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Sample records for beta chain gene

  1. Gene encoding the human beta-hexosaminidase beta chain: extensive homology of intron placement in the alpha- and beta-chain genes.

    PubMed Central

    Proia, R L

    1988-01-01

    Lysosomal beta-hexosaminidase (EC 3.2.1.52) is composed of two structurally similar chains, alpha and beta, that are the products of different genes. Mutations in either gene causing beta-hexosaminidase deficiency result in the lysosomal storage disease GM2-gangliosidosis. To enable the investigation of the molecular lesions in this disorder and to study the evolutionary relationship between the alpha and beta chains, the beta-chain gene was isolated, and its organization was characterized. The beta-chain coding region is divided into 14 exons distributed over approximately 40 kilobases of DNA. Comparison with the alpha-chain gene revealed that 12 of the 13 introns interrupt the coding regions at homologous positions. This extensive sharing of intron placement demonstrates that the alpha and beta chains evolved by way of the duplication of a common ancestor. PMID:2964638

  2. [Fibrinogen beta chain gene mutation contributes to one congenital afibrinogenemia].

    PubMed

    Xu, Xiu-cai; Zhou, Rong-fu; Wu, Jing-sheng; Fang, Yi; Wang, Xue-feng; Zhai, Zhi-min; Wang, Hong-li

    2005-03-01

    To identify the fibrinogen (Fg) gene mutations in a Chinese pedigree of congenital afibrinogenemia. The plasma Fg activity and protein of the proband and his family members were detected. Genomic DNA was isolated from the peripheral blood mononuclear cells. All the exons and exon-intron boundaries of fibrinogen gene were amplified by PCR and sequenced thereafter. Two mutations, 7972 del G in FGB and T2543A in FGG, were found in the proband. FGG2543 is a polymorphism site, which lead to the polymorphism of gamma144 I/K. The G deletion at base 7972 of FGB contributes to the frameshift mutation after amino acid 419, resulting in the truncated beta chain without the terminal 27 amino acids. The latter may contributes to the pathogenetic mechanisms in Chinese congenital afibrinogenemia patients. The G deletion at base 7972 of FGB is identified for the first time.

  3. Genomic organization of the mouse T-cell receptor beta-chain gene family.

    PubMed Central

    Lai, E; Barth, R K; Hood, L

    1987-01-01

    We have combined three different methods, deletion mapping of T-cell lines, field-inversion gel electrophoresis, and the restriction mapping of a cosmid clone, to construct a physical map of the murine T-cell receptor beta-chain gene family. We have mapped 19 variable (V beta) gene segments and the two clusters of diversity (D beta) and joining (J beta) gene segments and constant (C beta) genes. These members of the beta-chain gene family span approximately equal to 450 kilobases of DNA, excluding one potential gap in the DNA fragment alignments. Images PMID:3035555

  4. In vivo analysis of the murine beta-myosin heavy chain gene promoter.

    PubMed

    Rindt, H; Gulick, J; Knotts, S; Neumann, J; Robbins, J

    1993-03-05

    The 5' upstream region of the murine beta-myosin heavy chain (MHC) gene has been isolated and tested for its ability to drive gene expression in transgenic mice. Three classes of transgenic mice were generated. The constructs contained approximately 5000, 2500, and 600 base pairs of beta-MHC upstream sequence fused to the chloramphenicol acetyltransferase gene and were termed beta 5, beta 2.5, and beta .6, respectively. Muscle-specific expression was observed with all three constructs. However, only the beta 5 lines directed high levels of muscle-specific transgene expression in both pre- and postbirth mice. Expression driven by the two shorter constructs was two to three orders of magnitude lower when the chloramphenicol acetyltransferase specific activities were compared. These data suggest that a distal-positive element directs high levels of gene expression in the ventricle and in slow skeletal muscles. Analyses of transgene expression during heart maturation revealed that some of the beta 5 lines were not able to respond in an appropriate manner to developmental transcriptional cues. Unlike the endogenous beta-MHC gene, which is down regulated in the ventricles around the time of birth, reporter gene expression in the majority of the lines generated was not shut off in the ventricles of the adult animals. These data indicate that high levels of muscle-specific beta-MHC gene expression are dependent upon the combinatorial interactions of a number of sequence elements that are distributed over a large region of the gene's upstream sequence.

  5. Gene correction in the evolution of the T cell receptor beta chain

    PubMed Central

    1986-01-01

    Mutational mechanisms operating at the T cell receptor beta chain locus have been examined by comparison of the CT beta 1 and CT beta 2 gene sequences from Mus pahari, believed to be the oldest living species in the genus Mus, with those of inbred mice. Results indicate that a gene correction event independent of that suggested to have occurred in inbred mice has homogenized the M. pahari CT beta exon 1 sequences, minimizing diversity in this region of the molecule. These observations suggest that correction events such as gene conversion may occur frequently, even in pauci-gene families with as few as two members, and therefore play a significant role in gene diversification or homogenization of small as well as large gene families. PMID:3783089

  6. Allelic forms of the alpha- and beta-chain genes encoding DQw1-positive heterodimers.

    PubMed

    Turco, E; Care, A; Compagnone-Post, P; Robinson, C; Cascino, I; Trucco, M

    1987-01-01

    On chromosome 6, in the HLA region, the DQ subregion is located immediately centromeric to the DR subregion. Even though only three serological specificities to date have been officially recognized (DQw1, DQw2, and DQw3), it seems likely that the phenotypical polymorphism expressed by DQ molecules is much more complex. There are reasons to believe that fixed alpha-beta combinations exist, each of them associated with a different DR allele. DQw1 is a determinant present on DQ molecules that are found associated with DR1-, DR2-, and DRw6-positive haplotypes. By restriction fragment length polymorphism analysis, we recognized three allelic DQ-alpha and three allelic DQ-beta patterns associated with DQw1. In addition, one of these alpha/beta pairs associated with DR1, two with DR2, and a fourth with DRw6. We have obtained evidence using nucleotide sequencing that there are as many allelic forms of DQ-alpha and DQ-beta genes as there are different molecular DQ-alpha and DQ-beta patterns. The DQ-alpha and DQ-beta chains of DQw1-positive molecules each are encoded by at least three distinctly different allelic genes, and particular alpha/beta gene combinations are associated with the same DR alleles as their corresponding molecular alpha/beta pairs.

  7. Physical mapping of the human T-cell antigen receptor (TCR) {beta}-chain gene complex

    SciTech Connect

    Yashim, Y.; So, A.K.

    1994-09-01

    The genetic variation of the TCR loci and their contribution to autoimmune diseases is poorly defined, in direct contrast to the clear examples of disease association with the Class I and II alleles of the major histocompatibility complex. We have therefore started to determine the gene organization and polymorphism of the TCR {beta} locus. Yeast artificial chromosomes (YACs) were used to construct a physical map of the germline human TCR {beta}-chain gene complex. Variable gene (V{beta}) sequences for the 25 known V{beta} subfamilies were amplified by PCR and were used as probes to screen a YAC library. Five positive YACs were identified. YACs designated B3, E11 and H11 of sizes 820, 400 and 600 kbp, respectively, were analyzed for their V{beta} content by pulse-field gel electrophoresis (PFGE). YAC B3 was found to contain all 25 V{beta} subfamilies, E11 for 14 and H11 for 7. B3 was also positive for the constant region genes. Restriction enzyme mapping of B3 located V{beta} and C{beta} gene regions to four Sfi I fragments of 280, 110, 90 and 125 kbp, and was in accordance with published data. The data thus showed that YAC B3 encoded a complete and unrearranged TCR {beta}-gene locus. The map was further resolved by locating restriction sites for Sal I and Bssll II on B3. Fluorescent in situ hybridization to human metaphase chromosomes localized B3 to chromosome 7q35. However, two additional signals were obtained: one attributable to V{beta} orphon cluster on chromosome 9q21; the second to the long arm of chromosome 2. PCR amplification of a chromosome 2 somatic cell hybrid using primers for all 25 V{beta} gene families revealed the signal was not attributable to a second orphon cluster. It is suggested that B3 is a chimeric YAC with an intact TCR {beta} locus flanked by chromosome 2 sequences. The determination of the TCR genomic organization will help extend studies of the role T-cells play in autoimmune diseases.

  8. Missense mutation of the {beta}-cardiac myosin heavy-chain gene in hypertrophic cardiomyopathy

    SciTech Connect

    Arai, Shoichi; Matsuoka, Rumiko; Hirayama, Kenji; Sakurai, Hisanao

    1995-09-11

    Hypertrophic cardiomyopathy occurs as an autosomal dominant familial disorder or as a sporadic disease without familial involvement. We describe a missense mutation of the {beta}-cardiac myosin heavy chain (MHC) gene, a G to T transversion (741 Gly{r_arrow}Trp) identified by direct sequencing of exon 20 in four individuals affected with familial hypertrophic cardiomyopathy. Three individuals with sporadic hypertrophic cardiomyopathy, whose parents are clinically and genetically unaffected, had sequence variations of exon 34 of the {alpha}-cardiac MHC gene (a C to T transversion, 1658 Asp{r_arrow}Asp, resulting in FokI site polymorphism), of intron 33 of the {alpha}-cardiac MHC gene (a G to A and an A to T transversion), and also of intron 14 of the {beta}-cardiac MHC gene (a C to T transversion in a patient with Noonan syndrome). Including our case, 30 missense mutations of the {beta}-cardiac MHC gene in 49 families have been reported thus far worldwide. Almost all are located in the region of the gene coding for the globular head of the molecule, and only one mutation was found in both Caucasian and Japanese families. Missense mutations of the {Beta}-cardiac MHC gene in hypertrophic cardiomyopathy may therefore differ according to race. 29 refs., 6 figs., 3 tabs.

  9. In vivo regulation of the mouse beta myosin heavy chain gene.

    PubMed

    Knotts, S; Rindt, H; Neumann, J; Robbins, J

    1994-12-09

    The interactions of trans-acting factors with their respective cis-acting elements in the 5' upstream region of the beta myosin heavy chain gene (MyHC) regulate its tissue- and developmental stage-specific expression. The role of three conserved elements, an MCAT or TEF-1 binding site, a C-rich region, and a beta e3 region, in muscle-specific gene expression was analyzed in vivo. Each cis-acting site was ablated in the context of the beta MyHC promoter, fused to the chloramphenicol acetyltransferase reporter gene, and used to generate transgenic mice. In contrast to results obtained in vitro, the data demonstrate that mutating any one of these cis-acting elements does not affect the level or tissue specificity of transgene expression. Sequences upstream of -600 can functionally substitute for any one of these regulatory cassettes and are important both for high levels of expression as well as for controlled muscle specificity. Mutation of any two of the cis-acting elements also does not affect transgene expression. However, simultaneous mutation of the three sites significantly reduces expression, indicating that these conserved sequences do play an important role and that combinatorial interactions underlie the beta MyHC's regulation.

  10. A novel association of DQ alpha and DQ beta genes in the DRw10 haplotype. Determination of a DQw1 specificity by the DQ beta-chain.

    PubMed

    Merryman, P; Silver, J; Gregersen, P K; Solomon, G; Winchester, R

    1989-09-15

    The association of the class II genes of the DRw10 haplotype from a cell line, NASC, initiated from a member of a well characterized family, was analyzed by sequencing cDNA clones corresponding to DR beta I, DQ alpha, and DQ beta genes. An identical haplotype was also identified in the Raji cell line. In addition to typing as DRw10 and DQw1 with HLA typing sera both, the NASC and Raji cell lines were shown to react strongly with the monoclonal antibodies 109d6 (specific for DRw10 beta 1 and DRw53 beta 2 gene products) and Genox 3.5.3 (specific for DQw1) and exhibited the restriction fragment length polymorphism indicative of a DRw10, DQw1 haplotype. The DR beta 1 gene corresponding to the DRw10 specificity was found to have a first domain sequence different from all other DR beta I genes. Sequence analysis of the 3'-untranslated region of this DR beta-chain gene showed a significant divergence from the 3' untranslated region of the DRw53 family of haplotypes and a lesser divergence from that of the DRw52 and DR1/DR2 families. The sequence of the DQ beta genes corresponding to the DQw1 specificity in the DRw10 haplotype was found to be identical to the DQ beta gene from a DR1, DQw1 haplotype. Surprisingly, however, the DQ alpha gene did not resemble other DQw1-like DQ alpha genes, but was identical in sequence to the DQ alpha gene found in DR4 haplotypes. The novel association of DQ alpha and DQ beta genes in the DRw10 haplotype revealed in these studies may result from a double recombinational event. More consequentially, these studies strongly suggest that the DQw1 specificity recognized by Genox 3.5.3 is determined by the DQ beta chain and is not affected by the DQ alpha-chain.

  11. Characterization of horse (Equus caballus) T-cell receptor beta chain genes

    SciTech Connect

    Schrenzel, M.D.; Watson, J.L.; Ferrick, D.A.

    1994-12-31

    Genes encoding the horse (Equus caballus) T-cell receptor beta chain (TCRB) were cloned and characterized. Of 33 cDNA clones isolated from the mesenteric lymph node, 30 had functionally rearranged gene segments, and three contained germline sequences. Sixteen unique variable segments (TCRBV), 14 joining genes (TCRBJ), and two constant region genes (TCRBC) were identified. Horse TCRBV were grouped into nine families based on similarity to human sequences. TCRBV2 and TCRBV12 were the most commonly represented horse families. Analysis of predicted protein structure revealed the presence of conserved regions similar to those seen in TCRB of other species. A decanucleotide promoter sequence homologous to those found in humans and mice was located in the 5{prime} untranslated region of one horse gene. Germline sequences included the 5{prime} region of the TCRBD2 gene with flanking heptamer/nonamer recombination signals and portions of the TCRBJ2-C2 intro. Southern blot hybridizations demonstrated restriction fragment length polymorphisms at the TCRBC locus among different horse breeds.

  12. Haplotyping the human T-cell receptor. beta. -chain gene complex by use of restriction fragment length polymorphisms

    SciTech Connect

    Charmley, P.; Chao, A.; Gatti, R.A. ); Concannon, P. ); Hood, L. )

    1990-06-01

    The authors have studied the genetic segregation of human T-cell receptor {beta}-chain (TCR{beta}) genes on chromosome 7q in 40 CEPH (Centre d'Etude du Polymorphisme Humain) families by using restriction fragment length polymorphisms (RFLPs). They constructed haplotypes from eight RFLPs by using variable- and constant-region cDNA probes, which detect polymorphisms that span more than 600 kilobases of the TCR{beta} gene complex. Analysis of allele distributions between TCR{beta} genes revealed significant linkage disequilibrium between only 6 of the 28 different pairs of RFLPs. This linkage disequilibrium strongly influences the most efficient order to proceed for typing of these RFLPs in order to achieve maximum genetic informativeness, which in this study revealed a 97.3% level of heterozygosity within the TCR{beta} gene complex. The results should provide new insight into recent reports of disease associations with the TCR{beta} gene complex and should assist in designing future experiments to detect or confirm the existence of disease-susceptibility loci in this region of the human genome.

  13. Segregation of cardiac and skeletal muscle-specific regulatory elements of the beta-myosin heavy chain gene.

    PubMed Central

    Rindt, H; Knotts, S; Robbins, J

    1995-01-01

    The beta-myosin heavy chain (beta-MyHC) gene is expressed in cardiac and slow skeletal muscles. To examine the regulatory sequences that are required for the gene's expression in the two compartments in vivo, we analyzed the expression pattern of a transgene consisting of the beta-MyHC gene 5' upstream region linked to the chloramphenicol acetyltransferase reporter gene. By using 5600 bp of 5' upstream region, the transgene was expressed at high levels in the slow skeletal muscles. Decreased levels of thyroid hormone led to the up-regulation of the transgene in both cardiac and skeletal muscles, mimicking the behavior of the endogenous beta-MyHC gene. After deleting the distal 5000 bp, the level of reporter gene expression was strongly reduced. However, decreased levels of thyroid hormone led to an 80-fold skeletal muscle-specific increase in transgene expression, even upon the ablation of a conserved cis-regulatory element termed MCAT, which under normal (euthyroid) conditions abolishes muscle-specific expression. In contrast, cardiac-specific induction was not detected with the deletion construct. These observations indicate that the cardiac and skeletal muscle regulatory elements can be functionally segregated on the beta-MyHC gene promoter. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7878016

  14. Segregation of cardiac and skeletal muscle-specific regulatory elements of the beta-myosin heavy chain gene.

    PubMed

    Rindt, H; Knotts, S; Robbins, J

    1995-02-28

    The beta-myosin heavy chain (beta-MyHC) gene is expressed in cardiac and slow skeletal muscles. To examine the regulatory sequences that are required for the gene's expression in the two compartments in vivo, we analyzed the expression pattern of a transgene consisting of the beta-MyHC gene 5' upstream region linked to the chloramphenicol acetyltransferase reporter gene. By using 5600 bp of 5' upstream region, the transgene was expressed at high levels in the slow skeletal muscles. Decreased levels of thyroid hormone led to the up-regulation of the transgene in both cardiac and skeletal muscles, mimicking the behavior of the endogenous beta-MyHC gene. After deleting the distal 5000 bp, the level of reporter gene expression was strongly reduced. However, decreased levels of thyroid hormone led to an 80-fold skeletal muscle-specific increase in transgene expression, even upon the ablation of a conserved cis-regulatory element termed MCAT, which under normal (euthyroid) conditions abolishes muscle-specific expression. In contrast, cardiac-specific induction was not detected with the deletion construct. These observations indicate that the cardiac and skeletal muscle regulatory elements can be functionally segregated on the beta-MyHC gene promoter.

  15. Provocative pattern of rearrangements of the genes for the. gamma. and. beta. chains of the T-cell receptor in human leukemias

    SciTech Connect

    Goorha, R.; Bunin, N.; Mirro, J. Jr.; Murphy, S.B.; Cross, A.H.; Behm, F.G.; Quertermous, T.; Seidman, J.; Kitchingman, G.R.

    1987-07-01

    To examine the distribution of rearrangements of the ..gamma..- and ..beta..-chain T-cell receptor (TCR) genes in T- and non-T acute lymphoblastic leukemias (ALLs), and potentially to determine which genes rearrange first in ontogeny, the authors analyzed high molecular weight DNA from 102 patients with acute leukemia. Rearranged ..gamma..- and ..beta..-chain genes were found in all T-cell ALLs (22/22) examined. Overall, 27% (18/66) of B-lineage ALLs had ..beta..-chain gene rearrangements, and 41% (24/58) had ..gamma..-chain gene rearrangements, but the distribution of rearranged genes varied according to the stage of B-cell differentiation. The ..gamma..-chain genes were rearranged in 11% (1/9) of the B-lineage patients negative for the common acute lymphoblastic leukemia antigen (cALLA) and 50% (23/46) of cALLA/sup -/= ALL patients, while the ..beta..-chain genes were not rearranged in any of the 7 cALLA/sup -/ ALL patients examined but were rearranged in 32% (18/56) of the cALLA/sup +/ patients. Of the 44 cALLA/sup +/ patients in which a direct comparison of ..gamma..- and ..beta..-chain gene rearrangements could be made, 34% had both genes rearranged, 16% had only ..gamma..-chain gene rearrangements, and the remaining 50% had both genes in the germ-line configuration. ..beta..-Chain rearrangements have not been found in the absence of ..gamma..-chain rearrangements, thus supporting a proposed hierarchy of TCR gene rearrangements.

  16. Detection of beta-globin gene mutations among Kelantan Malay thalassaemia patients by polymerase chain reaction restriction fragment length polymorphism.

    PubMed

    Rozitah, R; Nizam, M Z; Nur Shafawati, A R; Nor Atifah, M A; Dewi, M; Kannan, T P; Ariffin, N; Norsarwany, M; Setianingsih, I; Harahap, A; Zilfalil, B A

    2008-12-01

    Beta-thalassaemia major is an autosomal recessive disorder that results in severe microcytic, hypochromic, haemolytic anaemia among affected patients. Beta-thalassaemia has emerged as one of the most common public health problems in Malaysia, particularly among Malaysian Chinese and Malays. This study aimed to observe the spectrum of mutations found in Kelantan Malay beta-thalassaemia major patients who attended the Paediatrics Daycare Unit, Hospital Universiti Sains Malaysia, Kelantan, Malaysia, the data of which was being used in establishing the prenatal diagnosis in this Human Genome Centre. This was a cross-sectional study conducted with 35 Kelantan Malay beta-thalassaemia major patients. DNA was extracted from the blood collected from the patients and subjected to polymerase chain reaction (PCR) amplification. Six restriction enzymes were used to digest the PCR products for the detection of mutations. Five out of the six beta-globin gene defects were detected, namely, IVS-1 nt5 (G>C), IVS-1 nt1 (G>T), codon 26 (G>A), codon 41-42 (4 bp del) and codon 19 (A>G). The mutation which was not observed in this study was in codon 15 (G>A). The two most common mutations observed were codon 26 (G>A) and IVS-1 nt5 (G>C), which was detected in 26 and 17 patients, respectively. Two patients did not show any of the six mutations. Our results added to the existing data on the common beta-globin gene defects in Kelantan Malay beta-thalassaemia patients.

  17. Association study of schizophrenia and IL-2 receptor {beta} chain gene

    SciTech Connect

    Nimgaonkar, V.L.; Yang, Z.W.; Zhang, X.R.; Brar, J.S.

    1995-10-09

    A case-control association study was conducted in Caucasian patients with schizophrenia (DSM-III-R, n = 42) and unaffected controls (n = 47) matched for ethnicity and area of residence. Serum interleukin-2 receptor (IL-2R) concentrations, as well as a dinucleotide repeat polymorphism in the IL-2RP chain gene, were examined in both groups. No significant differences in IL-2R concentrations or in the distribution of the polymorphism were noted. This study does not support an association between schizophrenia and the IL-2RP gene locus, contrary to the suggestive evidence from linkage analysis in multicase families. 17 refs., 2 tabs.

  18. Provocative pattern of rearrangements of the genes for the gamma and beta chains of the T-cell receptor in human leukemias.

    PubMed Central

    Goorha, R; Bunin, N; Mirro, J; Murphy, S B; Cross, A H; Behm, F G; Quertermous, T; Seidman, J; Kitchingman, G R

    1987-01-01

    To examine the distribution of rearrangements of the gamma- and beta-chain T-cell receptor (TCR) genes in T- and non-T acute lymphoblastic leukemias (ALLs), and potentially to determine which genes rearrange first in ontogeny, we analyzed high molecular weight DNA from 102 patients with acute leukemia. Rearranged gamma- and beta-chain genes were found in all T-cell ALLs (22/22) examined. Overall, 27% (18/66) of B-lineage ALLs had beta-chain gene rearrangements, and 41% (24/58) had gamma-chain gene rearrangements, but the distribution of rearranged genes varied according to the stage of B-cell differentiation. The gamma-chain genes were rearranged in 11% (1/9) of the B-lineage patients negative for the common acute lymphoblastic leukemia antigen (cALLA) and 50% (23/46) of cALLA+ ALL patients, while the beta-chain genes were not rearranged in any of the 7 cALLA- ALL patients examined but were rearranged in 32% (18/56) of the cALLA+ patients. Neither TCR gene was found to be rearranged in acute nonlymphoid leukemia patients (0/12) or in patients with B-cell (surface immunoglobulin-positive) leukemia (0/3). Of the 44 cALLA+ patients in which a direct comparison of gamma- and beta-chain gene rearrangements could be made, 34% had both genes rearranged, 16% had only gamma-chain gene rearrangements, and the remaining 50% had both genes in the germ-line configuration. beta-Chain rearrangements have not been found in the absence of gamma-chain rearrangements, thus supporting a proposed hierarchy of TCR gene rearrangements. A provocative finding was that only a small percentage (11%) of the patients with cALLA- B precursor cell ALLs had rearranged TCR genes, while 50% of the cALLA+ leukemia patients had at least gamma-chain rearrangement, raising a question as to whether indeed cALLA- cells are precursors to cALLA+ cells. Interestingly, 18% (2/11) of the cytoplasmic immunoglobulin (cIg)-positive cALLA+ (pre-B) ALLs involved TCR gene rearrangements, compared to 60% (21/35) of

  19. A homozygous nonsense mutation in the {beta}3 chain gene of laminin 5 (LAMB3) in herlitz junctional epidermolysis bullosa

    SciTech Connect

    Pulkkinen, L.; Christiano, A.M.; Uitto, J.

    1994-11-15

    Herlitz junctional epidermolysis bullosa (H-JEB) is a severe autosomal recessive disorder characterized by blister formation within the dermal-epidermal basement membrane. Based on immunofluorescence analysis recognizing laminin 5 epitopes (previously known as nicein/kalinin), the genes for this lamina lucida protein have been proposed as candidate genes in H-JEB. Amplification of mRNA by RT-PCR, followed by direct nucleotide sequencing, revealed a homozygous C-to T transition resulting in a premature termination codon (CGA{r_arrow}TGA) on both alleles. This mutation was verified at the genomic DNA level, and both parents were shown to be heterozygous carriers of the same mutation. This is the first description of a mutation in the laminin {beta}3 chain gene (LAMB3) of laminin 5 in an H-JEB patient. 15 refs., 2 figs.

  20. Analysis of T cell antigen receptor (TCR) expression by human peripheral blood CD4-8- alpha/beta T cells demonstrates preferential use of several V beta genes and an invariant TCR alpha chain

    PubMed Central

    1993-01-01

    CD4-CD8- (double negative [DN]) alpha/beta T cells are a largely uncharacterized subpopulation of unknown function. To investigate whether these cells are selected to recognize particular antigens or antigen-presenting molecules, DN alpha/beta T cells were purified from the peripheral blood of five normal donors and their T cell receptor (TCR) alpha and beta chains were examined. Random cloning of TCR alpha chains by single-sided polymerase chain reaction (PCR) amplification identified an invariant rearrangement between V alpha 24 and J alpha Q, with no N region diversity, which was expressed preferentially by DN alpha/beta T cells from all donors. Random cloning also identified a precise V alpha 7.2-J alpha (IGRJa14) rearrangement, with two variable amino acids encoded in the V-J junction, which was enriched in the DN alpha/beta T cell preparations from some, but not all, donors. Analysis of TCR beta chains by quantitative PCR amplification demonstrated that the expression of four V beta gene families, V beta 2, 8, 11, and 13, was markedly increased in these DN alpha/beta T cell preparations. The expression of particular TCRs by DN alpha/beta T cells from multiple donors indicates that these cells, or at least a subpopulation of cells with this phenotype, recognize a limited spectrum of antigens and suggests that they may use nonpolymorphic antigen-presenting molecules. PMID:8391057

  1. A novel gene of beta chain of the IFN-gamma receptor of Huiyang chicken: cloning, distribution, and CD assay.

    PubMed

    Han, Chun-Lai; Zhang, Wei; Dong, Hai-Tao; Han, Xue; Wang, Ming

    2006-07-01

    The beta chain of the interferon-gamma receptor (IFNGR-2) plays a critical role in signal transmission to the nucleus by IFN-gamma. Here, we cloned the full-length cDNA of IFNGR-2 of Huiyang chicken using RACE. mRNA transcripts of IFNGR-2 were detected in peripheral blood leukocytes (PBL) and various organs using Northern blot analysis. The extracellular region of IFNGR-2 (IFNGR-2EC) was expressed in Pichia pastoris, and its secondary structure was investigated by circular dichroism (CD). The Huiyang chicken IFNGR-2 gene is 2221 bp with a polyA+ tail, and it encodes 334 amino acids sharing 30%-33% identity with that of rat, mouse, and human IFNGR-2. IFNGR-2 is localized on chromosome 1 of chicken in tandem with IFNAR-1, interleukin- 10 receptor (IL-10R-2), and IFNAR-2. IFNGR-2 was highly expressed in PBL, muscle, spleen, thymus, and cecal tonsil, whereas its expression in cardiac muscle, cloacal bursa, liver, and kidney was comparatively low. Recombinant protein of IFNGR-2EC expressed in P. pastoris formed the secondary structure including 19.8% alpha-helix, 29.6% beta-sheet, 19.7% turn, and 30.9% random. The data show that Huiyang chicken IFNGR-2 shares properties of the IFN receptor family in gene structure and distribution in multiple tissues and PBL. CD analysis indicated that the recombinant protein of IFNGR-2EC resembles the known structure of human IFN receptors.

  2. Cloning of the {beta}3 chain gene (LAMB3) of human laminin 5, a candidate gene in junctional epidermolysis bullosa

    SciTech Connect

    Pulkkinen, L.; Christiano, A.M.; Uitto, J.

    1995-01-01

    Laminin 5 consists of three polypeptides, {alpha}3, {beta}3, and {gamma}2, encoded by the genes LAMA3, LAMB3, and LAMC2, respectively. In this study, we have elucidated the exon-intron organization of the human LAMB3 gene. Characterization of five overlapping {lambda} phage DNA clones revealed that the gene was approximately 29 kb in size. Subsequent sequence data revealed that the gene consisted of 23 exons that varied from 64 to 379 bp in size, accounting for the full-length cDNA with an open reading frame of 3516 hp encoding 1172 amino acids. Comparison of the LAMB3 gene structure with the previously characterized LAMB1 gene revealed that LAMB3 was considerably more compact. Knowledge of the exon-intron organization of the LAMB3 gene will facilitate elucidation of mutations in patients with the junctional forms of epidermolysis bullosa, some of which have been associated with mutations in the laminin 5 genes. 33 refs., 3 figs., 2 tabs.

