Albertella, M.R.; Jones, H.; Thomson, W. [Oxford Univ. (United Kingdom)] [and others

    1996-09-01

    A wide range of autoimmune and other diseases are known to be associated with the major histocompatibility complex. Many of these diseases are linked to the genes encoding the polymorphic histocompatibility complex. Many of these diseases are linked to the genes encoding the polymorphic histocompatibility antigens in the class I and class II regions, but some appear to be more strongly associated with genes in the central 1100-kb class III region, making it important to characterize this region fully for the presence of novel genes. An {approximately}220-kb segment of DNA in the class III region separating the Hsp70 (HSPA1L) and BAT1 (D6S8IE) genes, which was previously known to contain 14 genes. Genomic DNA fragments spanning the gaps between the known genes were used as probes to isolate cDNAs corresponding to five new genes within this region. Evidence from Northern blot analysis and exon trapping experiments that suggested the presence of at least two more new genes was also obtained. Partial cDNA and complete exonic genomic sequencing of one of the new genes has identified it as the casein kinase II{beta} subunit (CSNK2B). Two of the other novel genes lie within a region syntenic to that implicated in susceptibility to experimental allergic orchitis in the mouse, an autoimmune disease of the testis, and represent additional candidates for the Orch-1 locus associated with this disease. In addition, characterization of the 13-kb intergenic gap separating the RD (D6545) and G11 (D6S60E) genes has revealed the presence of a gene encoding a 1246-amino-acid polypeptide that shows significant sequence similarity to the yeast anti-viral Ski2p gene product. 49 refs., 8 figs.

  20. Structural organization of the human and mouse laminin beta2 chain genes, and alternative splicing at the 5' end of the human transcript

    DEFF Research Database (Denmark)

    Durkin, M E; Gautam, M; Loechel, F;

    1996-01-01

    We have determined the structural organization of the human and mouse genes that encode the laminin beta2 chain (s-laminin), an essential component of the basement membranes of the neuromuscular synapse and the kidney glomerulus. The human and mouse genes have a nearly identical exon......-intron organization and are the smallest laminin chain genes characterized to date, due to the unusually small size of their introns. The laminin beta2 chain genes of both species consist of 33 exons that span...

  1. Associations of a polymorphic AP-2 binding site in the 5'-flanking region of the bovine beta-lactoglobulin gene with milk proteins.

    Science.gov (United States)

    Kuss, A W; Gogol, J; Geidermann, H

    2003-06-01

    Studies on a polymorphic position (R10) in an Activator-Protein-2 (AP-2) binding site of the bovine beta-Lactoglobulin (beta-Lg) gene promoter region and quantitative traits of individual milk proteins were based on material from 79 German Holstein Friesian (HF) and 61 Simmental (Sm) cows. At least four milk samples per cow were analyzed with alkaline Urea-PAGE in combination with densitometry for quantification of individual milk proteins. The two alleles of the R10 single nucleotide polymorphism (SNP) carry either G or C in position -435 bp of the beta-Lg promoter region. G- and C-alleles were found in Sm with nearly equal frequencies, while in HF the C-allele frequency was higher (0.73) than that of the G-allele. In both breeds, the R10 G-homozygotes had higher (P beta-Lg secreted per day and proportion of beta-Lg in milk protein compared with the C-homozygotes. A similar association was found for alpha-lactalbumin, whereas the relative proportions and daily secreted amounts of caseins (alphaS1, beta, kappa) showed lower values in beta-Lg R10 G-homozygotes. A positive association (P beta-Lg locus to a candidate gene for this trait. The association between the SNP in the AP-2 binding site of the beta-Lg gene and its gene product can be explained as the result of differences in protein binding activity, and, therefore, allele specific differences in gene expression. Thus, our study clearly links a DNA polymorphism of molecular function very closely with in vivo expression parameters of the same locus.

  2. Candidate gene association study in type 2 diabetes indicates a role for genes involved in beta-cell function as well as insulin action.

    Directory of Open Access Journals (Sweden)

    Inês Barroso

    2003-10-01

    Full Text Available Type 2 diabetes is an increasingly common, serious metabolic disorder with a substantial inherited component. It is characterised by defects in both insulin secretion and action. Progress in identification of specific genetic variants predisposing to the disease has been limited. To complement ongoing positional cloning efforts, we have undertaken a large-scale candidate gene association study. We examined 152 SNPs in 71 candidate genes for association with diabetes status and related phenotypes in 2,134 Caucasians in a case-control study and an independent quantitative trait (QT cohort in the United Kingdom. Polymorphisms in five of 15 genes (33% encoding molecules known to primarily influence pancreatic beta-cell function-ABCC8 (sulphonylurea receptor, KCNJ11 (KIR6.2, SLC2A2 (GLUT2, HNF4A (HNF4alpha, and INS (insulin-significantly altered disease risk, and in three genes, the risk allele, haplotype, or both had a biologically consistent effect on a relevant physiological trait in the QT study. We examined 35 genes predicted to have their major influence on insulin action, and three (9%-INSR, PIK3R1, and SOS1-showed significant associations with diabetes. These results confirm the genetic complexity of Type 2 diabetes and provide evidence that common variants in genes influencing pancreatic beta-cell function may make a significant contribution to the inherited component of this disease. This study additionally demonstrates that the systematic examination of panels of biological candidate genes in large, well-characterised populations can be an effective complement to positional cloning approaches. The absence of large single-gene effects and the detection of multiple small effects accentuate the need for the study of larger populations in order to reliably identify the size of effect we now expect for complex diseases.

  3. Mutations in the latent TGF-beta binding protein 3 (LTBP3) gene cause brachyolmia with amelogenesis imperfecta.

    Science.gov (United States)

    Huckert, Mathilde; Stoetzel, Corinne; Morkmued, Supawich; Laugel-Haushalter, Virginie; Geoffroy, Véronique; Muller, Jean; Clauss, François; Prasad, Megana K; Obry, Frédéric; Raymond, Jean Louis; Switala, Marzena; Alembik, Yves; Soskin, Sylvie; Mathieu, Eric; Hemmerlé, Joseph; Weickert, Jean-Luc; Dabovic, Branka Brukner; Rifkin, Daniel B; Dheedene, Annelies; Boudin, Eveline; Caluseriu, Oana; Cholette, Marie-Claude; Mcleod, Ross; Antequera, Reynaldo; Gellé, Marie-Paule; Coeuriot, Jean-Louis; Jacquelin, Louis-Frédéric; Bailleul-Forestier, Isabelle; Manière, Marie-Cécile; Van Hul, Wim; Bertola, Debora; Dollé, Pascal; Verloes, Alain; Mortier, Geert; Dollfus, Hélène; Bloch-Zupan, Agnès

    2015-06-01

    Inherited dental malformations constitute a clinically and genetically heterogeneous group of disorders. Here, we report on four families, three of them consanguineous, with an identical phenotype, characterized by significant short stature with brachyolmia and hypoplastic amelogenesis imperfecta (AI) with almost absent enamel. This phenotype was first described in 1996 by Verloes et al. as an autosomal recessive form of brachyolmia associated with AI. Whole-exome sequencing resulted in the identification of recessive hypomorphic mutations including deletion, nonsense and splice mutations, in the LTBP3 gene, which is involved in the TGF-beta signaling pathway. We further investigated gene expression during mouse development and tooth formation. Differentiated ameloblasts synthesizing enamel matrix proteins and odontoblasts expressed the gene. Study of an available knockout mouse model showed that the mutant mice displayed very thin to absent enamel in both incisors and molars, hereby recapitulating the AI phenotype in the human disorder.

  4. [The cloning and expression of the gene for beta-galactosidase from Candida pseudotropicalis yeasts in Saccharomyces cerevisiae cells].

    Science.gov (United States)

    Tretiak, K A; Zakal'skiĭ, A E; Gudz', S P

    1998-01-01

    The gene of beta-galactosidase of lactose-assimilating yeast Candida pseudotropicalis was cloned in pG2 and pBG2-3 hybrid shuttle vectors and expressed in Saccharomyces cerevisiae laboratory strains under the control of own promoter. The plasmids were able to replicate autonomously with relative stability in transformants of baker's yeasts. The availability of glucose or lactose in the medium influenced the recombinant plasmid stability and the expression of the cloned gene. A number of experiments have shown that the LAC+ phenotype in pG2-transformed Saccharomyces cerevisiae was due to the expression of the Candida pseudotropicalis lactose permease gene that is probably located in SaIG1/XhoI DNA fragment about 4.3 kb long. Southern hybridization experiments showed that LAC(+)-transformants of Saccharomyces cerevisiae contained both autonomously-replicative, and integrative pG2 plasmid.

  5. The Evolution of the Globins: We Thought We Understood It

    Science.gov (United States)

    Lesk, Arthur M.

    Protein crystallography achieved its first results in the late 1950s with the structure determinations of sperm whale myoglobin and human haemoglobin. These gave us our first glimpse of the structural changes that take place during protein evolution. Many other structures of proteins in the globin family have continued to reveal interesting and important details of the coordinated divergence during evolution of amino acid sequences and protein structures and functions.

  6. Induction of the expression of genes encoding TGF-beta isoforms and their receptors by inositol hexaphosphate in human colon cancer cells.

    Science.gov (United States)

    Kapral, Małgorzata; Wawszczyk, Joanna; Hollek, Andrzej; Weglarz, Ludmiła

    2013-01-01

    Transforming growth factors-beta (TGF-beta) are multifunctional cytokines involved in the regulation of cell development, differentiation, survival and apoptosis. They are also potent anticancer agents that inhibit uncontrolled proliferation of cells. Incorrect TGF-beta regulation has been implicated in the pathogenesis of many diseases including inflammation and cancer. In humans, the TGF-beta family consists of three members (TGF-beta1, 2, 3) that show high similarity and homology. TGF-betas exert biological activities on various cell types including neoplastic cells via their specific receptors. Inositol hexaphosphate (phytic acid, IP6), a phytochemical has been reported to possess various health benefits. The aim of this study was to examine the effect of IP6 on the expression of genes encoding TGF-beta1, TGF-beta2, TGF-beta3 isoforms and their receptors TbetaRI, TbetaRII, TbetaRIII in human colorectal cancer cell line Caco-2. The cells were treated with 0.5, 1 and 2.5 mM IP6 for 3, 6 and 12 h. The untreated Caco-2 cells were used as the control. Quantification of genes expression was performed by real time QRT-PCR technique with a SYBR Green I chemistry. The experimental data revealed that the TGF-beta1 mRNA was the predominant isoform in Caco-2 cells and that IP6 enhanced transcriptional activity of genes of all three TGF-beta isoforms and their receptors TbetaRI, TbetaRII TbetaRIII in these cells. At concentrations up to 1 mM, IP6 over-expressed the genes in 6 h lasting cultures, and its higher dose (2.5 mM) caused successively increasing transcript level of TGF-beta isoforms and receptors with the duration of experiment up to 12 h. The findings of this study indicate that one of anti-cancer abilities of IP6 can be realized by enhancing the gene expression of TGF-beta isoforms and their receptors at the transcriptional level.

  7. Serotonin Transporter (5-HTT) and gamma-Aminobutyric Acid Receptor Subunit beta3 (GABRB3) Gene Polymorphisms are not Associated with Autism in the IMGSA Families

    DEFF Research Database (Denmark)

    Maestrini, E.; Lai, C.; Marlow, A.;

    1999-01-01

    Previous studies have suggested that the serotonin transporter (5-HTT) gene and the gamma-aminobutyric acid receptor subunit beta3 (GABRB3) gene, or other genes in the 15q11-q13 region, are possibly involved in susceptibility to autism. To test this hypothesis we performed an association study...... and the GABRB3 genes are unlikely to play a major role in the aetiology of autism in our family data set....

  8. Familial Congenital Hypothyroidism Caused by Abnormal and Bioinactive TSH due to Mutations in the beta-Subunit Gene.

    Science.gov (United States)

    Medeiros-Neto, G; de Lacerda, L; Wondisford, F E

    1997-01-01

    Hereditary TSH deficiency is a rare autosomal recessive disease described in inbred Japanese families and in Greek and Brazilian kindreds. The TSH-beta-subunit gene has been shown to be the site of mutations that will give rise to truncated proteins that cannot dimerize with the alpha subunit or, alternatively, will produce a mutated TSH that is present in the circulation of the affected patients, but it is biologically inactive. Characteristically, the patients with TSH-beta-subunit-defects are born with congenital hypothyroidism, with very low levels of serum thyroid hormones and serum thyroglobulin and, paradoxically, with serum TSH levels that are consistently undetectable or at very low levels. Goiter is not present at birth, but the low radioactive thyroid uptake will increase after bovine TSH stimulation. Other pituitary hormones responses to provocative tests are normal. The subunit levels are at high concentration and are significantly increased following TRH stimulation. In two kindreds, molecular biological studies have indicated mutations in two different sites of exon 2, generating a peptide that would not dimerize with subunits to synthesize TSH molecules. In one kindred, a truncated TSH-beta protein was translated that generated a biologically inactive but detectable serum TSH molecule. (c) 1997, Elsevier Science Inc. (Trends Endocrinol Metab 1997;8:15-20).

  9. Understanding the contrasting spatial haplotype patterns of malaria-protective β-globin polymorphisms.

    Science.gov (United States)

    Hockham, Carinna; Piel, Frédéric B; Gupta, Sunetra; Penman, Bridget S

    2015-12-01

    The malaria-protective β-globin polymorphisms, sickle-cell (β(S)) and β(0)-thalassaemia, are canonical examples of human adaptation to infectious disease. Occurring on distinct genetic backgrounds, they vary markedly in their patterns of linked genetic variation at the population level, suggesting different evolutionary histories. β(S) is associated with five classical restriction fragment length polymorphism haplotypes that exhibit remarkable specificity in their geographical distributions; by contrast, β(0)-thalassaemia mutations are found on haplotypes whose distributions overlap considerably. Here, we explore why these two polymorphisms display contrasting spatial haplotypic distributions, despite having malaria as a common selective pressure. We present a meta-population genetic model, incorporating individual-based processes, which tracks the evolution of β-globin polymorphisms on different haplotypic backgrounds. Our simulations reveal that, depending on the rate of mutation, a large population size and/or high population growth rate are required for both the β(S)- and the β(0)-thalassaemia-like patterns. However, whilst the β(S)-like pattern is more likely when population subdivision is high, migration low and long-distance migration absent, the opposite is true for β(0)-thalassaemia. Including gene conversion has little effect on the overall probability of each pattern; however, when inter-haplotype fitness variation exists, gene conversion is more likely to have contributed to the diversity of haplotypes actually present in the population. Our findings highlight how the contrasting spatial haplotype patterns exhibited by β(S) and β(0)-thalassaemia may provide important indications as to the evolution of these adaptive alleles and the demographic history of the populations in which they have evolved.

  10. Differential gene expression in human brain pericytes induced by amyloid-beta protein.

    NARCIS (Netherlands)

    Rensink, A.A.M.; Otte-Holler, I.; Donkelaar, H.J. ten; Waal, R.M.W. de; Kremer, H.P.H.; Verbeek, M.M.

    2004-01-01

    Cerebral amyloid angiopathy is one of the characteristics of Alzheimer's disease (AD) and this accumulation of fibrillar amyloid-beta (Alphabeta) in the vascular wall is accompanied by marked vascular damage. In vitro, Abeta1-40 carrying the 'Dutch' mutation (DAbeta1-40) induces degeneration of cult

  11. CLONING, SEQUENCING AND EXPRESSION STUDIES OF THE GENES ENCODING AMICYANIN AND THE BETA-SUBUNIT OF METHYLAMINE DEHYDROGENASE FROM THIOBACILLUS-VERSUTUS

    NARCIS (Netherlands)

    UBBINK, M; VANKLEEF, MAG; KLEINJAN, DJ; HOITINK, CWG; HUITEMA, F; BEINTEMA, JJ; DUINE, JA; CANTERS, GW

    1991-01-01

    The genes encoding amicyanin and the beta-subunit of methylamine dehydrogenase (MADH) from Thiobacillus versutus have been cloned and sequenced. The organization of these genes makes it likely that they are coordinately expressed and it supports earlier findings that the blue copper protein amicyani

  12. A forkhead gene related to HNF-3beta is required for gastrulation and axis formation in the ascidian embryo.

    Science.gov (United States)

    Olsen, C L; Jeffery, W R

    1997-09-01

    We have isolated a member of the HNF-3/forkhead gene family in ascidians as a means to determine the role of winged-helix genes in chordate development. The MocuFH1 gene, isolated from a Molgula oculata cDNA library, exhibits a forkhead DNA-binding domain most similar to zebrafish axial and rodent HNF-3beta. MocuFH1 is a single copy gene but there is at least one other related forkhead gene in the M. oculata genome. The MocuFH1 gene is expressed in the presumptive endoderm, mesenchyme and notochord cells beginning during the late cleavage stages. During gastrulation, MocuFH1 expression occurs in the prospective endoderm cells, which invaginate at the vegetal pole, and in the presumptive notochord and mesenchyme cells, which involute over the anterior and lateral lips of the blastopore, respectively. However, this gene is not expressed in the presumptive muscle cells, which involute over the posterior lip of the blastopore. MocuFH1 expression continues in the same cell lineages during neurulation and axis formation, however, during the tailbud stage, MocuFH1 is also expressed in ventral cells of the brain and spinal cord. The functional role of the MocuFH1 gene was studied using antisense oligodeoxynucleotides (ODNs), which transiently reduce MocuFH1 transcript levels during gastrulation. Embryos treated with antisense ODNs cleave normally and initiate gastrulation. However, gastrulation is incomplete, some of the endoderm and notochord cells do not enter the embryo and undergo subsequent movements, and axis formation is abnormal. In contrast, the prospective muscle cells, which do not express MocuFH1, undergo involution and later express muscle actin and acetylcholinesterase, markers of muscle cell differentiation. The results suggest that MocuFH1 is required for morphogenetic movements of the endoderm and notochord precursor cells during gastrulation and axis formation. The effects of inhibiting MocuFH1 expression on embryonic axis formation in ascidians are

  13. Increased renal gene transcription of protein kinase C-beta in human diabetic nephropathy: relationship to long-term glycaemic control

    DEFF Research Database (Denmark)

    Langham, R.G.; Kelly, D.J.; Gow, R.M.;

    2008-01-01

    was examined in renal biopsies (n=25) with classical histological features of diabetic nephropathy and compared with that in normal control tissue (n=6). Peptide localisation of PKC-alpha, PKC-beta and the activated forms phosphorylated PKC-alpha and -beta was also performed on matched paraffin......-embedded sections of renal biopsies using immunohistochemistry. The effects of high glucose on PRKC-beta expression and peptide production in cultured human proximal tubular epithelial cells were assessed. RESULTS: Quantitative real-time PCR demonstrated a 9.9-fold increase in PRKC-beta mRNA in kidney biopsies...... quantitative analysis of mRNA from archival, formalin-fixed, paraffin-embedded tissue sections. RNA was extracted from scraped 6 microm sections of biopsy tissue, and PRKC-alpha and PRKC-beta (also known as PRKCA and PRKCB) mRNA measured using real-time PCR. Expression of genes encoding PKC isoforms...

  14. Effect of transfection with human interferon-beta gene entrapped in cationic multilamellar liposomes in combination with 5-fluorouracil on the growth of human esophageal cancer cells in vitro.

    Science.gov (United States)

    Tsunoo, Hideo; Komura, Sadaaki; Ohishi, Nobuko; Yajima, Haruyoshi; Akiyama, Seiji; Kasai, Yasushi; Ito, Katsuki; Nakao, Akimasa; Yagi, Kunio

    2002-01-01

    When human esophageal cancer cells were transfected with the human interferon-beta (hIFN-beta) gene entrapped in cationic multilamellar liposomes, the growth of all cancer cells tested was suppressed in a dose-dependent manner. The 50% inhibitory concentration (IC50) of the hIFN-beta gene entrapped in the liposomes ranged from 16 to 176 ng plasmid DNA/ml culture medium. Among the 10 cell lines examined, NUEC3, NUEC4, TE-3 and WSSC cell lines were highly susceptible to transfection with this gene entrapped in the liposomes. The IC50 values of the hIFN-beta gene entrapped in the liposomes with respect to cell growth were positively-correlated with those of exogenous cytokine hIFN-beta, suggesting that the antiproliferative effect of hIFN-beta gene entrapped in the liposomes can be mainly ascribed to the function of hIFN-beta produced by cells transfected with the gene. Two days after transfection with the liposome-entrapped gene, the concentration of hIFN-beta secreted into the medium was determined. Even though the level of hIFN-beta observed in the medium was lower than that of the IC50 of exogenously added hIFN-beta, the inhibitory potency of the hIFN-beta gene entrapped in the liposomes on the cell growth was remarkable. When the esophageal cancer cells were treated with 5-fluorouracil (5-FU) in the presence of a low concentration of liposome-entrapped-gene, the rate of growth inhibition of these cells increased over that caused by either 5-FU or hIFN-beta gene entrapped in the liposomes alone. All these data suggest that combination therapy with the hIFN-beta gene entrapped in cationic multilamellar liposomes and the anticancer drug 5-FU would be beneficial for preoperative treatment of carcinoma of the esophagus.

  15. Effect of beta2-adrenoceptor agonists and other cAMP-elevating agents on inflammatory gene expression in human ASM cells: a role for protein kinase A.

    Science.gov (United States)

    Kaur, Manminder; Holden, Neil S; Wilson, Sylvia M; Sukkar, Maria B; Chung, Kian Fan; Barnes, Peter J; Newton, Robert; Giembycz, Mark A

    2008-09-01

    In diseases such as asthma, airway smooth muscle (ASM) cells play a synthetic role by secreting inflammatory mediators such as granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, or IL-8 and by expressing surface adhesion molecules, including ICAM-1. In the present study, PGE(2), forskolin, and short-acting (salbutamol) and long-acting (salmeterol and formoterol) beta(2)-adrenoceptor agonists reduced the expression of ICAM-1 and the release of GM-CSF evoked by IL-1beta in ASM cells. IL-1beta-induced IL-8 release was also repressed by PGE(2) and forskolin, whereas the beta(2)-adrenoceptor agonists were ineffective. In each case, repression of these inflammatory indexes was prevented by adenoviral overexpression of PKIalpha, a highly selective PKA inhibitor. These data indicate a PKA-dependent mechanism of repression and suggest that agents that elevate intracellular cAMP, and thereby activate PKA, may have a widespread anti-inflammatory effect in ASM cells. Since ICAM-1 and GM-CSF are highly NF-kappaB-dependent genes, we used an adenoviral-delivered NF-kappaB-dependent luciferase reporter to examine the effects of forskolin and the beta(2)-adrenoceptor agonists on NF-kappaB activation. There was no effect on luciferase activity measured in the presence of forskolin or beta(2)-adrenoceptor agonists. This finding is consistent with the observation that IL-1beta-induced expression of IL-6, a known NF-kappaB-dependent gene in ASM, was also unaffected by beta(2)-adrenoceptor agonists, forskolin, PGE(2), 8-bromo-cAMP, or rolipram. Collectively, these results indicate that repression of IL-1beta-induced ICAM-1 expression and GM-CSF release by cAMP-elevating agents, including beta(2)-adrenoceptor agonists, may not occur through a generic effect on NF-kappaB.

  16. The Effect of Estradiol-17(beta), Goitrogen (T3), and Flutamide on Gene Expression in Medaka, Oryzias latipes

    Energy Technology Data Exchange (ETDEWEB)

    E.Haut, J

    2005-09-06

    Concern has been generated over the discovery of endocrine disrupting chemicals in rivers near sewage outflows. The presence of endocrine disrupting chemicals such as estradiol-17{beta} has been associated with a reduction of reproductive success in fish and an increase in the female phenotype and gonadal intersex in fish downstream of sewage treatment facilities. Such effects are believed to result from a disruption in the normal estrogenic pathways since estrogen plays a vital role in reproduction, sexual differentiation, the developments of secondary sex characteristics, and ovulation. Most studies have focused on the effect of a single endocrine disruptor on a single gene which does not provide for the interaction between genes. Microarray technology has made it possible to put an entire genome on a single chip so that researchers can get a clearer picture of the interaction of genes expressed in a cell and changes of said interactions when those cells are exposed to various conditions. Medaka males were exposed to known endocrine disruptors, estradial-17{beta} and goitrogen, and medaka females were exposed to flutamide. All treatments were then compared to controls. Total RNA was extracted from the livers of both treated and untreated males and hybridized to a microarray chip designed to have EST sequences specific to medaka. ESTs were identified through two-channel microarray analysis and compared to GenBank using blastn searches to identify up regulated genes. Choriogenins H and L, zona radiata, and vitellogenin, previously shown to be estrogen-induced in male fish were identified. Heat shock proteins (hsp70, hsp90, and hsp8) were also induced by estradiol-17{beta}, as was choriogenin Hminor. Exposure to goitrogen (T3) resulted in the induced expression of glutathione S-transferase and a GABA receptor protein in male medaka. Treatment with flutamide, an antiandrogen, caused the up regulation of choriogenin L, choriogenin Hminor, and zona radiata-2 in female

  17. Abundances of tetracycline, sulphonamide and beta-lactam antibiotic resistance genes in conventional wastewater treatment plants (WWTPs) with different waste load

    DEFF Research Database (Denmark)

    Laht, Mailis; Karkman, Antti; Voolaid, Veiko

    2014-01-01

    Antibiotics and antibiotic resistant bacteria enter wastewater treatment plants (WWTPs), an environment where resistance genes can potentially spread and exchange between microbes. Several antibiotic resistance genes (ARGs) were quantified using qPCR in three WWTPs of decreasing capacity located...... conclude that there is neither considerable enrichment nor purification of antibiotic resistance genes in studied conventional WWTPs....... in Helsinki, Tallinn, and Tartu, respectively: sulphonamide resistance genes (sul1 and sul2), tetracycline resistance genes (tetM and tetC), and resistance genes for extended spectrum beta-lactams (blaoxa-58, blashv-34, and blactx-m-32). To avoid inconsistencies among qPCR assays we normalised the ARG...

  18. Pdx1 and BETA2/NeuroD1 participate in a transcriptional complex that mediates short-range DNA looping at the insulin gene.

    Science.gov (United States)

    Babu, Daniella A; Chakrabarti, Swarup K; Garmey, James C; Mirmira, Raghavendra G

    2008-03-28

    The activity of the insulin gene, Ins, in islet beta cells is thought to arise in part from the synergistic action of the transcription factors Pdx1 and BETA2/NeuroD1. We asked how the binding of these factors to A and E elements many tens or hundreds of base pairs upstream of the start site could influence activity of transcriptional machinery. We therefore tested the hypothesis that the complex of Pdx1 and BETA2/NeuroD1 maintains a DNA conformation such that distal regions of the gene are brought into proximity of the promoter and coding region. We show by coimmunoprecipitation that Pdx1 and BETA2/NeuroD1 exist within a complex and that the two physically interact with one another in this complex as assessed by fluorescence resonance energy transfer. Consistent with this interaction, we found that the two factors simultaneously occupy the same fragment of the Ins gene in beta cell lines using the chromatin immunoprecipitation/re-chromatin immunoprecipitation assay. Using a modification of the chromosome conformation capture assay in vitro and in beta cells, we observed that Pdx1 and BETA2/NeuroD1 mediate looping of a segment of Ins that brings EcoRI sites located at -623 and +761 bp (relative to the transcriptional start site) in proximity to one another. This looping appears to be dependent in vitro upon an intact A3 binding element, but not upon the E2 element. Based on our findings, we propose a model whereby Pdx1 and BETA2/NeuroD1 physically interact to form a nucleoprotein complex on the Ins gene that mediates formation of a short DNA loop. Our results suggest that such short loop conformations may be a general mechanism to permit interactions between transcription factors and basal transcriptional machinery.

  19. A Polymorphism Within the Promoter of the TGF{beta}1 Gene Is Associated With Radiation Sensitivity Using an Objective Radiologic Endpoint

    Energy Technology Data Exchange (ETDEWEB)

    Kelsey, Chris R., E-mail: kelse003@mc.duke.edu [Department of Radiation Oncology, Duke University Medical Center, Durham, NC (United States); Jackson, Lauren [Department of Pathology, Duke University Medical Center, Durham, NC (United States); Langdon, Scott [Department of Immunology, Duke University Medical Center, Durham, NC (United States); Owzar, Kouros [Department of Biostatistics and Bioinformatics, Duke University Medical Center, Durham, NC (United States); Hubbs, Jessica [Department of Radiation Oncology, University of North Carolina, Chapel Hill, NC (United States); Vujaskovic, Zeljko; Das, Shiva [Department of Radiation Oncology, Duke University Medical Center, Durham, NC (United States); Marks, Lawrence B. [Department of Radiation Oncology, University of North Carolina, Chapel Hill, NC (United States)

    2012-02-01

    Purpose: To evaluate whether single nucleotide polymorphisms (SNPs) in the transforming growth factor-{beta}1 (TGF{beta}1) gene are associated with radiation sensitivity using an objective radiologic endpoint. Methods and Materials: Preradiation therapy and serial postradiation therapy single photon emission computed tomography (SPECT) lung perfusion scans were obtained in patients undergoing treatment for lung cancer. Serial blood samples were obtained to measure circulating levels of TGF{beta}1. Changes in regional perfusion were related to regional radiation dose yielding a patient-specific dose-response curve, reflecting the patient's inherent sensitivity to radiation therapy. Six TGF{beta}1 SNPs (-988, -800, -509, 869, 941, and 1655) were assessed using high-resolution melting assays and DNA sequencing. The association between genotype and slope of the dose-response curve, and genotype and TGF{beta}1 ratio (4-week/preradiation therapy), was analyzed using the Kruskal-Wallis test. Results: 39 white patients with preradiation therapy and {>=}6-month postradiation therapy SPECT scans and blood samples were identified. Increasing slope of the dose-response curve was associated with the C(-509)T SNP (p = 0.035), but not the other analyzed SNPs. This SNP was also associated with higher TGF{beta}1 ratios. Conclusions: This study suggests that a polymorphism within the promoter of the TGF{beta}1 gene is associated with increased radiation sensitivity (defined objectively by dose-dependent changes in SPECT lung perfusion).