  3. Familial hypertrophic cardiomyopathy. Microsatellite haplotyping and identification of a hot spot for mutations in the beta-myosin heavy chain gene.

    PubMed

    Dausse, E; Komajda, M; Fetler, L; Dubourg, O; Dufour, C; Carrier, L; Wisnewsky, C; Bercovici, J; Hengstenberg, C; al-Mahdawi, S

    1993-12-01

    Familial hypertrophic cardiomyopathy (FHC) is a clinically and genetically heterogeneous disease. The first identified disease gene, located on chromosome 14q11-q12, encodes the beta-myosin heavy chain. We have performed linkage analysis of two French FHC pedigrees, 720 and 730, with two microsatellite markers located in the beta-myosin heavy chain gene (MYO I and MYO II) and with four highly informative markers, recently mapped to chromosome 14q11-q12. Significant linkage was found with MYO I and MYO II in pedigree 720, but results were not conclusive for pedigree 730. Haplotype analysis of the six markers allowed identification of affected individuals and of some unaffected subjects carrying the disease gene. Two novel missense mutations were identified in exon 13 by direct sequencing, 403Arg-->Leu and 403Arg-->Trp in families 720 and 730, respectively. The 403Arg-->Leu mutation was associated with incomplete penetrance, a high incidence of sudden deaths and severe cardiac events, whereas the consequences of the 403Arg-->Trp mutation appeared less severe. Haplotyping of polymorphic markers in close linkage to the beta-myosin heavy chain gene can, thus, provide rapid analysis of non informative pedigrees and rapid detection of carrier status. Our results also indicate that codon 403 of the beta-myosin heavy chain gene is a hot spot for mutations causing FHC.

  4. Characterization of the primary structure of T cell receptor beta chains in cells infiltrating the salivary gland in the sicca syndrome of HIV-1 infection. Evidence of antigen-driven clonal selection suggested by restricted combinations of V beta J beta gene segment usage and shared somatically encoded amino acid residues.

    PubMed Central

    Dwyer, E; Itescu, S; Winchester, R

    1993-01-01

    Infection with HIV-1 occasionally results in a sicca syndrome, termed the diffuse infiltrative lymphocytosis syndrome, characterized by infiltration of the salivary glands with a predominance of CD8 T cells. This response is strongly associated with certain MHC class I and class II alleles. To define the salivary gland T cell receptor (TCR) repertoire, the primary structure of the TCR beta-chains was determined using in situ cDNA synthesis followed by the "anchored" polymerase chain reaction. The sequences of 59 beta-chains from five individuals with diffuse infiltrative lymphocytosis syndrome shared structural features suggesting antigenic clonal selection. Certain combinations of V beta J beta gene segments were selectively overrepresented in the repertoire sample, demonstrating a common restricted usage of certain V beta and J beta gene segments. The beta-chains derived from these overrepresented V beta J beta combinations revealed a preference for specific amino acids at position 97 in the third complementarity-determining region, a residue postulated to contact peptide antigen. Moreover, the nucleotides encoding this position were not germline in origin. TCR beta-chains in nonoverrepresented V beta J beta combinations did not exhibit preferential usage of selected somatically encoded residues. The pattern of TCR beta-chains expressed in the salivary gland of a control person with primary Sjögren's syndrome was considerably more heterogeneous and different from that found in diffuse infiltrative lymphocytosis syndrome. Images PMID:8392093

  5. Interaction between T cell receptor beta chain and immunoglobulin heavy chain region genes in susceptibility to insulin-dependent diabetes mellitus.

    PubMed

    Field, L L; Stephure, D K; McArthur, R G

    1991-09-01

    Despite some reports of an association between insulin-dependent diabetes mellitus (IDDM) and a BglII RFLP in the T cell receptor beta chain (TCRB) constant region, results of several recent studies, including our own, have failed to support such an association. However, we here report evidence for an IDDM-TCRB relationship which is dependent on immunoglobulin heavy-chain-region genes. We analyzed 198 unrelated diabetics and 84 normal siblings (maximum one sibling per diabetic) typed for the BglII TCRB RFLP and Gm immunoglobulin allotypes Glm(1), Glm(2), G2m(23), and G3m(5), which identify the four common Gm haplotypes. The BglIII TCRB genotype frequencies were significantly different between diabetics positive and negative for G2m(23) (P = .017) and G3m(5) (P = .021) but were not different between normal siblings positive and negative for those allotypes (P = .94 and P = .77, respectively). Thus, there were significant interactions between TCRB, Gm, and IDDM for two of the four immunoglobulin allotypes examined. We have previously reported interactions between HLA, Gm (particularly G2m(23)), and IDDM and postulate that the TCRB-Gm-IDDM and HLA-Gm-IDDM interaction effects may be functionally related.

  6. Multiple muscle-specific regulatory elements are associated with a DNase I hypersensitive site of the cardiac beta-myosin heavy-chain gene.

    PubMed

    Huang, W Y; Chen, J J; Shih, N; Liew, C C

    1997-10-15

    Using nuclei isolated from neonatal cardiomyocytes, we have mapped the DNase I hypersensitive sites (DHSs) residing within the 5'-upstream regions of the hamster cardiac myosin heavy-chain (MyHC) gene. Two cardiac-specific DHSs within the 5 kb upstream region of the cardiac MyHC gene were identified. One of the DHSs was mapped to the -2.3 kb (beta-2.3 kb) region and the other to the proximal promoter region. We further localized the beta-2.3 kb site to a range of 250 bp. Multiple, conserved, muscle regulatory motifs were found within the beta-2.3 kb site, consisting of three E-boxes, one AP-2 site, one CArG motif, one CT/ACCC box and one myocyte-specific enhancer factor-2 site. This cluster of regulatory elements is strikingly similar to a cluster found in the enhancer of the mouse muscle creatine kinase gene (-1256 to -1050). The specific interaction of the motifs within the beta-2.3 kb site and the cardiac nuclear proteins was demonstrated using gel mobility-shift assays and footprinting analysis. In addition, transfection analysis revealed a significant increase in chloramphenicol acetyltransferase activity when the beta-2.3 kb site was linked to a heterologous promoter. These results suggest that previously undefined regulatory elements of the beta-MyHC gene may be associated with the beta-2.3 kb site.

  7. Sequences 5' of the basement membrane laminin beta 1 chain gene (LAMB1) direct the expression of beta-galactosidase during development of the mouse testis and ovary.

    PubMed

    Li, C; Gudas, L J

    1997-12-01

    The murine LAMB1 gene encoding laminin beta 1 is expressed in the developing male and female gonads and mesonephros. To identify the cis-acting elements regulating the expression of LAMB1, murine transgenic lines were generated by fusing regions of the LAMB1 gene to the Eschericia coli lacZ gene. The p3.9LAM beta gal construct contained approximately 4 kb of 5' flanking sequence and directed beta-galactosidase expression in many different organs including the kidney, mammary gland, and the male and female genital systems, the focus of this report. In male embryos, between gestational ages E 14.5 and birth beta-galactosidase was transiently expressed in the prospermatogonia cells of the testis and in the differentiating epithelial cells in the ductus deferens, ductus epididymis, and seminal vesicles. In female embryos, beta-galactosidase was not detected in the ovary until about 1 week after birth; at this time, beta-galactosidase was expressed by oocytes of primary and secondary follicles. In contrast, transgenic mice carrying the first 0.7 kb of LAMB1 fused to the lacZ gene expressed beta-galactosidase only in the prospermatogonia cells of the testis. Thus, the cis-acting element(s) necessary for the expression of the LAMB1 gene in prospermatogonia cells are located in the first 0.7 kb of LAMB1 5' flanking sequence; element(s) required for expression of the LAMB1 gene in oocytes and epithelial cells of the mesonephric ducts, mesonephric tubules, the ductus deferens, ductus epididymis, and seminal vesicles are located with 4 kb 5' of the transcription initiation site.

  8. In vivo regulation of the beta-myosin heavy chain gene in soleus muscle of suspended and weight-bearing rats

    NASA Technical Reports Server (NTRS)

    Giger, J. M.; Haddad, F.; Qin, A. X.; Baldwin, K. M.

    2000-01-01

    In the weight-bearing hindlimb soleus muscle of the rat, approximately 90% of muscle fibers express the beta-myosin heavy chain (beta-MHC) isoform protein. Hindlimb suspension (HS) causes the MHC isoform population to shift from beta toward the fast MHC isoforms. Our aim was to establish a model to test the hypothesis that this shift in expression is transcriptionally regulated through specific cis elements of the beta-MHC promoter. With the use of a direct gene transfer approach, we determined the activity of different length beta-MHC promoter fragments, linked to a firefly luciferase reporter gene, in soleus muscle of control and HS rats. In weight-bearing rats, the relative luciferase activity of the longest beta-promoter fragment (-3500 bp) was threefold higher than the shorter promoter constructs, which suggests that an enhancer sequence is present in the upstream promoter region. After 1 wk of HS, the reporter activities of the -3500-, -914-, and -408-bp promoter constructs were significantly reduced ( approximately 40%), compared with the control muscles. However, using the -215-bp construct, no differences in promoter activity were observed between HS and control muscles, which indicates that the response to HS in the rodent appears to be regulated within the -408 and -215 bp of the promoter.

  9. In vivo regulation of the beta-myosin heavy chain gene in soleus muscle of suspended and weight-bearing rats

    NASA Technical Reports Server (NTRS)

    Giger, J. M.; Haddad, F.; Qin, A. X.; Baldwin, K. M.

    2000-01-01

    In the weight-bearing hindlimb soleus muscle of the rat, approximately 90% of muscle fibers express the beta-myosin heavy chain (beta-MHC) isoform protein. Hindlimb suspension (HS) causes the MHC isoform population to shift from beta toward the fast MHC isoforms. Our aim was to establish a model to test the hypothesis that this shift in expression is transcriptionally regulated through specific cis elements of the beta-MHC promoter. With the use of a direct gene transfer approach, we determined the activity of different length beta-MHC promoter fragments, linked to a firefly luciferase reporter gene, in soleus muscle of control and HS rats. In weight-bearing rats, the relative luciferase activity of the longest beta-promoter fragment (-3500 bp) was threefold higher than the shorter promoter constructs, which suggests that an enhancer sequence is present in the upstream promoter region. After 1 wk of HS, the reporter activities of the -3500-, -914-, and -408-bp promoter constructs were significantly reduced ( approximately 40%), compared with the control muscles. However, using the -215-bp construct, no differences in promoter activity were observed between HS and control muscles, which indicates that the response to HS in the rodent appears to be regulated within the -408 and -215 bp of the promoter.

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

    SciTech Connect

    Wewer, U.M.; Durkin, M.E.; Albrechtsen, R.

    1994-11-15

    Overlapping cDNA clones that encode the full-length human laminin {beta}2 chain, formerly called the S chain, were isolated. The cDNA of 5680 nt contains a 5391-nt open reading frame encoding 1797 amino acids. At the amino terminus is a 32-amino-acid signal peptide that is followed by the mature {beta}2 chain polypeptide of 1765 amino acids with a calculated molecular mass of 192,389 Da. The human {beta}2 chain is predicted to have all of the seven structural domains typical of the {beta} chains of laminin, including the short cysteine-rich {alpha} region. The amino acid sequence of human {beta}2 chain showed 86.1% sequence identity to the rat {beta}2 chain, 50.0% to human {beta}1 chain, and 36.3% to the human {beta}3 chain. The greatest sequence identity was in domains VI, V, and III. The sequence of a 24-amino-acid peptide fragment isolated from the {beta}2 chain of laminin purified from human amniotic basement membrane matched the sequence predicted from the cDNA, confirming that the cDNA encodes human {beta}2 laminin. The cDNA was used to assign the gene (LAMB2) to human chromosome 3p21 by in situ hybridization. It is not linked to genes for human laminin chains {alpha}1, {beta}1, and {gamma}1 or other known laminin genes. Immunostaining showed that the {beta}2 chain is localized to the smooth muscle basement membranes of the arteries, while the homologous {beta}1 chain is confined to the subendothelial basement membranes. The {beta}2 chain was found in the basement membranes of ovarian carcinomas but not colon carcinomas. These results indicate that the expression of the {beta}2 chain gene is tightly regulated in normal human tissues and in disease. 43 refs., 6 figs., 1 tab.

  11. Human beta-hexosaminidase alpha chain: coding sequence and homology with the beta chain.

    PubMed Central

    Myerowitz, R; Piekarz, R; Neufeld, E F; Shows, T B; Suzuki, K

    1985-01-01

    We have isolated a cDNA clone, p beta H alpha-5, from an adult human liver library that contains the entire coding sequence of the alpha chain of beta-hexosaminidase. The cDNA insert of p beta H alpha-5 is 1944 base pairs long and contains a 168-base-pair 5' untranslated region, a 186-base-pair 3' untranslated region, and an open reading frame of 1587 base pairs corresponding to 529 amino acids (Mr, 60,697). The first 17-22 amino acids satisfy the requirements of a signal sequence. A striking sequence homology with a published partial amino acid sequence for the beta chain [O'Dowd, B. F., Quan, F., Willard, H. F., Lamhonwah, A. M., Korneluk, R. G., Lowden, J. A., Gravel, R. A. & Mahuran, D. J. (1985) Proc. Natl. Acad. Sci. USA 82, 1184-1188] suggests that both chains may have evolved from a common ancestor. A shorter alpha-chain cDNA was found to hybridize to the long arm of chromosome 15, the known location for the alpha-chain gene. In addition, we isolated another alpha-chain cDNA clone, p beta H alpha-4, from a simian virus 40-transformed human fibroblast library that contained an extra 453-base-pair piece at its 3' end. A probe consisting of this additional sequence hybridized exclusively to a single mRNA species (2.6 kilobases) in mRNA preparations from cultured human fibroblasts. In contrast, p beta H alpha-5 hybridized to both a 2.1-kilobase major and a 2.6-kilobase minor mRNA species in these same mRNA preparations, indicating the presence of two distinct alpha-chain mRNA species differing at the 3' end. Fibroblasts from an Ashkenazi Jewish patient with classic Tay-Sachs disease were deficient in both species of mRNA, confirming their genetic relationship. Images PMID:2933746

  12. Identification of an immunodominant region on the I-A beta chain using site-directed mutagenesis and DNA-mediated gene transfer

    PubMed Central

    1988-01-01

    To identify which polymorphic residues determine the allospecific antibody binding sites on A beta polypeptides, mutant Ak beta genes were constructed encoding single or multiple amino acids of the d allele at 14 polymorphic positions in the beta 1 domain. Cell lines expressing these genes were analyzed by quantitative immunofluorescence using 16 mAbs reactive to Ak beta or Ad beta. Substitution of d allele residues at positions 63 and 65-67 in the Ak beta polypeptide resulted in the loss of binding of all Ak beta-reactive antibodies and the gain of binding of most Ad beta-reactive antibodies. Two Ad beta-reactive mAbs bound to the mutant Ak beta polypeptide containing d allele- characteristic residue at position 40. In contrast, substitution of the other polymorphic residues in the NH2-terminal and COOH-terminal regions of the beta 1 domain did not alter antibody binding. PMID:2450160

  13. Position independent expression and developmental regulation is directed by the beta myosin heavy chain gene's 5' upstream region in transgenic mice.

    PubMed

    Knotts, S; Rindt, H; Robbins, J

    1995-08-25

    Transgenic mice generated with constructs containing 5.6 kb of the beta myosin heavy chain (MyHC) gene's 5' flanking region linked to the cat reporter gene express the transgene at high levels. In all 47 lines analyzed, tissue-specific accumulation of chloramphenicol acetyltransferase was found at levels proportional to the number of integrated transgene copies. Deletion constructs containing only 0.6 kb of 5' upstream region showed position effects in transgenic mice and did not demonstrate copy number dependence although transgene expression remained muscle-specific. The 5.6 kb 5' upstream region conferred appropriate developmental control of the transgene to the cardiac compartment and directs copy number dependent and position independent expression. Lines generated with a construct in which three proximal cis-acting elements were mutated showed reduced levels of transgene expression, but all maintained their position independence and copy number dependence, suggesting the presence of distinct regulatory mechanisms.

  14. Position independent expression and developmental regulation is directed by the beta myosin heavy chain gene's 5' upstream region in transgenic mice.

    PubMed Central

    Knotts, S; Rindt, H; Robbins, J

    1995-01-01

    Transgenic mice generated with constructs containing 5.6 kb of the beta myosin heavy chain (MyHC) gene's 5' flanking region linked to the cat reporter gene express the transgene at high levels. In all 47 lines analyzed, tissue-specific accumulation of chloramphenicol acetyltransferase was found at levels proportional to the number of integrated transgene copies. Deletion constructs containing only 0.6 kb of 5' upstream region showed position effects in transgenic mice and did not demonstrate copy number dependence although transgene expression remained muscle-specific. The 5.6 kb 5' upstream region conferred appropriate developmental control of the transgene to the cardiac compartment and directs copy number dependent and position independent expression. Lines generated with a construct in which three proximal cis-acting elements were mutated showed reduced levels of transgene expression, but all maintained their position independence and copy number dependence, suggesting the presence of distinct regulatory mechanisms. Images PMID:7667107

  15. Ginger extract mitigates ethanol-induced changes of alpha and beta - myosin heavy chain isoforms gene expression and oxidative stress in the heart of male wistar rats.

    PubMed

    Shirpoor, Alireza; Zerehpoosh, Mitra; Ansari, Mohammad Hasan Khadem; Kheradmand, Fatemeh; Rasmi, Yousef

    2017-09-01

    The association between ethanol consumption and heart abnormalities, such as chamber dilation, myocyte damage, ventricular hypertrophy, and hypertension is well known. However, underlying molecular mediators involved in ethanol-induced heart abnormalities remain elusive. The aim of this study was to investigate the effect of chronic ethanol exposure on alpha and beta - myosin heavy chain (MHC) isoforms gene expression transition and oxidative stress in rats' heart. It was also planned to find out whether ginger extract mitigated the abnormalities induced by ethanol in rats' heart. Male wistar rats were divided into three groups of eight animals as follows: Control, ethanol, and ginger extract treated ethanolic (GETE) groups. After six weeks of treatment, the results revealed a significant increase in the β-MHC gene expression, 8- OHdG amount, and NADPH oxidase level. Furthermore, a significant decrease in the ratio of α-MHC/β-MHC gene expression to the amount of paraoxonase enzyme in the ethanol group compared to the control group was found. The consumption of Ginger extract along with ethanol ameliorated the changes in MHC isoforms gene expression and reduced the elevated amount of 8-OHdG and NADPH oxidase. Moreover, compared to the consumption of ethanol alone, it increased the paraoxonase level significantly. These findings indicate that ethanol-induced heart abnormalities may in part be associated with MHC isoforms changes mediated by oxidative stress, and that these effects can be alleviated by using ginger extract as an antioxidant molecule. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Conserved structure of amphibian T-cell antigen receptor beta chain.

    PubMed Central

    Fellah, J S; Kerfourn, F; Guillet, F; Charlemagne, J

    1993-01-01

    All jawed vertebrates possess well-differentiated thymuses and elicit T-cell-like cell-mediated responses; however, no surface T-cell receptor (TCR) molecules or TCR genes have been identified in ectothermic vertebrate species. Here we describe cDNA clones from an amphibian species, Ambystoma mexicanum (the Mexican axolotl), that have sequences highly homologous to the avian and mammalian TCR beta chains. The cloned amphibian beta chain variable region (V beta) shares most of the structural characteristics with the more evolved vertebrate V beta and presents approximately 56% amino acid identities with the murine V beta 14 and human V beta 18 families. The two different cloned axolotl beta chain joining regions (J beta) were found to have conserved all the invariant mammalian J beta residues, and in addition, the presence of a conserved glycine at the V beta-J beta junction suggests the existence of diversity elements. The extracellular domains of the two axolotl beta chain constant region isotypes C beta 1 and C beta 2 show an impressively high degree of identity, thus suggesting that a very efficient mechanism of gene correction has been in operation to preserve this structure at least from the early tetrapod evolution. The transmembrane axolotl C beta domains have been less well conserved when compared to the mammalian C beta but they do maintain the lysine residue that is thought to be involved in the charged interaction between the TCR alpha beta heterodimer and the CD3 complex. Images Fig. 1 PMID:8341702

  17. DNA segment containing C/sub. beta. 1/, a gene for the constant region of the. beta. chain of the T-cell antigen receptor, was inserted into chromosome 6 in cells from one patients with human T-cell leukemia

    SciTech Connect

    Ino, T.; Kurosawa, Y.; Yoshida, M.C.; Hirano, M.

    1987-06-01

    DNA rearrangements that occurred in the vicinity of T-cell antigen receptor ..beta..-chain gene clusters residing on chromosome 7 were examined in human T-cell acute lymphoblastic leukemia cells. In one patient, it was observed that, for the T-cell receptor ..beta..-chain genes, a D/sub ..beta.. 1/-J/sub ..beta..2.3/ (where D is diversity and J is joining) junction was found on one chromosome, while the other chromosome kept the germ-line configuration. If this D/sub ..beta../-J/sub ..beta../ junction was formed by the customary deletion mechanism, the C/sub ..beta..1/ gene (where C is constant) located between the D/sub ..beta..1/ and J/sub BETA2.3/ loci should have disappeared from this chromosome. The C/sub ..beta..1/ gene indeed was absent from the rearranged chromosome 7, but it was found on chromosome 6 as an inserted segment. The implications of the observations are discussed.

  18. Search for genetic variants in the retinoid X receptor-gamma-gene by polymerase chain reaction-single-strand conformation polymorphism in patients with resistance to thyroid hormone without mutations in thyroid hormone receptor beta gene.

    PubMed

    Romeo, Stefano; Menzaghi, Claudia; Bruno, Rocco; Sentinelli, Federica; Fallarino, Mara; Fioretti, Francesca; Filetti, Sebastiano; Balsamo, Armando; Di Mario, Umberto; Baroni, Marco G

    2004-05-01

    Resistance to thyroid hormone (RTH) is an inherited disease characterized by reduced tissue sensitivity to thyroid hormone. Approximately 90% of subjects with RTH have mutation in the thyroid hormone receptor beta (TRbeta) gene. Approximately 10% of subjects diagnosed as having RTH do not carry mutation in the TRbeta gene. A possible linkage was reported with the retinoid X receptor-gamma (RXR-gamma) gene in two families. The aim of this study is to search for mutation within the RXR-gamma gene in unrelated subjects with diagnosed RTH without mutations in the TRbeta gene. Four subjects with RTH were studied, and sequence variants in the RXR-gamma gene were searched by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP). Analysis of all the 10 exons of the RXR-gamma gene, including intron-exon boundaries, promoter region and 3' untranslated region (UTR) reveled two variant bands in subjects II and III. Sequencing of these variants showed two single nucleotide polymorphisms (SNPs): 447C > T in exon 3 for patients II and IVS9 + 6A > G for patient III. Both SNPs were also present at high frequency in a group of normal subjects and in nonaffected relatives of subject III. In conclusion, in patients with RTH we have found two SNPs in the RXR-gamma gene; these SNPS are common in the general population, thus excluding a role for the RXR-gamma gene in these patients.

  19. Comprehensive analysis of the T-cell receptor beta chain gene in rhesus monkey by high throughput sequencing

    PubMed Central

    Li, Zhoufang; Liu, Guangjie; Tong, Yin; Zhang, Meng; Xu, Ying; Qin, Li; Wang, Zhanhui; Chen, Xiaoping; He, Jiankui

    2015-01-01

    Profiling immune repertoires by high throughput sequencing enhances our understanding of immune system complexity and immune-related diseases in humans. Previously, cloning and Sanger sequencing identified limited numbers of T cell receptor (TCR) nucleotide sequences in rhesus monkeys, thus their full immune repertoire is unknown. We applied multiplex PCR and Illumina high throughput sequencing to study the TCRβ of rhesus monkeys. We identified 1.26 million TCRβ sequences corresponding to 643,570 unique TCRβ sequences and 270,557 unique complementarity-determining region 3 (CDR3) gene sequences. Precise measurements of CDR3 length distribution, CDR3 amino acid distribution, length distribution of N nucleotide of junctional region, and TCRV and TCRJ gene usage preferences were performed. A comprehensive profile of rhesus monkey immune repertoire might aid human infectious disease studies using rhesus monkeys. PMID:25961410

  20. An experimentally validated panel of subfamily-specific oligonucleotide primers (V alpha 1-w29/V beta 1-w24) for the study of human T cell receptor variable V gene segment usage by polymerase chain reaction.

    PubMed

    Genevée, C; Diu, A; Nierat, J; Caignard, A; Dietrich, P Y; Ferradini, L; Roman-Roman, S; Triebel, F; Hercend, T

    1992-05-01

    We report here the characterization of a series of T cell receptor (TcR) V alpha or V beta subfamily-specific oligonucleotide primers. Criteria that have guided the design of each oligonucleotide include appropriate thermodynamic parameters as well as differential base-pairing scores with related and unrelated target sequences. The specificity of the oligonucleotides for each V alpha or V beta subfamily was tested by polymerase chain reaction (PCR) on both a series of TcR encoding plasmid DNA and clonal T cell populations. Unexpected cross-reactivities were observed with plasmid cDNA sequences corresponding to unrelated subfamily gene segments. This led to the synthesis of additional series of oligonucleotides to obtain a relevant panel. A series of V alpha 1-w29/V beta 1-w24 TcR subfamily-specific oligonucleotides was eventually selected which generates little, if any, cross-reactivity. The use of C alpha or C beta primers for the amplification of internal positive control templates (i.e. C beta for the V alpha series and C alpha for the V beta series) has been tested in PCR performed with cDNA derived from peripheral blood lymphocytes; it was shown not to alter the amplification of the V subfamily-specific DNA fragments. This panel of oligonucleotides will be helpful in the study of TcRV gene segment usage and, thus, may lead to a better characterization of T cell responses in physiological and pathological situations.

  1. A phorbol ester response element within the human T-cell receptor beta-chain enhancer.

    PubMed Central

    Prosser, H M; Wotton, D; Gegonne, A; Ghysdael, J; Wang, S; Speck, N A; Owen, M J

    1992-01-01

    The activity of the T-cell receptor beta-chain gene enhancer is increased by activators of the protein kinase C pathway during T-cell activation. Analysis of mutant enhancer constructs identified two elements, beta E2 and beta E3, conferring phorbol ester inducibility. Multimerized beta E2 acted in isolation as a phorbol ester-responsive element. Both beta E2 and beta E3, which contain a consensus Ets-binding site, were shown to bind directly to the product of the c-ets-1 protooncogene. Both regions also bound a second factor, core-binding factor. Mutation of the beta E2 Ets site abolished the inducibility of the beta E2 multimer. beta E2 and beta E3 Ets site mutations also profoundly affected activity and inducibility of the enhancer. In contrast, enhancer activity but not its inducibility was affected by mutation of the beta E2 core-binding factor site. Cotransfection studies showed that Ets-1 specifically repressed activity of the multimerized beta E2 element and the complete T-cell receptor beta-chain enhancer. These data show that the T-cell receptor beta-chain enhancer responds to protein kinase C-mediated activation signals via a functional domain, composed of two elements, which contains binding sites for Ets transcription factors and which is negatively regulated by Ets-1. Images PMID:1409722

  2. Betaxolol inhibits extracellular signal-regulated kinase and P70S6 kinase activities and gene expressions of platelet-derived growth factor A-chain and transforming growth factor-beta1 in Dahl salt-sensitive hypertensive rats.

    PubMed

    Kobayashi, Naohiko; Nakano, Shigefumi; Mori, Yousuke; Mita, Shin-ichiro; Kobayashi, Tsutomu; Honda, Takeaki; Tsubokou, Yusuke; Matsuoka, Hiroaki

    2002-03-01

    We evaluated the protective effects of long-term treatment with betaxolol, a specific beta-antagonist, on platelet-derived growth factor (PDGF) A-chain and transforming growth factor (TGF)-beta1 gene expression in the left ventricle of Dahl salt-sensitive hypertensive rats fed a high-salt diet. In addition, we evaluated the relations between these effects and coronary microvascular remodeling, expression of extracellular signal-regulated kinases (ERK) belonging to one subfamily of mitogen-activated protein kinases, and expression of p70S6 kinase belonging to one subfamily of ribosomal S6 kinases. Betaxolol (0.9 mg/kg/day, subdepressor dose) was administered for 5 weeks, from 6 weeks of age to the left ventricular hypertrophy stage at 11 weeks of age. Increased PDGF A-chain and TGF-beta1 mRNA and protein expression were suppressed by betaxolol. Upregulated activities of ERK1/2 and p70S6 kinase phosphorylations were decreased by betaxolol. Betaxolol administration resulted in significant improvements in the wall-to-lumen ratio, perivascular fibrosis and myocardial fibrosis. Thus, we conclude that ERK1/2 and p70S6 kinase activities may play a key role in coronary microvascular remodeling of Dahl salt-sensitive hypertensive rats, and that beneficial effects of betaxolol on cardiovascular remodeling may be at least partially mediated by decreased PDGF A-chain and TGF-beta1 expression in the left ventricle.

  3. Isolation of chicken major histocompatibility complex class II (B-L) beta chain sequences: comparison with mammalian beta chains and expression in lymphoid organs.

    PubMed Central

    Bourlet, Y; Béhar, G; Guillemot, F; Fréchin, N; Billault, A; Chaussé, A M; Zoorob, R; Auffray, C

    1988-01-01

    By cross-hybridization in low stringency conditions, using a probe derived from an HLA-DQ beta cDNA clone, we have isolated several chicken genomic DNA clones. These clones were mapped to the major histocompatibility complex (MHC) of the chick (B complex) by virtue of their ability to detect restriction enzyme length polymorphisms between congenic lines of chicken. Evidence was obtained for the presence of at least three B-L beta genes in the chicken genome. The B-L beta genes are transcribed specifically in tissues containing cells of the B lymphocyte and myeloid lineages and expressing the B-L antigens. Exons encoding the beta 1, beta 2 and transmembrane domains of a B-L beta chain have been identified with 63, 66 and 62% similarity with the HLA-DQ beta sequence. This first isolation of an MHC class II gene outside of the mammalian class provides insight into the evolution of MHC genes based on the comparison of avian and mammalian class II beta chain amino acid and nucleotide sequences. Images PMID:2841107

  4. Chicken T-cell receptor beta-chain diversity: an evolutionarily conserved D beta-encoded glycine turn within the hypervariable CDR3 domain.

    PubMed Central

    McCormack, W T; Tjoelker, L W; Stella, G; Postema, C E; Thompson, C B

    1991-01-01

    Unlike mammals, chickens generate an immunoglobulin (Ig) repertoire by a developmentally regulated process of intrachromosomal gene conversion, which results in nucleotide substitutions throughout the variable regions of the Ig heavy- and light-chain genes. In contrast to chicken Ig genes, we show in this report that diversity of the rearranged chicken T-cell receptor (TCR) beta-chain gene is generated by junctional heterogeneity, as observed in rearranged mammalian TCR genes. This junctional diversity increases during chicken development as a result of an increasing base-pair addition at the V beta-D beta and D beta-J beta joints (where V, D, and J are the variable, diversity, and joining gene segments). Despite the junctional hypervariability, however, almost all functional V beta-D beta-J beta junctions appear to encode a glycine-containing beta-turn. Such a turn may serve to position the amino acid side chains of a hypervariable TCR beta-chain loop with respect to the antigen-binding groove of the major histocompatibility complex molecule. Consistent with this hypothesis, the germ-line D beta nucleotide sequences of chickens, mice, rabbits, and humans have been highly conserved and encode a glycine in all three reading frames. Images PMID:1652759

  5. Maple syrup urine disease. Complete primary structure of the E1 beta subunit of human branched chain alpha-ketoacid dehydrogenase complex deduced from the nucleotide sequence and a gene analysis of patients with this disease.

    PubMed Central

    Nobukuni, Y; Mitsubuchi, H; Endo, F; Akaboshi, I; Asaka, J; Matsuda, I

    1990-01-01

    A defect in the E1 beta subunit of the branched chain alpha-ketoacid dehydrogenase (BCKDH) complex is one cause of maple syrup urine disease (MSUD). In an attempt to elucidate the molecular basis of MSUD, we isolated and characterized a 1.35 kbp cDNA clone encoding the entire precursor of the E1 beta subunit of BCKDH complex from a human placental cDNA library. Nucleotide sequence analysis revealed that the isolated cDNA clone (lambda hBE1 beta-1) contained a 5'-untranslated sequence of four nucleotides, the translated sequence of 1,176 nucleotides and the 3'-untranslated sequence of 169 nucleotides. Comparison of the amino acid sequence predicted from the nucleotide sequence of the cDNA insert of the clone with the NH2-terminal amino acid sequence of the purified mature bovine BCKDH-E1 beta subunit showed that the cDNA insert encodes for a 342-amino acid subunit with a Mr = 37,585. The subunit is synthesized as the precursor with a leader sequence of 50 amino acids and is processed at the NH2 terminus. A search for protein homology revealed that the primary structure of human BCKDH-E1 beta was similar to the bovine BCKDH-E1 beta and to the E1 beta subunit of human pyruvate dehydrogenase complex, in all regions. The structures and functions of mammalian alpha-ketoacid dehydrogenase complexes are apparently highly conserved. Genomic DNA from lymphoblastoid cell lines derived from normal and five MSUD patients, in whom E1 beta was not detected by immunoblot analysis, gave the same restriction maps on Southern blot analysis. The gene has at least 80 kbp. Images PMID:2365818

  6. Isolation of cDNA clones coding for the alpha and beta chains of human propionyl-CoA carboxylase: chromosomal assignments and DNA polymorphisms associated with PCCA and PCCB genes.

    PubMed Central

    Lamhonwah, A M; Barankiewicz, T J; Willard, H F; Mahuran, D J; Quan, F; Gravel, R A

    1986-01-01

    Propionyl-CoA carboxylase [PCC, propanoyl-CoA:carbon-dioxide ligase (ADP-forming), EC 6.4.1.3] is a biotin-dependent enzyme involved in the degradation of branched-chain amino acids, fatty acids with odd-numbered chain lengths, and other metabolites. Inherited deficiency of the enzyme results in propionic acidemia, an autosomal recessive disorder showing considerable clinical heterogeneity. To facilitate investigations of enzyme structure and the nature of mutation in propionic acidemia, we have isolated cDNA clones coding for the alpha and beta polypeptides of human PCC. Sequences of two peptides derived from human liver PCC were used to specify oligonucleotide probes that were then used to screen a human fibroblast cDNA library. Two classes of cDNA clones were thus identified. One class contained the anticipated Ala-Met-Lys-Met sequence, corresponding to the biotin binding site found in several biotin-dependent carboxylases, thus confirming the alpha-chain assignment of these clones. In addition, they contained the deduced amino acid sequence of two of the sequenced peptides, including that of one of the oligonucleotide probes. The second class, coding for the beta polypeptide, contained the sequences of four peptides, including the sequence corresponding to the other oligonucleotide probe. Blot hybridization of RNA from normal human fibroblasts revealed a single mRNA species of 2.9 kilobases coding for the alpha polypeptide and two species of 4.5 and 2.0 kilobases detected for the beta polypeptide. By use of a panel of somatic mouse-human hybrids, the human gene encoding the alpha polypeptide (PCCA) was localized to chromosome 13, while the gene encoding the beta polypeptide (PCCB) was assigned to chromosome 3. Restriction fragment length polymorphisms were identified, at both PCCA and PCCB, that should prove useful to individual families at risk for propionic acidemia. Images PMID:3460076

  7. MicroRNA-27a Regulates Beta Cardiac Myosin Heavy Chain Gene Expression by Targeting Thyroid Hormone Receptor β1 in Neonatal Rat Ventricular Myocytes▿

    PubMed Central

    Nishi, Hitoo; Ono, Koh; Horie, Takahiro; Nagao, Kazuya; Kinoshita, Minako; Kuwabara, Yasuhide; Watanabe, Shin; Takaya, Tomohide; Tamaki, Yodo; Takanabe-Mori, Rieko; Wada, Hiromichi; Hasegawa, Koji; Iwanaga, Yoshitaka; Kawamura, Teruhisa; Kita, Toru; Kimura, Takeshi

    2011-01-01

    MicroRNAs (miRNAs), small noncoding RNAs, are negative regulators of gene expression and play important roles in gene regulation in the heart. To examine the role of miRNAs in the expression of the two isoforms of the cardiac myosin heavy chain (MHC) gene, α- and β-MHC, which regulate cardiac contractility, endogenous miRNAs were downregulated in neonatal rat ventricular myocytes (NRVMs) using lentivirus-mediated small interfering RNA (siRNA) against Dicer, an essential enzyme for miRNA biosynthesis, and MHC expression levels were examined. As a result, Dicer siRNA could downregulate endogenous miRNAs simultaneously and the β-MHC gene but not α-MHC, which implied that specific miRNAs could upregulate the β-MHC gene. Among 19 selected miRNAs, miR-27a was found to most strongly upregulate the β-MHC gene but not α-MHC. Moreover, β-MHC protein was downregulated by silencing of endogenous miR-27a. Through a bioinformatics screening using TargetScan, we identified thyroid hormone receptor β1 (TRβ1), which negatively regulates β-MHC transcription, as a target of miR-27a. Moreover, miR-27a was demonstrated to modulate β-MHC gene regulation via thyroid hormone signaling and to be upregulated during the differentiation of mouse embryonic stem (ES) cells or in hypertrophic hearts in association with β-MHC gene upregulation. These findings suggested that miR-27a regulates β-MHC gene expression by targeting TRβ1 in cardiomyocytes. PMID:21149577

  8. Complete nucleotide sequence of primitive vertebrate immunoglobulin light chain genes.

    PubMed Central

    Shamblott, M J; Litman, G W

    1989-01-01

    Antibody to Heterodontus francisci (horned shark) immunoglobulin light chain was used to screen a spleen cDNA expression library, and recombinant clones encoding light chain genes were isolated. The complete sequences of the mature coding regions of two light chain genes in this phylogenetically distant vertebrate have been determined and are reported here. Comparisons of the sequences are consistent with the presence of mammalian-like framework and complementarity-determining regions. The predicted amino acid sequences of the genes are more related to mammalian lambda than to kappa light chains. The nucleotide sequences of the genes are most related to mammalian T-cell antigen receptor beta chain. Heterodontus light chain genes may reflect characteristics of the common ancestor of immunoglobulin and T-cell antigen receptors before its evolutionary diversification. PMID:2499889

  9. Complete nucleotide sequence of primitive vertebrate immunoglobulin light chain genes.

    PubMed

    Shamblott, M J; Litman, G W

    1989-06-01

    Antibody to Heterodontus francisci (horned shark) immunoglobulin light chain was used to screen a spleen cDNA expression library, and recombinant clones encoding light chain genes were isolated. The complete sequences of the mature coding regions of two light chain genes in this phylogenetically distant vertebrate have been determined and are reported here. Comparisons of the sequences are consistent with the presence of mammalian-like framework and complementarity-determining regions. The predicted amino acid sequences of the genes are more related to mammalian lambda than to kappa light chains. The nucleotide sequences of the genes are most related to mammalian T-cell antigen receptor beta chain. Heterodontus light chain genes may reflect characteristics of the common ancestor of immunoglobulin and T-cell antigen receptors before its evolutionary diversification.

  10. Energetic, Structural, and Antimicrobial Analyses of [beta]-Lactam Side Chain Recognition by [beta]-Lactamases

    SciTech Connect

    Caselli, E.; Powers, R.A.; Blaszczak, L.C.; Wu, C.Y.E.; Prati, F.; Shoichet, B.K.

    2010-03-05

    Penicillins and cephalosporins are among the most widely used and successful antibiotics. The emergence of resistance to these {beta}-lactams, most often through bacterial expression of {beta}-lactamases, threatens public health. To understand how {beta}-lactamases recognize their substrates, it would be helpful to know their binding energies. Unfortunately, these have been difficult to measure because {beta}-lactams form covalent adducts with {beta}-lactamases. This has complicated functional analyses and inhibitor design. To investigate the contribution to interaction energy of the key amide (R1) side chain of {beta}-lactam antibiotics, eight acylglycineboronic acids that bear the side chains of characteristic penicillins and cephalosporins, as well as four other analogs, were synthesized. These transition-state analogs form reversible adducts with serine {beta}-lactamases. Therefore, binding energies can be calculated directly from K{sub i} values. The K{sub i} values measured span four orders of magnitude against the Group I {beta}-lactamase AmpC and three orders of magnitude against the Group II {beta}-lactamase TEM-1. The acylglycineboronic acids have K{sub i} values as low as 20 nM against AmpC and as low as 390 nM against TEM-1. The inhibitors showed little activity against serine proteases, such as chymotrypsin. R1 side chains characteristic of {beta}-lactam inhibitors did not have better affinity for AmpC than did side chains characteristic of {beta}-lactam substrates. Two of the inhibitors reversed the resistance of pathogenic bacteria to {beta}-lactams in cell culture. Structures of two inhibitors in their complexes with AmpC were determined by X-ray crystallography to 1.90 {angstrom} and 1.75 {angstrom} resolution; these structures suggest interactions that are important to the affinity of the inhibitors. Acylglycineboronic acids allow us to begin to dissect interaction energies between {beta}-lactam side chains and {beta}-lactamases. Surprisingly

  11. Luteal expression of cytochrome P450 side-chain cleavage, steroidogenic acute regulatory protein, 3beta-hydroxysteroid dehydrogenase, and 20alpha-hydroxysteroid dehydrogenase genes in late pregnant rats: effect of luteinizing hormone and RU486.

    PubMed

    Stocco, C O; Chedrese, J; Deis, R P

    2001-10-01

    A decrease in serum progesterone at the end of pregnancy is essential for the induction of parturition in rats. We have previously demonstrated that LH participates in this process through: 1) inhibiting 3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity and 2) stimulating progesterone catabolism by inducing 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) activity. The objective of this investigation was to determine the effect of LH and progesterone on the luteal expression of the steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage (P450(scc)), 3beta-HSD, and 20alpha-HSD genes. Gene expression was analyzed by Northern blot analysis 24 and 48 h after administration of LH or vehicle on Day 19 of pregnancy. StAR and 3beta-HSD mRNA levels were lower in LH-treated rats than in rats administered with vehicle at both time points studied. P450(scc) mRNA levels were unaffected by LH. The 20alpha-HSD mRNA levels were not different between LH and control rats 24 h after treatment; however, greater expression of 20alpha-HSD, with respect to controls, was observed in LH-treated rats 48 h after treatment. Luteal progesterone content dropped in LH-treated rats at both time points studied, whereas serum progesterone decreased after 48 h only. In a second set of experiments, the anti-progesterone RU486 was injected intrabursally on Day 20 of pregnancy. RU486 had no effect on 3beta-HSD or P450(scc) expression but increased 20alpha-HSD mRNA levels after 8 h treatment. In conclusion, the luteolytic effect of LH is mediated by a drop in StAR and 3beta-HSD expression without effect on P450(scc) expression. We also provide the first in vivo evidence indicating that a decrease in luteal progesterone content may be an essential step toward the induction of 20alpha-HSD expression at the end of pregnancy in rats.

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

    PubMed

    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.

  13. Characterization of the beta2-microglobulin gene of the horse.

    PubMed

    Tallmadge, Rebecca L; Lear, Teri L; Johnson, Amanda K; Guérin, Gérard; Millon, Lee V; Carpenter, Susan L; Antczak, Douglas F

    2003-01-01

    A clone containing beta(2)-microglobulin (beta(2)-m), the light chain of the major histocompatibility complex class I cell surface molecule, was isolated from an equine bacterial artificial chromosome library. This clone was used as a template for polymerase chain reaction (PCR) and unidirectional sequencing to elucidate the genomic sequence and intron/exon boundaries. We obtained 7,000 bases of sequence, extending from 1,100 nucleotides (nt) upstream of the coding region start through 1,698 nt downstream of the stop codon. The sequence contained regulatory elements in the region upstream of the coding sequence similar to those of the beta(2)-m gene of other species. The beta(2)-m gene was localized to horse chromosome ECA1q23-q25 by fluorescent in situ hybridization. This was confirmed by synteny mapping on a (horse x mouse) somatic cell hybrid panel. The sequence and intron/exon boundaries determined were used to design PCR primers to amplify and sequence the coding region of the beta(2)-m gene in other equids, including five breeds of domestic horse, one Przewalski's horse, five domestic donkeys and five zebras. A high degree of conservation was found among equids, illustrated by >98% (349/354) identity at the nucleotide level and 95% (113/118) at the amino acid level, because of non-synonymous nucleotide substitutions. The promoter detected in the region upstream of the coding sequence was subcloned and used in chloramphenicol acetyl transferase (CAT) assays to demonstrate the presence of a functional promoter. This study provides tools for the analysis of regulation of not only the horse beta(2)-m gene, but also for any genes dependent upon beta(2)-m for expression.

  14. Bone marrow and peripheral blood globin chain synthesis in sickle cell beta zero thalassaemia.

    PubMed Central

    Costa, F F; Zago, M A

    1986-01-01

    A similar imbalance of globin chain synthesis, with low non-alpha/alpha ratios, was shown in peripheral blood and in bone marrow of compound heterozygotes for both the Hb S and beta zero thalassaemia genes (S/beta zero thalassaemia). Previous purification of whole cell globin obtained from the bone marrow did not change the non-alpha/alpha ratio. The mean non-alpha/alpha ratios were 0.57 +/- 0.13 (means +/- SD) for the peripheral blood of 12 patients, 0.52 +/- 0.10 for five patients using bone marrow globin purified on Sephadex G100, and 0.55 +/- 0.16 for the unfiltered bone marrow globin of five patients. The data show that patients with S/beta zero thalassaemia have a similar beta chain deficiency in reticulocytes and in bone marrow cells, provided whole cell globin is used which avoids the removal of the free alpha chains. The non-alpha/alpha ratios in the peripheral blood of an S/beta zero thalassaemia patient and a beta thalassaemia heterozygote from the same family were compared in seven families and no significant difference was found. PMID:3723554

  15. Surface expression of the beta T cell receptor (TCR) chain in the absence of other TCR or CD3 proteins on immature T cells.

    PubMed Central

    Kishi, H; Borgulya, P; Scott, B; Karjalainen, K; Traunecker, A; Kaufman, J; von Boehmer, H

    1991-01-01

    T cell receptor (TCR) beta genes are rearranged prior to TCR alpha genes. A productively rearranged TCR beta gene suppresses further V beta gene rearrangement. Here we show that in beta TCR transgenic mice the TCR beta-chain can be expressed on the surface of immature CD4-8- thymocytes, but not on mature T cells, in the absence of any other known TCR chain and proteins of the CD3 complex. Analysis by NEPHGE and SDS-PAGE showed that at least some beta TCR exists on the surface as a large disulfide-linked complex with unknown acidic molecules. The introduction of the beta TCR gene into scid mice resulted in the expression of the beta TCR on the cell surface of thymocytes and induced the expression of CD4 and CD8 co-receptors as well as transcription of the alpha TCR locus. Images PMID:1703490

  16. The C-terminal region of laminin beta chains modulates the integrin binding affinities of laminins.

    PubMed

    Taniguchi, Yukimasa; Ido, Hiroyuki; Sanzen, Noriko; Hayashi, Maria; Sato-Nishiuchi, Ryoko; Futaki, Sugiko; Sekiguchi, Kiyotoshi

    2009-03-20

    Laminins are major cell-adhesive proteins in basement membranes that are capable of binding to integrins. Laminins consist of three chains (alpha, beta, and gamma), in which three laminin globular modules in the alpha chain and the Glu residue in the C-terminal tail of the gamma chain have been shown to be prerequisites for binding to integrins. However, it remains unknown whether any part of the beta chain is involved in laminin-integrin interactions. We compared the binding affinities of pairs of laminin isoforms containing the beta1 or beta2 chain toward a panel of laminin-binding integrins, and we found that beta2 chain-containing laminins (beta2-laminins) bound more avidly to alpha3beta1 and alpha7X2beta1 integrins than beta1 chain-containing laminins (beta1-laminins), whereas alpha6beta1, alpha6beta4, and alpha7X1beta1 integrins did not show any preference toward beta2-laminins. Because alpha3beta1 contains the "X2-type" variable region in the alpha3 subunit and alpha6beta1 and alpha6beta4 contain the "X1-type" region in the alpha6 subunit, we hypothesized that only integrins containing the X2-type region were capable of discriminating between beta1-laminins and beta2-laminins. In support of this possibility, a putative X2-type variant of alpha6beta1 was produced and found to bind preferentially to beta2-laminins. Production of a series of swap mutants between the beta1 and beta2 chains revealed that the C-terminal 20 amino acids in the coiled-coil domain were responsible for the enhanced integrin binding by beta2-laminins. Taken together, the results provide evidence that the C-terminal region of beta chains is involved in laminin recognition by integrins and modulates the binding affinities of laminins toward X2-type integrins.

  17. Localization of a site on bacterial superantigens that determines T cell receptor beta chain specificity

    PubMed Central

    1993-01-01

    A defining characteristic of superantigens is their ability to stimulate T cells based predominantly on the type of variable segment of the T cell receptor (TCR) beta chain (V beta). The V beta specificity of these toxins most likely results from direct contact between the toxin and the TCR, although the low affinity nature of this binding has prevented direct assessment of this interaction. To identify important functional sites on the toxin, we created chimeric enterotoxin genes between staphylococcal enterotoxins A and E (SEA and SEE) and tested the V beta specificity of the chimeric toxins. This approach allowed us to identify three amino acid residues in the extreme COOH terminus of these toxins that are largely responsible for their ability to stimulate either human V beta 5- or V beta 8-bearing T cells, or mouse V beta 3 or V beta 11. We also found that residues in the NH2 terminus were required for wild-type levels of V beta-specific T cell activation, suggesting that the NH2 and COOH ends of these superantigens may come together to form the full TCR V beta contact site. SEA and SEE also differ with respect to their class II binding characteristics. Using the same chimeric molecules, we demonstrate that the first third of the molecule controls the class II binding phenotype. These data lead us to propose that for SEA and SEE, and perhaps for all bacterial-derived superantigens, the COOH and NH2 termini together form the contact sites for the TCR and therefore largely determine the V beta specificity of the toxin, while the NH2 terminus alone binds major histocompatibility complex class II molecules. The predominant role of the COOH terminus of bacterial superantigens in determining V beta specificity resembles current models being proposed for virally encoded superantigens, suggesting that these molecules may demonstrate some structural relationship not seen at the amino acid level. PMID:7678849

  18. Developmental Modulation of a beta myosin heavy chain promoter-driven transgene.

    PubMed

    Knotts, S; Sánchez, A; Rindt, H; Robbins, J

    1996-06-01

    The molecular mechanisms underlying heart and skeletal muscle-specific gene expression during development and in response to physioloic stimuli are largely unknown. Using a novel immunohistochemical procedure to detect chloramphenicol acetyltransferase (CAT), we have investigated, in vivo at high resolution, the ability of cis-acting DNA sequences within the 5' flanking region of the mouse beta myosin heavy chain (MyHC) gene (beta-MyHC) to direct appropriate gene expression throughout development. A 5.6-kb fragment 5' to the beta-MyHC's transcriptional start site was linked to the reporter gene encoding CAT (cat) and used to generate transgenic mice. The anti-CAT in situ assay described in this report allowed us to define the ability of the promoter fragment to direct appropriate temporal, tissue- and muscle fiber type-specific gene expression throughout early development. In skeletal muscles, the transgene expression profile mimics the endogenous beta-myHC's at all developmental stages and is appropriately restricted to slow (type I) skeletal fibers in the adult. Surprisingly, transgene expression was detected in both the atria and ventricles during embryonic and fetal development, indicating that ventricular specification involves elements outside the 5.6-kb fragment. In contrast, in the adult, hypothyroid conditions led to transgene induction specifically in the ventricles, suggesting that distinct regulatory mechanisms control fetal versus adult beta-MyHC expression in the cardiac compartment.

  19. First molecular cloning and gene expression analysis of teleost CD42 (glycoprotein Ib beta chain) GPIb-IX-V subunit from rock bream, Oplegnathus fasciatus.

    PubMed

    Jeong, Ji-Min; Kim, Ju-Won; Kim, Do-Hyung; Park, Chan-Il

    2015-04-01

    CD42 is a platelet membrane glycoprotein Ib that plays a key role in haemostasis and thrombin-induced platelet activation. Here, we report the molecular cloning and sequence analysis of the CD42c gene from rock bream (Oplegnathus fasciatus). Rock bream CD42 (RbCD42c) gene expression profiles were determined after infection with Streptococcus iniae, Edwardsiella tarda and red seabream iridovirus (RSIV). The full-length RbCD42c cDNA contained an open reading frame of 624 bp encoding 207 amino acids. The deduced amino acid sequences of the leucine-rich repeat (LRR)-N terminal and LRR-C terminal were conserved between fish and mammals. RbCD42c was highly expressed in red blood cells, spleen, gill, liver and kidney of healthy rock bream. The RbCD42c gene was not significantly up- or downregulated after E. tarda exposure. However, RbCD42c gene expression was upregulated in kidney, spleen and gill after S. iniae infection. RbCD42c was upregulated in spleen, liver and gill, but downregulated in kidney 24 and 48 h after RSIV infection. These results suggest that RbCD42c has different expression patterns after infection with bacterial or viral pathogens. This gene may be directly involved in haemostasis.

  20. Modeling of polypeptide chains as C alpha chains, C alpha chains with C beta, and C alpha chains with ellipsoidal lateral chains.

    PubMed Central

    Fogolari, F; Esposito, G; Viglino, P; Cattarinussi, S

    1996-01-01

    In an effort to reduce the number of degrees of freedom necessary to describe a polypeptide chain we analyze the statistical behavior of polypeptide chains when represented as C alpha chains, C alpha chains with C beta atoms attached, and C alpha chains with rotational ellipsoids as models of side chains. A statistical analysis on a restricted data set of 75 unrelated protein structures is performed. The comparison of the database distributions with those obtained by model calculation on very short polypeptide stretches allows the dissection of local versus nonlocal features of the distributions. The database distribution of the bend angles of polypeptide chains of pseudo bonded C alpha atoms spans a restricted range of values and shows a bimodal structure. On the other hand, the torsion angles of the C alpha chain may assume almost all possible values. The distribution is bimodal, but with a much broader probability distribution than for bend angles. The C alpha - C beta vectors may be taken as representative of the orientation of the lateral chain, as the direction of the bond is close to the direction of the vector joining C alpha to the ad hoc defined center of the "steric mass" of the side chain. Interestingly, both the bend angle defined by C alpha i-C alpha i+1-C beta i+1 and the torsional angle offset of the pseudo-dihedral C alpha i-C alpha i+1-C alpha i+2-C beta i+2 with respect to C alpha i-C alpha i+1-C alpha i+2-C alpha i+3 span a limited range of values. The latter results show that it is possible to give a more realistic representation of polypeptide chains without introducing additional degrees of freedom, i.e., by just adding to the C alpha chain a C beta with given side-chain properties. However, a more realistic description of side chains may be attained by modeling side chains as rotational ellipsoids that have roughly the same orientation and steric hindrance. To this end, we define the steric mass of an atom as proportional to its van der

  1. V(D)J recombination and allelic exclusion of a TCR {beta}-chain minilocus occurs in the absence of a functional promoter

    SciTech Connect

    Alvarez, J.D.; Anderson, S.J.; Loh, D.Y.

    1995-08-01

    Transcriptional activation of rearranging Ag receptor gene segments has been hypothesized to regulate their accessibility to V(D)J recombination. We analyzed the role of a functional promoter in the rearrangement of the murine TCR {beta}-chain locus using two transgenic minilocus constructs. These miniloci each contain an unrearranged V{beta}8.3 gene. One has a wild-type V{beta}8.3 gene, but the other has a V{beta}8.3 gene with a promoter mutation that was previously shown to abrogate transcription in tissue culture. FACS analysis of thymus and lymph node cells from transgenic mouse lines showed that only the lines with the wild-type V{beta}8.3 gene promoter express an 8.3 TCR {beta}-chain. Consistent with the protein expression data, V{beta}8.3 gene transcripts were found only in the transgenic lines with the wild-type promoter. Using a quantitative PCR-based assay, it was shown that both types of transgenic lines recombine the V{beta}8.3 gene at similar levels. Rearrangement of the transgenes was normal with respect to thymic development and junctional reading frame. Interestingly, both types of miniloci also underwent allelic exclusion in that recombination was blocked by the expression of a rearranged TCR {beta}-chain transgene. We conclude that a functional V{beta} gene promoter is not necessary for proper V(D)J recombination to occur.

  2. Hemoglobinopathies among the Gond tribal groups of central India; interaction of alpha- and beta-thalassemia with beta chain variants.

    PubMed

    Gupta, R B; Tiwary, R S; Pande, P L; Kutlar, F; Oner, C; Oner, R; Huisman, T H

    1991-01-01

    We have investigated the frequencies and types of alpha-thal, beta-thal, and Hb variants among nearly 200 inhabitants of villages in the Mandla and Jabalpur districts of Madhya Pradesh in Central India. Over 85% were tribals of the Gond group. alpha-Thal, as -alpha 3.7/and -alpha 4.2/, and the nondeletional Koya Dora mutation were present at the combined frequency of 0.54. There were indications for the presence of other nondeletional types of alpha-thal. alpha-Globin gene triplications were not observed. Four of the six beta-thal alleles observed were in the tribal groups; two (G----C at codon 30 and G----A at IVS-I-1) were found for the first time. The simultaneous presence of an alpha-thal (-alpha/alpha alpha or -alpha/-alpha) greatly improved the clinical and hematological condition of the patients with Hb S-beta(+)-thal (IVS-I-5; G----C). The lower frequency of alpha-thal among the beta-thal heterozygotes (f = 0.32) may indicate that some of the beta-thal alleles in the tribal populations originated from an outside source. Forty-one subjects had SS; all but one had beta S with haplotype #31, while one chromosome had haplotype #17. The presence of an alpha-thal-2 (f = 0.53) in the SS patients did not affect hematological data. The Hb F levels varied between 7.5% and 42.5% with high G gamma values. No difference in Hb F level between males and females was observed. Lower Hb F levels were present in 10 SS patients with an alpha-thal-2 homozygosity (average 16% versus 23.5% for eight SS patients with alpha alpha/alpha alpha) suggesting a decreased formation of alpha gamma dimers in severe alpha chain deficiency. Several younger SS patients (less than 10 years) also had high Hb F levels (32-42%). Variations in the sequence at -530 of the beta-globin gene; i.e. in the so-called silencer sequence, were present in all beta S chromosomes with haplotype #31, but were not considered important for understanding the variability in the Hb F level. gamma-Globin gene

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

    SciTech Connect

    Sleasman, J.W.; Harville, T.O.; White, G.B.; Barrett, D.J. ); George, J.F. ); Goodenow, M.M. Univ. of Alabama, Birmingham, AL )

    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.