  20. Development a diagnostic pan-dermatophyte TaqMan probe real-time PCR assay based on beta tubulin gene.

    Science.gov (United States)

    Mirhendi, Hossein; Motamedi, Marjan; Makimura, Koichi; Satoh, Kazuo

    2016-08-01

    Early differentiation of dermatophytosis from other cutaneous mycoses is essential to avoid inaccurate therapy. DNA-based techniques including real-time PCR have increasingly been considered for detection of fungal elements in clinical specimens. In this study, after partial sequence analysis of beta tubulin (BT2) gene in 13 common and rare pathogenic dermatophyte species, a pan-dermatophyte primer and probe set was designed in a TaqMan probe-based PCR format. The sensitivity and specificity of the system was tested with 22 reference strains of dermatophytes, 234 positive clinical specimens, 32 DNA samples extracted from normal nails, several fungi other than dermatophytes and human DNAs. Analytical detection limit of the designed PCR on serially diluted DNAs of prepared recombinant plasmid indicated that only five molecules per sample are the minimum number for reliable detection by the assay. A total of 226 out of 234 (96.5%) DNAs extracted from clinical samples, but none of the 32 nail samples, from healthy volunteers were positive in PCR. The real-time PCR targeted beta tubulin gene established in this study could be a sensitive diagnostic tool which is significantly faster than the conventional culture method and should be useful in the clinical settings, in large-scale epidemiological studies and in clinical trials of antifungal therapy.

  1. Hemoglobina C em homozigose e interação com talassemia beta Homozygous hemoglobin C and its interaction with beta thalassemia

    Directory of Open Access Journals (Sweden)

    Ivan L. Angulo

    2009-01-01

    Full Text Available A hemoglobina C (Hb C é originária do oeste da África e é detectada por migração lenta na eletroforese alcalina em acetato de celulose. Consiste na mutação do gene da globina beta no códon 6 (GAG-AAG, resultando na substituição do sexto aminoácido da cadeia beta da hemoglobina humana, o ácido glutâmico, pelo aminoácido lisina. A cromatografia de alto desempenho (HPLC separa completamente as frações C e A2, permitindo caracterizar a presença da interação com talassemia beta. Esta entidade (Hb CC, em homozigoze é considerada benigna em relação à doença falciforme, já que a falcização não faz parte de sua fisiopatologia. A raridade do diagnóstico C homozigoto e C talassemia beta nos pacientes portadores de hemoglobinopatias nos alertou para a necessidade de se conhecer melhor e estudar aspectos clínicos e hematológicos dos casos dessa mutação em homozigose e na interação com a talassemia beta no ambulatório de anemias do Centro Regional de Hematologia e Hemoterapia de Ribeirão Preto, SP, Brasil.Hemoglobin C (Hb C originated in the west of Africa and is detected by alkaline electrophoresis by slow migration in cellulose acetate. It consists of a mutation of the beta globin gene in codon 6 (GAG-AAG, resulting in a substitution of glutamic acid, the sixth amino acid of the beta string of the human hemoglobin, for lysine. High performance chromatography (HPLC separates the C and A2 fractions completely, allowing the characterization of the presence of interactions with thalassemia beta. This entity (Hb CC is considered benign in respect to sickle cell disease, as sickle cells are not part of its physiopathology. The rarity of the diagnosis of homozygous C and beta thalassemia in patients with hemoglobinopathies showed the necessity of studying clinical and hematologic aspects of the cases of this mutation in homozygosis carriers and the interaction with beta thalassemia in the anemias clinic of the Regional Blood

  2. Detection of four beta-thalassemia point mutations in Iranians using a PCR-ELISA genotyping system.

    Science.gov (United States)

    Gill, Pooria; Forouzandeh, Mehdi; Eshraghi, Naser; Ghalami, Mostafa; Safa, Majid; Noori-Daloii, Mohammad-Reza

    2008-04-01

    Development of molecular techniques with analytical capability of mutation detection can realize the medical diagnosis of diseases and improve people's health. beta-Thalassemia is one of the most prevalent genetic disorders in Iran and using a simple and rapid test in laboratories for the mass screening and prenatal diagnosis is essential. Here, we described a simple method for rapid detection of four common beta-thalassemia point mutations in Iranians (IVS-II-1 (G-->A), IVS-I-5 (G-->C), FSC 8/9 (+G), IVS-I-110 (G-->A)) using a PCR-ELISA genotyping system. After DNA isolation from whole blood, a segment of beta-globin gene was amplified by DIG-labeling PCR. The DIG-labeled PCR amplicons were denatured and added to biotinylated normal probe (for normal gene allele) and mutant probe (for mutant gene allele). The hybrids were detected by colorimetric ELISA method. The optical densities obtained using normal and mutant probes with heterozygous PCR products were very similar. The optical densities obtained using mutant probes were higher than normal probes with homozygous PCR products. In vice versa, the optical densities obtained using normal probes were higher than mutant probes with normal PCR products. All the results demonstrated that the PCR-ELISA has similar specificity in comparison to the amplification refractory mutation system.

  3. An X11alpha/FSBP complex represses transcription of the GSK3beta gene promoter.

    LENUS (Irish Health Repository)

    Lau, Kwok-Fai

    2010-08-04

    X11alpha is a neuronal adaptor protein that interacts with the amyloid precursor protein (APP) through a centrally located phosphotyrosine binding domain to inhibit the production of Abeta peptide that is deposited in Alzheimer\\'s disease brains. X11alpha also contains two C-terminal postsynaptic density-95, large discs, zona occludens 1 (PDZ) domains, and we show here that through its PDZ domains, X11alpha interacts with a novel transcription factor, fibrinogen silencer binding protein. Moreover, we show that an X11alpha\\/fibrinogen silencer binding protein complex signals to the nucleus to repress glycogen synthase kinase-3beta promoter activity. Glycogen synthase kinase-3beta is a favoured candidate kinase for phosphorylating tau in Alzheimer\\'s disease. Our findings show a new function for X11alpha that may impact on Alzheimer\\'s disease pathogenesis.

  4. Beta-D-glycan synthases and the CesA gene family: lessons to be learned from the mixed-linkage (1-->3),(1-->4)beta-D-glucan synthase.

    Science.gov (United States)

    Vergara, C E; Carpita, N C

    2001-09-01

    Cellulose synthase genes (CesAs) encode a broad range of processive glycosyltransferases that synthesize (1-->4)beta-D-glycosyl units. The proteins predicted to be encoded by these genes contain up to eight membrane-spanning domains and four 'U-motifs' with conserved aspartate residues and a QxxRW motif that are essential for substrate binding and catalysis. In higher plants, the domain structure includes two plant-specific regions, one that is relatively conserved and a second, so-called 'hypervariable region' (HVR). Analysis of the phylogenetic relationships among members of the CesA multi-gene families from two grass species, Oryza sativa and Zea mays, with Arabidopsis thaliana and other dicotyledonous species reveals that the CesA genes cluster into several distinct sub-classes. Whereas some sub-classes are populated by CesAs from all species, two sub-classes are populated solely by CesAs from grass species. The sub-class identity is primarily defined by the HVR, and the sequence in this region does not vary substantially among members of the same sub-class. Hence, we suggest that the region is more aptly termed a 'class-specific region' (CSR). Several motifs containing cysteine, basic, acidic and aromatic residues indicate that the CSR may function in substrate binding specificity and catalysis. Similar motifs are conserved in bacterial cellulose synthases, the Dictyostelium discoideum cellulose synthase, and other processive glycosyltransferases involved in the synthesis of non-cellulosic polymers with (1-->4)beta-linked backbones, including chitin, heparan, and hyaluronan. These analyses re-open the question whether all the CesA genes encode cellulose synthases or whether some of the sub-class members may encode other non-cellulosic (1-->4)beta-glycan synthases in plants. For example, the mixed-linkage (1-->3)(1-->4)beta-D-glucan synthase is found specifically in grasses and possesses many features more similar to those of cellulose synthase than to those of

  5. Identification of Klebsiella pneumoniae strains harboring inactive extended-spectrum beta-lactamase antibiotic-resistance genes

    Institute of Scientific and Technical Information of China (English)

    Xu Li; Zhai Yao; Lyu Yuan; Wang Qi; An Shuchang; Chen Jichao; Chen Yusheng

    2014-01-01

    Background The extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae has increasingly become a major contributor to nosocomial infections and can exhibit multiple antibiotic resistance.Previous studies have focused on the resistance genes in ESBL-producing strains,and the resistance-associated genetic environment of non-ESBL-producing strains has been ignored until now.Here,we investigated the occurrence and characteristics of non-ESBL-producing K.pneumoniae,which potentially carries unexpressed resistance genes.Methods K.pneumoniae strains were collected from five medical institutions in China from February 2010 to August 2013.The VITEK-2 ESBL detection system was used as a primary screen to identify the ESBL-producing phenotype,and the three primary types of ESBL-associated genes (CTX,SHV,and TEM) were detected by polymerase chain reaction (PCR) to confirm the strains presenting with a non-ESBL-producing phenotype.mRNA expression in the non-ESBL-producing strains was further screened by reverse-transcription PCR (RT-PCR) to validate their transcriptional efficiency.Results Out of 224 clinically isolated antibiotic-sensitive K.pneumoniae strains with a non-ESBL-producing phenotype,5 (2.2%) were identified to carry inactivated ESBL blaSHV genes with intact upstream promoter regions and resistance gene sequences.Interestingly,three of the five antibiotic-sensitive K.pneumoniae strains containing ESBL blaSHV genes still exhibited mRNA transcription of blasHv,while the other two exhibited no mRNA transcription.Conclusion These findings suggest that inactivated ESBL genes exist in non-ESBL-producing antibiotic-sensitive K.pneumoniae strains,which have the potential to transform the strain into an ESBL phenotype if an inappropriate application or overdose of antibiotics is implemented during clinical management.

  6. [The effect of CSN1 S2, CSN3 and beta-lg genes on milk performance in Xinong Saanen dairy goat].

    Science.gov (United States)

    Chen, Hong; Lan, Xian-Yong; Li, Rui-Biao; Lei, Chu-Zhao; Sun, Wei-Bin; Zhang, Run-Feng; Zheng, Yuan-Lin; Zhu, Bi-Cai

    2005-08-01

    PCR-RFLP technique was applied to analyze correlation between the polymorphisms of CSN1 S2 (alpha(s2) casein), CSN3 (kappa casein) and beta-lg (beta-lactoglobulin) genes and milk performance in 69 individuals of Xinong Saanen dairy goat. The results showed that there was significant correlation between different genotypes of CSN1 S2 locus and milk yield:average milk yield of individuals with genotype FF was less than that of genotype NN (P gene digested with endonuclease Hind III cleavage showed that no significant difference of milk yield between genotype DE and genotype EE was detected in first, second, third and fourth lactation milk yield and average milk yield (P > 0.05). The results of CSN3 gene with endonuclease Taq I cleavage showed that no significant difference of milk yield among individuals with genotype TT, TC and CC was detected (P > 0.05). No polymorphism was detected in PCR products of CSN3 gene digested with endonuclease Hae III. The analysis of beta-lg gene's 5' flanking region (710 bp) by PCR-RFLP in Xinong Saanen dairy goat showed that milk yield of individuals with genotype AA was higher than that with genotype AB in second, third lactation milk yield and average milk yield (P beta-lg gene's 5' flanking region is probably related to high milk yield.

  7. Expression of Beta-Human Chorionic Gonadotropin Genes in Renal Cell Cancer and Benign Renal Disease Tissues

    Institute of Scientific and Technical Information of China (English)

    姜永光; 曾甫清; 肖传国; 刘俊敏

    2003-01-01

    To study the expression of beta-human chorionic gonadotropin (βhCG) genes in renal cellcarcinomas (RCC) and benign renal disease tissues, nested reverse transcription-polymerase chainreaction (RT-PCR) and restriction endonuclease analysis were employed to detect the expression ofβhCG genes in 44 cases of RCC tissues and 24 cases of benign renal disease tissues. It was foundthat 52% RCC samples revealed positive for βhCG mRNA expression. Positive rate in advancedstage and poorly differentiated RCC was higher, but there was no significant difference. The posi-tive rate of βhCG mRNA expression was 54% in 24 cases of benign renal tissues, including 3 casesout of 6 polycystic kidneys, 7 cases out of 13 renal atrophies, 2 cases out of 2 oncocytomas and 1case out of 2 pyonephrotic kidneys. β7 was most frequently transcribed subtype gene independent onthe histology. These findings suggested βhCG gene transcription is not only involved in RCC but al-so in benign renal diseases.

  8. Computational identification and characterization of conserved miRNAs and their target genes in beet (Beta vulgaris).

    Science.gov (United States)

    Li, J L; Cui, J; Cheng, D Y

    2015-08-07

    Highly conserved endogenous non-coding microRNAs (miRNAs) play important roles in plants and animals by silencing genes via destruction or blocking of translation of homologous mRNA. Sugar beet, Beta vulgaris, is one of the most important sugar crops in China, with properties that include wide adaptability and strong tolerance to salinity and impoverished soils. Seedlings of B. vulgaris can grow in soils containing up to 0.6% salt; it is important to understand the molecular mechanisms of salt tolerance to enrich genetic resources for breeding salt-tolerant sugar beets. Here, we report 13 mature miRNAs from 12 families, predicted using an in silico approach from 29,857 expressed sequence tags and 279,223 genome survey sequences. The psRNATarget server predicted 25 target genes for the 13 miRNAs. Most of the target genes appeared to encode transcription factors or were involved in metabolism, signal transduction, stress response, growth, and development. These results improve our understanding of the molecular mechanisms of miRNA in beet and may aid in the development of novel and precise techniques for understanding post-transcriptional gene-silencing mechanisms in response to stress tolerance.

  9. Low insulin resistance and preserved beta-cell function contribute to human longevity but are not associated with TH-INS genes.