  4. Tissue distribution of the laminin beta1 and beta2 chain during embryonic and fetal human development.

    PubMed

    Roediger, Matthias; Miosge, Nicolai; Gersdorff, Nikolaus

    2010-04-01

    Laminins are the major glycoproteins present in all basement membranes. Previously, we showed that perlecan is present during human development. Although an overview of mRNA-expression of the laminin beta1 and beta2 chains in various developing fetal organs is already available, a systematic localization of the laminin beta1 and beta2 chains on the protein level during embryonic and fetal human development is missing. Therefore, we studied the immunohistochemical expression and tissue distribution of the laminin beta1 and beta2 chains in various developing embryonic and fetal human organs between gestational weeks 8 and 12. The laminin beta1 chain was ubiquitously expressed in the basement membrane zones of the brain, ganglia, blood vessels, liver, kidney, skin, pancreas, intestine, heart and skeletal system. Furthermore, the laminin beta2 chain was present in the basement membrane zones of the brain, ganglia, skin, heart and skeletal system. The findings of this study support and expand upon the theory that these two laminin chains are important during human development.

  5. HLA-DQ beta chain can present mouse endogenous provirus MTV-9 product and clonally delete Tcr V beta 5+ and V beta 11+ T cells in transgenic mice.

    PubMed

    Zhou, P; Smart, M K; Cheng, S; Savarirayan, S; Inoko, H; David, C S

    1992-01-01

    The elusive Mls gene(s) are mouse mammary tumor virus genes. The endogenous cotolerogen involved in the clonal deletion of Tcr V beta 5.1, 5.2, and 11 in H-2E+ mouse strains has been narrowed down to MTV-9. We demonstrate that similar to H-2E alpha molecules, human DQw6 beta chain mediated clonal deletion of Tcr V beta 5.1, 5.2, and 11 also requires the MTV-9 gene product. This shows that human class II molecules can present mouse retroviral antigen. Further, backcross analysis involving [B10.M(DQb) x DBA/1] suggest a second cotolerogen in the B10.M background in the clonal deletion of V beta 5-bearing T cells.

  6. Haemoglobins of the shark, Heterodontus portusjacksoni. III. Amino acid sequence of the beta-chain.

    PubMed

    Fisher, W K; Nash, A R; Thompson, E O

    1977-12-01

    The amino acid sequence of the beta-chain of the principal haemoglobin from the shark H. portusjacksoni has been determined. The chain has 141 residues, the same as that of mammalian alpha-chains and less than the 146 residues of mammalian beta-chains or the 148 residues of the alpha-chain from the tetrameric shark haemoglobin. The sequence was deduced from the sequences of peptides obtained by digestion of the globin or its cyanogen bromide fragments with trypsin, chymotrypsin, pepsin and papain. The difference in length of the beta-chain is most readily accounted for by the absence of the D helix. This small helical section is normally present in myoglobins and beta-globins but absent in alpha-chains. The deduction that it is absent from shark beta-chain is based on consideration of homology. The beta-chain shows the insertion of histidine beta2 and the deletions corresponding to residues A17 and AB1 relative to alpha-and myoglobin chains. The reactive thiol group in shark haemoglobin was shown by radioactive labelling to be residue 51 in the beta-chain, immediately preceding the E helix. The amino acid sequence of shark beta-chain shows 92 differences from human beta-chain, significantly more differences than shown by chicken or frog beta-chains, in line with its earlier time of divergence. If the tertiary structure of the shark beta-chain is the same as that of the horse then there are two changes in the alpha1beta2 contact site in oxyhaemoglobin and an additional one in deoxyhaemoglobin. When both alpha- and beta-chain contacts are considered there is a total of nine changes in residues involved in the alpha1beta2 contacts. There is no Bohr effect in shark haemoglobin, and of the residues normally involved in this effect the C-terminal histidine residue of the beta-chain is present, but the aspartyl (FG1) residue to which it is salt-linked is not, being replaced by a glutamyl residue.

  7. Beta globin gene cluster haplotypes of the beta thalassemia mutations observed in Denizli province of Turkey.

    PubMed

    Bahadır, Anzel; Öztürk, Onur; Atalay, Ayfer; Atalay, Erol Ömer

    2009-09-05

    Our aim is to identify the beta globin gene cluster haplotypes for the beta thalassemia mutations in Turkey at regional basis. Beta thalassemia mutations included in this study were IVS-I-110 (G>A), FSC 8/9 (+G), IVS-II-1 (G>A), IVS-I-5 (G>C), IVS-I-1 (G>A), IVS-I-6 (T>C) and FSC 8 (-AA). We studied 22 unrelated patients with β-thalassemia major and 72 unrelated healthy subjects from our Department's DNA bank. Haplotype analysis was done by polymerase chain reaction (PCR)-based restriction enzyme digestion for the beta globin gene cluster of the following polymorphic restriction sites: Hinc II 5' to ε, Hind III 5' to Gγ, Hind III in the IVS-II 5' to Aγ, Hinc II in pseudo β, Hinc II 3' to pseudo β, Ava II in β, Hinf I 3' to β. Associated haplotypes for the normal control samples (72 individuals, 144 chromosomes) were determined by Arlequin 3.1 software with unknown gametic phase. According to the results obtained, the most frequent beta globin gene cluster haplotypes in the normal population are (+----++), (+----+-), (-+-++++), (+-----+) with the frequencies of 28.6 %, 17.2 %, 9.8 % and 8.3 % respectively. IVS-I-110 mutation is linked with the haplotypes (+----++) and (+-----+). Observed haplotypes are (+----++) for FSC 8/9 (+G), (-+-+++-) for IVS-II-1 (G>A), (-+-++-+ and -+-++++) for IVS-I-5 (G>C), (+----+- and +------) for IVS-I-1 (G>A), (-++---+) for IVS-I-6 (T>C) and (+-----+) for FSC 8 (-AA). In conclusion, our region shows the Mediterranean character for the beta thalassemia mutations. According to the obtained results, IVS-I-110 (G>A) mutation linked with haplotype VII (+-----+), IVS-I-5 (G>C) mutation with haplotype IV (-+-++-+), codon 8/9 (+G) linked with haplotype I (+----++) were shown for the first time in Turkish population. The linkage of haplotype (+------) with the IVS-I-1 (G>A) mutation is reported for the first time in the published literature. In Denizli province of Turkey, beta globin gene cluster haplotypes of the normal population are

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

    PubMed

    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.

  9. Two monoclonal rat antibodies with specificity for the beta-chain variable region V beta 6 of the murine T-cell receptor.

    PubMed Central

    Payne, J; Huber, B T; Cannon, N A; Schneider, R; Schilham, M W; Acha-Orbea, H; MacDonald, H R; Hengartner, H

    1988-01-01

    Two rat monoclonal antibodies (mAbs), 44-22-1 and 46-6B5, which recognize an alloreactive cytotoxic clone, 3F9, have been further tested on a panel of T hybridomas and cytotoxic T-cell clones for binding and functional activities. The mAbs recognized only those cells sharing the expression of the T-cell receptor beta-chain variable region gene V beta 6 with 3F9. All V beta 6+ cells were activated by these mAbs under cross-linking conditions and their antigen-specific activation was blocked by soluble mAb. Furthermore, depletion of 46-6B5+ normal lymph node T cells eliminated all cells expressing the epitope recognized by 44-22-1 and V beta 6 mRNA. Images PMID:2459713

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

    SciTech Connect

    Patrick, N.; Miyakawa, F.; Hunt, J.A.

    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].

  11. Proliferation of thymocytes in relation to T-cell receptor beta-chain expression.

    PubMed

    Parkin, I G; Owen, J J; Jenkinson, E J

    1988-05-01

    During proliferation and differentiation of maturing thymocytes, T-cell receptor beta-chain products are first expressed in the cytoplasm. Only subsequently are they expressed on the cell surface, presumably as part of the alpha beta/CD3 receptor complex. This study uses double immunofluorescence labelling to identify these cytoplasmic and surface phases separately in relationship to cell-cycle parameters. The use of a mitotic arrest agent and tritiated thymidine autoradiography both show that cells with cytoplasmic beta-chains are in cell cycle, whereas cells with surface beta-chains are cycling slowly, if at all.

  12. Expression of 3beta-HSD and P5betaR, genes respectively coding for Delta5-3beta-hydroxysteroid dehydrogenase and progesterone 5beta-reductase, in leaves and cell cultures of Digitalis lanata EHRH.

    PubMed

    Ernst, Mona; de Padua, Rodrigo Maia; Herl, Vanessa; Müller-Uri, Frieder; Kreis, Wolfgang

    2010-06-01

    Plants of the genus Digitalis produce 5 beta-cardenolides that are used in the therapy of cardiac insufficiency in humans. 3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) and progesterone 5 beta-reductase (P5 betaR) are both supposed to be important enzymes in the biosynthesis of these natural products. Activity and gene expression were demonstrated for both enzymes in cardenolide-accumulating leaves of Digitalis lanata but also in cardenolide-free permanent cell suspension cultures initiated from D. lanata leaf tissue. Enzyme activities were determined and quantified by HPLC and GC-MS methods. Expression of the respective genes, namely AY585867.1 (P5betaR gene) and DQ466890.1 (3beta-HSD gene), was made evident by real-time polymerase chain reaction (qPCR) analysis. We demonstrate for the first time that the P5betaR gene, encoding an enzyme described as a key enzyme in cardenolide biosynthesis, is also expressed in cardenolide-free tissues of cardenolide-containing plants.

  13. Domains of the TCR beta-chain required for early thymocyte development

    PubMed Central

    1996-01-01

    The T cell receptor beta (TCR beta) chain controls the developmental transition from CD4-CD8- to CD4+8+thymocytes. We show that the extracellular constant region and the transmembrane region, but not the variable domain or cytoplasmic tail of the TCR beta chain are required for this differentiation step. TCR beta mutant chains lacking the cytoplasmic tail can be found at the cell surface both in functional TCR/CD3 complexes and in a GPI-anchored monomeric form indicating that the cytoplasmic tail of the TCR beta chain functions as an ER retention signal. The concordance between cell surface expression of the mutant chains as TCR/CD3 complexes and their capacity to mediate thymocyte differentiation supports the CD3 mediated feedback model in which preTCR/CD3 complexes control the developmental transition from CD4-CD8- to CD4+CD8+thymocytes. PMID:8920871

  14. Aspartic acid at position 57 of the HLA-DQ beta chain protects against type I diabetes: a family study.

    PubMed Central

    Morel, P A; Dorman, J S; Todd, J A; McDevitt, H O; Trucco, M

    1988-01-01

    One hundred seventy-two members from 27 randomly selected multiple case Caucasian families of patients with insulin-dependent diabetes mellitus (IDDM) were studied at the DNA level to ascertain the reliability of codon 57 of the HLA-DQ beta-chain gene as a disease protection/susceptibility marker. The analysis was carried out by polymerase chain reaction amplification of DNA encoding the first domain of the DQ beta chain and by dot blot analysis of the amplified material with allele-specific oligonucleotide probes. One hundred twenty-three randomly selected healthy Caucasian donors were also tested. The results demonstrated that haplotypes carrying an aspartic acid in position 57 (Asp-57) of their DQ beta chain were significantly increased in frequency among nondiabetic haplotypes (23/38), while non-Asp-57 haplotypes were significantly increased in frequency among diabetic haplotypes (65/69). Ninety-six percent of the diabetic probands in our study were homozygous non-Asp/non-Asp as compared to 19.5% of healthy unrelated controls. This conferred a relative risk of 107 (chi 2 = 54.97; P = 0.00003) for non-Asp-57 homozygous individuals. Even though the inheritance and genetic features of IDDM are complex and are not necessarily fully explained by DQ beta chain polymorphism, this approach is much more sensitive than HLA serolog in assessing risk for IDDM. PMID:3186714

  15. Retroviral transfer of a human beta-globin/delta-globin hybrid gene linked to beta locus control region hypersensitive site 2 aimed at the gene therapy of sickle cell disease.

    PubMed Central

    Takekoshi, K J; Oh, Y H; Westerman, K W; London, I M; Leboulch, P

    1995-01-01

    Human gamma-globin and delta-globin chains have been previously identified as strong inhibitors of the polymerization of hemoglobin S, in contrast to the beta-globin chain, which exerts only a moderate antisickling effect. However, gamma-globin and delta-globin are normally expressed at very low levels in adult erythroid cells, in contrast to beta-globin. We report the design of a beta-globin/delta-globin hybrid gene, beta/delta-sickle cell inhibitor 1 (beta/delta-SCI1) and its transduction by retrovirus-mediated gene transfer. The beta/delta-SCI1-encoding gene retains the overall structure of the human beta-globin gene, while incorporating specific amino acid residues from the delta chain previously found responsible for its enhanced antisickling properties. To achieve high expression levels of beta/delta-SCI1 in adult erythrocytes, the hybrid gene was placed under the transcriptional control of the human beta-globin promoter and the DNase I hypersensitive site 2 of the human beta locus control region. High-titer retroviruses were generated, and stable proviral transmission was achieved in infected cells. The mRNA expression levels of the beta/delta-SCI1 gene in infected, dimethyl sulfoxide-induced murine erythroleukemia cells approached 85% of the endogenous murine beta maj-globin mRNA, on a per gene basis, evidence that high gene expression levels were achieved in adult erythroid cells. Further evaluation of this strategy in transgenic animal models of sickle cell disease should assess its efficacy for the gene therapy of human patients. Images Fig. 4 Fig. 5 PMID:7708766

  16. Crystal Structures of T Cell Receptor (Beta) Chains Related to Rheumatoid Arthritis

    SciTech Connect

    Li,H.; van Vranken, S.; Zhao, Y.; Li, Z.; Guo, Y.; Eisele, L.; Li, Y.

    2005-01-01

    The crystal structures of the V{beta}17+ {beta} chains of two human T cell receptors (TCRs), originally derived from the synovial fluid (SF4) and tissue (C5-1) of a patient with rheumatoid arthritis (RA), have been determined in native (SF4) and mutant (C5-1{sub F104{yields}Y/C187{yields}S}) forms, respectively. These TCR {beta} chains form homo-dimers in solution and in crystals. Structural comparison reveals that the main-chain conformations in the CDR regions of the C5-1 and SF4 V{beta}17 closely resemble those of a V{beta}17 JM22 in a bound form; however, the CDR3 region shows different conformations among these three V{beta}17 structures. At the side-chain level, conformational differences were observed at the CDR2 regions between our two ligand-free forms and the bound JM22 form. Other significant differences were observed at the V{beta} regions 8-12, 40-44, and 82-88 between C5-1/SF4 and JM22 V{beta}17, implying that there is considerable variability in the structures of very similar {beta} chains. Structural alignments also reveal a considerable variation in the V{beta}-C{beta} associations, and this may affect ligand recognition. The crystal structures also provide insights into the structure basis of T cell recognition of Mycoplasma arthritidis mitogen (MAM), a superantigen that may be implicated in the development of human RA. Structural comparisons of the V{beta} domains of known TCR structures indicate that there are significant similarities among V{beta} regions that are MAM-reactive, whereas there appear to be significant structural differences among those V{beta} regions that lack MAM-reactivity. It further reveals that CDR2 and framework region (FR) 3 are likely to account for the binding of TCR to MAM.

  17. Human globin gene analysis for a patient with beta-o/delta beta-thalassemia.

    PubMed Central

    Ottolenghi, S; Lanyon, W G; Williamson, R; Weatherall, D J; Clegg, J B; Pitcher, C S

    1975-01-01

    Complementary DNA (cDNA) was prepared with RNA-dependent DNA polymerase from human globin messenger RNA (mRNA). Annealing and translation experimenta with total mRNA from circulating cells from a patient with heterozygous beta/heterozygous beta-delta-o thalassemia (beta-o/delta beta-o-thalassemia) demonstrated no detectable mRNA for beta-globin. cDNA enriched in sequences homologous to beta-globin mRNA was prepared by hydroxylapatite fractionation of hybrids formed between beta-o/delta beta-o-thalassemic mRNA and cDNA made from mRNA from a patient with alpha-thalassemia (hemoglobin H disease). The rate of annealing of this beta-enriched cDNA to normal human nuclear DNA was that of a sequence present as only a single copy per haploid genome. The beta-enriched cDNA annealed to the beta-o-delta beta-o-thalassemia total DNA with approximately the same kinetics as to normal DNA, indicating that no total gene deletion of beta-globin genes from the diploid genome has occurred, although the accuracy of the technique could not exclude with certainty a partial deletion or a deletion of a beta-globin gene from only one of the haploid genomes. This demonstrates that at least one of the beta-o- or the delta beta-o-thalassemia haploid genomes in this case contains a substantially intact beta-globin gene. PMID:49057

  18. Human beta-globin gene expression in transgenic mice is enhanced by a distant DNase I hypersensitive site.

    PubMed Central

    Curtin, P T; Liu, D P; Liu, W; Chang, J C; Kan, Y W

    1989-01-01

    Several lines of evidence suggest that erythroid-specific DNase I hypersensitive sites (HS) located far upstream of the human beta-globin gene are important in regulating beta-globin gene expression. We used the polymerase chain reaction technique to amplify and clone an 882-base-pair DNA fragment spanning one of these HS, designated HSII, which is located 54 kilobases upstream of the beta-globin gene. The cloned HSII fragment was linked to a human beta-globin gene in either the genomic (HSII-beta) or antigenomic (HSII-beta) orientation. These two constructs and a beta-globin gene alone (beta) were injected into fertilized mouse eggs, and expression was analyzed in liver and brain from day-16 transgenic fetuses. Five of 7 beta-transgenic fetuses expressed human beta-globin mRNA, but the level of expression per gene copy was low, ranging from 0.93 to 22.4% of mouse alpha-globin mRNA (average 9.9%). In contrast, 11 of 12 HSII-beta transgenic fetuses expressed beta-globin mRNA at levels per gene copy ranging from 31.3 to 336.6% of mouse alpha-globin mRNA (average 139.5%). Only three fetuses containing intact copies of the HSII-beta construct were produced. Two of three expressed human beta-globin mRNA at levels per gene copy of 179.2 and 387.1%. Expression of human beta-globin mRNA was tissue-specific in all three types of transgenic fetuses. These studies demonstrate that a small DNA fragment containing a single erythroid-specific HS can stimulate high-level human beta-globin gene expression in transgenic mice. Images PMID:2780563

  19. The nucleotide sequence of the human beta-globin gene.

    PubMed

    Lawn, R M; Efstratiadis, A; O'Connell, C; Maniatis, T

    1980-10-01

    We report the complete nucleotide sequence of the human beta-globin gene. The purpose of this study is to obtain information necessary to study the evolutionary relationships between members of the human beta-like globin gene family and to provide the basis for comparing normal beta-globin genes with those obtained from the DNA of individuals with genetic defects in hemoglobin expression.

  20. Hemoglobinopathies in the Dogon Country: presence of beta S, beta C, and delta A' genes.

    PubMed

    Ducrocq, R; Bennani, M; Bellis, G; Baby, M; Traore, K; Nagel, R L; Krishnamoorthy, R; Chaventre, A

    1994-07-01

    The population of the Dogon, located in Mali, is divided in an endogamic Noble class and two endogamic servant castes (Tanners and Blacksmiths). We find that the polymorphic frequencies of beta c, beta S, and, unexpectedly, a mutation of the delta-chain (delta A'), are geographically (valley vs. plateau) as well as social status dependent.

  1. Coexpression of the platelet-derived growth factor (PDGF) B chain and the PDGF. beta. receptor in isolated pancreatic islet cells stimulates DNA synthesis

    SciTech Connect

    Welsh, M.; Hallberg, A.; Welsh, N.; Arkhammar, P.; Nilsson, T.; Berggren, P.O. ); Claesson-Welsh, L.; Heldin, C.H. ); Betsholtz, C. ); Berggren, P.O. )

    1990-08-01

    Suspensions rich in pancreatic {beta} cells were transfected by means of electroporation or by using the liposome technique with DNA constructs coding for the {beta} chain of platelet-derived growth factor (PDGF) and the PDGF {alpha} and {beta} receptors to induce a mitotic response in this slowly replicating cell type. Transfection with the B-chain construct induced synthesis of the PDGF B-chain homodimer (PDGF-BB) as assessed by the presence of {sup 125}I-labeled PDGF-BB competing activity in the conditioned medium of the transfected islet cells. Moreover, islet cells transfected with the PDGF {beta}-receptor construct exhibited increased immunofluorescence staining with a PDGF {beta}-receptor antibody. These cells also displayed increased {sup 125}I-labeled PDGF-BB binding compared with control transfected cells. The {beta} cells exhibited elevated levels of ({sup 3}H)inositol trisphosphate after transfection with the B-chain and {beta}-receptor constructs, indicating activation of phospholipase C. Islet cells transfected with the different receptor constructs exhibited different patterns of tyrosine phosphorylation upon ligand activation. The results demonstrate that pancreatic islet cells can be stimulated to increase DNA synthesis by transfection with the PDGF {beta}-receptor gene, whereas cotransfection with the {alpha}-receptor gene may attenuate the growth response.

  2. Independent origin of identical [beta] cardiac myosin heavy-chain mutations in hypertrophic cardiomyopathy

    SciTech Connect

    Watkins, H. Harvard Medical School, Boston, MA St. George's Hospital Medical School, London ); Thierfelder, L.; Anan, R.; Jarcho, J.; Seidman, C.E. Harvard Medical School, Boston, MA ); Matsumori, Akira ); McKenna, W. ); Seidman, J.G. Howard Hughes Medical Inst., Boston, MA )

    1993-12-01

    The origins of the [beta] cardiac myosin heavy-chain (MHC) gene missense mutations that cause familial hypertrophic cardiomyopathy (FHC) in 14 families have been evaluated. Of eight different mutations, four were present in single families, while four occurred in two or more families. To investigate the origins of the four shared mutations, the authors defined the [beta] cardiac MHC haplotypes of each of the mutation-bearing chromosomes by determining the alleles present at three intragenic polymorphic loci. Two of the mutations (Arg453Cys and Val606Met) have arisen independently in each of three families, being found on different chromosomal backgrounds. A third mutation (Gly584Arg) is associated with identical haplotypes in two families with Portuguese ancestors, suggesting a founder effect. Haplotype analysis was uninformative for the fourth mutation (Arg403Gln). Thus, FHC-causing mutations have arisen independently in at least 12 of the 14 families studied, suggesting that the majority have arisen relatively recently as new mutations. This finding predicts the prevalence of disease-causing [beta] cardiac MHC mutations to be comparable in all population groups. 21 refs., 4 figs., 1 tab.

  3. The spectrum of beta-globin gene mutations in children with beta-thalassaemia major from Kota Kinabalu, Sabah, Malaysia.

    PubMed

    Thong, M K; Soo, T L

    2005-07-01

    Beta-thalassaemia major is one of the commonest genetic disorders in South East Asia. The strategy for the community control of beta-thalassaemia major requires the characterisation of the spectrum of beta-globin gene mutations in any multi-ethnic population. There is only a single report of mutation analyses of the beta-globin gene in an isolated Kadazandusun community in Kota Belud, Sabah, Malaysia, which showed the presence of a common 45 kb deletion. To confirm the observation that this large deletion is the commonest beta-globin gene mutation among the Kadazandusun and other indigenous populations in Sabah, Malaysia, we performed polymerase chain reaction (PCR) analysis of the beta-globin gene in ten children with beta-thalassaemia major attending the Thalassaemia Centre, Queen Elizabeth Hospital, the major paediatric referral centre in Kota Kinabalu, Sabah. The 45 kb deletion was confirmed to be the commonest mutation found in the Kadazandusun, Bajau and Murut populations, whereby it was detected in 19 out of the 20 (95 percent) alleles analysed. The other mutation was due to an IVS-1 position 1 G > T mutation. This finding confirmed the deletion in the homozygous state was associated with a severe phenotype. The reason for the predominance of this mutation in Kota Kinabalu is most likely to be due to founder effects and possibly intermarriages between the various ethnic groups. Prenatal diagnosis using PCR for this common mutation is feasible in this community. Medical workers and scientists at molecular diagnostic centres serving large South East Asian populations should incorporate a diagnostic strategy for this deletion in the appropriate population. Future studies on these indigenous ethnic groups in other areas and other groups in Sabah are required.

  4. Differential gene expression in response to transforming growth factor-beta1 by fetal and postnatal dermal fibroblasts.

    PubMed

    Rolfe, Kerstin J; Irvine, Laurie M; Grobbelaar, Addie O; Linge, Claire

    2007-01-01

    The multipotent growth factor transforming growth factor (TGF)-beta1 is consistently linked with fibrosis and scarring. The perfect (scarless) healing of cutaneous wounds in early gestational age fetuses is proposed to be due to this tissue's predominance of the TGF-beta3 isoform over the profibrotic TGF-beta1 and 2. Nevertheless, TGF-beta1 is present during wound healing in the early fetus and recently we demonstrated that relevant intracellular signaling pathways are activated (albeit transiently) on TGF-beta1 stimulation. This study aimed to determine whether TGF-beta1 has different effects on gene transcription in human fetal (<14 weeks) vs. human postnatal dermal fibroblasts, using real-time polymerase chain reaction. The regulation pattern of a number of TGF-beta response genes differed dramatically between the two cell sources. The typical autocrine loop of TGF-beta1 autoinduction did not occur in fetal fibroblasts and genes that are normally up-regulated, connective tissue growth factor and collagen type I were actually down-regulated. Furthermore, other response genes responded in a delayed fashion (TGF-beta3) compared with that seen in the more developmentally mature postnatal fibroblasts. Finally, genes unaltered by TGF-beta stimulation in postnatal cells, TGF-beta2 and collagen III, were up-regulated in fetal cells. These developmentally related differences in fibroblast response to TGF-beta1 may influence wound-healing outcome, i.e., perfect regeneration or fibrosis.

  5. Isolation of monotreme T-cell receptor alpha and beta chains.

    PubMed

    Belov, Katherine; Miller, Robert D; Ilijeski, Aron; Hellman, Lars; Harrison, Gavan A

    2004-06-01

    Monotremes are an ancient mammalian lineage that last shared a common ancestor with the marsupial and eutherian (placental) mammals about 170 million years ago. Characterization of their immune genes is allowing us to gain insights into the evolutionary processes that lead to the 'mammalian' immune response. Here we describe the characterization of the first cDNA clones encoding T-cell receptors from a monotreme. Two TCR alpha-chain cDNAs ( TCRA) from the short-beaked echidna, Tachyglossus aculeatus, containing complete variable, joining and constant regions were isolated. The echidna TCRA constant region shares approximately 37% amino acid identity with other mammalian TCRA constant region sequences. The two variable regions belong to the TCRAV group C, which also contains V genes from humans, mice, cattle and chickens. One echidna TCR beta-chain cDNA ( TCRB) containing the entire constant region was isolated and sequenced. It shares about 63% identity with other mammalian TCRB constant region sequences. The echidna TCRBV belongs to TCRBV group A, which also contains V genes from various eutherian species. Southern blot analysis indicates that, like in other mammalian species, there is only one TCRA constant region copy in the echidna genome, but at least two TCRB constant regions.

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

    PubMed

    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.

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

    SciTech Connect

    Pestov, Nikolay B.; Zhao, Hao; Basrur, Venkatesha; Modyanov, Nikolai N.

    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

  8. Cloning and expression analysis of the murine lymphotoxin beta gene.

    PubMed Central

    Pokholok, D K; Maroulakou, I G; Kuprash, D V; Alimzhanov, M B; Kozlov, S V; Novobrantseva, T I; Turetskaya, R L; Green, J E; Nedospasov, S A

    1995-01-01

    Tumor necrosis factor alpha (TNF-alpha) and soluble lymphotoxin (LT) (also called LT-alpha or TNF-beta) are cytokines with similar biological activities that are encoded by related and closely linked genes. TNF-alpha, a mediator of the inflammatory response, exists in soluble and transmembrane forms. LT-alpha can be secreted or retained at the cell surface by binding to a 33-kDa transmembrane subunit, LT-beta. The recently cloned human LT-beta gene encodes another TNF family member and is linked to the TNF/LT locus within the major histocompatibility complex locus. The cell surface LT is a heterotrimer consisting of LT-alpha and LT-beta, whose physiological function is not yet clearly defined. We now report the sequence analysis of the genomic region and cDNA of murine LT-beta gene, which is closely associated with the TNF-alpha and LT-alpha genes within the murine major histocompatibility complex locus. Unlike the TNF-alpha, LT-alpha, and human LT-beta genes, which contain four exons, the murine LT-beta contains three exons and encodes a 244-amino acid polypeptide with a 66-amino acid insert that is absent from the human homologue. In situ hybridization demonstrates constitutive expression of LT-beta in lymphoid and hematopoietic tissues. LT-beta transcription is maximal in the thymic medulla and in splenic white pulp. LT-beta mRNA is also detected in the skin and in specific regions of the brain. The LT-beta promoter region contains putative Ets-binding sites, suggesting that the expression of LT-beta may be regulated in part by Ets transcription factors whose pattern of lymphoid expression overlaps that of LT-beta. Images Fig. 3 Fig. 4 PMID:7846035

  9. Characterization of a beta-tubulin gene and a beta-tubulin gene products of Brugia pahangi.

    PubMed

    Guénette, S; Prichard, R K; Klein, R D; Matlashewski, G

    1991-02-01

    A genomic clone containing a beta-tubulin gene from the parasitic nematode Brugia pahangi was isolated. This gene was sequenced to determine its size, structural organization, and corresponding primary amino acid sequence. The coding sequence of the beta-tubulin gene spans 3.8 kb, is organized into 9 exons and expresses an mNRA of 1.8 kb which codes for a protein of 448 amino acids. The predicted beta-tubulin amino acid sequence is 89%, 94%, 90% and 88% identical to the chicken beta 2, and the Caenorhabditis elegans ben-1, tub-1 and mec-7 gene products, respectively. Southern hybridization analyses demonstrated that there is only one copy of this gene isotype but that other distinct beta-tubulin genes may exist in the Brugia pahangi genome. A nematode specific antipeptide rabbit antiserum raised against the predicted amino acid sequence of the extreme carboxy-terminal region of the B. pahangi beta-tubulin was used to identify beta-tubulin isoforms in adult nematodes and microfilariae. Isoforms detected by this nematode-specific antipeptide antiserum were identical in both adult worms and microfilariae and did not differ from the isoform patterns detected by a monoclonal antibody recognizing a conserved beta-tubulin epitope. This suggests that this carboxy-terminal peptide is highly represented in the beta-tubulin isoforms of B. pahangi.

  10. Analysis of cDNA coding MHC class II beta chain of the chimpanzee (Pan troglodytes).

    PubMed

    Hatta, Yuki; Kanai, Tomoko; Matsumoto, Yoshitsugu; Kyuwa, Shigeru; Hayasaka, Ikuo; Yoshikawa, Yasuhiro

    2002-04-01

    The chimpanzee (Pan troglodytes, Patr) is the closest zoological living relative of humans and shares approximately 98.6% genetic homology to human beings. Although major histocompatibility complex (MHC) plays a critical role in T cell-mediated immune responses in vertebrates, the information on Patr MHC remains at a relatively poor level. Therefore, we attempted to isolate Patr MHC class II genes and determine their nucleotide sequences. The cDNAs encoding Patr MHC class II DP, DQ and DR beta chains were isolated from the cDNA library of a chimpanzee B lymphocyte cell line Bch261. As a result of screening, the clone 6-3-1 as a representative of Patr DP clone, clone 30-1 as a Patr DQ clone, and clones 4-7-1 and 55-1 having different sequences as Patr DR clones were detected. The clone 6-3-1 consisted of 1,062 nucleotides including an open reading frame (ORF) of 777 bp. In the same way, clone 30-1 consisted of 1,172 nucleotides including ORF of 786 bp, clones 4-7-1 and 55-1 consisted of 1,163 nucleotides including ORF of 801 bp. Except for five nucleotide changes, clones 4-7-1 and 55-1 were the same sequence. By comparison with the nucleotide sequences already reported on chimpanzee MHC class II beta 1 genes, clones 6-3-1, 30-1, 4-7-1 and 55-1 were classified as PatrDPB1*16, PatrDQB1*0302, PatrDRB1*0201 and PatrDRB1*0204, respectively. This is the first report to describe complete cDNA sequences of Patr DP and DQ molecules. The nucleotide sequence data of Patr MHC class II genes obtained in this study will be useful for the genotyping of Patr MHC class II genes in individual chimpanzees.

  11. Evolutionary ecology of beta-lactam gene clusters in animals.

    PubMed

    Suring, Wouter; Meusemann, Karen; Blanke, Alexander; Mariën, Janine; Schol, Tim; Agamennone, Valeria; Faddeeva-Vakhrusheva, Anna; Berg, Matty P; Brouwer, Abraham; van Straalen, Nico M; Roelofs, Dick

    2017-06-01

    Beta-lactam biosynthesis was thought to occur only in fungi and bacteria, but we recently reported the presence of isopenicillin N synthase in a soil-dwelling animal, Folsomia candida. However, it has remained unclear whether this gene is part of a larger beta-lactam biosynthesis pathway and how widespread the occurrence of penicillin biosynthesis is among animals. Here, we analysed the distribution of beta-lactam biosynthesis genes throughout the animal kingdom and identified a beta-lactam gene cluster in the genome of F. candida (Collembola), consisting of isopenicillin N synthase (IPNS), δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine synthetase (ACVS), and two cephamycin C genes (cmcI and cmcJ) on a genomic scaffold of 0.76 Mb. All genes are transcriptionally active and are inducible by stress (heat shock). A beta-lactam compound was detected in vivo using an ELISA beta-lactam assay. The gene cluster also contains an ABC transporter which is coregulated with IPNS and ACVS after heat shock. Furthermore, we show that different combinations of beta-lactam biosynthesis genes are present in over 60% of springtail families, but they are absent from genome- and transcript libraries of other animals including close relatives of springtails (Protura, Diplura and insects). The presence of beta-lactam genes is strongly correlated with an euedaphic (soil-living) lifestyle. Beta-lactam genes IPNS and ACVS each form a phylogenetic clade in between bacteria and fungi, while cmcI and cmcJ genes cluster within bacteria. This suggests a single horizontal gene transfer event most probably from a bacterial host, followed by differential loss in more recently evolving species. © 2017 John Wiley & Sons Ltd.

  12. The alpha 4 integrin chain is a ligand for alpha 4 beta 7 and alpha 4 beta 1

    PubMed Central

    1995-01-01

    The heterodimeric alpha 4 integrins alpha 4 beta 7 lymphocyte Peyer's patch adhesion molecule ([LPAM]-1) and alpha 4 beta 1 (very late antigen-4) are cell surface adhesion molecules involved in lymphocyte trafficking and lymphocyte-cell and matrix interactions. Known cellular ligands include vascular cell adhesion molecule (VCAM)-1, which binds to alpha 4 beta 1 and alpha 4 beta 7, and the mucosal addressin cell adhesion molecule (MAdCAM)-1, which binds to alpha 4 beta 7. Here we show that the alpha 4 chain of these integrins can itself serve as a ligand. The alpha 4 chain, immunoaffinity purified and immobilized on glass slides, binds thymocytes and T lymphocytes. Binding exhibits divalent cation requirements and temperature sensitivity which are characteristic of integrin-mediated interactions, and is specifically inhibited by anti-alpha 4 integrin antibodies, which exert their effect at the cell surface. Cells expressing exclusively alpha 4 beta 7 (TK-1) or alpha 4 beta 1 (L1-2) both bound avidly, whereas alpha 4-negative cells did not. A soluble 34-kD alpha 4 chain fragment retained binding activity, and it inhibited lymphocyte adhesion to alpha 4 ligands. It has been shown that alpha 4 integrin binding to fibronectin involves an leucine-aspartic acid-valine (LDV) motif in the HepII/IIICS region of fibronectin (CS-1 peptide), and homologous sequences are important in binding to VCAM-1 and MAdCAM-1. Three conserved LDV motifs occur in the extracellular sequence of alpha 4. A synthetic LDV-containing alpha 4- derived oligopeptide supports alpha 4-integrin-dependent lymphocyte adhesion and blocks binding to the 34-kD alpha 4 chain fragment. Our results suggest that alpha 4 beta 7 and alpha 4 beta 1 integrins may be able to bind to the alpha 4 subunit on adjacent cells, providing a novel mechanism for alpha 4 integrin-mediated and activation-regulated lymphocyte interactions during immune responses. PMID:7629498

  13. Beta-thalassemia genes in French-Canadians: haplotype and mutation analysis of Portneuf chromosomes.

    PubMed Central

    Kaplan, F; Kokotsis, G; DeBraekeleer, M; Morgan, K; Scriver, C R

    1990-01-01

    beta-Thalassemia minor occurs at approximately 1% frequency in French-Canadians--in families residing in Portneuf County (population approximately 40,000) of Quebec province. We found eight different RFLP haplotypes at the beta-globin gene cluster in 37 normal persons and in 12 beta-thalassemia heterozygotes from six families. beta-Thalassemia genes in these families associated with two haplotypes only: Mediterranean I and Mediterranean II. There were two different beta-thalassemia mutations segregating in the Portneuf population: an RNA processing mutation (beta(+)IVS-1,nt110) on haplotype I (five families) and a point mutation leading to chain termination (beta(0) nonsense codon 39) on haplotype II (one family). The distribution of 5' haplotypes on normal beta A Portneuf chromosomes compared with other European populations was most similar to that in British subjects (data for French subjects have not yet been reported). Genealogical reconstructions traced the ancestry of carrier couples to settlers emigrating from several different regions of France to New France in the 17th century. These findings indicate genetic diversity of a greater degree among French-Canadians than recognized heretofore. Images Figure 4 PMID:1967205

  14. Beta-thalassemia genes in French-Canadians: haplotype and mutation analysis of Portneuf chromosomes.

    PubMed

    Kaplan, F; Kokotsis, G; DeBraekeleer, M; Morgan, K; Scriver, C R

    1990-01-01

    beta-Thalassemia minor occurs at approximately 1% frequency in French-Canadians--in families residing in Portneuf County (population approximately 40,000) of Quebec province. We found eight different RFLP haplotypes at the beta-globin gene cluster in 37 normal persons and in 12 beta-thalassemia heterozygotes from six families. beta-Thalassemia genes in these families associated with two haplotypes only: Mediterranean I and Mediterranean II. There were two different beta-thalassemia mutations segregating in the Portneuf population: an RNA processing mutation (beta(+)IVS-1,nt110) on haplotype I (five families) and a point mutation leading to chain termination (beta(0) nonsense codon 39) on haplotype II (one family). The distribution of 5' haplotypes on normal beta A Portneuf chromosomes compared with other European populations was most similar to that in British subjects (data for French subjects have not yet been reported). Genealogical reconstructions traced the ancestry of carrier couples to settlers emigrating from several different regions of France to New France in the 17th century. These findings indicate genetic diversity of a greater degree among French-Canadians than recognized heretofore.

  15. Endogenous beta-cell CART regulates insulin secretion and transcription of beta-cell genes.

    PubMed

    Shcherbina, L; Edlund, A; Esguerra, J L S; Abels, M; Zhou, Y; Ottosson-Laakso, E; Wollheim, C B; Hansson, O; Eliasson, L; Wierup, N

    2017-05-15

    Impaired beta-cell function is key to the development of type 2 diabetes. Cocaine- and amphetamine-regulated transcript (CART) is an islet peptide with insulinotropic and glucagonostatic properties. Here we studied the role of endogenous CART in beta-cell function. CART silencing in INS-1 (832/13) beta-cells reduced insulin secretion and production, ATP levels and beta-cell exocytosis. This was substantiated by reduced expression of several exocytosis genes, as well as reduced expression of genes important for insulin secretion and processing. In addition, CART silencing reduced the expression of a network of transcription factors essential for beta-cell function. Moreover, in RNAseq data from human islet donors, CARTPT expression levels correlated with insulin, exocytosis genes and key beta-cell transcription factors. Thus, endogenous beta-cell CART regulates insulin expression and secretion in INS-1 (832/13) cells, via actions on the exocytotic machinery and a network of beta-cell transcription factors. We conclude that CART is important for maintaining the beta-cell phenotype. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Monoclonal anti-mouse laminin antibodies: AL-1 reacts with laminin alpha1 chain, AL-2 with laminin beta1 chain, and AL-4 with the coiled-coil domain of laminin beta1 chain.

    PubMed

    Schéele, Susanne; Sasaki, Takako; Arnal-Estapé, Anna; Durbeej, Madeleine; Ekblom, Peter

    2006-07-01

    We analyzed the reactivity of three different commercially available rat monoclonal antibodies raised against mouse laminin-alpha1beta1gamma1 (laminin-111), AL-1, AL-2, and AL-4. Using ELISA assays, Western blot analysis and immunostainings we present refined epitope maps for these three laminin monoclonals. AL-1 reacted, as predicted with laminin alpha1 chain. AL-4 has also been marketed as an alpha1 chain specific probe, but we show here that AL-4 detects mouse laminin beta1 chain, in the distal part of the coiled-coil region. AL-2 was predicted to react with all three chains near the cross-region, but seems to primarily react with laminin beta1 chain.

  17. Peroxisomal very long chain fatty acid beta-oxidation activity is determined by the level of adrenodeukodystrophy protein (ALDP) expression.

    PubMed

    Braiterman, L T; Watkins, P A; Moser, A B; Smith, K D

    1999-02-01

    Impaired peroxisomal beta-oxidation of saturated very long chain fatty acids (VLCFA, >/=C22:0) results in increased VLCFA levels in the tissues and body fluids of patients with disorders of peroxisomal biogenesis (i.e., Zellweger syndrome and neonatal adrenoleukodystrophy) and single peroxisomal protein defects (i.e., X-linked adrenoleukodystrophy (X-ALD) and acyl-CoA oxidase deficiency). We show that SV40T transformation also results in impaired peroxisomal beta-oxidation and VLCFA accumulation despite the presence of abundant peroxisomes. To explore the mechanism responsible for this observation, we have examined expression of key components of peroxisomal VLCFA beta-oxidation. We found that expression of both acyl-CoA oxidase, the rate limiting enzyme of peroxisomal VLCFA beta-oxidation and the adrenoleukodystrophy protein (ALDP), the defective gene product in X-ALD, are reduced after SV40T transformation. Surprisingly, ALDP overexpression by itself restores peroxisomal VLCFA beta-oxidation in SV40T-transformed control and X-ALD cells. These results demonstrate that ALDP is a fundamental component in VLCFA peroxisomal beta-oxidation and may serve as a "gatekeeper" for VLCFA homeostasis. Copyright 1999 Academic Press.

  18. Phosphorylation/dephosphorylation of the beta light chain of clathrin from rat liver coated vesicles.

    PubMed

    Loeb, J E; Cantournet, B; Vartanian, J P; Goris, J; Merlevede, W

    1989-06-01

    The phosphorylation in vitro, on serine residues by endogenous casein kinase 2, of the clathrin beta light chain (33 kDa) of rat liver coated vesicles requires the presence of poly(L-lysine) which acts through binding to the beta light chain. The phosphorylation of other proteins is also increased in the presence of poly(L-lysine) and casein kinase 2. In contrast, the phosphorylation of the upper band of the 50-kDa protein doublet from rat liver coated vesicles is inhibited. Rat liver coated vesicles display a protein phosphatase activity which preferentially dephosphorylates clathrin beta light chain. This activity is different from the protein phosphatase which dephosphorylates the 50-kDa protein. This enzyme seems to be unrelated to the ATP/Mg-dependent protein phosphatase, or the polycation-stimulated protein phosphatases, which dephosphorylate the 50-kDa protein and beta light chain very efficiently, but with a different specificity. After dissociation of coated vesicles the beta-light-chain phosphatase activity is recovered in the membrane fraction. This phosphatase activity is inhibited by 50 microM orthovanadate and 5 mM p-nitrophenyl phosphate but not by 10 mM EDTA.

  19. No evidence of the human chorionic gonadotropin-beta gene 5 betaV79M polymorphism in Mexican women.

    PubMed

    Dominguez-Lopez, Pablo; Diaz-Cueto, Laura; Ulloa-Aguirre, Alfredo; Lopez-Valle, Miguel Angel; Arechavaleta-Velasco, Fabian

    2008-01-01

    Human chorionic gonadotropin (hCG) is a placental hormone essential for the maintenance of pregnancy. Previous studies have shown a G to A transition in exon 3 of the hCGbeta gene 5, which changes the naturally occurring valine to methionine in codon 79. The frequency of this transition varies among different ethnic groups, being high in USA women, and less common, or absent, in various European populations. The purpose of the present study was to determine the frequency of the betaV79M allelic variant of the beta-subunit of hCG in a Mexican population, and to compare this frequency with those found in other ethnic groups. Placental DNA from 161 pregnant Mexican women was genotyped for the betaV79M by polymerase chain reaction (PCR)-restriction fragments length polymorphism analysis. No polymorphic betaV79M alleles were identified in the population studied. The allele and genotypic frequencies of betaV79M polymorphism in Mexican Mestizo women were significantly different from those reported for the US population, but not from five different European populations. In contrast to what has been found in women from the USA, it seems that the hCGbeta V79M polymorphism is absent or extremely rare in Mexican Mestizo women.

  20. Expression of cytokine genes in human cardiac allografts: correlation of IL-6 and transforming growth factor-beta (TGF-beta) with histological rejection.

    PubMed Central

    Zhao, X M; Frist, W H; Yeoh, T K; Miller, G G

    1993-01-01

    Cytokines may play critical roles in allograft rejection. Currently, a clear pattern of cytokine production that correlates with rejection has not emerged. Our preliminary studies suggested a trend toward increased IL-6 and TGF-beta gene expression in cardiac allografts during rejection. We have extended these studies using reverse transcriptase/polymerase chain reaction (RT/PCR) to detect the expression of IL-6, TGF-beta, and T cell receptor beta chain constant region (TCR-beta) genes in 21 additional consecutive myocardial biopsies obtained from six heart transplant patients and from five pre-transplant donor hearts. Cytokine gene expression was compared with histological diagnosis of rejection. There was strong correlation between IL-6 as well as TGF-beta gene expression, and histological rejection (6/8 biopsies with versus 0/7 without rejection (P = 0.006) and 7/9 biopsies with versus 0/7 without rejection (P = 0.003) respectively). Neither IL-6 nor TGF-beta transcripts were detected in any pre-transplant donor heart. TCR-beta chain mRNA was found in all allograft biopsies regardless of the presence of rejection, but was absent in pre-transplant donor hearts. Our results indicate that expression of IL-6 and TGF-beta is highly correlated with allograft rejection and thus may play an important role in regulation of cardiac allograft rejection. T cell infiltration of allografted myocardium is invariably detected by PCR regardless of histological rejection. The long-term functional significance of these cells in transplanted hearts needs further investigation. Images Fig. 1 PMID:8370174

  1. The IL-2 receptor alpha-chain alters the binding of IL-2 to the beta-chain.

    PubMed

    Arima, N; Kamio, M; Okuma, M; Ju, G; Uchiyama, T

    1991-11-15

    The binding of IL-2 to its high affinity receptor results in the formation of the ternary complex consisting of IL-2, alpha-chain (p55, Tac) and beta-chain (p75). We studied the role of alpha-chain in IL-2 binding to the high affinity receptor using IL-2 analog Lys20 which was made by the substitution of Lys for Asp20 of wild-type rIL-2. Lys20 bound to MT-1 cells solely expressing alpha-chain at low affinity, but did not bind to YT-2C2 cells which solely expressed beta-chain. However, direct binding of radiolabeled Lys20 to ED515-D cells, an HTLV-I-infected and IL-2-dependent T cell line, revealed both high affinity and low affinity binding although the Kd value of high affinity binding was 50 to 100 times higher than that of the high affinity binding of wild-type rIL-2. High affinity binding of Lys20 was completely blocked by 2R-B mAb recognizing IL-2R beta-chain. Anti-Tac mAb recognizing IL-2R alpha-chain abolished all of the specific Lys20 bindings. In contrast to the replacement of cell bound 2R-B mAb with wild-type rIL-2 at 37 degrees C, the addition of an excess of Lys20 did not cause the detachment of cell-bound radiolabeled or FITC-labeled 2R-B mAb. Consistent with the results of binding studies, Lys20 induced the proliferation of ED515-D cells, but not large granular lymphocyte leukemic cells. The growth of ED-515D cells was completely suppressed by either anti-Tac mAb or 2R-B mAb. These results strongly suggest that coexpression of the IL-2R alpha- and beta-chains alters the binding affinity of Lys20 and that the interaction between IL-2 and the alpha-chain is a key event in the formation of the IL-2/IL-2R ternary complex.

  2. Biological activities of (1-->3)-beta-D-glucans with reducing glucose side chains.

    PubMed

    Kiho, T; Matsushita, M; Usui, S; Ukai, S

    1998-03-01

    Newly synthesized (1-->3)-beta-D-glucans with reducing glucose side chains (6-O-glucopyranosylated curdlan and 3-O-glucopyranosylated curdlan, with glucose linked directly (except for anomeric carbon) had antitumor activity against mice sarcoma 180 in mice. The two glucans potentiated the reticuloendotheliai system and activated macrophages (increased their glucose consumption). The activity inducing tumor regressing factor of the glucan derivatives was stronger than a linear (1-->3)-beta-D-glucan (curdlan).

  3. Presence and diversity of the beta-lactamase gene in cat and dog staphylococci.

    PubMed

    Malik, Seidu; Christensen, Henrik; Peng, Haihong; Barton, Mary D

    2007-07-20

    Staphylococci are part of the normal microflora of humans and animals and some are potential pathogens that have become resistant to almost all known antibiotics. Despite the widespread reports of penicillin resistance in cat and dog staphylococci, the mechanism underlying penicillin resistance has not been examined. This study was aimed at investigating the molecular basis of resistance to penicillin in cat and dog staphylococcal isolates that showed phenotypic resistance to beta-lactam antibiotics. An 861 bp fragment of the structural blaZ gene which codes for beta-lactamase production in staphylococci was amplified by polymerase chain reaction (PCR) and the products were sequenced. Sequenced fragments were analysed by protein signature typing and sequences were compared to published blaZ sequences of human and bovine staphylococcal strains held in a public database. Four known protein signature types (1, 3, 5 and 6) and one new type (12) were identified in this study. When sequences were compared with published blaZ sequences, gene phylogenetic analysis revealed three major groups. The four variants of beta-lactamases types (A, B, C and D) belonged to each major group except for types A and D which were both in group II. These findings confirm that the blaZ gene is responsible for beta-lactamase production leading to subsequent resistance to beta-lactam antibiotics in feline and canine staphylococci and that the gene shows similar diversity and relatedness as found with blaZ sequences obtained from human and bovine staphylococci.

  4. Physical association of JAK1 and JAK2 tyrosine kinases with the interleukin 2 receptor beta and gamma chains.

    PubMed Central

    Tanaka, N; Asao, H; Ohbo, K; Ishii, N; Takeshita, T; Nakamura, M; Sasaki, H; Sugamura, K

    1994-01-01

    The functional interleukin 2 (IL-2) receptors contain the beta and gamma chains which are necessary for the transduction of cell growth signals. Monoclonal antibodies specific for the beta chain and gamma chain coimmunoprecipitated JAK1 and 114-kDa JAK2 tyrosine kinases, respectively. Tyrosine phosphorylation of JAK1 and JAK2 was induced upon IL-2 stimulation, and IL-2 activated the JAK2 kinase. These results demonstrate that the JAK1 and JAK2 tyrosine kinases are physically associated with the beta chain and gamma chain, respectively, and suggest that regulation of the kinases may be linked to IL-2-induced signal transduction. Images PMID:8041779

  5. Ca2+-dependent dephosphorylation of kinesin heavy chain on beta-granules in pancreatic beta-cells. Implications for regulated beta-granule transport and insulin exocytosis

    NASA Technical Reports Server (NTRS)

    Donelan, Matthew J.; Morfini, Gerardo; Julyan, Richard; Sommers, Scott; Hays, Lori; Kajio, Hiroshi; Briaud, Isabelle; Easom, Richard A.; Molkentin, Jeffery D.; Brady, Scott T.; hide

    2002-01-01

    The specific biochemical steps required for glucose-regulated insulin exocytosis from beta-cells are not well defined. Elevation of glucose leads to increases in cytosolic [Ca2+]i and biphasic release of insulin from both a readily releasable and a storage pool of beta-granules. The effect of elevated [Ca2+]i on phosphorylation of isolated beta-granule membrane proteins was evaluated, and the phosphorylation of four proteins was found to be altered by [Ca2+]i. One (a 18/20-kDa doublet) was a Ca2+-dependent increase in phosphorylation, and, surprisingly, three others (138, 42, and 36 kDa) were Ca2+-dependent dephosphorylations. The 138-kDa beta-granule phosphoprotein was found to be kinesin heavy chain (KHC). At low levels of [Ca2+]i KHC was phosphorylated by casein kinase 2, but KHC was rapidly dephosphorylated by protein phosphatase 2B beta (PP2Bbeta) as [Ca2+]i increased. Inhibitors of PP2B specifically reduced the second, microtubule-dependent, phase of insulin secretion, suggesting that dephosphorylation of KHC was required for transport of beta-granules from the storage pool to replenish the readily releasable pool of beta-granules. This is distinct from synaptic vesicle exocytosis, because neurotransmitter release from synaptosomes did not require a Ca2+-dependent KHC dephosphorylation. These results suggest a novel mechanism for regulating KHC function and beta-granule transport in beta-cells that is mediated by casein kinase 2 and PP2B. They also implicate a novel regulatory role for PP2B/calcineurin in the control of insulin secretion downstream of a rise in [Ca2+]i.

  6. Ca2+-dependent dephosphorylation of kinesin heavy chain on beta-granules in pancreatic beta-cells. Implications for regulated beta-granule transport and insulin exocytosis

    NASA Technical Reports Server (NTRS)

    Donelan, Matthew J.; Morfini, Gerardo; Julyan, Richard; Sommers, Scott; Hays, Lori; Kajio, Hiroshi; Briaud, Isabelle; Easom, Richard A.; Molkentin, Jeffery D.; Brady, Scott T.; Rhodes, Christopher J.

    2002-01-01

    The specific biochemical steps required for glucose-regulated insulin exocytosis from beta-cells are not well defined. Elevation of glucose leads to increases in cytosolic [Ca2+]i and biphasic release of insulin from both a readily releasable and a storage pool of beta-granules. The effect of elevated [Ca2+]i on phosphorylation of isolated beta-granule membrane proteins was evaluated, and the phosphorylation of four proteins was found to be altered by [Ca2+]i. One (a 18/20-kDa doublet) was a Ca2+-dependent increase in phosphorylation, and, surprisingly, three others (138, 42, and 36 kDa) were Ca2+-dependent dephosphorylations. The 138-kDa beta-granule phosphoprotein was found to be kinesin heavy chain (KHC). At low levels of [Ca2+]i KHC was phosphorylated by casein kinase 2, but KHC was rapidly dephosphorylated by protein phosphatase 2B beta (PP2Bbeta) as [Ca2+]i increased. Inhibitors of PP2B specifically reduced the second, microtubule-dependent, phase of insulin secretion, suggesting that dephosphorylation of KHC was required for transport of beta-granules from the storage pool to replenish the readily releasable pool of beta-granules. This is distinct from synaptic vesicle exocytosis, because neurotransmitter release from synaptosomes did not require a Ca2+-dependent KHC dephosphorylation. These results suggest a novel mechanism for regulating KHC function and beta-granule transport in beta-cells that is mediated by casein kinase 2 and PP2B. They also implicate a novel regulatory role for PP2B/calcineurin in the control of insulin secretion downstream of a rise in [Ca2+]i.

  7. Ca2+-dependent dephosphorylation of kinesin heavy chain on beta-granules in pancreatic beta-cells. Implications for regulated beta-granule transport and insulin exocytosis.

    PubMed

    Donelan, Matthew J; Morfini, Gerardo; Julyan, Richard; Sommers, Scott; Hays, Lori; Kajio, Hiroshi; Briaud, Isabelle; Easom, Richard A; Molkentin, Jeffery D; Brady, Scott T; Rhodes, Christopher J

    2002-07-05

    The specific biochemical steps required for glucose-regulated insulin exocytosis from beta-cells are not well defined. Elevation of glucose leads to increases in cytosolic [Ca2+]i and biphasic release of insulin from both a readily releasable and a storage pool of beta-granules. The effect of elevated [Ca2+]i on phosphorylation of isolated beta-granule membrane proteins was evaluated, and the phosphorylation of four proteins was found to be altered by [Ca2+]i. One (a 18/20-kDa doublet) was a Ca2+-dependent increase in phosphorylation, and, surprisingly, three others (138, 42, and 36 kDa) were Ca2+-dependent dephosphorylations. The 138-kDa beta-granule phosphoprotein was found to be kinesin heavy chain (KHC). At low levels of [Ca2+]i KHC was phosphorylated by casein kinase 2, but KHC was rapidly dephosphorylated by protein phosphatase 2B beta (PP2Bbeta) as [Ca2+]i increased. Inhibitors of PP2B specifically reduced the second, microtubule-dependent, phase of insulin secretion, suggesting that dephosphorylation of KHC was required for transport of beta-granules from the storage pool to replenish the readily releasable pool of beta-granules. This is distinct from synaptic vesicle exocytosis, because neurotransmitter release from synaptosomes did not require a Ca2+-dependent KHC dephosphorylation. These results suggest a novel mechanism for regulating KHC function and beta-granule transport in beta-cells that is mediated by casein kinase 2 and PP2B. They also implicate a novel regulatory role for PP2B/calcineurin in the control of insulin secretion downstream of a rise in [Ca2+]i.