    Science.gov (United States)

    Paolisso, G; Barbieri, M; Rizzo, M R; Carella, C; Rotondi, M; Bonafè, M; Franceschi, C; Rose, G; De Benedictis, G

    2001-12-01

    Tyrosine Hydroxylase (TH) and Insulin (INS) genes lie extremely close in the 11p15.5 chromosomal region. An STR marker of the TH gene had revealed this locus associated with longevity. Thus, it seemed of interest to investigate the association between the TH-STR and INS gene variability (FokI-RFLP) with a phenotypic trait, such as the degree of insulin resistance (IR) and beta-cell function in centenarians (C). We analyzed age-related trajectories of IR and beta-cell function in a large sample (n=466) of individuals whose age ranged from 28 to more than 100 years; furthermore, allele average effects on IR and beta-cell function relevant to TH-STR and INS-FokI polymorphisms were estimated in C. Both IR and beta-cell function increased with advancing age and declined in subjects older than 90 years (p for trend <0.001). C had lower IR (1.5+/-0.7 vs. 3.9+/-1.7, p<0.001) and beta-cell function (26.1+/-8.5 vs. 55.4+/-16, p<0.001) than nC. In nC, but not in C, IR and beta-cell function correlated with the main anthropometric and metabolic confounders. Nevertheless, significant allele average effects by TH-STR and INS-FokI polymorphisms on IR and beta-cell function were not observed in C. In conclusion, C has a lower degree of IR and a preserved beta-cell function in comparison to nC, but the cause of such metabolic differences, which are likely does not lie in this genomic region.

  10. The Polymorphisms of Ser49Gly and Gly389Arg in Beta-1-Adrenergic Receptor Gene in Major Depression

    Science.gov (United States)

    KOKUT, Süleyman; ATAY, İnci Meltem; UZ, Efkan; AKPINAR, Abdullah; DEMİRDAŞ, Arif

    2015-01-01

    Introduction It was reported that the genetic susceptibility of major depressive disorder (MDD) is related with genetic polymorphisms. The aim of this study was to investigate the possible association of the genotype and allele frequencies of Ser49Gly and Arg389Gly polymorphisms in MDD by comparing them with healthy subjects. Methods A total of 144 patients with MDD diagnosed according to Diagnostic and Statistical Manual of Mental Disorders, 4th Edition (DSM-IV) criteria and 105 healthy controls were included in the study. Polymerase chain reaction (PCR) with restriction fragment length polymorphism (RFLP) was used for genotyping. Results Of the 144 participants in the MDD group, 77 (53.5%) had homozygous wild type (AA), 57 (39.6%) had heterozygous type (AG), and 10 (6.9%) had mutant (GG) genotype for Ser49Gly, whereas 75 (52.1%) had homozygous wild type (GG), 59 (41.0%) had heterozygous (GC) type, and 10 (6.9%) had mutant homozygous (CC) genotype for Gly386Arg. There were no significant difference in the allele and genotype frequencies of the beta-1-adrenergic receptor (ADRB1) gene for Ser49Gly and Arg389Gly polymorphisms after comparing with healthy controls (p=0.626; p=0.863 and p=0.625; p=0.914). Conclusion The results of our study did not reveal a major effect of the polymorphism of Ser49Gly and Gly389Arg in the ADRB1 gene in MDD. Further studies with larger sample size are required to elucidate the role of other beta-1 adrenergic gene polymorphisms in MDD.

  11. Selection for the α-Thalassemia Genes

    OpenAIRE

    Yokoyama, Shozo

    1983-01-01

    Extremely high incidences of single and double deletions of α-globin genes are known among Asian populations. To study possible mechanisms for the maintenance of such deletions, mathematical analyses have been conducted. It has been shown that a stable polymorphism can be achieved easily through heterozygote advantage using deterministic models. The results strongly suggest that high incidences of single and double deletion of α-globin genes among Asian populations are maintained by some type...

  12. Highly efficient modification of beta-lactoglobulin (BLG) gene via zinc-finger nucleases in cattle

    Institute of Scientific and Technical Information of China (English)

    Shengli Yu; Junjie Luo; Zhiyuan Song; Fangrong Ding; Yunping Dai; Ning Li

    2011-01-01

    Dear Editor,Gene targeting is in widespread use as a gold standard for determining the function of genes in mice and human embryonic stem cells [1].However,the poor efficiency of this technology has hindered its application to domestic animals,for which embryonic stem cells are not available.Although gene-targeted large domestic animals have been produced successfully by combination of homologous recombination-based targeting strategy and cloning [2-4],the efficiency is very low and,more importantly,the disruption of the targeted gene is usually mono-allelic.It thus takes a long time to obtain a null mutant.

  13. Chromosomal integration of a cephalosporinase gene from Acinetobacter baumannii into Oligella urethralis as a source of acquired resistance to beta-lactams.

    Science.gov (United States)

    Mammeri, Hedi; Poirel, Laurent; Mangeney, Nicole; Nordmann, Patrice

    2003-05-01

    Clinical Oligella urethralis isolate COH-1, which was uncommonly resistant to penicillins and narrow-spectrum cephalosporins, was recovered from a 55-year-old patient with a urinary tract infection. Shotgun cloning into Escherichia coli and expression experiments gave recombinant clones expressing either an AmpC beta-lactamase-type phenotype of resistance or a carbenicillin-hydrolyzing beta-lactamase-type phenotype of resistance. The AmpC beta-lactamase identified (ABA-1), which had a pI value of 8.2, had 98% amino acid identity with a chromosomally encoded cephalosporinase of Acinetobacter baumannii. A 820-bp insertion sequence element, ISOur1, belonging to the IS6 family of insertion sequence elements, was identified immediately upstream of bla(ABA-1), providing a -35 promoter sequence and likely giving rise to a hybrid promoter region. The carbenicillin-hydrolyzing beta-lactamase identified (CARB-8), which had a pI value of 6.4, differed from CARB-5 by two amino acid substitutions. Hybridization of CeuI fragment I-restricted DNA fragments of O. urethralis COH-1 with bla(ABA-1)-, bla(CARB-8)-, and 16S rRNA-specific probes indicated the chromosomal integration of the beta-lactamase genes. PCR and hybridization experiments failed to detect bla(CARB-8)- and bla(ABA-1)-like genes in three O. urethralis reference strains, indicating that the beta-lactamase genes identified were the source of acquired resistance in O. urethralis COH-1. This is one of the few examples of the interspecies transfer and the chromosomal integration of a gene encoding a naturally occurring beta-lactamase.

  14. A new alpha-globin variant with increased oxygen affinity in a Swiss family: Hb Frauenfeld [alpha 138(H21)Ser-->Phe, TCC>TTC (alpha 2)].

    Science.gov (United States)

    Hochuli, Michel; Zurbriggen, Karin; Schmid, Marlis; Speer, Oliver; Rochat, Philippe; Frauchiger, Beat; Kleinert, Peter; Schmugge, Markus; Troxler, Heinz

    2009-01-01

    A new alpha-globin mutation [alpha 138(H21)Ser-->Phe] was found in a 55-year-old male proband with an erythrocytosis known since his youth. Cation exchange high performance liquid chromatography (HPLC) revealed an additional peak eluting slightly before Hb A indicating the presence of a variant. The peak area of the variant was approximately one-third that of Hb A suggesting an alpha-globin variant. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis confirmed the mutation at the protein level. The variant is also detectable with isoelectric focusing and reversed phase HPLC. DNA analysis revealed a heterozygous sequence mutation at codon 138 of the alpha2 gene. A C>T transition at the second nucleotide of the codon indicated a Ser-->Phe exchange. The variant showed increased oxygen affinity and was named Hb Frauenfeld.

  15. Molecular Cloning and Sequencing of Hemoglobin-Beta Gene of Channel Catfish, Ictalurus Punctatus Rafinesque

    Science.gov (United States)

    : Hemoglobin-y gene of channel catfish , lctalurus punctatus, was cloned and sequenced . Total RNA from head kidneys was isolated, reverse transcribed and amplified . The sequence of the channel catfish hemoglobin-y gene consists of 600 nucleotides . Analysis of the nucleotide sequence reveals one o...

  16. Characterization of beta-thalassemia mutations in patients from the state of Rio Grande do Norte, Brazil

    Directory of Open Access Journals (Sweden)

    Zama Messala Luna da Silveira

    2011-01-01

    Full Text Available 35 unrelated individuals were studied for characterization as either heterozygous or homozygous for beta-thalassemia. Molecular analysis was done by PCR/RFLP to detect the mutations most commonly associated with beta-thalassemia (β0IVS-I-1, β+IVS-I-6, and β039. In the patients who showed none of these mutations, the beta-globin genes were sequenced. Of the 31 heterozygous patients, 13 (41.9% had the β+IVS-I-6 mutation, 15 (48.4% the β0IVS-I-1 mutation, 2 (6.5% the β+IVS-I-110 mutation and 1 (3.2% the β+IVS-I-5 mutation. IVS-I-6 was detected in the four homozygotes. The mutation in codon 39, often found in previous studies in Brazil, was not detected in the present case. This is the first study aiming at identifying mutations that determine beta-thalassemia in the state of Rio Grande do Norte.

  17. Rearrangement and expression of beta-T-cell receptor and immunoglobulin genes in established Ph1 chronic myelogenous leukemia cell lines.

    Science.gov (United States)

    Berenson, J; Koeffler, H P

    1989-01-01

    We have determined the arrangement and expression of immunoglobulin (Ig) and beta-T-cell receptor (TCR) genes in six established Philadelphia chromosome-positive (Ph1) chronic myelogenous leukemia (CML) cell lines, and correlated these results with their phenotypic characteristics. Three cell lines with nonlymphoid characteristics, EM2, EM3, and K562, did not demonstrate rearrangement or expression of Ig or beta-TCR genes. A new cell line, MB, with a mature B-cell phenotype recently established in our laboratory, contained light and heavy chain immunoglobulin gene rearrangements and expressed mature Ig RNA. In a cell line with an early lymphoid phenotype, BV173, this analysis showed rearrangement of Ig heavy chain and beta-TCR genes, unrearranged Ig light chain DNA, and expression of only an immature beta-TCR transcript. This line provides evidence for T-cell lineage involvement in Ph1 CML. One cell line without markers of any cell type, KCL-22, demonstrated rearranged, unexpressed Ig heavy chain genes, suggesting these cells are at the very earliest stages of lymphoid differentiation. These lines should provide valuable tools to dissect the molecular biology of differentiation in CML and in early lymphocytes.

  18. Haplotypes in tribal Indians bearing the sickle gene: evidence for the unicentric origin of the beta S mutation and the unicentric origin of the tribal populations of India.

    Science.gov (United States)

    Labie, D; Srinivas, R; Dunda, O; Dode, C; Lapoumeroulie, C; Devi, V; Devi, S; Ramasami, K; Elion, J; Ducrocq, R

    1989-08-01

    To determine the origin of sickle cell anemia (SS) in India, we analyzed haplotypes of the beta gene cluster in beta S-carrying individuals belonging to tribal populations living in the Nilgiris region of southern India and complemented the available data on tribes of east-central India. We found that in the Nilgiris tribes chromosomes bearing the beta S gene are linked in 91% of the cases to the "Asian" (Arab-Indian) haplotype (although 25% of the haplotypes had the epsilon polymorphic site negative, making the 5' portion of the haplotype identical with the African Senegal haplotype). These XmnI (+) chromosomes were associated with high G gamma expression (67.2 +/- 5.9%) and a high percentage of Hb F (15.5 +/- 7.9%; range, 6-25.3%). We have similar findings for tribal groups from west-central India (Gujarat). In east-central India we have confirmed the data of others, finding the same haplotype linked to beta S in tribes living in the east (Orissa, Andhra Pradesh). We conclude that the beta S gene in presently isolated and disperse tribal populations in India is associated with one predominant typical haplotype, suggesting a unicentric origin of the mutation in India. In addition, this finding implies a unicentric origin of the tribal populations themselves: The gene must have arisen and spread before tribal dispersion. Furthermore, we find extremely high frequencies of the (-alpha) haplotype in the Nilgiris (0.89) and in Gujarat (0.95). The beta S gene linkage to a high Hb F-expressing haplotype and the high incidence of alpha-thalassemia predict a mild phenotypical expression of sickle cell anemia in India.

  19. Poly purine.pyrimidine sequences upstream of the beta-galactosidase gene affect gene expression in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Brahmachari Samir K

    2001-10-01

    Full Text Available Abstract Background Poly purine.pyrimidine sequences have the potential to adopt intramolecular triplex structures and are overrepresented upstream of genes in eukaryotes. These sequences may regulate gene expression by modulating the interaction of transcription factors with DNA sequences upstream of genes. Results A poly purine.pyrimidine sequence with the potential to adopt an intramolecular triplex DNA structure was designed. The sequence was inserted within a nucleosome positioned upstream of the β-galactosidase gene in yeast, Saccharomyces cerevisiae, between the cycl promoter and gal 10Upstream Activating Sequences (UASg. Upon derepression with galactose, β-galactosidase gene expression is reduced 12-fold in cells carrying single copy poly purine.pyrimidine sequences. This reduction in expression is correlated with reduced transcription. Furthermore, we show that plasmids carrying a poly purine.pyrimidine sequence are not specifically lost from yeast cells. Conclusion We propose that a poly purine.pyrimidine sequence upstream of a gene affects transcription. Plasmids carrying this sequence are not specifically lost from cells and thus no additional effort is needed for the replication of these sequences in eukaryotic cells.

  20. Transforming growth factor-beta1 regulation of ATF-3 and identification of ATF-3 target genes in breast cancer cells.

    Science.gov (United States)

    Kwok, Sukyee; Rittling, Susan R; Partridge, Nicola C; Benson, Chellakkan S; Thiyagaraj, Mayuranathan; Srinivasan, Narasimhan; Selvamurugan, Nagarajan

    2009-10-01

    Transforming growth factor-beta1 (TGF-beta1) is a crucial molecule for stimulation of breast cancer invasion and formation of bone metastases. The molecular mechanisms of how TGF-beta1 mediates these effects have yet to be completely determined. We have found that activating transcription factor-3 (ATF-3) is strongly stimulated and its level is sustained by TGF-beta1 in highly invasive and metastatic human breast cancer (MDA-MB231) and in mouse mammary pad tumor cells (r3T). ATF-3 is also overexpressed in human primary breast cancer tissue. Overexpression of ATF-3 increased normal human mammary epithelial cell number and DNA synthesis suggesting a role for ATF-3 in cell proliferation. The functional role of ATF-3 in breast cancer progression was determined by the RNA interference technique. Knockdown of ATF-3 by ATF-3 shRNA in MDA-MB231 cells decreased expression of cell cycle gene, cyclin A1 in MDA-MB231 cells. ATF-3 shRNA also decreased expression of an invasive and metastatic gene, matrix metalloproteinase-13 (MMP-13; collagenase-3) in these cells. Chromatin immunoprecipitation experiments identified the direct physical interaction of ATF-3 protein on the human MMP-13 promoter. Thus, the dysregulation of ATF-3 by TGF-beta1 is likely to activate cyclin A1 and MMP-13 genes in breast cancer cells and that would be key to the subsequent cancer cell invasion and metastasis.

  1. Genomic organization of the mouse peroxisome proliferator-activated receptor beta/delta gene

    DEFF Research Database (Denmark)

    Larsen, Leif K; Amri, Ez-Zoubir; Mandrup, Susanne

    2002-01-01

    containing eight upstream AUG codons. We show that the presence of the 548 nt leader resulted in a low translational efficiency of the corresponding PPARbeta/delta mRNA and propose, based on structural features of the 5'-untranslated region, that translational initiation may be mediated via an internal...... gene encoding PPARbeta/delta. The gene spans approx. 41 kb and comprises 11 exons of which the six exons located in the 3'-end of the gene are included in all transcripts. Primer-extension and 5'-rapid amplification of cDNA ends experiments revealed the presence of multiple transcription start points...