  8. Structure of a Ruminococcus albus endo-1,4-beta-glucanase gene.

    PubMed Central

    Ohmiya, K; Kajino, T; Kato, A; Shimizu, S

    1989-01-01

    A chromosomal DNA fragment encoding an endo-1,4-beta-glucanase I (Eg I) gene from Ruminococcus albus cloned and expressed in Escherichia coli with pUC18 was fully sequenced by the dideoxy-chain termination method. The sequence contained a consensus promoter sequence and a structural amino acid sequence. The initial 43 amino acids of the protein were deduced to be a signal sequence, since they are missing in the mature protein (Eg I). High homology was found when the amino acid sequence of the Eg I was compared with that of endoglucanase E from Clostridium thermocellum. Codon usage of the gene was not biased. These results suggested that the properties of the Eg I gene from R. albus was specified from the known beta-glucanase genes of the other organisms. Images PMID:2687251

  9. Distinct tyrosine residues within the interleukin-2 receptor beta chain drive signal transduction specificity, redundancy, and diversity.

    PubMed

    Gaffen, S L; Lai, S Y; Ha, M; Liu, X; Hennighausen, L; Greene, W C; Goldsmith, M A

    1996-08-30

    To explore the basis for interleukin (IL)-2 receptor (IL-2R) signaling specificity, the roles of tyrosine-based sequences located within the cytoplasmic tails of the beta and gammac chains were examined in the murine helper T cell line HT-2. Activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, cellular proliferation, and the induction of various genes were monitored. All four of the cytoplasmic tyrosine residues as well as the distal portion of the gammac proved dispensable for the entire spectrum of IL-2R signaling responses studied. Conversely, select tyrosine residues within the beta chain were essential and differentially required for various signaling events. Specifically, activation of c-fos gene expression was found to occur exclusively through the most membrane proximal tyrosine, Tyr-338, whereas proliferation and the activation of STAT-5 were induced either through Tyr-338 or through the two C-terminal tyrosine residues, Tyr-392 and Tyr-510. These tyrosine residues mediated the induction of two different STAT-5 isoforms, which were found to form heterodimers upon receptor activation. In contrast to the tyrosine dependence of c-fos and STAT-5 induction, bcl-2 gene induction proceeded independently of all IL-2Rbeta tyrosine residues. Thus, the tyrosine-based modules present within the IL-2Rbeta cytoplasmic tail play a critical role in IL-2R signaling, mediating specificity, redundancy, and multifunctionality.

  10. Gamma-chain heterogeneity in Greek (delta beta)zero-thalassemia.

    PubMed

    Georgiou, I; Seferiadis, K; Lolis, D; Tsolas, O; Bourantas, K L

    1995-02-01

    A molecular and biochemical population study of (delta beta)zero thalassemia in central Greece is described. The molecular study was focused on the type of the deletion and the status of G gamma-XmnI polymorphism, whereas the biochemical approach was centered on the G gamma/A gamma ratio as well as the frequency of the A gamma T chain in the fetal hemoglobin of 19 delta beta-thalassemia heterozygotes and 3 homozygotes. This study includes individuals from the mountainous district of Epirus (northwestern Greece) where the trait was found to be concentrated along the river Arachthos. The Sicilian (delta beta)zero thalassemia deletion was found in all subjects tested by direct PCR. The levels for the G gamma-chain presented values ranging from 29 to 83% of the total gamma-chain content. Thirteen heterozygotes had the adult G gamma/A gamma ratio (mean G gamma: 35% +/- 10) of whom 10 were XmnI-negative (- / -), 6 had the newborn ratio (mean G gamma: 70% +/- 9) and were XmnI-positive, while homozygotes had equal amounts of G gamma and A gamma. Five of the 19 heterozygotes were A gamma T-positive with low levels of this A gamma-chain variant, suggesting an in-trans to the delta beta-thalassemia determinant production.

  11. Beta-amylase gene variability in introgressive wheat lines.

    PubMed

    Antonyuk, Maksym; Navalikhina, Anastasiia; Ternovska, Tamara

    2017-05-01

    Variability of the beta-amylase gene in bread wheat, artificial amphidiploids, and derived introgression wheat lines was analyzed. Variation in homeologous beta-amylase sequences caused by the presence of MITE (Miniature Inverted-Repeat Transposable Element) and its footprint has been identified in bread wheat. The previously unknown location of MITE in Triticum urartu and T. aestivum L. beta-amylase gene has been found. These species have a MITE sequence in the third intron of beta-amylase, as opposed to Aegilops comosa and a number of other Triticeae species, which have it in the fourth intron. These two MITEs from Ae. comosa and T. aestivum were shown to have low identity scores. Miosa, an artificial amphidiploid, which has the M genome from Ae. comosa was shown to lose the MITE sequences. This loss might be caused by genomic shock due to allopolyploidization.

  12. Nucleotide sequence of SHV-2 beta-lactamase gene

    SciTech Connect

    Garbarg-Chenon, A.; Godard, V.; Labia, R.; Nicolas, J.C. )

    1990-07-01

    The nucleotide sequence of plasmid-mediated beta-lactamase SHV-2 from Salmonella typhimurium (SHV-2pHT1) was determined. The gene was very similar to chromosomally encoded beta-lactamase LEN-1 of Klebsiella pneumoniae. Compared with the sequence of the Escherichia coli SHV-2 enzyme (SHV-2E.coli) obtained by protein sequencing, the deduced amino acid sequence of SHV-2pHT1 differed by three amino acid substitutions.

  13. Expression of the human. beta. -globin gene following retroviral-mediated transfer into multipotential hematopoietic progenitors of mice

    SciTech Connect

    Karlsson, S.; Bodine, D.M.; Perry, L.; Papayannopoulou, T.; Nienhuis, A.W. )

    1988-08-01

    Efficient transfer of the {beta}-globin gene into primitive hematopoietic progenitors was achieved with consistent and significant expression in the progeny of those cells. Retroviral vectors containing the intact genomic human {beta}-globin gene and the neomycin (G418)-resistance (neo{sup R}) gene were constructed. These gave titers of 10{sup 6} or more neo{sup R} colony-forming units/ml when packaged in {psi}2 cells. Mouse bone marrow cells were infected by coculture with producer cells and injected into lethally irradiated animals. Several parameters were varied to enhance infection frequency of colony-forming units, spleen (CFU-S); overall 41% of 116 foci studied contained an intact proviral genome. The human {beta}-globin gene was expressed in 31 of 35 CFU-S-derived spleen colonies that contained the intact vector genome at levels ranging from 1% to 5% of that of the mouse {beta}-globin genes. Infected bone marrow cells were also injected into genetically anemic W/W{sup v} recipients without prior irradiation. Human {beta}-globin chains were detected in circulating erythrocytes by immunofluorescent staining with a specific monoclonal antibody. All animals injected with donor cells that had been cultured in G418 (1 mg/ml) for 48 hr after retroviral infection had circulating erythrocytes containing human {beta}-globin chains between 3 and 8 weeks after transplantation.

  14. Inhibition of autologous mixed lymphocyte reaction by monoclonal antibodies specific for the beta chain of HLA-DR antigens.

    PubMed

    Kasahara, M; Ikeda, H; Ogasawara, K; Ishikawa, N; Okuyama, T; Fukasawa, Y; Kojima, H; Kunikane, H; Hawkin, S; Ohhashi, T

    1984-09-01

    Recent studies using rabbit antisera to the separated HLA-DR alpha and beta subunits have suggested that alpha chain-specific, but not beta chain-specific, antisera inhibit T cell proliferative responses in primary and secondary human autologous mixed lymphocyte reaction (AMLR). In the present study, with the aid of sequential co-precipitation assays and Western blotting methods, a monoclonal rat alloantibody 1E4, specific for the beta chain of rat class II molecules carrying an Ia determinant Ba-2.7, was characterized to recognize a monomorphic determinant located on the beta chain of DR antigens. This antibody and a murine monoclonal antibody HU-4, also specific for the beta chain of DR antigens, strongly inhibited both primary and secondary AMLR through a mechanism distinct from an antibody-dependent cell-mediated cytotoxicity reaction. These results indicate that the inhibition of AMLR is not a unique feature of DR alpha-specific antibodies.

  15. Profiling the T-cell receptor beta-chain repertoire by massively parallel sequencing.

    PubMed

    Freeman, J Douglas; Warren, René L; Webb, John R; Nelson, Brad H; Holt, Robert A

    2009-10-01

    T-cell receptor (TCR) genomic loci undergo somatic V(D)J recombination, plus the addition/subtraction of nontemplated bases at recombination junctions, in order to generate the repertoire of structurally diverse T cells necessary for antigen recognition. TCR beta subunits can be unambiguously identified by their hypervariable CDR3 (Complement Determining Region 3) sequence. This is the site of V(D)J recombination encoding the principal site of antigen contact. The complexity and dynamics of the T-cell repertoire remain unknown because the potential repertoire size has made conventional sequence analysis intractable. Here, we use 5'-RACE, Illumina sequencing, and a novel short read assembly strategy to sample CDR3(beta) diversity in human T lymphocytes from peripheral blood. Assembly of 40.5 million short reads identified 33,664 distinct TCR(beta) clonotypes and provides precise measurements of CDR3(beta) length diversity, usage of nontemplated bases, sequence convergence, and preferences for TRBV (T-cell receptor beta variable gene) and TRBJ (T-cell receptor beta joining gene) gene usage and pairing. CDR3 length between conserved residues of TRBV and TRBJ ranged from 21 to 81 nucleotides (nt). TRBV gene usage ranged from 0.01% for TRBV17 to 24.6% for TRBV20-1. TRBJ gene usage ranged from 1.6% for TRBJ2-6 to 17.2% for TRBJ2-1. We identified 1573 examples of convergence where the same amino acid translation was specified by distinct CDR3(beta) nucleotide sequences. Direct sequence-based immunoprofiling will likely prove to be a useful tool for understanding repertoire dynamics in response to immune challenge, without a priori knowledge of antigen.

  16. Assignment of an intron-containing human heat-shock protein gene (hsp90[beta], HSPCB) to chromosome 6 near TCTE1 (6p21) and two intronless pseudogenes to chromosomes 4 and 15 by polymerase chain reaction amplification from a panel of hybrid cell lines

    SciTech Connect

    Durkin, A.S.; Nierman, W.C.; Maglott, D.R. ); Vamvakopoulos, N.C. ); Zoghbi, H.Y. )

    1993-11-01

    We report here the successful application of designing primers from intronic sequences to map a structural hsp90[beta] gene to a unique human chromosome distinct from potential pseudogenes or rodent background. Also, by designing primers that bracket an intron and detecting products from intronless genes, we localized two hsp90[beta] pseudogenes to human chromosomes 4 and 15. PCR primers were designed from the published human hsp90[beta] DNA sequence from exon 11 (nucleotides 7066-7085, 7181-7198), intron A (1659-1678, 1722-1741), intron B (8109, 8170-8187), and exons 3 and 4 to amplify across intron C (3391-3412, 3731-3752).

  17. Development of a Quantitative Methylation-Specific Polymerase Chain Reaction Method for Monitoring Beta Cell Death in Type 1 Diabetes

    PubMed Central

    Husseiny, Mohamed I.; Kuroda, Akio; Kaye, Alexander N.; Nair, Indu; Kandeel, Fouad; Ferreri, Kevin

    2012-01-01

    DNA methylation is a mechanism by which cells control gene expression, and cell-specific genes often exhibit unique patterns of DNA methylation. We previously reported that the mouse insulin-2 gene (Ins2) promoter has three potential methylation (CpG) sites, all of which are unmethylated in insulin-producing cells but methylated in other tissues. In this study we examined Ins2 exon 2 and found a similar tissue-specific methylation pattern. These methylation patterns can differentiate between DNA from insulin-producing beta cells and other tissues. We hypothesized that damaged beta cells release their DNA into circulation at the onset of type 1 diabetes mellitus (T1DM) and sought to develop a quantitative methylation-specific polymerase chain reaction (qMSP) assay for circulating beta cell DNA to monitor the loss of beta cells. Methylation-specific primers were designed to interrogate two or more CpG in the same assay. The cloned mouse Ins2 gene was methylated in vitro and used for development of the qMSP assay. We found the qMSP method to be sensitive and specific to differentiate between insulin-producing cells and other tissues with a detection limit of 10 copies in the presence of non-specific genomic DNA background. We also compared different methods for data analysis and found that the Relative Expression Ratio method is the most robust method since it incorporates both a reference value to normalize day-to-day variability as well as PCR reaction efficiencies to normalize between the methylation-specific and bisulfite-specific components of the calculations. The assay was applied in the streptozotocin-treated diabetic mouse model and detected a significant increase in circulating beta cell DNA before the rise in blood glucose level. These results demonstrate that this qMSP assay can be used for monitoring circulating DNA from insulin-producing cells, which will provide the basis for development of assays to detect beta cell destruction in early T1DM. PMID

  18. 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.

    PubMed

    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.

  19. Immunomodulation by mucosal gene transfer using TGF-beta DNA.

    PubMed Central

    Kuklin, N A; Daheshia, M; Chun, S; Rouse, B T

    1998-01-01

    This report evaluates the efficacy of DNA encoding TGF-beta administered mucosally to suppress immunity and modulate the immunoinflammatory response to herpes simplex virus (HSV) infection. A single intranasal administration of an eukaryotic expression vector encoding TGF-beta1 led to expression in the lung and lymphoid tissue. T cell-mediated immune responses to HSV infection were suppressed with this effect persisting as measured by the delayed-type hypersensitivity reaction for at least 7 wk. Treated animals were more susceptible to systemic infection with HSV. Multiple prophylactic mucosal administrations of TGF-beta DNA also suppressed the severity of ocular lesions caused by HSV infection, although no effects on this immunoinflammatory response were evident after therapeutic treatment with TGF-beta DNA. Our results demonstrate that the direct mucosal gene transfer of immunomodulatory cytokines provides a convenient means of modulating immunity and influencing the expression of inflammatory disorders. PMID:9664086

  20. BetaSCPWeb: side-chain prediction for protein structures using Voronoi diagrams and geometry prioritization

    PubMed Central

    Ryu, Joonghyun; Lee, Mokwon; Cha, Jehyun; Laskowski, Roman A.; Ryu, Seong Eon; Kim, Deok-Soo

    2016-01-01

    Many applications, such as protein design, homology modeling, flexible docking, etc. require the prediction of a protein's optimal side-chain conformations from just its amino acid sequence and backbone structure. Side-chain prediction (SCP) is an NP-hard energy minimization problem. Here, we present BetaSCPWeb which efficiently computes a conformation close to optimal using a geometry-prioritization method based on the Voronoi diagram of spherical atoms. Its outputs are visual, textual and PDB file format. The web server is free and open to all users at http://voronoi.hanyang.ac.kr/betascpweb with no login requirement. PMID:27151195

  1. Hemoglobin from the antarctic fish Notothenia coriiceps neglecta. Amino acid sequence of the beta chain.

    PubMed

    D'Avino, R; Caruso, C; Schinina, M E; Rutigliano, B; Romano, M; Camardella, L; Bossa, F; Barra, D; di Prisco, G

    1990-01-01

    1. Notothenia coriiceps neglecta is a cold-adapted notothenioid teleost, widely distributed in the Antarctic waters. 2. In comparison with fishes from temperate waters, the blood of this teleost contains a reduced number of erythrocytes and concentration of hemoglobin; the erythrocytes contain two hemoglobins, Hb1 and Hb2, respectively accounting for approximately 90, and 5% of the total. 3. The two components differ by the alpha chain; the amino acid sequence of the beta chain in common to the two hemoglobins has been established, thus completing the elucidation of the primary structure of the major component Hb 1.

  2. The linkage arrangement of four rabbit beta-like globin genes.

    PubMed

    Lacy, E; Hardison, R C; Quon, D; Maniatis, T

    1979-12-01

    Four different regions of rabbit beta-like globin gene sequences designated beta 1, beta 2, beta 3 and beta 4 were identified in a set of clones isolated from a bacteriophage lambda library of chromosomal DNA fragments (Maniatis et al., 1978). Restriction mapping and blot hybridization (Southern, 1975) studies indicate that a subset of these clones containing beta 1 and beta 2 hybridizes to an adult beta-globin cDNA clone (Maniatis et al., 1976) more efficiently than to a human gamma-globin cDNA clone (Wilson et al., 1978), while another subset containing beta 3 and beta 4 displays the converse hybridization specificity. beta 1 was identified as the adult beta-globin gene, while beta 2, beta 3 and beta 4 have not been identified with any known rabbit globin polypeptides. Cross-hybridization and transcriptional orientation experiments indicate that the set of beta-like gene clones contains overlapping restriction fragments encompassing 44 kb of rabbit chromosomal DNA. In addition, all four genes have the same transcriptional orientation and are arranged in the order 5'-beta 4-beta 3-beta 2-beta 1-3'.

  3. Molecular characterization of the gene for human interleukin-1[beta] converting enzyme (IL1BC)

    SciTech Connect

    Cerretti, D.P.; Hollingsworth, L.T.; Kozlosky, C.J.; Nelson, N. ); Valentine, M.B. ); Shapiro, D.N.; Morris, S.W. Univ. of Tennessee College of Medicine, Memphis, TN )

    1994-04-01

    Interleukin-1[beta] (IL-1[beta]) mediates a wide range of immune and inflammatory responses. The active cytokine is generated by proteolytic cleavage of an inactive precursor by a protease called the IL-1[beta] converting enzyme (ICE). A cDNA encoding this protease was recently isolated. A human genomic clone containing the ICE gene (IL1BC) was isolated using the cDNA as a probe. The gene consists of 10 exons spanning at least 10.6 kb. 5[prime]-anchored polymerase chain reaction indicated a single transcription start site [approximately]33 bp upstream of the initiator Met codon. The 5[prime]-flanking region does not have an apparent TATA box but may contain an initiator (Inr) promotor element. However, transcriptional activity could not be detected with a fusion gene containing the 5[prime]-flanking region linked to the bacterial chloramphenicol acetyltransferase gene (CAT) when transfected into the human acute monocytic leukemia cell line THP-1. Using the genomic IL1BC clone, the authors have confirmed the localization of the gene to chromosome 11 band q22.2-q22.3 by fluorescence in situ hybridization. 34 refs., 2 figs., 1 tab.

  4. Endomyocardial upregulation of beta1 adrenoreceptor gene expression and myocardial contractile reserve following cardiac resynchronization therapy.

    PubMed

    Vanderheyden, Marc; Mullens, Wilfried; Delrue, Leen; Goethals, Marc; Verstreken, Sofie; Wijns, William; de Bruyne, Bernard; Bartunek, Jozef

    2008-03-01

    Congestive heart failure (CHF) is associated with a blunted force-frequency relation (FFR) and myocardial contractile reserve (MCR) partially from a downregulation of beta1-adrenoreceptors (beta1-AR). We investigated whether acute and chronic cardiac resynchronization therapy (CRT) was capable of reversing the blunted FFR and MCR and if this was associated with upregulation of beta1-AR. Left ventricle dP/dtmax was invasively measured in 10 CHF patients (New York Heart Association class > or =3; ejection fraction <25%) during incremental dual chamber (DDD)-CRT pacing at 70, 90, 110, and 130 beats/min, with and without continuous infusion of intravenous dobutamine, immediately after CRT implantation (BL) and 4 months later (FU). In a subgroup of 5 patients, serial left ventricle beta1 and beta2-AR gene expression was measured using reverse transcriptase-polymerase chain reaction. Four months after the initiation of resynchronization therapy, DDD-CRT pacing results in a significant upward shift of the heart rate versus LV dP/dtmax relationship (P < .01) with force frequency amplification as evidenced by the steeper slope of the force frequency response (P = .04). Infusion of dobutamine recruits myocardial contractile reserve and increases the heart rate versus LV dP/dtmax relationship at BL and at FU (both P < .05). However, only at follow-up was an additional force frequency amplification noticed (P < .05) during dobutamine infusion. This observation was paralleled by a significant upregulation of beta1-AR gene expression (P = .02). Chronic CRT is associated with a partial restoration of the FFR and with a recruitment in myocardial contractile reserve, which is paralleled by upregulation of beta1-AR.

  5. Correction of human. beta. sup S -globin gene by gene targeting

    SciTech Connect

    Shesely, E.G.; Hyungsuk Kim; Shehee, W.R.; Smithies, O. ); Papayannopoulou, T. ); Popovich, B.W. )

    1991-05-15

    As a step toward using gene targeting for gene therapy, the authors have corrected a human {beta}{sup S}-globin gene to the normal {beta}{sup A} allele by homologous recombination in the mouse-human hybrid cell line BSM. BSM is derived from a mouse erythroleukemia cell line and carries a single human chromosome 11 with the {beta}{sup S}-globin allele. A {beta}{sup A}-globin targeting construct containing a unique oligomer and a neomycin-resistance gene was electroporated into the BSM cells, which were then placed under G418 selection. Then 126 resulting pools containing a total {approx}29,000 G418-resistant clones were screened by PCR for the presence of a targeted recombinant: 3 positive pools were identified. A targeted clone was isolated by replating one of the positive pools into smaller pools and rescreening by PCR, followed by dilution cloning. Southern blot analysis demonstrated that the isolated clone had been targeted as planned. The correction of the {beta}{sup S} allele to {beta}{sup A} was confirmed both by allele-specific PCR and by allele-specific antibodies. Expression studies comparing the uninduced and induced RNA levels in unmodified BSM cells and in the targeted clone showed no significant alteration in the ability of the targeted clone to undergo induction, despite the potentially disrupting presence of a transcriptionally active neomycin gene 5{prime} to the human {beta}{sup A}-globin gene. Thus gene targeting can correct a {beta}{sup S} allele to {beta}{sup A}, and the use of a selectable helper gene need not significantly interfere with the induction of the corrected gene.

  6. Gene expression of type 2 17 beta hydroxysteroid dehydrogenase in scalp hairs of hirsute women.

    PubMed

    Oliveira, Isabel O; Lhullier, Cintia; Brum, Ilma S; Spritzer, Poli Mara

    2003-09-01

    Androgens are the main hormonal regulators of human hair growth and they are related to clinical conditions such as hirsutism. The aim of this study was to analyze the gene expression of androgen receptor (AR) and type 2 17 beta hydroxysteroid dehydrogenase (17 beta-HSD) in keratinocytes of plucked scalp hairs from hirsute patients and normal subjects. We studied 58 women with hirsutism (31 with polycystic ovary syndrome (PCOS), 27 with idiopathic hirsutism (IH)); 15 control women; and 10 control men. Hirsutism was assessed by a modified Ferriman-Gallwey method. Hormonal status was assessed between days 2 and 10 of the menstrual cycle or on any day when the patients were amenorrheic. AR and type 2 17 beta-HSD mRNA levels were estimated by reverse transcription-polymerase chain reaction (RT-PCR). AR expression was similar in all groups. Type 2 17 beta-HSD gene expression in untreated hirsute patients was lower (2.1+/-0.10) than in normal women (3.1+/-0.17), and similar to men (1.8+/-0.22). Comparing hirsute patients, type 2 17 beta-HSD expression was higher in treated PCOS (3.0+/-0.34 versus 2.2+/-0.13) and IH patients (2.5+/-0.19 versus 2.0+/-0.15); hirsutism score was lower (P=0.003, PCOS; P=0.003, IH); and SHBG levels were higher (P=0.001, PCOS; P=0.024, IH) in treated patients. The free androgen index was lower in treated women (P=0.024 for the IH group). In conclusion, the lower expression of type 2 17 beta-HSD mRNA in scalp hairs of untreated hirsute patients suggests androgen metabolism disturbances with predominance of more potent androgens, as occurs in men. The enzyme's higher gene expression in treated hirsute patients could be an indirect evidence of restored enzyme activity and intracellular androgen metabolism.

  7. Detection of genes mediating beta-lactamase production in isolates of enterobacteria recovered from wild pets in Saudi Arabia

    PubMed Central

    Hassan, Sabry A.; Shobrak, Mohammed Y.

    2015-01-01

    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 TEM 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. PMID:27047051

  8. The genomic structure of the gene encoding the human transforming growth factor {beta} type II receptor (TGF-{beta} RII)

    SciTech Connect

    Takenoshita, Seiichi; Hagiwara, Koichi; Nagashima, Makoto; Gemma, Akihiko

    1996-09-01

    The genomic structure of the human transforming growth factor-{beta} type II receptor gene (TGF-{beta} RII) was determined by two PCR-based methods, the {open_quotes}long distance sequencer{close_quotes} method and the {open_quotes}promoter finder{close_quotes} method. Genomic fragments containing exons and adjacent introns were amplified by PCR, and the nucleotide sequences were determined by direct sequencing and subcloning sequencing. The TGF-{beta} RII protein is encoded by 567 codons in 7 exons. This is the first report about the genomic structure of a gene that belongs to the serine/threonine kinase type II receptor subfamily. Knowledge of the genomic structure of the TGF-{beta} RII gene will facilitate investigation of the TGF-{beta} RII gene will facilitate investigation of the TGF-{beta} signaling pathway in normal human cells and of the aberrations occurring during carcinogenesis. 18 refs., 2 figs., 1 tab.

  9. Effect of side-chain amide thionation on turnover of beta-lactam substrates by beta-lactamases. Further evidence on the question of side-chain hydrogen-bonding in catalysis.

    PubMed Central

    Pratt, R F; Krishnaraj, R; Xu, H

    1992-01-01

    Two side-chain-thionated beta-lactams, a penicillin and a cephalosporin, have been prepared and found to be not significantly poorer as substrates of typical serine (classes A and C) beta-lactamases than are their oxo analogues. This result is interpreted to mean that any hydrogen-bonding site on these enzymes for the beta-lactam side-chain amide carbonyl group must be flexible and is more likely to be a passive rather than active or essential feature of the active site. Previously, data from crystal structures and site-directed mutagenesis had suggested that the side chain of Asn-132 of class-A beta-lactamases, a component of the conserved SDN loop, forms a hydrogen bond with the side-chain carbonyl of the beta-lactam substrate and may provide significant transition-state stabilization during catalysis. The thionocephalosporin was also equally as good as its oxo analogue as a substrate of the class-B beta-lactamase II of Bacillus cereus and not significantly less effective as an inhibitor of the Streptomyces R61 DD-peptidase; a tight hydrogen-bond donor site for the beta-lactam side-chain amide is apparently not present in these enzymes either. PMID:1417747

  10. Effect of side-chain amide thionation on turnover of beta-lactam substrates by beta-lactamases. Further evidence on the question of side-chain hydrogen-bonding in catalysis.

    PubMed

    Pratt, R F; Krishnaraj, R; Xu, H

    1992-09-15

    Two side-chain-thionated beta-lactams, a penicillin and a cephalosporin, have been prepared and found to be not significantly poorer as substrates of typical serine (classes A and C) beta-lactamases than are their oxo analogues. This result is interpreted to mean that any hydrogen-bonding site on these enzymes for the beta-lactam side-chain amide carbonyl group must be flexible and is more likely to be a passive rather than active or essential feature of the active site. Previously, data from crystal structures and site-directed mutagenesis had suggested that the side chain of Asn-132 of class-A beta-lactamases, a component of the conserved SDN loop, forms a hydrogen bond with the side-chain carbonyl of the beta-lactam substrate and may provide significant transition-state stabilization during catalysis. The thionocephalosporin was also equally as good as its oxo analogue as a substrate of the class-B beta-lactamase II of Bacillus cereus and not significantly less effective as an inhibitor of the Streptomyces R61 DD-peptidase; a tight hydrogen-bond donor site for the beta-lactam side-chain amide is apparently not present in these enzymes either.

  11. Characterization of the mouse lymphotoxin-beta gene.

    PubMed

    Lawton, P; Nelson, J; Tizard, R; Browning, J L

    1995-01-01

    Lymphotoxin-beta (LT-beta) is a member of the TNF family of ligands which when expressed with lymphotoxin-alpha (LT-alpha, i.e., the original LT or TNF-beta) forms a heteromeric complex with LT-alpha on the cell surface. The mouse gene structure was determined by both cDNA cloning and analysis of a genomic DNA fragment encompassing the TNF/LT locus in the H-2 region of chromosome 17. The mouse and human genomic structures were found to be similar in terms of location in the class III region of the MHC; however, the mouse gene lacks one intron found in most members of the family. Both the cDNA and the genomic sequences revealed an altered splice donor in the conventional intron 2 position, rendering it nonfunctional. The altered gene retains an open reading frame such that an additional 66 amino acids are inserted into the stalk region connecting the transmembrane domain with the receptor binding domain encoded by exon 4 in this type II membrane protein. Northern analysis showed that this gene is expressed predominantly in lymphoid organs. The outlining of the complete mouse TNF locus will further studies of the relationship between these genes and immune function.

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

    PubMed

    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 < 0.05). Cows with LL genotype of GH produced more milk than cows with LVgenotype (P < 0.05). Also, for Pit-1 gene, animals with AB genotype produced more milk than BB genotype (P < 0.05). In the case of beta-LG gene, milk yield of animals with AA genotype was more than BB genotype (P < 0.01). Therefore, it might be concluded that homozygote genotypes of GH (LL) and beta-LG (AA) were superior compared to heterozygote genotypes, whereas, the heterozygote genotype of Pit-1 gene (AB) was desirable.

  13. Flow cytometric analysis of expression of interleukin-2 receptor beta chain (p70-75) on various leukemic cells

    SciTech Connect

    Hoshino, S.; Oshimi, K.; Tsudo, M.; Miyasaka, M.; Teramura, M.; Masuda, M.; Motoji, T.; Mizoguchi, H. )

    1990-08-15

    We analyzed the expression of the interleukin-2 receptor (IL-2R) beta chain (p70-75) on various leukemic cells from 44 patients by flow cytometric analysis using the IL-2R beta chain-specific monoclonal antibody, designated Mik-beta 1. Flow cytometric analysis demonstrated the expression of the IL-2R beta chain on granular lymphocytes (GLs) from all eight patients with granular lymphocyte proliferative disorders (GLPDs), on adult T-cell leukemia (ATL) cells from all three patients with ATL, and on T-cell acute lymphoblastic leukemia (T-ALL) cells from one of three patients with T-ALL. Although GLs from all the GLPD patients expressed the IL-2R beta chain alone and not the IL-2R alpha chain (Tac-antigen: p55), ATL and T-ALL cells expressing the beta chain coexpressed the alpha chain. In two of seven patients with common ALL (cALL) and in both patients with B-cell chronic lymphocytic leukemia, the leukemic cells expressed the alpha chain alone. Neither the alpha chain nor the beta chain was expressed on leukemic cells from the remaining 28 patients, including all 18 patients with acute nonlymphocytic leukemia, five of seven patients with cALL, all three patients with multiple myeloma, and two of three patients with T-ALL. These results indicate that three different forms of IL-2R chain expression exist on leukemic cells: the alpha chain alone; the beta chain alone; and both the alpha and beta chains. To examine whether the results obtained by flow cytometric analysis actually reflect functional aspects of the expressed IL-2Rs, we studied the specific binding of 125I-labeled IL-2 (125I-IL-2) to leukemic cells in 18 of the 44 patients. In addition, we performed 125I-IL-2 crosslinking studies in seven patients. The results of IL-2R expression of both 125I-IL-2 binding assay and crosslinking studies were in agreement with those obtained by flow cytometric analysis.

  14. Transcriptional activation of cloned human beta-globin genes by viral immediate-early gene products.

    PubMed

    Green, M R; Treisman, R; Maniatis, T

    1983-11-01

    When the human beta-globin gene is transfected into Hela cells, no beta-globin RNA is detected unless the gene is linked to a viral transcription enhancer. In this paper we show that trans-acting adenovirus and herpesvirus (pseudorabies) transcriptional regulatory proteins can circumvent this enhancer requirement for detectable beta-globin transcription in transient expression assays. The viral gene products can be provided by constitutively expressed, integrated viral genes in established cell lines, by viral infection of permissive cells, or by transfection of cells with bacterial plasmids carrying the viral immediate-early genes. These results demonstrate the utility of transient expression assays for studying regulatory mechanisms involving trans-acting factors. Analysis of beta-globin promoter mutants indicates that between 75 and 128 bp of sequence 5' to the mRNA cap site is required for enhancer-dependent transcription in Hela cells. In contrast, beta-globin transcription in the presence of viral immediate-early gene products requires only 36 bp of 5'-flanking sequence, which includes the TATA box. Thus both cis and trans-acting viral factors activate beta-globin gene transcription in transient expression experiments, but the mechanisms by which they act appear to be fundamentally different.

  15. Cloning of the elk TSH beta-subunit cDNA and seasonal expression of the pituitary glycoprotein hormone genes.

    PubMed

    Clark, Rena J; Furlan, Michael A; Chedrese, P Jorge

    2005-06-01

    We report the elk (Cervus elaphus) thyroid stimulating hormone (TSH) beta-subunit cDNA cloning, nucleotide and deduced amino acid sequences. The TSH beta-subunit cDNA was obtained by RT-PCR of polyadenylated pituitary RNA. The deduced elk TSH beta-subunit peptide chain shares between 93 to 99% sequence similarities with the reported TSH beta-subunit of a sub-set of related species. The TSH beta-subunit gene is expressed in the elk pituitary gland as a mature transcript of approximately 600 bases, which corresponds to the size of the mRNA expressed in the sheep pituitary gland. Seasonal expression of the pituitary gonadotropin genes was investigated by Northern blot analyses. Samples of elk pituitary glands collected during the breeding season showed elevated steady state levels of common alpha-subunit and FSH and LH beta-subunit gene expression, consistent with the seasonal reproductive cycling of this species. Samples collected before the breeding season demonstrated decreased expression of the gonadotropin genes. TSH, which is not directly tied to reproduction, had similar levels of expression, regardless of the animal's reproductive status.

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

    PubMed

    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.

  17. The beta104-109 sequence is essential for the secretion of correctly folded single-chain beta alpha horse LH/CG and for its FSH activity.

    PubMed

    Galet, Colette; Guillou, Florian; Foulon-Gauze, Florence; Combarnous, Yves; Chopineau, Maryse

    2009-10-01

    The dual LH and FSH activity of the equine LH (eLH)/equine chorionic gonadotropin (eCG) in heterologous species makes eLH/CG a good model to study structure/function relationships of gonadotropins. In order to bypass the problem of intracellular association of the heterodimer, a recombinant single-chain beta alpha eLH/CG was used to identify sequences in the beta-subunit involved in the secretion and activities of the hormone. The C-terminal region of the beta-subunit was progressively truncated. All resulting truncated single-chains were secreted in the media as detected by an anti-beta peptide antibody in reducing conditions. However, using a conformation sensitive ELISA we show that the truncated single-chains were differently recognized: deletion of the last 40 amino acids of the beta-subunit (beta109alpha eLH/CG) resulted in a 90% decrease in the recognized correctly folded hormone compared with the full-length beta alpha eLH/CG single-chain and no properly folded hormone was detected in the secretion medium when the last 46 amino acids of the beta-subunit were deleted (beta103alpha eLH/CG). We thus focused on the six amino acids sequence 104-109, which belongs to the seat-belt region. Mutation of the 104-109 sequence in alanines in the full-length beta alpha eLH/CG (beta104-109Ala alpha) led to a 50% decrease in the production of properly folded hormone in COS-7 as well as in alphaT3 pituitary cells. Moreover, the FSH activity of this mutant was decreased by 70% whereas its LH activity remained intact. These data lead us to conclude that the 104-109 region of the beta eLH/CG subunit is essential for the secretion of a fully folded beta alpha eLH/CG and for its FSH activity but not for its LH activity.

  18. Allelic polymorphism in IL-1 beta and IL-1 receptor antagonist (IL-1Ra) genes in inflammatory bowel disease.

    PubMed Central

    Bioque, G; Crusius, J B; Koutroubakis, I; Bouma, G; Kostense, P J; Meuwissen, S G; Peña, A S

    1995-01-01

    Recent reports have shown that allele 2 of the IL-1 receptor antagonist (IL-1Ra) gene is over-represented in ulcerative colitis (UC). Healthy individuals carrying allele 2 of this gene have increased production of IL-1Ra protein. Since the final outcome of the biological effects of IL-1 beta may depend on the relative proportion of these two cytokines, we have studied if a TaqI polymorphism in the IL-1 beta gene, which is relevant to IL-1 beta protein production, may be involved in the genetic susceptibility to UC and Crohn's disease (CD), in association with the established IL-1Ra gene polymorphism. Polymorphisms in the closely linked genes for IL-1 beta and IL-1Ra were typed in 100 unrelated Dutch patients with UC, 79 with CD, and 71 healthy controls. The polymorphic regions in exon 5 of the IL-1 beta gene and in intron 2 of the IL-1Ra gene, were studied by polymerase chain reaction (PCR)-based methods. The IL-1 beta allele frequencies in UC and CD patients did not differ from those in healthy controls. In order to study if the IL-1 beta gene polymorphism might participate synergistically with the IL-1Ra gene polymorphism in susceptibility to UC and CD, individuals were distributed into carriers and non-carriers of allele 2 of the genes encoding IL-1 beta and IL-1Ra, in each of the patient groups and controls. Results indicated a significant association of this pair of genes, estimated by the odds ratio (OR) after performing Fisher's exact test, in the UC group (P = 0.023, OR = 2.81), as well as in the CD group (P = 0.01, OR = 3.79). Thus, non-carriers of IL-1 beta allele 2 were more often present in the subgroup of patients carrying the IL-1Ra allele 2. By contrast, no association of these alleles was detected in the group of healthy controls (P = 1.00, OR = 0.92). These results suggest that the IL-1 beta/IL-1Ra allelic cluster may participate in defining the biological basis of predisposition to chronic inflammatory bowel diseases. PMID:7586694

  19. Medium-chain fatty acids undergo elongation before {beta}-oxidation in fibroblasts

    SciTech Connect

    Jones, Patricia M. . E-mail: Patti.Jones@childrens.com; Butt, Yasmeen; Messmer, Bette; Boriak, Richard; Bennett, Michael J.

    2006-07-21

    Although mitochondrial fatty acid {beta}-oxidation (FAO) is considered to be well understood, further elucidation of the pathway continues through evaluation of patients with FAO defects. The FAO pathway can be examined by measuring the 3-hydroxy-fatty acid (3-OHFA) intermediates. We present a unique finding in the study of this pathway: the addition of medium-chain fatty acids to the culture media of fibroblasts results in generation of 3-OHFAs which are two carbons longer than the precursor substrate. Cultured skin fibroblasts from normal and LCHAD-deficient individuals were grown in media supplemented with various chain-length fatty acids. The cell-free medium was analyzed for 3-OHFAs by stable-isotope dilution gas-chromatography/mass-spectrometry. Our finding suggests that a novel carbon chain-length elongation process precedes the oxidation of medium-chain fatty acids. This previously undescribed metabolic step may have important implications for the metabolism of medium-chain triglycerides, components in the dietary treatment of a number of disorders.

  20. Expression of the leukemia-associated CBF{beta}/SMMHC chimeric gene causes transformation of 3T3 cells

    SciTech Connect

    Hajra, A.; Liu, P.; Collins, E.S.

    1994-09-01

    A pericentric inversion of chromosome 16 (inv(16)(p13;q22)) is consistently seen in acute myeloid leukemia of the M4Eo subtype. This inversion fuses almost the entire coding region of the gene encoding of the {beta} subunit of the heterodimeric transcription factor CBF/PEBP2 to the region of the MYH11 gene encoding the rod domain for the smooth muscle myosin heavy chain (SMMHC). To investigate the biological properties of the CBF{beta}/SMMHC fusion protein, we have generated 3T3 cell lines that stably express the CBF{beta}/SMMHC chimeric cDNA or the normal, nonchimeric CBF{beta} and SMMHC cDNAs. 3T3 cells expressing CBF{beta}/SMMHC acquire a transformed phenotype, as indicated by altered cell morphology, formation of foci, and growth in soft agar. Cells constitutively overexpressing the normal CBF{beta} cDNA or the rod region of SMMHC remain nontransformed. Western blot analysis using antibodies to CBF{beta} and the SMMHC rod demonstrates that stably transfected cells express the appropriate chimeric or normal protein. Electrophoretic mobility shift assays reveal that cells transformed by the chimeric cDNA do not have a CBF-DNA complex of the expected mobility, but instead contain a large complex with CBF DNA-binding activity that fails to migrate out of the gel wells. In order to define the regions of CBF{beta}/SMMHC necessary for 3T3 transformation, we have stably transfected cells with mutant CBF{beta}/SMMHC cDNAs containing various deletions of the coding region. Analysis of these cell lines indicates that the transformation property of CBF{beta}/SMMHC requires regions of CBF{beta} known to be necessary for association with the DNA-binding CBF{alpha} subunit, and also requires an intact SMMHC carboxyl terminus, which is necessary for formation of the coiled coil domain of the myosin rod.

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

    PubMed

    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.

  2. Light chain replacement: a new model for antibody gene rearrangement

    PubMed Central

    1995-01-01

    A functional B cell antigen receptor is thought to regulate antibody gene rearrangement either by stopping further rearrangement (exclusion) or by promoting additional rearrangement (editing). We have developed a new model to study the regulation of antibody gene rearrangement. In this model, we used gene targeting to replace the J kappa region with a functional V kappa-J kappa light chain gene. Two different strains of mice were created; one, V kappa 4R, has a V kappa 4-J kappa 4 rearrangement followed by a downstream J kappa 5 segment, while the other, V kappa 8R, has a V kappa 8-J kappa 5 light chain. Here, we analyze the influence of these functional light chains on light chain rearrangement. We show that some V kappa 4R and V kappa 8R B cells only have the V kappa R light chain rearrangement, whereas others undergo additional rearrangements. Additional rearrangement can occur not only at the other kappa allele or isotype (lambda), but also at the targeted locus in both V kappa 4R and V kappa 8R. Rearrangement to the downstream J kappa 5 segment is observed in V kappa 4R, as is deletion of the targeted locus in both V kappa 4R and V kappa 8R. The V kappa R models illustrate that a productively rearranged light chain can either terminate further rearrangement or allow further rearrangement. We attribute the latter to editing of autoantibodies and to corrections of dysfunctional receptors. PMID:7629511

  3. Distribution of beta-glucosidase and beta-glucuronidase activity and of beta-glucuronidase gene gus in human colonic bacteria.

    PubMed

    Dabek, Marta; McCrae, Sheila I; Stevens, Valerie J; Duncan, Sylvia H; Louis, Petra

    2008-12-01

    beta-Glycosidase activities present in the human colonic microbiota act on glycosidic plant secondary compounds and xenobiotics entering the colon, with potential health implications for the human host. Information on beta-glycosidases is currently limited to relatively few species of bacteria from the human colonic ecosystem. We therefore screened 40 different bacterial strains that are representative of dominant bacterial groups from human faeces for beta-glucosidase and beta-glucuronidase activity. More than half of the low G+C% Gram-positive firmicutes harboured beta-glucosidase activity, while beta-glucuronidase activity was only found in some firmicutes within clostridial clusters XIVa and IV. Most of the Bifidobacterium spp. and Bacteroides thetaiotaomicron carried beta-glucosidase activity. A beta-glucuronidase gene belonging to family 2 glycosyl hydrolases was detected in 10 of the 40 isolates based on degenerate PCR. These included all nine isolates that gave positive assays for beta-glucuronidase activity, suggesting that the degenerate PCR could provide a useful assay for the capacity to produce beta-glucuronidase in the gut community. beta-Glucuronidase activity was induced by growth on d-glucuronic acid, or by addition of 4-nitrophenol-glucuronide, in Roseburia hominis A2-183, while beta-glucosidase activity was induced by 4-nitrophenol-glucopyranoside. Inducibility varied between strains.

  4. Beta-oxidation in hepatocyte cultures from mice with peroxisomal gene knockouts.

    PubMed

    Dirkx, Ruud; Meyhi, Els; Asselberghs, Stanny; Reddy, Janardan; Baes, Myriam; Van Veldhoven, Paul P

    2007-06-08

    Beta-oxidation of carboxylates takes place both in mitochondria and peroxisomes and in each pathway parallel enzymes exist for each conversion step. In order to better define the substrate specificities of these enzymes and in particular the elusive role of peroxisomal MFP-1, hepatocyte cultures from mice with peroxisomal gene knockouts were used to assess the consequences on substrate degradation. Hepatocytes from mice with liver selective elimination of peroxisomes displayed severely impaired oxidation of 2-methylhexadecanoic acid, the bile acid intermediate trihydroxycholestanoic acid (THCA), and tetradecanedioic acid. In contrast, mitochondrial beta-oxidation rates of palmitate were doubled, despite the severely affected inner mitochondrial membrane. As expected, beta-oxidation of the branched chain compounds 2-methylhexadecanoic acid and THCA was reduced in hepatocytes from mice with inactivation of MFP-2. More surprisingly, dicarboxylic fatty acid oxidation was impaired in MFP-1 but not in MFP-2 knockout hepatocytes, indicating that MFP-1 might play more than an obsolete role in peroxisomal beta-oxidation.

  5. Chronic tonsillitis is not associated with beta defensin 1 gene polymorphisms in Turkish population.

    PubMed

    Arslan, Fatih; Babakurban, Seda Turkoglu; Erbek, Selim S; Sahin, Feride I; Terzi, Yunus Kasım

    2015-04-01

    Defensins are antimicrobial peptides expressed on mucosal surfaces. They function as part of the innate immune system. Palatine tonsils play important roles in innate immune system. However, our knowledge on the pathophysiology of chronic tonsils is limited. The aim of this study was to investigate the association between beta defensin 1 gene single nucleotide polymorphisms and chronic tonsillitis. Prospective, non-randomized, controlled clinical study. Tertiary referral center. Eighty six patients with chronic tonsillitis and eighty controls without history of chronic tonsillitis were enrolled in this study. Genotypes were determined by restriction fragment length polymorphism analyses after polymerase chain reaction. Genotype and allele frequencies of the -20G/A (rs11362), -44C/G (rs1800972) and -52G/A (rs1799946) single nucleotide polymorphisms were not statistically different between patients and control groups (p>0.05). In this study, we found that DEFB1 gene -20G/A, -44C/G and -52G/A single nucleotide polymorphisms were not associated with chronic tonsillitis. Studies, which analyse other polymorphism of the beta defensin 1 gene in large case series, should be conducted to understand the role of DEFB1 gene on chronic tonsillitis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  6. Salt, phosphate and the Bohr effect at the hemoglobin beta chain C terminus studied by hydrogen exchange.

    PubMed

    Louie, G; Englander, J J; Englander, S W

    1988-06-20

    Hydrogen exchange experiments using functional labeling and fragment separation methods were performed to study interactions at the C terminus of the hemoglobin beta subunit that contribute to the phosphate effect and the Bohr effect. The results show that the H-exchange behavior of several peptide NH at the beta chain C terminus is determined by a transient, concerted unfolding reaction involving five or more residues, from the C-terminal His146 beta through at least Ala142 beta, and that H-exchange rate can be used to measure the stabilization free energy of interactions, both individually and collectively, at this locus. In deoxy hemoglobin at pH 7.4 and 0 degrees C, the removal of 2,3-diphosphoglycerate (DPG) or pyrophosphate (loss of a salt to His143 beta) speeds the exchange of the beta chain C-terminal peptide NH protons by 2.5-fold (at high salt), indicating a destabilization of the C-terminal segment by 0.5 kcal of free energy. Loss of the His146 beta 1 to Asp94 beta 1 salt link speeds all these protons by 6.3-fold, indicating a bond stabilization free energy of 1.0 kcal. When both these salt links are removed together, the effect is found to be strictly additive; all the protons exchange faster by 16-fold indicating a loss of 1.5 kcal in stabilization free energy. Added salt is slightly destabilizing when DPG is present but provides some increased stability, in the 0.2 kcal range, when DPG is absent. The total allosteric stabilization energy at each beta chain C terminus in deoxy hemoglobin under these conditions is measured to be 3.8 kcal (pH 7.4, 0 degrees C, with DPG). In oxy hemoglobin at pH 7.4 and 0 degrees C, stability at the beta chain C terminus is essentially independent of salt concentration, and the NES modification, which in deoxy hemoglobin blocks the His146 beta to Asp94 beta salt link, has no destabilizing effect, either at high or low salt. These results appear to show that the His146 beta salt link, which participates importantly in the

  7. Molecular analysis of abnormal hemoglobins in beta chain in Aegean region of Turkey and first reports of hemoglobin Andrew-Minneapolis and Hb Hinsdale from Turkey.

    PubMed

    Aykut, Ayça; Onay, Hüseyin; Durmaz, Asude; Karaca, Emin; Vergin, Canan; Aydınok, Yeşim; Özkınay, Ferda

    2015-07-01

    The Agean is one of the regions in Turkey where thalassemias and abnormal hemoglobins (Hbs) are prevalent. Combined heterozygosity of thalassemia mutations with a variety of structural Hb variants lead to an extremely wide spectrum of clinical and hematological phenotypes which is of importance for prenatal diagnosis. One hundred and seventeen patients and carriers diagnosed by hemoglobin electrophoresis (HPLC), at risk for abnormal hemoglobinopathies were screened for mutational analysis of the beta-globin gene. The full coding the 5' UTR, and the 3' UTR sequences of beta-globin gene (GenBank accession no. U01317) were amplified and sequenced. In this study, a total of 118 (12.24%) structural Hb variant alleles were identified in 1341 mutated beta-chain alleles in Medical Genetics Department of Ege University between January 2006 and November 2013. Here, we report the mutation spectrum of abnormal Hbs associated with the beta-globin gene in Aegean region of Turkey. In the present study, the Hb Hinsdale and Hb Andrew-Minneapolis variants are demonstrated for the first time in the Turkish population.

  8. Position-independent human beta-globin gene expression mediated by a recombinant adeno-associated virus vector carrying the chicken beta-globin insulator.

    PubMed

    Inoue, T; Yamaza, H; Sakai, Y; Mizuno, S; Ohno, M; Hamasaki, N; Fukumaki, Y

    1999-01-01

    The position-independent expression of transgenes in target cells is an essential subject for determining effective gene therapies. The chicken beta-globin insulator blocks the effects of regulatory sequences on transcriptional units at differential domains. We prepared a recombinant adeno-associated virus (rAAV) containing various combinations of the DNase I-hypersensitive site 2 (HS2), 3 (HS3), and 4 (HS4) core elements from the human beta-globin locus control region (LCR), the human beta-globin gene, and the herpes virus thymidine kinase promoter driven neomycin-resistant gene (neoR) (rHS432, rHS43, rHS42, rHS32, and rHS2), and also rAAV containing two copies of the 250-bp core sequence of the chicken beta-globin insulator on both sides of the rHS2 (rIns/HS2/2Ins). After isolating neomycin-resistant mouse erythroleukemia (MEL) cells infected with each rAAV, we analyzed the rAAV genome by Southern blots and polymerase chain reaction (PCR), using primers specific for HS core elements and the human beta-globin gene. All clones contained a single copy of the rAAV genome in the chromosome, however, some of them had a rearranged proviral genome. In five clones with a single unrearranged rAAV genome for each rAAV construct, we assayed the expression of the human b-globin gene relative to the endogenous mouse beta maj-globin gene, using quantitative reverse transcriptase (RT)-PCR. In clones infected with rHS432, the expression level of the human beta-globin gene ranged from 51.6% to 765.6% of that in the mouse beta maj-globin gene. Likewise, in rHS43, the expression level ranged from 36.7% to 259.0%; in rHS42, from 47.8% to 207.0%; in rHS32, from 47.9% to 105.4%; and in rHS2, from 6.1% to 172.1%, indicating a high variability of expression level in clones infected with recombinant virus lacking the insulator. In contrast, in clones infected with rIns/HS2/Ins, the range of expression of the human beta-globin gene ranged from 52.8% to 58.3% of that in the mouse beta maj

  9. A chain reaction approach to modelling gene pathways

    PubMed Central

    Cheng, Gary C.; Chen, Dung-Tsa; Chen, James J.; Soong, Seng-jaw; Lamartiniere, Coral; Barnes, Stephen

    2012-01-01

    Background Of great interest in cancer prevention is how nutrient components affect gene pathways associated with the physiological events of puberty. Nutrient-gene interactions may cause changes in breast or prostate cells and, therefore, may result in cancer risk later in life. Analysis of gene pathways can lead to insights about nutrient-gene interactions and the development of more effective prevention approaches to reduce cancer risk. To date, researchers have relied heavily upon experimental assays (such as microarray analysis, etc.) to identify genes and their associated pathways that are affected by nutrient and diets. However, the vast number of genes and combinations of gene pathways, coupled with the expense of the experimental analyses, has delayed the progress of gene-pathway research. The development of an analytical approach based on available test data could greatly benefit the evaluation of gene pathways, and thus advance the study of nutrient-gene interactions in cancer prevention. In the present study, we have proposed a chain reaction model to simulate gene pathways, in which the gene expression changes through the pathway are represented by the species undergoing a set of chemical reactions. We have also developed a numerical tool to solve for the species changes due to the chain reactions over time. Through this approach we can examine the impact of nutrient-containing diets on the gene pathway; moreover, transformation of genes over time with a nutrient treatment can be observed numerically, which is very difficult to achieve experimentally. We apply this approach to microarray analysis data from an experiment which involved the effects of three polyphenols (nutrient treatments), epigallo-catechin-3-O-gallate (EGCG), genistein, and resveratrol, in a study of nutrient-gene interaction in the estrogen synthesis pathway during puberty. Results In this preliminary study, the estrogen synthesis pathway was simulated by a chain reaction model. By

  10. A chain reaction approach to modelling gene pathways.

    PubMed

    Cheng, Gary C; Chen, Dung-Tsa; Chen, James J; Soong, Seng-Jaw; Lamartiniere, Coral; Barnes, Stephen

    2012-08-01

    BACKGROUND: Of great interest in cancer prevention is how nutrient components affect gene pathways associated with the physiological events of puberty. Nutrient-gene interactions may cause changes in breast or prostate cells and, therefore, may result in cancer risk later in life. Analysis of gene pathways can lead to insights about nutrient-gene interactions and the development of more effective prevention approaches to reduce cancer risk. To date, researchers have relied heavily upon experimental assays (such as microarray analysis, etc.) to identify genes and their associated pathways that are affected by nutrient and diets. However, the vast number of genes and combinations of gene pathways, coupled with the expense of the experimental analyses, has delayed the progress of gene-pathway research. The development of an analytical approach based on available test data could greatly benefit the evaluation of gene pathways, and thus advance the study of nutrient-gene interactions in cancer prevention. In the present study, we have proposed a chain reaction model to simulate gene pathways, in which the gene expression changes through the pathway are represented by the species undergoing a set of chemical reactions. We have also developed a numerical tool to solve for the species changes due to the chain reactions over time. Through this approach we can examine the impact of nutrient-containing diets on the gene pathway; moreover, transformation of genes over time with a nutrient treatment can be observed numerically, which is very difficult to achieve experimentally. We apply this approach to microarray analysis data from an experiment which involved the effects of three polyphenols (nutrient treatments), epigallo-catechin-3-O-gallate (EGCG), genistein, and resveratrol, in a study of nutrient-gene interaction in the estrogen synthesis pathway during puberty. RESULTS: In this preliminary study, the estrogen synthesis pathway was simulated by a chain reaction model. By

  11. Cloning of alpha-beta fusion gene from Clostridium perfringens and its expression.

    PubMed

    Bai, Jia-Ning; Zhang, Yan; Zhao, Bao-Hua

    2006-02-28

    To study the cloning of alpha-beta fusion gene from Clostridium perfringens and the immunogenicity of alpha-beta fusion expression. Cloning was accomplished after PCR amplification from strains NCTC64609 and C58-1 of the protective antigen genes of alpha-toxin and beta-toxin. The fragment of the gene was cloned using plasmid pZCPAB. This fragment coded for the gene with the stable expression of alpha-beta fusion gene binding. In order to verify the exact location of the alpha-beta fusion gene, domain plasmids were constructed. The two genes were fused into expression vector pBV221. The expressed alpha-beta fusion protein was identified by ELISA, SDS-PAGE, Western blotting and neutralization assay. The protective alpha-toxin gene (cpa906) and the beta-toxin gene (cpb930) were obtained. The recombinant plasmid pZCPAB carrying alpha-beta fusion gene was constructed and transformed into BL21(DE3). The recombinant strain BL21(DE3)(pZCPAB) was obtained. After the recombinant strain BL21(DE3)(pZCPAB) was induced by 42 degC, its expressed product was about 22.14% of total cellular protein at SDS-PAGE and thin-layer gel scanning analysis. Neutralization assay indicated that the antibody induced by immunization with alpha-beta fusion protein could neutralize the toxicity of alpha-toxin and beta-toxin. The obtained alpha-toxin and beta-toxin genes are correct. The recombinant strain BL21(DE3)(pZCPAB) could produce alpha-beta fusion protein. This protein can be used for immunization and is immunogenic. The antibody induced by immunization with alpha-beta fusion protein could neutralize the toxicity of alpha-toxin and beta-toxin.

  12. Thrombospondin interaction with fibrinogen. Evidence for binding to the A alpha- and B beta-chains of fibrinogen.

    PubMed

    Bacon-Baguley, T; Kudryk, B J; Walz, D A

    1987-02-15

    Platelet thrombospondin interacts with fibrinogen in a specific and saturable manner. Thrombospondin was found to specifically bind to the A alpha- and B beta-chains of fibrinogen; binding was independent of divalent ions. Binding could be blocked either by preincubation of thrombospondin with 9.4 microM fibrinogen or by preincubation of fibrinogen with 1.1 nM thrombospondin. Thrombospondin bound only to the beta-chain component of the D and DD plasmin fragment of fibrinogen. Thrombospondin interaction with fibrinogen was not blocked by preincubation with synthetic peptides which have previously been identified as either the fibrinogen receptor (alpha 572-575, the synthetic tetrapeptide arginyl-glycyl-aspartyl-serine) or cell attachment (gamma 400-411) domains. Fibrinogen, therefore, possesses at least two unique and distinct sites, within the A alpha- and B beta-chains, for its interaction with thrombospondin.