  2. Expression of gibberellin 3 beta-hydroxylase gene in a gravi-response mutant, weeping Japanese flowering cherry

    Science.gov (United States)

    Sugano, Mami; Nakagawa, Yuriko; Nyunoya, Hiroshi; Nakamura, Teruko

    2004-01-01

    Expressions of the gibberellin biosynthesis gene were investigated in a normal upright type and a gravi-response mutant, a weeping type of Japanese flowering cherry (Prunus spachiana), that is unable to support its own weight and elongates downward. A segment of the gibberellin 3 beta-hydroxylase cDNA of Prunus spachiana (Ps3ox), which is responsible for active gibberellin synthesis, was amplified by using real-time RT-PCR. The content of Ps3ox mRNA in the weeping type was much greater than that in the upright type, while the endogenous gibberellin level was much higher in the elongating zone of the weeping type. These results suggest that the amount and distribution of synthesized gibberellin regulate secondary xylem formation, and the unbalanced distribution of gibberellin affects the gravi-response of the Prunus tree.

  3. Ventilator-associated pneumonia caused by carbapenem-resistant Enterobacteriaceae carrying multiple metallo-beta-lactamase genes

    Directory of Open Access Journals (Sweden)

    Dwivedi Mayank

    2009-07-01

    Full Text Available Context: Ventilator-associated pneumonia (VAP is a leading nosocomial infection in the intensive care unit (ICU. Members of Enterobacteriaceae are the most common causative agents and carbapenems are the most commonly used antibiotics. Metallo-beta-lactamase (MBL production leading to treatment failure may go unnoticed by routine disc diffusion susceptibility testing. Moreover, there is not much information on association of MBL-producing Enterobacteriaceae with ICU-acquired VAP. Therefore, a study was undertaken to find out the association of MBL-producing Enterobacteriaceae with VAP. Settings: This study was conducted in a large tertiary care hospital of North India with an eight-bed critical care unit. Materials and Methods: The respiratory samples (bronchoalveolar lavage, protected brush catheter specimens and endotracheal or transtracheal aspirates obtained from VAP patients (during January 2005-December 2006 were processed, isolated bacteria identified and their antibiotic susceptibilities tested as per standard protocols. The isolates of Enterobacteriaceae resistant to carbapenem were subjected to phenotypic and genotypic tests for the detection of MBLs. Results: Twelve of 64 isolates of Enterobacteriaceae were detected as MBL producers, bla IMP being the most prevalent gene. Additionally, in three strains, simultaneous coexistence of multiple MBL genes was detected. Conclusion: The coexistence of multiple MBL genes in Enterobacteriaceae is an alarming situation. As MBL genes are associated with integrons that can be embedded in transposons, which in turn can be accommodated on plasmids thereby resulting in a highly mobile genetic apparatus, the further spread of these genes in different pathogens is likely to occur.

  4. Thymosin beta 4 protects cardiomyocytes from oxidative stress by targeting anti-oxidative enzymes and anti-apoptotic genes.

    Directory of Open Access Journals (Sweden)

    Chuanyu Wei

    Full Text Available BACKGROUND: Thymosin beta-4 (Tβ4 is a ubiquitous protein with many properties relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory mediators. The mechanism by which Tβ4 modulates cardiac protection under oxidative stress is not known. The purpose of this study is to dissect the cardioprotective mechanism of Tβ4 on H(2O(2 induced cardiac damage. METHODS: Rat neonatal cardiomyocytes with or without Tβ4 pretreatment were exposed to H(2O(2 and expression of antioxidant, apoptotic, and anti-inflammatory genes was evaluated by quantitative real-time PCR and western blotting. ROS levels were estimated by DCF-DA using fluorescent microscopy and fluorimetry. Selected antioxidant, anti-inflammatory and antiapoptotic genes were silenced by siRNA transfections in neonatal cardiomyocytes and effect of Tβ4 on H(2O(2-induced cardiac damage was evaluated. RESULTS: Pre-treatment of Tβ4 resulted in reduction of the intracellular ROS levels induced by H(2O(2 in cardiomyocytes. Tβ4 pretreatment also resulted in an increase in the expression of antiapoptotic proteins and reduction of Bax/BCl(2 ratio in the cardiomyocytes. Pretreatment with Tβ4 resulted in stimulating the expression of antioxidant enzymes copper/zinc SOD and catalase in cardiomyocytes at both transcription and translation levels. Tβ4 treatment resulted in the increased expression of anti-apoptotic and anti-inflammatory genes. Silencing of Cu/Zn SOD and catalase gene resulted in apoptotic cell death in the cardiomyocytes which was prevented by treatment with Tβ4. CONCLUSION: This is the first report that demonstrates the effect of Tβ4 on cardiomyocytes and its capability to selectively upregulate anti-oxidative enzymes, anti-inflammatory genes, and antiapoptotic enzymes in the neonatal cardiomyocytes thus preventing cell death thereby protecting the myocardium. Tβ4 treatment resulted in decreased oxidative stress and inflammation in the

  5. Correlation of phenotypic tests with the presence of the blaZ gene for detection of beta-lactamase

    Directory of Open Access Journals (Sweden)

    Adriano Martison Ferreira

    Full Text Available Abstract Staphylococcus aureus and Staphylococcus saprophyticus are the most common and most important staphylococcal species associated with urinary tract infections. The objective of the present study was to compare and to evaluate the accuracy of four phenotypic methods for the detection of beta-lactamase production in Staphylococcus spp. Seventy-three strains produced a halo with a diameter ≤28 mm (penicillin resistant and all of them were positive for the blaZ gene. Among the 28 susceptible strain (halo ≥29 mm, 23 carried the blaZ gene and five did not. The zone edge test was the most sensitive (90.3%, followed by MIC determination (85.5%, but the specificity of the former was low (40.0%. The nitrocefin test was the least sensitive (28.9%. However, the nitrocefin test together with the disk diffusion method showed the highest specificity (100%. The present results demonstrated that the zone edge test was the most sensitive phenotypic test for detection of beta-lactamase, although it is still not an ideal test to detect this type of resistance since its specificity was low. However, the inhibition halo diameter of the penicillin disk can be used together with the zone edge test since the same disk is employed in the two tests. Combined analysis of the two tests shows a sensitivity of 90.3% and specificity of 100%, proving better sensitivity, especially for S. saprophyticus. This is a low-cost test of easy application and interpretation that can be used in small and medium-sized laboratories where susceptibility testing is usually performed by the disk diffusion method.

  6. Correlation of phenotypic tests with the presence of the blaZ gene for detection of beta-lactamase.

    Science.gov (United States)

    Ferreira, Adriano Martison; Martins, Katheryne Benini; Silva, Vanessa Rocha da; Mondelli, Alessandro Lia; Cunha, Maria de Lourdes Ribeiro de Souza da

    Staphylococcus aureus and Staphylococcus saprophyticus are the most common and most important staphylococcal species associated with urinary tract infections. The objective of the present study was to compare and to evaluate the accuracy of four phenotypic methods for the detection of beta-lactamase production in Staphylococcus spp. Seventy-three strains produced a halo with a diameter ≤28mm (penicillin resistant) and all of them were positive for the blaZ gene. Among the 28 susceptible strain (halo ≥29mm), 23 carried the blaZ gene and five did not. The zone edge test was the most sensitive (90.3%), followed by MIC determination (85.5%), but the specificity of the former was low (40.0%). The nitrocefin test was the least sensitive (28.9%). However, the nitrocefin test together with the disk diffusion method showed the highest specificity (100%). The present results demonstrated that the zone edge test was the most sensitive phenotypic test for detection of beta-lactamase, although it is still not an ideal test to detect this type of resistance since its specificity was low. However, the inhibition halo diameter of the penicillin disk can be used together with the zone edge test since the same disk is employed in the two tests. Combined analysis of the two tests shows a sensitivity of 90.3% and specificity of 100%, proving better sensitivity, especially for S. saprophyticus. This is a low-cost test of easy application and interpretation that can be used in small and medium-sized laboratories where susceptibility testing is usually performed by the disk diffusion method.

  7. Hydrocephalus caused by conditional ablation of the Pten or beta-catenin gene

    Directory of Open Access Journals (Sweden)

    Ohtoshi Akihira

    2008-10-01

    Full Text Available Abstract To investigate the roles of Pten and β-Catenin in the midbrain, either the Pten gene or the β-catenin gene was conditionally ablated, using Dmbx1 (diencephalon/mesencephalon-expressed brain homeobox gene 1-Cre mice. Homozygous disruption of the Pten or β-catenin gene in Dmbx1-expressing cells caused severe hydrocephalus and mortality during the postnatal period. Conditional deletion of Pten resulted in enlargement of midbrain structures. β-catenin conditional mutant mice showed malformation of the superior and inferior colliculi and stenosis of the midbrain aqueduct. These results demonstrate that both Pten and β-Catenin are essential for proper midbrain development, and provide the direct evidence that mutations of both Pten and β-catenin lead to hydrocephalus.

  8. Type 1 Diabetes Candidate Genes Linked to Pancreatic Islet Cell Inflammation and Beta-Cell Apoptosis

    Science.gov (United States)

    Størling, Joachim; Pociot, Flemming

    2017-01-01

    Type 1 diabetes (T1D) is a chronic immune-mediated disease resulting from the selective destruction of the insulin-producing pancreatic islet β-cells. Susceptibility to the disease is the result of complex interactions between environmental and genetic risk factors. Genome-wide association studies (GWAS) have identified more than 50 genetic regions that affect the risk of developing T1D. Most of these susceptibility loci, however, harbor several genes, and the causal variant(s) and gene(s) for most of the loci remain to be established. A significant part of the genes located in the T1D susceptibility loci are expressed in human islets and β cells and mounting evidence suggests that some of these genes modulate the β-cell response to the immune system and viral infection and regulate apoptotic β-cell death. Here, we discuss the current status of T1D susceptibility loci and candidate genes with focus on pancreatic islet cell inflammation and β-cell apoptosis. PMID:28212332

  9. Type 1 Diabetes Candidate Genes Linked to Pancreatic Islet Cell Inflammation and Beta-Cell Apoptosis.

    Science.gov (United States)

    Størling, Joachim; Pociot, Flemming

    2017-02-16

    Type 1 diabetes (T1D) is a chronic immune-mediated disease resulting from the selective destruction of the insulin-producing pancreatic islet β-cells. Susceptibility to the disease is the result of complex interactions between environmental and genetic risk factors. Genome-wide association studies (GWAS) have identified more than 50 genetic regions that affect the risk of developing T1D. Most of these susceptibility loci, however, harbor several genes, and the causal variant(s) and gene(s) for most of the loci remain to be established. A significant part of the genes located in the T1D susceptibility loci are expressed in human islets and β cells and mounting evidence suggests that some of these genes modulate the β-cell response to the immune system and viral infection and regulate apoptotic β-cell death. Here, we discuss the current status of T1D susceptibility loci and candidate genes with focus on pancreatic islet cell inflammation and β-cell apoptosis.

  10. Genes, Gender, Environment, and Novel Functions of Estrogen Receptor Beta in the Susceptibility to Neurodevelopmental Disorders

    Directory of Open Access Journals (Sweden)

    Mukesh Varshney

    2017-02-01

    Full Text Available Many neurological disorders affect men and women differently regarding prevalence, progression, and severity. It is clear that many of these disorders may originate from defective signaling during fetal or perinatal brain development, which may affect males and females differently. Such sex-specific differences may originate from chromosomal or sex-hormone specific effects. This short review will focus on the estrogen receptor beta (ERβ signaling during perinatal brain development and put it in the context of sex-specific differences in neurodevelopmental disorders. We will discuss ERβ’s recent discovery in directing DNA de-methylation to specific sites, of which one such site may bear consequences for the susceptibility to the neurological reading disorder dyslexia. We will also discuss how dysregulations in sex-hormone signaling, like those evoked by endocrine disruptive chemicals, may affect this and other neurodevelopmental disorders in a sex-specific manner through ERβ.

  11. Genes, Gender, Environment, and Novel Functions of Estrogen Receptor Beta in the Susceptibility to Neurodevelopmental Disorders

    Science.gov (United States)

    Varshney, Mukesh; Nalvarte, Ivan

    2017-01-01

    Many neurological disorders affect men and women differently regarding prevalence, progression, and severity. It is clear that many of these disorders may originate from defective signaling during fetal or perinatal brain development, which may affect males and females differently. Such sex-specific differences may originate from chromosomal or sex-hormone specific effects. This short review will focus on the estrogen receptor beta (ERβ) signaling during perinatal brain development and put it in the context of sex-specific differences in neurodevelopmental disorders. We will discuss ERβ’s recent discovery in directing DNA de-methylation to specific sites, of which one such site may bear consequences for the susceptibility to the neurological reading disorder dyslexia. We will also discuss how dysregulations in sex-hormone signaling, like those evoked by endocrine disruptive chemicals, may affect this and other neurodevelopmental disorders in a sex-specific manner through ERβ. PMID:28241485

  12. Alternative-splicing in the exon-10 region of GABA(A receptor beta(2 subunit gene: relationships between novel isoforms and psychotic disorders.

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    Cunyou Zhao

    Full Text Available BACKGROUND: Non-coding single nucleotide polymorphisms (SNPs in GABRB2, the gene for beta(2-subunit of gamma-aminobutyric acid type A (GABA(A receptor, have been associated with schizophrenia (SCZ and quantitatively correlated to mRNA expression and alternative splicing. METHODS AND FINDINGS: Expression of the Exon 10 region of GABRB2 from minigene constructs revealed this region to be an "alternative splicing hotspot" that readily gave rise to differently spliced isoforms depending on intron sequences. This led to a search in human brain cDNA libraries, and the discovery of two novel isoforms, beta(2S1 and beta(2S2, bearing variations in the neighborhood of Exon-10. Quantitative real-time PCR analysis of postmortem brain samples showed increased beta(2S1 expression and decreased beta(2S2 expression in both SCZ and bipolar disorder (BPD compared to controls. Disease-control differences were significantly correlated with SNP rs187269 in BPD males for both beta(2S1 and beta(2S2 expressions, and significantly correlated with SNPs rs2546620 and rs187269 in SCZ males for beta(2S2 expression. Moreover, site-directed mutagenesis indicated that Thr(365, a potential phosphorylation site in Exon-10, played a key role in determining the time profile of the ATP-dependent electrophysiological current run-down. CONCLUSION: This study therefore provided experimental evidence for the importance of non-coding sequences in the Exon-10 region in GABRB2 with respect to beta(2-subunit splicing diversity and the etiologies of SCZ and BPD.

  13. Energy expenditure, body composition and insulin response to glucose in male twins discordant for the Trp64Arg polymorphism of the beta3-adrenergic receptor gene

    DEFF Research Database (Denmark)

    Højlund, K; Christiansen, C; Bjørnsbo, K S;

    2006-01-01

    and environmental background, the Trp64Arg polymorphism of the beta3AR gene is associated with lower fat mass, fasting insulin levels and an appropriate insulin response to glucose. Thus, heterozygosity for the Trp64Arg variant is unlikely to increase the risk of obesity, insulin resistance or type 2 diabetes.......AIM: The tryptophan to arginine change in position 64 (Trp64Arg) polymorphism of the beta3-adrenergic receptor (beta3AR) gene has been associated with an increased prevalence of obesity, insulin resistance and type 2 diabetes. In this, decreased rates of energy expenditure and impaired insulin......-ray absorptiometry scanning and energy expenditure by indirect and direct calorimetry. RESULTS: Twins heterozygous for the Trp64Arg polymorphism showed significantly lower fat mass independent of the method used, and significantly lower fasting insulin and glucose concentrations compared with their homozygous wild...

  14. VIGS技术在甜菜上的应用%Application of Virus-Induced Gene Silencing in Beta Vulgaris

    Institute of Scientific and Technical Information of China (English)

    龚攀; 崔杰; 李俊良; 罗成飞; 杨虎臣

    2015-01-01

    Virus⁃induced gene silencing is an effective method of reverse genetics research for gene function analysis in plants. Compared with gene knockout, transgene and some other methods, it has many advantages such as faster, low⁃cost and high⁃throughput. With the accomplishment of Beta vulgaris′ complete genome sequencing,the functional characterization of a large number of sequences need to be identified . It is important to develop a rapid method of VIGS functional analysis system of beet genes. In this paper, we mediated the TRV ( Tobacco rattle virus) infection of sugar beet seedlings by Agrobacterium while Tobacco rattle virus is currently the most widely used viral vectors. Total RNA was extracted from the beet leaf and reverse transcribed into cDNA, we designed specific primers used in RT⁃PCR to detect the virus on the infected beet. The results demonstrated that the virus could infect beet, which laid the foundation for the next VIGS system established in Beta vulgaris.%病毒诱导基因沉默( VIGS)是一种应用于研究植物基因功能的反向遗传学手段。相对于基因敲除及转基因等方法,它具有时间短、成本低、高通量等优点。随着甜菜全基因组的测序完成,为尽早对大量序列信息进行注释和功能鉴定,急需建立甜菜VIGS体系。本文采用目前应用最广泛的烟草脆裂病毒载体在农杆菌的介导下侵染甜菜幼苗叶片,同时设立对照。提取甜菜幼苗叶片总RNA,并反转录成cDNA,设计特异引物进行RT⁃PCR检测。结果表明:在侵染植株上检测到了烟草脆裂病毒特异条带,证明该病毒可以对甜菜进行侵染,这一结果为下一步建立甜菜VIGS体系奠定了基础。

  15. The 3D chromatin structure of the mouse β-haemoglobin gene cluster

    NARCIS (Netherlands)

    M.P.C. van de Corput (Mariëtte); T.A. Knoch (Tobias); E. de Boer (Ernie); W.A. van Cappellen (Gert); M. Lesnussa (Michael); H.J.F.M.M. Eussen (Bert)

    2010-01-01

    textabstractHere we show a 3D DNA-FISH method to visualizes the 3D structure of the β-globin locus. Geometric size and shape measurements of the 3D rendered signals (128Kb) show that the volume of the β-globin locus decreases almost two fold upon gene activation. A decrease in length and a distinc

  16. Use of a non-radioactive hybridisation assay for direct detection of gram-negative bacteria carrying TEM beta-lactamase genes in infected urine.

    Science.gov (United States)

    Carter, G I; Towner, K J; Pearson, N J; Slack, R C

    1989-02-01

    DNA in infected urines from 81 patients with urinary tract infection was hybridised directly with a non-radioactive DNA probe specific for bacterial genes coding for TEM-type beta-lactamase. The results were assessed by means of a computerised image analysis system and compared with those obtained following isolation of the infecting organism, conventional sensitivity testing and isoelectric focusing (IEF) procedures for the detection of TEM-type beta-lactamase. Of the 27 ampicillin-resistant gram-negative organisms isolated in pure culture from the urines, 14 were shown by both hybridisation and IEF to carry a gene for TEM beta-lactamase production. Only four discordant results were obtained: three "false positive" direct hybridisation results, one due to urine pigmentation, and one, possibly, to a TEM beta-lactamase gene which was not being expressed, and one "false negative" result due to insufficient cell numbers in the urine. The system is capable of screening large numbers of samples and is applicable to any gene for which a suitable DNA probe is available.

  17. Thiabendazole resistance and mutations in the beta-tubulin gene of Penicillium expansum strains isolated from apples and pears with blue mold decay.

    Science.gov (United States)

    Cabañas, Romualdo; Castellá, Gemma; Abarca, M Lourdes; Bragulat, M Rosa; Cabañes, F Javier

    2009-08-01

    Penicillium expansum is the causal agent of blue mold rot, a postharvest decay of stored fruits. This fungus also produces the mycotoxins patulin and citrinin. Control of P. expansum still relies mainly on the use of fungicides such as thiabendazole. Since its introduction, resistant strains have been reported. The aim of this work was to investigate the thiabendazole resistance and mutations in the beta-tubulin gene of P. expansum strains isolated from apples and pears with blue mold decay from Spain. A total of 71 strains of P. expansum were scored for resistance to thiabendazole and the beta-tubulin gene was sequenced. Out of 71 strains, 37 were sensitive and 34 were resistant to thiabendazole. Regarding the beta-tubulin gene sequence, 10 different genetic types were determined, with a 99.7-100% similarity. When the amino acid sequence was deduced, five different amino acid sequences were found. All except one of the sensitive strains lacked mutations in the region sequenced. Of the 34 resistant strains, only eight had mutations that involved the residues 198 and 240. All the strains with mutations at position 198 always corresponded to resistant isolates. However, a high percentage of resistant strains had no mutations in the region of the beta-tubulin gene sequenced, and so other mechanisms may be involved in thiabendazole resistance.

  18. Characterization of a Beta vulgaris PGIP defense gene promoter in transgenic plants

    Science.gov (United States)

    Polygalacturonase-inhibiting protein (BvPGIP) genes were cloned from a sugar beet breeding line F1016 with increased tolerance to the sugar beet root maggot. Polygalacturonase-inhibiting proteins are cell wall leucine-rich repeat (LRR) proteins with crucial roles in development, pathogen defense an...

  19. Beta vulgaris L. serine proteinase inhibitor gene expression correlates to insect pest resistance in sugar beet

    Science.gov (United States)

    Analyzing genes that can be used for improving sugar beet resistance to the sugar beet root maggot (SBRM, Tetanops myopaeformis Roder), one of the most destructive insect pests of sugar beet in North America, was a major goal in our investigation. We report on the expression patterns of a sugar beet...

  20. A PUTATIVE BETA-GLUCANASE PSEUDOGENE BEHIND THE POTATO GBSS GENE

    NARCIS (Netherlands)

    VANDERLEIJ, FR; ABELN, ECA; HESSELINGMEINDERS, A; FEENSTRA, WJ

    1993-01-01

    We identified an open reading frame (ORF) which is located closely behind the gene encoding granule-bound starch synthase (GBSS) of potato (Solanum tuberosum L.). The ORF ends with a perfect 43 bp direct repeat, which carries the stop triplet precisely at the beginning of the second repeat. The dedu

  1. The origin of HTLV-1 in southern Bahia by phylogenetic, mtDNA and β-globin analysis

    Directory of Open Access Journals (Sweden)

    Milena Magalhães Aleluia

    2015-12-01

    Full Text Available Different hypotheses have been elaborated to explain how the HTLV spread throughout the world. It has been proposed that the virus was introduced in Bahia, during the slave-trade period from the 16th century to 19th century. However, there is no information about the HTLV evolutionary history in southern Bahia. The phylogeny is fundamental in order to clarify its introduction and dispersion. The DNA of 29 samples was extracted, followed by nested-PCR assay for the LTR and DNA sequencing. These sequences were analyzed by phylogenic methods. The mtDNA ancestry markers and βA-globin haplotypes were analyzed by PCR/RFLP. In relation to HTLV subtyping, all samples were classified as cosmopolitan subtype and transcontinental subgroup. Results suggest an ancient post-Columbian introduction of HTLV-1a-A associated with the slave trade between the XVI and late XIX centuries in southern Bahia. As regards the ethnicity of HTLV-infected women, the haplotype characterization of β-globin gene and the mtDNA ethnicity of HTLV-infected women, we have detected a major African contribution, with a predominance of Benin and Bantu types. HTLV-1 infection is spread in Bahia and the point of origin was possibly Salvador.

  2. Cloning of the astaxanthin synthase gene from Xanthophyllomyces dendrorhous (Phaffia rhodozyma) and its assignment as a beta-carotene 3-hydroxylase/4-ketolase.

    Science.gov (United States)

    Ojima, Kazuyuki; Breitenbach, Jürgen; Visser, Hans; Setoguchi, Yutaka; Tabata, Kazuyuki; Hoshino, Tatsuo; van den Berg, Johan; Sandmann, Gerhard

    2006-02-01

    A gene has been cloned from Xanthophyllomyces dendrorhous by complementation of astaxanthin formation in a beta-carotene accumulating mutant. It consists of 3,166 bp and contains 17 introns. For the beta-carotene mutant ATCC 96815, a single point mutation in the splicing sequence of intron 8 was found. The resulting improper splicing of the mRNA results in an inactive protein. The cDNA of this beta-carotene oxygenase encodes a cytochrome P450 monooxygenase belonging to the 3A subfamily. P450-specific domains were identified including a cytochrome P450 and an oxygen binding motif. Electrons are provided by a cytochrome P450 reductase. Functional characterization of the enzyme by genetic modification of X. dendrorhous demonstrated that this P450 monooxygenase is multifunctional catalyzing all steps from beta-carotene to astaxanthin formation by oxygenation of carbon 3 and 4. The reaction sequence is first 4-ketolation of beta-carotene followed by 3-hydroxylation. A hydroxylation mechanism at allylic carbon atoms has been proposed for the generation of 4-keto and 3-hydroxy groups at both beta-ionone ends.

  3. Transforming growth factor beta stimulation of biglycan gene expression is potentially mediated by sp1 binding factors

    DEFF Research Database (Denmark)

    Heegaard, Anne-Marie; Xie, Zhongjian; Young, Marian Frances;

    2004-01-01

    Biglycan is a small leucine-rich proteoglycan which is localized in the extracellular matrix of bone and other specialized connective tissues. Both biglycan mRNA and protein are up-regulated by transforming growth factor-beta(1) (TGF-beta(1)) and biglycan appears to influence TGF-beta(1) activity...

  4. Cloning and Expression Analysis of a Gene Encoding for Ascorbate Peroxidase and Responsive to Salt Stress in Beet (Beta vulgaris).

    Science.gov (United States)

    Dunajska-Ordak, Kamila; Skorupa-Kłaput, Monika; Kurnik, Katarzyna; Tretyn, Andrzej; Tyburski, Jarosław

    2014-01-01

    BvpAPX is a full-length cDNA-encoding peroxisomal ascorbate peroxidase isolated from leaves of salt-stressed beet (Beta vulgaris) plants. A high level of identity has been reported between the deduced amino acid sequence of BvpAPX and other known ascorbate peroxidases. The genomic sequence of BvpAPX revealed a gene composed of 5 exons and 4 introns. Several sequence motifs revealed in the 5'UTR region of the gene confer to BvpAPX a putative responsiveness to various abiotic stresses. We determined the effect of salt stress on BvpAPX expression in leaves of the cultivated beet varieties, Huzar and Janosik, and their wild salt-tolerant relative B. vulgaris ssp. maritima. Plants were subjected to salt stress during a 32-day culture period (long-term salt treatment). An alternative salinization protocol consisted of an 18-h incubation of detached beet leaves in media supplemented with toxic salt concentrations (short-term salt treatment). RT-Q-PCR analysis revealed that BvpAPX expression markedly increased in leaves of plants subjected to conditions of long-term treatment with salinity, whereas BvpAPX transcript levels remained unaffected in detached leaves during short-term salt treatment. In addition, several leaf redox system parameters, such as ascorbate peroxidase activity or ascorbic acid, hydrogen peroxide, and lipid hydroperoxide concentration, were determined in the leaves of beet plants subjected to salt stress conditions.

  5. Differential effects of histone deacetylase inhibitors on phorbol ester- and TGF-beta1 induced murine tissue inhibitor of metalloproteinases-1 gene expression.

    Science.gov (United States)

    Young, David A; Billingham, Olivia; Sampieri, Clara L; Edwards, Dylan R; Clark, Ian M

    2005-04-01

    Expression of the tissue inhibitor of metalloproteinases-1 (Timp-1) gene can be induced by either phorbol myristate acetate (PMA) or transforming growth factor beta1 (TGF-beta1), although the signalling pathways involved are not clearly defined. Canonically, histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA) or sodium butyrate (NaB) increase total cellular histone acetylation and activate expression of susceptible genes. Remarkably, PMA and TGF-beta1 stimulation of Timp-1 show a differential response to TSA or NaB. TSA or NaB potentiate PMA-induced Timp-1 expression but repress TGF-beta1-induced Timp-1 expression. The repression of TGF-beta1-induced Timp-1 by TSA was maximal at 5 ng.mL(-1), while for the superinduction of PMA-induced Timp-1 expression, the maximal dose is > 500 ng x mL(-1) TSA. A further HDACi, valproic acid, did not block TGF-beta1-induced Timp-1 expression, demonstrating that different HDACs impact on the induction of Timp-1. For either PMA or TGF-beta1 to induce Timp-1 expression, new protein synthesis is required, and the induction of AP-1 factors closely precedes that of Timp-1. The effects of the HDACi can be reiterated in transient transfection using Timp-1 promoter constructs. Mutation or deletion of the AP-1 motif (-59/-53) in the Timp-1 promoter diminishes PMA-induction of reporter constructs, however, the further addition of TSA still superinduces the reporter. In c-Jun-/- cells, PMA still stimulates Timp-1 expression, but TSA superinduction is lost. Transfection of a series of Timp-1 promoter constructs identified three regions through which TSA superinduces PMA-induced Timp-1 and we have demonstrated specific protein binding to two of these regions which contain either an avian erythroblastosis virus E26 (v-ets) oncogene homologue (Ets) or Sp1 binding motif.

  6. Renal tubular dysfunction with nephrocalcinosis in a patient with beta thalassemia minor

    Directory of Open Access Journals (Sweden)

    Prabahar Murugesan

    2008-01-01

    Full Text Available Thalassemia is a hereditary anemia resulting from defect in hemoglobin production. Beta thalassemia is due to impaired production of beta globin chains, leading to a relative excess of alpha globin chains. The term beta thalassemia minor is used to describe heterozygotes, who carry one normal beta globin allele and one beta thalassemic allele. The vast majority of these patients are asymptomatic. However, a variety of renal tubular abnormalities including hypercalciuria, hypo-magnesemia with renal magnesium wasting, decreased tubular absorption of phosphorus, hypo-uricemia with renal uric acid wasting, renal glycosuria and tubular proteinuria have been described even in patients with beta thalassemia minor. We here in report a 24-year old female patient who was found to have thalassemia minor and nephrocalcinosis with evidence of renal tubular dysfunction. Investigations revealed normal renal function, hypercalciuria, reduced tubular reabsorption of phos-phorus, hypomagnesemia and renal magnesium wasting. Screening for aminoaciduria was found to be negative. An acid loading test revealed normal urinary acidification. Ultrasonogram of the abdomen revealed nephrocalcinosis and splenomegaly. Detailed work up for anemia showed normal white cell and platelet count while peripheral smear showed microcytic hypochromic anemia with few target cells. Hemoglobin electrophoresis revealed hemoglobin A of 92%, hemoglobin A2 of 6.2% and hemo-globin F of 1.8% consistent with beta thalassemia minor. Her parental screening was normal. A diag-nosis of beta thalassemia minor with renal tubular dysfunction was made and the patient was started on thiazide diuretics to reduce hypercalciuria and advised regular follow-up.

  7. Searching for Beta-Haemolysin hlb Gene in Staphylococcus pseudintermedius with Species-Specific Primers.

    Science.gov (United States)

    Kmieciak, Wioletta; Szewczyk, Eligia M; Ciszewski, Marcin

    2016-07-01

    The paper presents an analysis of 51 Staphylococcus pseudintermedius clinically isolated strains from humans and from animals. Staphylococcus pseudintermedius strains' ability to produce β-haemolysin was evaluated with phenotypic methods (hot-cold effect, reverse CAMP test). In order to determine the hlb gene presence (coding for β-haemolysin) in a genomic DNA, PCR reactions were conducted with two different pairs of primers: one described in the literature for Staphylococcus aureus and recommended for analysing SIG group staphylococci and newly designed one in CLC Main Workbench software. Only reactions with newly designed primers resulted in product amplification, the presence of which was fully compatible with the results of phenotypic β-haemolysin test. Negative results for S. aureus and S. intermedius reference ATCC strains suggest that after further analysis the fragment of hlb gene amplified with primers described in this study might be included in the process of S. pseudintermedius strains identification.

  8. Sequence analysis of a 9873 bp fragment of the left arm of yeast chromosome XV that contains the ARG8 and CDC33 genes, a putative riboflavin synthase beta chain gene, and four new open reading frames.

    Science.gov (United States)

    Casas, C; Aldea, M; Casamayor, A; Lafuente, M J; Gamo, F J; Gancedo, C; Ariño, J; Herrero, E

    1995-09-15

    The DNA sequence of a 9873 bp fragment located near the left telomere of chromosome XV has been determined. Sequence analysis reveals seven open reading frames. One is the ARG8 gene coding for N-acetylornithine aminotransferase. Another corresponds to CDC33, which codes for the initiation factor 4E or cap binding protein. The open reading frame AOE169 can be considered as the putative gene for the Saccharomyces cerevisiae riboflavin synthase beta chain, since its translation product shows strong homology with four prokaryotic riboflavin synthase beta chains.

  9. Characterization of the human laminin beta2 chain locus (LAMB2): linkage to a gene containing a nonprocessed, transcribed LAMB2-like pseudogene (LAMB2L) and to the gene encoding glutaminyl tRNA synthetase (QARS)

    DEFF Research Database (Denmark)

    Durkin, M E; Jäger, A C; Khurana, T S;

    1999-01-01

    7q22-->q31 region that contains the laminin beta1 chain gene locus (LAMB1). Several CpG islands and a novel polymorphic microsatellite marker (D3S4594) were identified. The 3' end of LAMB2 lies 16 kb from the 5' end of the glutaminyl tRNA synthetase gene (QARS). About 20 kb upstream of LAMB2 we...... found a gene encoding a transcribed, non-processed LAMB2-like pseudogene (LAMB2L). The sequence of 1.75 kb of genomic DNA at the 3' end of LAMB2L was similar to exons 8-12 of the laminin beta2 chain gene. The LAMB2L-LAMB2-QARS cluster lies telomeric to the gene encoding the laminin-binding protein...

  10. Upregulation of B7 molecules (CD80 and CD86) and exacerbated eosinophilic pulmonary inflammatory response in mice lacking the IFN-beta gene

    DEFF Research Database (Denmark)

    Matheu, Victor; Treschow, Alexandra; Navikas, Vaidrius

    2003-01-01

    )-sensitized and OVA-challenged mice. RESULTS: OVA-sensitized and OVA-challenged mice with lack of the IFNB gene had more severe pulmonary inflammation with increased lung local response, including IL-4, IL-5, IL-13, IgE, eosinophilia, and goblet cells, than their litter mates (IFN-beta+/-), whereas no differences...... were observed in regard to local levels of IFN-gamma. Moreover, systemic response with IgE production is also enhanced. Lack of IFN-beta also results in significantly higher antigen-specific T cells, with higher levels of IL-4, IL-5, and IL-13, whereas no significant differences in IFN-gamma response...

  11. Imputing Variants in HLA-DR Beta Genes Reveals That HLA-DRB1 Is Solely Associated with Rheumatoid Arthritis and Systemic Lupus Erythematosus.

    Directory of Open Access Journals (Sweden)

    Kwangwoo Kim

    Full Text Available The genetic association of HLA-DRB1 with rheumatoid arthritis (RA and systemic lupus erythematosus (SLE is well documented, but association with other HLA-DR beta genes (HLA-DRB3, HLA-DRB4 and HLA-DRB5 has not been thoroughly studied, despite their similar functions and chromosomal positions. We examined variants in all functional HLA-DR beta genes in RA and SLE patients and controls, down to the amino-acid level, to better understand disease association with the HLA-DR locus. To this end, we improved an existing HLA reference panel to impute variants in all protein-coding HLA-DR beta genes. Using the reference panel, HLA variants were inferred from high-density SNP data of 9,271 RA-control subjects and 5,342 SLE-control subjects. Disease association tests were performed by logistic regression and log-likelihood ratio tests. After imputation using the newly constructed HLA reference panel and statistical analysis, we observed that HLA-DRB1 variants better accounted for the association between MHC and susceptibility to RA and SLE than did the other three HLA-DRB variants. Moreover, there were no secondary effects in HLA-DRB3, HLA-DRB4, or HLA-DRB5 in RA or SLE. Of all the HLA-DR beta chain paralogs, those encoded by HLA-DRB1 solely or dominantly influence susceptibility to RA and SLE.

  12. Imputing Variants in HLA-DR Beta Genes Reveals That HLA-DRB1 Is Solely Associated with Rheumatoid Arthritis and Systemic Lupus Erythematosus

    Science.gov (United States)

    Kim, Kwangwoo; Bang, So-Young; Yoo, Dae Hyun; Cho, Soo-Kyung; Choi, Chan-Bum; Sung, Yoon-Kyoung; Kim, Tae-Hwan; Jun, Jae-Bum; Kang, Young Mo; Suh, Chang-Hee; Shim, Seung-Cheol; Lee, Shin-Seok; Lee, Jisoo; Chung, Won Tae; Kim, Seong-Kyu; Choe, Jung-Yoon; Nath, Swapan K.; Lee, Hye-Soon; Bae, Sang-Cheol

    2016-01-01

    The genetic association of HLA-DRB1 with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) is well documented, but association with other HLA-DR beta genes (HLA-DRB3, HLA-DRB4 and HLA-DRB5) has not been thoroughly studied, despite their similar functions and chromosomal positions. We examined variants in all functional HLA-DR beta genes in RA and SLE patients and controls, down to the amino-acid level, to better understand disease association with the HLA-DR locus. To this end, we improved an existing HLA reference panel to impute variants in all protein-coding HLA-DR beta genes. Using the reference panel, HLA variants were inferred from high-density SNP data of 9,271 RA-control subjects and 5,342 SLE-control subjects. Disease association tests were performed by logistic regression and log-likelihood ratio tests. After imputation using the newly constructed HLA reference panel and statistical analysis, we observed that HLA-DRB1 variants better accounted for the association between MHC and susceptibility to RA and SLE than did the other three HLA-DRB variants. Moreover, there were no secondary effects in HLA-DRB3, HLA-DRB4, or HLA-DRB5 in RA or SLE. Of all the HLA-DR beta chain paralogs, those encoded by HLA-DRB1 solely or dominantly influence susceptibility to RA and SLE. PMID:26919467

  13. Retrovirus-mediated gene transfer of the cytokine genes interleukin-1beta and tumor necrosis factor-alpha into human neuroblastoma cells: consequences for cell line behavior and immunomodulatory properties.

    Science.gov (United States)

    Coze, C; Leimig, T; Jimeno, M T; Mannoni, P

    2001-03-01

    We have investigated the value of a gene therapy approach for neuroblastoma (NB), based on retroviral transduction of the IL-1beta or TNF-alpha cytokine genes into human NB lines. Secretion of the corresponding cytokine, was demonstrated in all lines, although with considerable quantitative variations. Cytokine gene expression significantly reduced the proliferation index (p = 0.0001); this effect was associated with either terminal neuronal (one TNF-alpha line) or fibroblast-like differentiation (two IL-1beta lines), leading to growth arrest after a few weeks. Cell surface levels of CD54 and HLA class II remained unaffected, but HLA class I (p < 0.001) and CD58 expression (p = 0.01) increased on SKNSH after TNF-alpha gene transfer. Mononuclear cells from normal allogeneic donors cocultured with both IL-1beta (p < 0.001) and TNF-alpha lines (p < 0.01), showed a significant increase in the proportion of activated T cells (CD3+DR+); however, their cytotoxicity and proliferation rate remained unchanged. Immunotherapy of neuroblastoma will require identification of transduced lines in which cytokine secretion induces phenotypic changes in such a way as to augment their likely immunomodulatory properties without impeding cell growth. Because of the limited efficacy of IL-1beta or TNF-alpha gene transfer alone, further studies should focus on combination with other immunomodulatory agents, to improve their potential efficacy in neuroblastoma.

  14. Commensal Enterobacteriaceae as reservoirs of extended-spectrum beta-lactamases, integrons and sul genes in Portugal

    Directory of Open Access Journals (Sweden)

    Elisabete eMachado

    2013-04-01

    Full Text Available Bacteria colonizing the human intestine have a relevant role in the spread of antimicrobial resistance. We investigated the faecal carriage of extended-spectrum beta-lactamase (ESBL-producing Enterobacteriaceae in healthy humans from Portugal and analysed the distribution of sul genes and class 1 and 2 integrons. Faecal samples (n=113 were recovered from healthy persons (North/Centre of Portugal, 2001-04 and plated on MacConkey agar with and without ceftazidime (1mg/L or cefotaxime (1mg/L. Isolates representing different morphotypes/plate and antibiotic susceptibility patterns (n=201 were selected. Isolates resistant to sulfonamides and/or streptomycin, gentamicin and trimethoprim were screened (PCR, sequencing for sul genes (sul1, sul2, sul3 and class 1 and 2 integrons. Presence of ESBLs was inferred using the DDST and further confirmed by PCR and sequencing. ESBL producers were selected for clonal analysis, plasmid characterization and conjugation assays by standard methods. ESBL-producing isolates were found in 1.8% (2/113 of samples, corresponding to Escherichia coli of phylogroups A (n=1 and B1 (n=1 carrying transferable blaCTX-M-14 and the new blaTEM-153, respectively. A 80kb IncK-blaCTX-M-14 was found, being highly related to that widely spread among CTX-M-14 producers of humans and animals from Portugal and other European countries. sul genes were found in 88% (22/25;sul2-60%, sul1-48%, sul3-4% of the sulfonamide resistant isolates. Class 1 integrons were more frequently found than class 2 (7% vs 3%. Interestingly, gene cassette arrangements within these platforms were identical to those commonly observed among Enterobacteriaceae from Portuguese food-producing animals, although aadA13 is here firstly described in Morganella morganii. These results reinforce the relevance of human commensal flora as reservoir of clinically relevant antibiotic resistance genes including blaESBLs, and highly transferable genetic platforms as IncK epidemic

  15. Thymosin beta 4 prevents oxidative stress by targeting antioxidant and anti-apoptotic genes in cardiac fibroblasts.

    Directory of Open Access Journals (Sweden)

    Sandeep Kumar

    Full Text Available RATIONALE: Thymosin beta-4 (Tβ4 is a ubiquitous protein with diverse functions relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory responses. The effecter molecules targeted by Tβ4 for cardiac protection remains unknown. The purpose of this study is to determine the molecules targeted by Tβ4 that mediate cardio-protection under oxidative stress. METHODS: Rat neonatal fibroblasts cells were exposed to hydrogen peroxide (H(2O(2 in presence and absence of Tβ4 and expression of antioxidant, apoptotic and pro-fibrotic genes was evaluated by quantitative real-time PCR and western blotting. Reactive oxygen species (ROS levels were estimated by DCF-DA using fluorescent microscopy and fluorimetry. Selected antioxidant and antiapoptotic genes were silenced by siRNA transfections in cardiac fibroblasts and the effect of Tβ4 on H(2O(2-induced profibrotic events was evaluated. RESULTS: Pre-treatment with Tβ4 resulted in reduction of the intracellular ROS levels induced by H(2O(2 in the cardiac fibroblasts. This was associated with an increased expression of antioxidant enzymes Cu/Zn superoxide dismutase (SOD and catalase and reduction of Bax/Bcl(2 ratio. Tβ4 treatment reduced the expression of pro-fibrotic genes [connective tissue growth factor (CTGF, collagen type-1 (Col-I and collagen type-3 (Col-III] in the cardiac fibroblasts. Silencing of Cu/Zn-SOD and catalase gene triggered apoptotic cell death in the cardiac fibroblasts, which was prevented by treatment with Tβ4. CONCLUSION: This is the first report that exhibits the targeted molecules modulated by Tβ4 under oxidative stress utilizing the cardiac fibroblasts. Tβ4 treatment prevented the profibrotic gene expression in the in vitro settings. Our findings indicate that Tβ4 selectively targets and upregulates catalase, Cu/Zn-SOD and Bcl(2, thereby, preventing H(2O(2-induced profibrotic changes in the myocardium. Further studies are

  16. Gene Network Analysis of Metallo Beta Lactamase Family Proteins Indicates the Role of Gene Partners in Antibiotic Resistance and Reveals Important Drug Targets.

    Science.gov (United States)

    Parimelzaghan, Anitha; Anbarasu, Anand; Ramaiah, Sudha

    2016-06-01

    Metallo Beta (β) Lactamases (MBL) are metal dependent bacterial enzymes that hydrolyze the β-lactam antibiotics. In recent years, MBL have received considerable attention because it inactivates most of the β-lactam antibiotics. Increase in dissemination of MBL encoding antibiotic resistance genes in pathogenic bacteria often results in unsuccessful treatments. Gene interaction network of MBL provides a complete understanding on the molecular basis of MBL mediated antibiotic resistance. In our present study, we have constructed the MBL network of 37 proteins with 751 functional partners from pathogenic bacterial spp. We found 12 highly interconnecting clusters. Among the 37 MBL proteins considered in the present study, 22 MBL proteins are from B3 subclass, 14 are from B1 subclass and only one is from B2 subclass. Global topological parameters are used to calculate and compare the probability of interactions in MBL proteins. Our results indicate that the proteins associated within the network have a strong influence in antibiotic resistance mechanism. Interestingly, several drug targets are identified from the constructed network. We believe that our results would be helpful for researchers exploring MBL-mediated antibiotic resistant mechanisms.

  17. Recent advances in gene therapy for thalassemia

    Directory of Open Access Journals (Sweden)

    J V Raja

    2012-01-01

    Full Text Available Thalassemias are genetically transmitted disorders. Depending upon whether the genetic defects or deletion lies in transmission of α or β globin chain gene, thalassemias are classified into α and β-thalassemias. Thus, thalassemias could be cured by introducing or correcting a gene into the hematopoietic compartment or a single stem cell. Initial attempts at gene transfer have proved unsuccessful due to limitations of available gene transfer vectors. The present review described the newer approaches to overcome these limitations, includes the introduction of lentiviral vectors. New approaches have also focused on targeting the specific mutation in the globin genes, correcting the DNA sequence or manipulating the development in DNA translocation and splicing to restore globin chain synthesis. This review mainly discusses the gene therapy strategies for the thalassemias, including the use of lentiviral vectors, generation of induced pluripotent stem (iPS cells, gene targeting, splice-switching and stop codon readthrough.

  18. Recent advances in gene therapy for thalassemia.

    Science.gov (United States)

    Raja, J V; Rachchh, M A; Gokani, R H

    2012-07-01

    Thalassemias are genetically transmitted disorders. Depending upon whether the genetic defects or deletion lies in transmission of α or β globin chain gene, thalassemias are classified into α and β-thalassemias. Thus, thalassemias could be cured by introducing or correcting a gene into the hematopoietic compartment or a single stem cell. Initial attempts at gene transfer have proved unsuccessful due to limitations of available gene transfer vectors. The present review described the newer approaches to overcome these limitations, includes the introduction of lentiviral vectors. New approaches have also focused on targeting the specific mutation in the globin genes, correcting the DNA sequence or manipulating the development in DNA translocation and splicing to restore globin chain synthesis. This review mainly discusses the gene therapy strategies for the thalassemias, including the use of lentiviral vectors, generation of induced pluripotent stem (iPS) cells, gene targeting, splice-switching and stop codon readthrough.

  19. Transforming growth factor-beta 1 downregulates dexamethasone-induced tetranectin gene expression during the in vitro mineralization of the human osteoblastic cell line SV-HFO

    DEFF Research Database (Denmark)

    Iba, K; Sawada, N; Chiba, H;

    1995-01-01

    treatment as evidenced by Northern blotting. When transforming growth factor-beta 1 (TGF-beta 1) was added together with dexamethasone to the SV-HFO cell cultures, the mineralization process was markedly suppressed and the expression of tetra nectin and alkaline phosphatase was downregulated in a dose......-dependent manner. These results demonstrate that the expression of tetranectin in these osteoblastic cells is regulated by dexamethasone and TGF-beta 1 and that tetranectin expression is tightly linked to the process of mineralization.......In the present study, we examined the regulation of tetranectin gene expression using a human osteoblastic cell line, SV-HFO, that undergoes mineralization upon treatment with dexamethasone. We found that the expression of tetranectin and alkaline phosphatase mRNA was induced by dexamethasone...

  20. T-cell receptor v-alpha and v-Beta gene usage in interleukin-2-cultured tumor-infiltrating lymphocytes from patients with breast-cancer

    DEFF Research Database (Denmark)

    Andersen, E; Scholler, J; Straten, P;

    1994-01-01

    surface through the T cell receptor (TCR) complex. We have studied the phenotype, cytotoxicity, and expression of TCR variable (V) alpha and beta chain on in vitro IL-2-cultured TIL isolated from primary malignant breast tumors from 11 patients. 10/11 cultures were dominated by CD4(+) (T-helper) cells....... The different TIL cultures exhibited varying levels of cytotoxicity against the natural killer (NK)-sensitive cell line K562 and breast cancer cell line T47D. The level of clonality, as measured by PCR-based analyses of usage of the different V segments was low, as only a few tumors showed patterns...... of restricted V gene expression. The mean number of V alpha segments per TIL culture was higher than the number of V beta segments per culture. A significant negative correlation was observed between the number of CD4+ cells and the number of V beta segments per culture, and no other correlations between...

  1. In vitro and in vivo gene therapy with CMV vector-mediated presumed dog beta-nerve growth factor in pyridoxine-induced neuropathy dogs.

    Science.gov (United States)

    Chung, Jin Young; Choi, Jung Hoon; Shin, Il Seob; Choi, Eun Wha; Hwang, Cheol Yong; Lee, Sang Koo; Youn, Hwa Young

    2008-12-01

    Due to the therapeutic potential of gene therapy for neuronal injury, many studies of neurotrophic factors, vectors, and animal models have been performed. The presumed dog beta-nerve growth factor (pdbeta-NGF) was generated and cloned and its expression was confirmed in CHO cells. The recombinant pdbeta-NGF protein reacted with a human beta-NGF antibody and showed bioactivity in PC12 cells. The pdbeta-NGF was shown to have similar bioactivity to the dog beta-NGF. The recombinant pdbeta-NGF plasmid was administrated into the intrathecal space in the gene therapy group. Twenty-four hours after the vector inoculation, the gene therapy group and the positive control group were intoxicated with excess pyridoxine for seven days. Each morning throughout the test period, the dogs' body weight was taken and postural reaction assessments were made. Electrophysiological recordings were performed twice, once before the experiment and once after the test period. After the experimental period, histological analysis was performed. Dogs in the gene therapy group had no weight change and were normal in postural reaction assessments. Electrophysiological recordings were also normal for the gene therapy group. Histological analysis showed that neither the axons nor the myelin of the dorsal funiculus of L4 were severely damaged in the gene therapy group. In addition, the dorsal root ganglia of L4 and the peripheral nerves (sciatic nerve) did not experience severe degenerative changes in the gene therapy group. This study is the first to show the protective effect of NGF gene therapy in a dog model.

  2. cDNA sequence and gene locus of the human retinal phosphoinositide-specific phospholipase-C{beta}4 (PLCB4)

    Energy Technology Data Exchange (ETDEWEB)

    Alvarez, R.A.; Ghalayini, A.J.; Anderson, R.E. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1995-09-01

    Defects in the Drosophila norpA (no receptor potential A) gene encoding a phosphoinositide-specific phospholipase C (PLC) block invertebrate phototransduction and lead to retinal degeneration. The mammalian homolog, PLCB4, is expressed in rat brain, bovine cerebellum, and the bovine retina in several splice variants. To determine a possible role of PLCB4 gene defects in human disease, we isolated several overlapping cDNA clones from a human retina library. The composite cDNA sequence predicts a human PLC{beta}4 polypeptide of 1022 amino acid residues (MW 117,000). This PLC{beta}4 variant lacks a 165-amino-acid N-terminal domain characteristic for the rat brain isoforms, but has a distinct putative exon 1 unique for human and bovine retina isoforms. A PLC{beta}4 monospecific antibody detected a major (130 kDa) and a minor (160 kDa) isoform in retina homogenates. Somatic cell hybrids and deletion panels were used to localize the PCLB4 gene to the short arm of chromosome 20. The gene was further sublocalized to 20p12 by florescence in situ hybridization. 4 refs., 5 figs.

  3. Analysis of the beta-tubulin gene and morphological changes of the microsporidium Anncaliia algerae both suggest albendazole sensitivity.

    Science.gov (United States)

    Santiana, Marianita; Pau, Cyrilla; Takvorian, Peter M; Cali, Ann

    2015-01-01

    The Microsporidium, Anncaliia algerae, an obligate intracellular parasite, has been identified as an opportunistic human pathogen, but treatment has not been evaluated for infections with this organism. Albendazole, an antitubulin polymerization drug used against parasitic worm infections, has been the medication of choice used to treat some microsporidial infections affecting humans, with varying results ranging from clearing infection (Encephalitozoon) to resistance (Enterocytozoon). This study illustrates the effect of albendazole treatment on A. algerae infection in Rabbit Kidney (RK13) cells and Human Fetal Lung (HFL-1) fibroblasts. Albendazole appears to have an attenuating effect on A. algerae infection and albendazole's IC50 in RK13 cells is 0.1 μg/ml. Long-term treatment inhibits up to 98% of spore production, but interrupting treatment reestablishes the infection without new exposure to the parasite as supported by microscopic observations. The parasite's beta-tubulin gene was purified, cloned, and sequenced. Five of the six specific amino acids, associated with benzimidazole sensitivity, are conserved in A. algerae. These findings suggest that A. algerae is sensitive to albendazole; however, the organism is not completely cleared from cultures.

  4. Beta-thymosin gene polymorphism associated with freshwater invasiveness of alewife (Alosa pseudoharengus)

    Science.gov (United States)

    Michalak, Katarzyna; Czesny, Sergiusz J.; Epifanio, John; Snyder, Randal J.; Schultz, Eric T.; Velotta, Jonathan P.; McCormick, Stephen D.; Brown, Bonnie L.; Santopietro, Graciela; Michalak, Pawel

    2014-01-01

    Predicting the success of a species’ colonization into a novel environment is routinely considered to be predicated on niche-space similarity and vacancy, as well as propagule pressure. The role genomic variation plays in colonization success (and the interaction with environment) may be suggested, but has not rigorously been documented. To test an hypothesis that previously observed ecotype-specific polymorphisms between anadromous and landlocked alewife (Alosa pseudoharengus) populations are an adaptive response to osmoregulatory challenges rather than a result of allele sampling at founding, we examined multiple anadromous and landlocked (colonized) populations for their allelic profiles at a conserved region (3’-UTR end) of a β-thymosin gene whose protein product plays a central role in the organization of cytoskeleton. The putatively ancestral β-thymosin allele was prevalent in anadromous populations, whereas a newly derived allele was overrepresented in landlocked populations; a third allele was exclusive to the anadromous populations. We also conducted a complementary set of salinity exposure experiments to test osmoregulatory performance of the alewife ecotypes in contrasting saline environments. The pattern of variation and results from these challenges indicate a strong association of β-thymosin with colonization success and a transition for species with an anadromous life-history to one with only a freshwater component.

  5. Emergence of Acinetobacter pittii harboring New Delhi metallo-beta-lactamase genes in Daejeon, Korea.

    Science.gov (United States)

    Sung, Ji Youn; Koo, Sun Hoe; Kim, Semi; Kwon, Gye Cheol

    2015-09-01

    Carbapenemase production has been reported worldwide in gram-negative bacteria, including Acinetobacter species. We detected carbapenemase-producing Acinetobacter pittii in clinical isolates in Daejeon, Korea. Twenty-one ertapenem-resistant A. pittii isolates screened with a disk diffusion method were characterized by using the Epsilon test, four multiplex PCR assays, and a multilocus sequence typing (MLST) scheme. A total of 21 A. pittii isolates harbored the metallo-β-lactamase (MBL) gene bla(IMP-1) or bla(NDM-1). Nineteen isolates containing bla(IMP-1) were resistant to imipenem and meropenem, but two isolates harboring bla(NDM-1) were susceptible to them. The sequence types (STs) of the two New Delhi MBL (NDM-1)-producing A. pittii isolates were ST70 and ST207, which differed from the STs (ST63, ST119, ST396, and a novel ST) of the IMP-1-producing A. pittii. This is the first report on NDM-1-producing A. pittii isolates in Korea. Our results emphasize that the study of NDM-1-producing gram-negative bacteria should involve carbapenem-susceptible as well as carbapenem-resistant isolates.

  6. Polymorphisms within beta-catenin encoding gene affect multiple myeloma development and treatment.

    Science.gov (United States)

    Butrym, Aleksandra; Rybka, Justyna; Łacina, Piotr; Gębura, Katarzyna; Frontkiewicz, Diana; Bogunia-Kubik, Katarzyna; Mazur, Grzegorz

    2015-12-01

    Recent studies have suggested that cereblon (CRBN) is essential for the anti-myeloma (MM) activity of immunomodulatory drugs (IMiDs), such as thalidomide and lenalidomide, and that dysregulation of Wnt/β-catenin pathway may be one of possible reasons of lenalidomide resistance. This prompted us to analyze the effect of polymorphisms within the genes coding for cereblon (CRBN (rs121918368 C>T)) and β-catenin (CTNNB1 (rs4135385 A>G; rs4533622 A>C)). MM patients (n=142) and healthy individuals (n=123) were genotyped using the Light SNiP assays. The presence of the CTNNB1 (rs4533622) A allele was more frequently detected in patients presented with stage II-III disease according to International Staging System (63/82 vs. 26/44, p=0.043) and Durie-Salmon criteria (75/99 vs. 14/26, p=0.049). The CTNNB1 (rs4135385) AA homozygosity was more frequent among patients with better response to CTD, i.e., cyclophosphamide-thalidomide-dexamethasone (18/23 vs. 32/60, p=0.047). Patients carrying the CTNNB1 (rs4533622) AA genotype were better responders to the first line therapy with thalidomide containing regimens (pCRBN polymorphism. These results suggest that the CTNNB1 polymorphisms may affect the clinical course and response to chemotherapy in patients with multiple myeloma.

  7. A combined approach for β-thalassemia based on gene therapy-mediated adult hemoglobin (HbA) production and fetal hemoglobin (HbF) induction.

    Science.gov (United States)

    Zuccato, Cristina; Breda, Laura; Salvatori, Francesca; Breveglieri, Giulia; Gardenghi, Sara; Bianchi, Nicoletta; Brognara, Eleonora; Lampronti, Ilaria; Borgatti, Monica; Rivella, Stefano; Gambari, Roberto

    2012-08-01

    Gene therapy might fall short in achieving a complete reversion of the β-thalassemic phenotype due to current limitations in vector design and myeloablative regimen. Following gene transfer, all or a large proportion of erythroid cells might express suboptimal levels of β-globin, impairing the therapeutic potential of the treatment. Our aim was to evaluate whether, in absence of complete reversion of the β-globin phenotype upon gene transfer, it is possible to use fetal hemoglobin induction to eliminate the residual α-globin aggregates and achieve normal levels of hemoglobin. Transgenic K562 cell lines and erythroid precursor cells from β(0)39-thalassemia patients were employed. Gene therapy was performed with the lentiviral vector T9W. Induction of fetal hemoglobin was obtained using mithramycin. Levels of mRNA and hemoglobins were determined by qRT-PCR and HPLC. First, we analyzed the effect of mithramycin on K562 transgenic cell lines harboring different copies of a lentiviral vector carrying the human β-globin gene, showing that γ-globin mRNA expression and HbF production can be induced in the presence of high levels of β-globin gene expression and HbA accumulation. We then treated erythroid pr