  13. Gene Prioritization by Compressive Data Fusion and Chaining

    PubMed Central

    Žitnik, Marinka; Nam, Edward A.; Dinh, Christopher; Kuspa, Adam; Shaulsky, Gad; Zupan, Blaž

    2015-01-01

    Data integration procedures combine heterogeneous data sets into predictive models, but they are limited to data explicitly related to the target object type, such as genes. Collage is a new data fusion approach to gene prioritization. It considers data sets of various association levels with the prediction task, utilizes collective matrix factorization to compress the data, and chaining to relate different object types contained in a data compendium. Collage prioritizes genes based on their similarity to several seed genes. We tested Collage by prioritizing bacterial response genes in Dictyostelium as a novel model system for prokaryote-eukaryote interactions. Using 4 seed genes and 14 data sets, only one of which was directly related to the bacterial response, Collage proposed 8 candidate genes that were readily validated as necessary for the response of Dictyostelium to Gram-negative bacteria. These findings establish Collage as a method for inferring biological knowledge from the integration of heterogeneous and coarsely related data sets. PMID:26465776

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

    SciTech Connect

    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.

  15. DNP-specific/class I MHC-restricted suppressor molecules bear determinants of the T cell receptor alpha- and beta-chains. The V beta 8+ chain dictates restriction to either K or D.

    PubMed

    Fairchild, R L; Kubo, R T; Moorhead, J W

    1990-10-01

    To examine in greater detail the relationship between DNP-specific/class I MHC-restricted suppressor molecules (SSF) that inhibit contact sensitivity to 2,4-dinitrofluorobenzene and the receptors on the T cells that produce them, we have generated two T cell hybridomas that can be induced to produce and secrete these molecules. In order to become activated to produce SSF, the Ts 15.15 and 15.31 cells required recognition of complexes of DNP/Dd on presenting cells. The suppressor molecules produced by each of the Ts hybrids had the same specificity, recognizing DNP/Dd on cells in the immune lymph node cell target population. The activation of the Ts hybrids was blocked when the cells were treated with the anti-V beta 8 antibody F23.1 before coculture with the DNP-presenting cells. Reduction of the 15.15 and 15.31 SSF followed by affinity chromatography on DNP-bovine-gamma-globulin-Sepharose beads indicated that these molecules are dimers and that one of the chains (Ag-binding(AgB] binds to cellfree DNP and one (non-Ag-binding (NAgB) chain) does not. The AgB chain was found to express an epitope bound by a mAb specific for a TCR alpha-chain-constant region determinant. Alternatively, the NAgB chain expressed an epitope bound by the anti-V beta 8 mAb F23.1. Active hybrid suppressor molecules were generated by combining the NAgB chain from a DNP-specific/H-2Kd-restricted SSF (produced by Ts hybridoma 3-10) with the AgB chain from Ts 15.31 and by combining the NAgB chain from Ts cell 15.15 with the 3-10 AgB chain. In each case, the class I MHC element (i.e., Kd or Dd) restricting the activity of these hybrid SSF correlated with the source of the V beta 8+, NAgB chain. Thus, these secreted immunoregulatory molecules have the Ag/MHC specificity of the T cells producing them and are structurally and serologically related to the TCR-alpha/beta. The results further suggest that for some hapten-specific/class I MHC-restricted TCR, the alpha-chain may have avidity for the

  16. Isolation and identification of a cDNA clone corresponding to an HLA-DR antigen beta chain.

    PubMed

    Wiman, K; Larhammar, D; Claesson, L; Gustafsson, K; Schenning, L; Bill, P; Böhme, J; Denaro, M; Dobberstein, B; Hammerling, U; Kvist, S; Servenius, B; Sundelin, J; Peterson, P A; Rask, L

    1982-03-01

    The HLA-D locus in the major histocompatibility complex controls the expression of the genetically polymorphic HLA-DR antigens. mRNA coding for the beta chains of these antigens was partially purified from the human lymphoblastoid cell line Raji. The mRNA was copied into double-stranded cDNA and cloned in Escherichia coli. One clone, pDR-beta-1, obtained by hybrid selection, carries a 1070-base-pair insert comprising all of the coding region except the signal sequence and a substantial portion of the untranslated region. To identify pDR-beta-1, highly purified HLA-DR antigen beta chains derived from Raji cells were subjected to NH2-terminal amino acid sequence determination. This sequence displayed extensive homology with that deduced from the nucleotide sequence at the 5' end of the pDR-beta-1 coding region. Taken together, the amino acid and nucleotide sequences strongly argue in favor of Raji cells containing at least two beta-chain loci.

  17. Evidence for beta1-adrenergic receptor involvement in amygdalar corticotropin-releasing factor gene expression: implications for cocaine withdrawal.

    PubMed

    Rudoy, Carla A; Reyes, Arith-Ruth S; Van Bockstaele, Elisabeth J

    2009-04-01

    We previously showed that betaxolol, a selective beta(1)-adrenergic receptor antagonist, administered during early phases of cocaine abstinence, ameliorated withdrawal-induced anxiety and blocked increases in amygdalar beta(1)-adrenergic receptor expression in rats. Here, we report the efficacy of betaxolol in reducing increases in gene expression of amygdalar corticotropin-releasing factor (CRF), a peptide known to be involved in mediating 'anxiety-like' behaviors during initial phases of cocaine abstinence. We also demonstrate attenuation of an amygdalar beta(1)-adrenergic receptor-mediated cell-signaling pathway following this treatment. Male rats were administered betaxolol at 24 and 44 h following chronic cocaine administration. Animals were euthanized at the 48-h time point and the amygdala was microdissected and processed for quantitative reverse transcriptase-polymerase chain reaction and/or western blot analysis. Results showed that betaxolol treatment during early cocaine withdrawal attenuated increases in amygdalar CRF gene expression and cyclic adenosine monophosphate-dependent protein kinase regulatory and catalytic subunit (nuclear fraction) protein expression. Our data also reveal that beta(1)-adrenergic receptors are on amygdalar neurons, which are immunoreactive for CRF. The present findings suggest that the efficacy of betaxolol treatment on cocaine withdrawal-induced anxiety may be related, in part, to its effect on amygdalar beta(1)-adrenergic receptor, modulation of its downstream cell-signaling elements and CRF gene expression.

  18. The amino acid sequence of Canada goose (Branta canadensis) and mute swan (Cygnus olor) hemoglobins. Two different species with identical beta-chains.

    PubMed

    Oberthür, W; Godovac-Zimmermann, J; Braunitzer, G; Wiesner, H

    1982-08-01

    The amino acid sequences of the alpha- and beta-chains from the major hemoglobin component (HbA) of Canada goose (Branta canadensis) and mute swan (Cygnus olor) are given. The alpha-chains are of the alpha A-type, since alpha D-type was expressed but only found in low concentrations. By homologous comparison, greylag goose hemoglobin (Anser anser) and Canada goose hemoglobin alpha-chains differ by two exchanges, and beta-chains by three exchanges. A valine substitution for threonine was found at position alpha 34 (B15). This exchange is a result of a two point mutation. Thus, there are three nucleotide mutations in alpha-chains, as in beta-chains. Substitutions in positions alpha 34 (B15) and beta 125 (H3) have modified intersubunit contacts (alpha 1 beta 1-contacts). A comparison of mute swan hemoglobin with greylag goose hemoglobin shows four exchanges in alpha-chains and three in beta-chains. Canada goose and mute swan have identical beta-chains, while alpha-chains differ in two amino acids. One of these exchanges is implicated in one of the alpha 1 beta 1-contact points (alpha 34) where isoleucine substitution for valine was found. Comparison of hemoglobins from different species in the same tribe (Anserini) shows a high homology between Canada goose and mute swan hemoglobins.

  19. Biased Immunoglobulin Light Chain Gene Usage in the Shark.

    PubMed

    Iacoangeli, Anna; Lui, Anita; Naik, Ushma; Ohta, Yuko; Flajnik, Martin; Hsu, Ellen

    2015-10-15

    This study of a large family of κ L chain clusters in nurse shark completes the characterization of its classical Ig gene content (two H chain isotypes, μ and ω, and four L chain isotypes, κ, λ, σ, and σ-2). The shark κ clusters are minigenes consisting of a simple VL-JL-CL array, where V to J recombination occurs over an ~500-bp interval, and functional clusters are widely separated by at least 100 kb. Six out of ~39 κ clusters are prerearranged in the germline (germline joined). Unlike the complex gene organization and multistep assembly process of Ig in mammals, each shark Ig rearrangement, somatic or in the germline, appears to be an independent event localized to the minigene. This study examined the expression of functional, nonproductive, and sterile transcripts of the κ clusters compared with the other three L chain isotypes. κ cluster usage was investigated in young sharks, and a skewed pattern of split gene expression was observed, one similar in functional and nonproductive rearrangements. These results show that the individual activation of the spatially distant κ clusters is nonrandom. Although both split and germline-joined κ genes are expressed, the latter are prominent in young animals and wane with age. We speculate that, in the shark, the differential activation of the multiple isotypes can be advantageously used in receptor editing.

  20. Biased immunoglobulin light chain gene usage in the shark1

    PubMed Central

    Iacoangeli, Anna; Lui, Anita; Naik, Ushma; Ohta, Yuko; Flajnik, Martin; Hsu, Ellen

    2015-01-01

    This study of a large family of kappa light (L) chain clusters in nurse shark completes the characterization of its classical immunoglobulin (Ig) gene content (two heavy chain classes, mu and omega, and four L chain isotopes, kappa, lambda, sigma, and sigma-2). The shark kappa clusters are minigenes consisting of a simple VL-JL-CL array, where V to J recombination occurs over a ~500 bp interval, and functional clusters are widely separated by at least 100 kb. Six out of ca. 39 kappa clusters are pre-rearranged in the germline (GL-joined). Unlike the complex gene organization and multistep assembly process of Ig in mammals, each shark Ig rearrangement, somatic or in the germline, appears to be an independent event localized to the minigene. This study examined the expression of functional, non-productive, and sterile transcripts of the kappa clusters compared to the other three L chain isotypes. Kappa cluster usage was investigated in young sharks, and a skewed pattern of split gene expression was observed, one similar in functional and non-productive rearrangements. These results show that the individual activation of the spatially distant kappa clusters is non-random. Although both split and GL-joined kappa genes are expressed, the latter are prominent in young animals and wane with age. We speculate that, in the shark, the differential activation of the multiple isotypes can be advantageously used in receptor editing. PMID:26342033

  1. Association of single nucleotide gene polymorphism at interleukin-1beta +3954, -511, and -31 in chronic periodontitis and aggressive periodontitis in Dravidian ethnicity.

    PubMed

    Shete, Abhijeet Rajendra; Joseph, Rosamma; Vijayan, Neetha N; Srinivas, Lekshmy; Banerjee, Moinak

    2010-01-01

    Interleukin (IL)-1beta gene polymorphisms are considered a potential risk factor for periodontal disease. The aim of this study is to identify the association of IL-1beta gene polymorphisms with chronic periodontitis and aggressive periodontitis in a Malayalam-speaking Dravidian population from South India. The case-control study consisted of 43 patients with chronic periodontitis and 54 patients with aggressive periodontitis as cases, and the control group consisted of 101 healthy subjects. All subjects were genotyped for IL-1beta +3954, -511, and -31 loci by polymerase chain reaction amplification followed by restriction enzyme digestion and gel electrophoresis. Genotype, allele, and haplotype analyses were done. Analyses for allele and genotypes showed a high frequency of the C allele and CC genotype for single nucleotide polymorphism IL-1beta +3954 in the group with chronic periodontitis and no difference for patients with aggressive periodontitis compared to controls (P <0.05). Haplotype analysis showed that IL-1beta -31 and -511 were in strong linkage disequilibrium in all groups. The IL-1beta -31 allele T was in linkage with allele T of IL-1beta +3954 in the control group. In the Malayalam-speaking Dravidian population, allele C of IL-1beta +3954 appeared to be an important risk factor for chronic periodontitis. The IL-1beta -31 allele T was in linkage with allele T of IL-1beta +3954 in the control group. No gene polymorphisms were found in patients with aggressive periodontitis. More studies with a larger sample size involving the entire cluster of the IL-1beta gene are necessary to determine the exact role of IL-1beta gene polymorphisms in periodontal disease.

  2. Crystal Structure of Staphylococcal Enterotoxin G (SEG) in Complex with a Mouse T-cell Receptor Beta Chain

    SciTech Connect

    Fernandez, M.M.; Robinson, H.; Cho, S.; De Marzi, M. C.; Kerzic, M. C.; Mariuzza, R. A.; Malchiodi, E. L.

    2011-01-14

    Superantigens (SAgs) are bacterial or viral toxins that bind MHC class II (MHC-II) molecules and T-cell receptor (TCR) in a nonconventional manner, inducing T-cell activation that leads to inflammatory cytokine production, which may result in acute toxic shock. In addition, the emerging threat of purpura fulminans and community-associated meticillin-resistant Staphylococcus aureus emphasizes the importance of a better characterization of SAg binding to their natural ligands that may allow the development of reagents to neutralize their action. The three-dimensional structure of the complex between a mouse TCR {beta} chain (mV{beta}8.2) and staphylococcal enterotoxin G (SEG) at 2.0 {angstrom} resolution revealed a binding site that does not conserve the 'hot spots' present in mV{beta}8.2-SEC2, mV{beta}8.2-SEC3, mV{beta}8.2-SEB, and mV{beta}8.2-SPEA complexes. Analysis of the mV{beta}8.2-SEG interface allowed us to explain the higher affinity of this complex compared with the others, which may account for the early activation of T-cells bearing mV{beta}8.2 by SEG. This mode of interaction between SEG and mV{beta}8.2 could be an adaptive advantage to bestow on the pathogen a faster rate of colonization of the host.

  3. Characterization of two divergent beta-tubulin genes from Colletotrichum graminicola.

    PubMed

    Panaccione, D G; Hanau, R M

    1990-02-14

    We have cloned and sequenced two beta-tubulin genes, TUB1 and TUB2, from the phytopathogenic fungus, Colletotrichum graminicola. The nucleotide sequences of the coding regions of the two genes are only 72.8% homologous. This divergence is reflected in the deduced amino acid (aa) sequences which differ at 94 aa residues. Comparison with the aa sequences of other fungal beta-tubulins indicates that the C. graminicola TUB2 gene encodes a conserved isotype, whereas the C. graminicola TUB1 product is highly divergent. Both genes contain six identically placed introns and the position of each intron is conserved in other fungal beta-tubulin genes. Also typical of other fungal beta-tubulin genes, there is a pronounced bias in codon usage in the C. graminicola TUB2 gene; there is a lesser codon bias in TUB1 from C. graminicola. Both C. graminicola beta-tubulin genes are transcribed and yield similar sized messages.

  4. Lactogenic hormonal induction of long distance interactions between beta-casein gene regulatory elements

    USDA-ARS?s Scientific Manuscript database

    Lactogenic hormone regulation of beta-casein gene expression in mammary epithelial cells provides, an excellent model in which to study the mechanisms by which steroid and peptide hormone signaling control gene expression. Prolactin- and glucocorticoid-mediated induction of beta-casein gene express...

  5. Differentiation phenotypes of pancreatic islet beta- and alpha-cells are closely related with homeotic genes and a group of differentially expressed genes.

    PubMed

    Mizusawa, Noriko; Hasegawa, Tomoko; Ohigashi, Izumi; Tanaka-Kosugi, Chisato; Harada, Nagakatsu; Itakura, Mitsuo; Yoshimoto, Katsuhiko

    2004-04-28

    To identify the genes that determine differentiation phenotypes, we compared gene expression of pancreatic islet beta- and alpha-cells, which are derived from the common precursor and secrete insulin and glucagon, respectively. The expression levels of homeotic genes including Hox genes known to determine region specificity in the antero-posterior (AP) body axis, tissue-specific homeobox genes, and other 8,734 genes were compared in a beta- and alpha-cell line of MIN6 and alpha TC1.6. The expression of homeotic genes were surveyed with reverse transcription-polymerase chain reaction (RT-PCR) using degenerate primers corresponding to invariant amino acid sequences within the homeodomain and subsequently with specific primers. Expression of Hoxc6, Hoxc9, Hoxc10, Pdx1, Cdx2, Gbx2, Pax4, and Hlxb9 genes in MIN6 was higher than those in alpha TC1.6, while expression of Hoxa2, Hoxa3, Hoxa5, Hoxa6, Hoxa7, Hoxa9, Hoxa10, Hoxa13, Hoxb3, Hoxb5, Hoxb6, Hoxb13, Hoxb8, and Brain4 genes in alpha TC1.6 was higher than those in MIN6. Out of 8,734 mouse genes screened with high-density mouse cDNA microarrays for MIN6- and alpha TC1.6-derived cDNA, 58 and 25 genes were differentially over- and under-expressed in MIN6, respectively. GLUTag, which is derived from a large bowel tumor and expresses the proglucagon gene, showed a comparatively similar expression profile to that of alpha TC1.6 in both homeotic and other genes analyzed in cDNA microarray. Our results are consistent with the interpretation that not only the tissue-specific homeotic genes, but also Hox genes are related to differentiation phenotypes of pancreatic beta- and alpha-cells rather than their regional specification of the body in vertebrates.

  6. Genomic evidence for independent origins of beta-like globin genes in monotremes and therian mammals.

    PubMed

    Opazo, Juan C; Hoffmann, Federico G; Storz, Jay F

    2008-02-05

    Phylogenetic reconstructions of the beta-globin gene family in vertebrates have revealed that developmentally regulated systems of hemoglobin synthesis have been reinvented multiple times in independent lineages. For example, the functional differentiation of embryonic and adult beta-like globin genes occurred independently in birds and mammals. In both taxa, the embryonic beta-globin gene is exclusively expressed in primitive erythroid cells derived from the yolk sac. However, the "epsilon-globin" gene in birds is not orthologous to the epsilon-globin gene in mammals, because they are independently derived from lineage-specific duplications of a proto beta-globin gene. Here, we report evidence that the early and late expressed beta-like globin genes in monotremes and therian mammals (marsupials and placental mammals) are the products of independent duplications of a proto beta-globin gene in each of these two lineages. Results of our analysis of genomic sequence data from a large number of vertebrate taxa, including sequence from the recently completed platypus genome, reveal that the epsilon- and beta-globin genes of therian mammals arose via duplication of a proto beta-globin gene after the therian/monotreme split. Our analysis of genomic sequence from the platypus also revealed the presence of a duplicate pair of beta-like globin genes that originated via duplication of a proto beta-globin gene in the monotreme lineage. This discovery provides evidence that, in different lineages of mammals, descendent copies of the same proto beta-globin gene may have been independently neofunctionalized to perform physiological tasks associated with oxygen uptake and storage during embryonic development.

  7. [Perissodactyla: the primary structure of hemoglobins from the lowland tapir (Tapirus terrestris): glutamic acid in position 2 of the beta chains].

    PubMed

    Mazur, G; Braunitzer, G

    1984-09-01

    The hemoglobins from a lowland tapir (Tapirus terrestris) were analysed and the complete primary structure is described. The globin chains were separated on CM cellulose column in 8M urea and the amino-acid sequences were determined in the liquid phase sequenator. The results show that globin consists of two alpha chains (alpha I and alpha II) and beta major and beta minor components. The alpha chains differ only at one position: alpha I contains aspartic acid and alpha II glycine. The beta chains are heterogeneous: aspartic and glutamic acid were found at position beta 21 and beta 73 of the beta major components and asparagine and serine at position beta 139. In the beta minor components four positions were found with more than one amino acid, namely beta 2, beta 4, beta 6 and beta 56. The sequences are compared with those of man, horse and rhinoceros. Four residues of horse methemoglobin, which are involved in the alpha 1 beta 1 contacts are substituted in tapir hemoglobins. In the alpha chains: alpha 107(G14)Ser----Val, alpha 111-(G18) Val----Leu, alpha 115(GH3) Asn----Asp or Gly; in the beta chains: beta 116(G18) Arg----Gln. The amino acid at beta 2 of the major components is glutamic acid while glutamine and histidine are found in the minor components. Although glutamic acid, a binding site for ATP, does not interact with 2,3-bisphosphoglycerate, glutamine and histidine in the minor components are responsible for the slight effect of 2,3-bisphosphoglycerate on tapir hemoglobin.

  8. Stimulatory effect of Crocus sativus (saffron) on beta2-adrenoceptors of guinea pig tracheal chains.

    PubMed

    Nemati, H; Boskabady, M H; Ahmadzadef Vostakolaei, H

    2008-12-01

    To study the mechanism(s) of the relaxant effects of Crocus sativus (Iridaceae), the stimulatory effect of aqueous-ethanolic extracts of this plant and one of its constituent, safranal was examined on beta-adrenoceptors in tracheal chains of guinea pigs. The beta(2)-adrenergic stimulatory was tested by performing the cumulative concentration-response curves of isoprenaline-induced relaxation of pre-contracted isolated guinea pig tracheal chains. The studied solutions were included two concentrations of aqueous-ethanolic extract from Crocus sativus (0.1 and 0.2g%), safranal (1.25 and 2.5 microg), 10nM propranolol, and saline. The study was done in two different conditions including: non-incubated (group 1, n=9) and incubated tissues with 1 microM chlorpheniramine (group 2, n=6). The results showed clear leftward shifts in isoprenaline curves obtained in the presence of only higher concentration of the extract in group 1 and its both concentrations in group 2 compared with that of saline. The EC(50) (the effective concentration of isoprenaline, causing 50% of maximum response) obtained in the presence of both concentrations of the extract (0.17+/-0.06 and 0.12+/-0.02) and safranal (0.22+/-0.05 and 0.22+/-0.05) in group 1 and only in the presence of two concentrations of the extract (1.16+/-0.31 and 0.68+/-0.21) in group 2 was significantly lower compared to saline (1.00+/-0.22 and 4.06+/-1.04 for groups 1 and 2, respectively) (p<0.05-0.001). The maximum responses obtained in the presence of both concentrations of the extract and safranal in group 1 were significantly lower than that of saline (p<0.005 for all cases). All values (CR-1=(EC(50) obtained in the presence of active substances/EC(50), obtained in the presence of saline)-1) obtained in the presence of higher concentrations of extract in group 1, its both concentrations and higher concentration of safranal in group 2 were negative and there were significant differences in this value between propranolol and

  9. cAMP-mediated beta-adrenergic signaling negatively regulates Gq-coupled receptor-mediated fetal gene response in cardiomyocytes.

    PubMed

    Patrizio, Mario; Vago, Valerio; Musumeci, Marco; Fecchi, Katia; Sposi, Nadia Maria; Mattei, Elisabetta; Catalano, Liviana; Stati, Tonino; Marano, Giuseppe

    2008-12-01

    The treatment with beta-blockers causes an enhancement of the norepinephrine-induced fetal gene response in cultured cardiomyocytes. Here, we tested whether the activation of cAMP-mediated beta-adrenergic signaling antagonizes alpha(1)-adrenergic receptor (AR)-mediated fetal gene response. To address this question, the fetal gene program, of which atrial natriuretic peptide (ANP) and the beta-isoform of myosin heavy chain are classical members, was induced by phenylephrine (PE), an alpha(1)-AR agonist. In cultured neonatal rat cardiomyocytes, we found that stimulation of beta-ARs with isoproterenol, a beta-AR agonist, inhibited the fetal gene expression induced by PE. Similar results were also observed when cardiomyocytes were treated with forskolin (FSK), a direct activator of adenylyl cyclase, or 8-CPT-6-Phe-cAMP, a selective activator of protein kinase A (PKA). Conversely, the PE-induced fetal gene expression was further upregulated by H89, a selective PKA inhibitor. To evaluate whether these results could be generalized to Gq-mediated signaling and not specifically to alpha(1)-ARs, cardiomyocytes were treated with prostaglandin F(2)alpha, another Gq-coupled receptor agonist, which is able to promote fetal gene expression. This treatment caused an increase of both ANP mRNA and protein levels, which was almost completely abolished by FSK treatment. The capability of beta-adrenergic signaling to regulate the fetal gene expression was also evaluated in vivo conditions by using beta1- and beta2-AR double knockout mice, in which the predominant cardiac beta-AR subtypes are lacking, or by administering isoproterenol (ISO), a beta-AR agonist, at a subpressor dose. A significant increase of the fetal gene expression was found in beta(1)- and beta(2)-AR gene deficient mice. Conversely, we found that ANP, beta-MHC and skACT mRNA levels were significantly decreased in ISO-treated hearts. Collectively, these data indicate that cAMP-mediated beta-adrenergic signaling

  10. Inhibition of bacterial cell wall-induced leukocyte recruitment and hepatic granuloma formation by TGF-beta gene transfer.

    PubMed

    Song, X; Zeng, L; Pilo, C M; Zagorski, J; Wahl, S M

    1999-10-01

    Intraperitoneal injection of streptococcal cell walls (SCW) into Lewis rats results in dissemination of SCW to the liver, spleen, bone marrow, and peripheral joints. The uptake of SCW by Kupffer cells in the liver initiates a chain of events largely mediated by T lymphocytes and macrophages. Local synthesis and secretion of cytokines and growth factors in response to the persistent SCW lead to the evolution and maintenance of a chronic T cell-dependent granulomatous response and result in granuloma formation and irreversible hepatic fibrosis. In an attempt to impede the development of the chronic granulomatous lesions in the liver, we injected a plasmid DNA encoding TGF-beta 1 i.m. to the SCW animals to determine the effect of TGF-beta 1 gene transfer on the course of liver inflammation and fibrosis. A single injection of plasmid DNA encoding TGF-beta 1 resulted in virtual abolition of the development of the SCW-induced hepatic granuloma formation and matrix expansion. TGF-beta 1 DNA not only reduced key proinflammatory cytokines including TNF-alpha, IL-1 beta, IFN-gamma, and IL-18, but also inhibited both CXC and CC chemokine production, thereby blocking inflammatory cell recruitment and accumulation in the liver. Moreover, TGF-beta 1 gene delivery inhibited its own expression in the liver tissue, which is otherwise up-regulated in SCW-injected animals. Our study suggests that TGF-beta 1 gene transfer suppresses hepatic granuloma formation by blocking the recruitment of inflammatory cells to the liver, and thus may provide a new approach to the control of hepatic granulomatous and fibrotic diseases.

  11. Signaling through the interleukin 2 receptor beta chain activates a STAT-5-like DNA-binding activity.

    PubMed Central

    Gaffen, S L; Lai, S Y; Xu, W; Gouilleux, F; Groner, B; Goldsmith, M A; Greene, W C

    1995-01-01

    To explore the possible involvement of STAT factors ("signal transducers and activators of transcription") in the interleukin 2 receptor (IL-2R) signaling cascade, murine HT-2 cells expressing chimeric receptors composed of the extracellular domain of the erythropoietin receptor fused to the cytoplasmic domains of the IL-2R beta or -gamma c chains were prepared. Erythropoietin or IL-2 activation of these cells resulted in rapid nuclear expression of a DNA-binding activity that reacted with select STAT response elements. Based on reactivity with specific anti-STAT antibodies, this DNA-binding activity was identified as a murine homologue of STAT-5. Induction of nuclear expression of this STAT-5-like factor was blocked by the addition of herbimycin A, a tyrosine kinase inhibitor, but not by rapamycin, an immunophilin-binding antagonist of IL-2-induced proliferation. The IL-2R beta chain appeared critical for IL-2-induced activation of STAT-5, since a mutant beta chain lacking all cytoplasmic tyrosine residues was incapable of inducing this DNA binding. In contrast, a gamma c mutant lacking all of its cytoplasmic tyrosine residues proved fully competent for the induction of STAT-5. Physical binding of STAT-5 to functionally important tyrosine residues within IL-2R beta was supported by the finding that phosphorylated, but not nonphosphorylated, peptides corresponding to sequences spanning Y392 and Y510 of the IL-2R beta tail specifically inhibited STAT-5 DNA binding. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7543676

  12. Lack of neighborhood effects from a transcriptionally active phosphoglycerate kinase-neo cassette located between the murine beta-major and beta-minor globin genes.

    PubMed

    Kaufman, R M; Lu, Z H; Behl, R; Holt, J M; Ackers, G K; Ley, T J

    2001-07-01

    For the treatment of beta-globin gene defects, a homologous recombination-mediated gene correction approach would provide advantages over random integration-based gene therapy strategies. However, "neighborhood effects" from retained selectable marker genes in the targeted locus are among the key issues that must be taken into consideration for any attempt to use this strategy for gene correction. An Ala-to-Ile mutation was created in the beta6 position of the mouse beta-major globin gene (beta(6I)) as a step toward the development of a murine model system that could serve as a platform for therapeutic gene correction studies. The marked beta-major gene can be tracked at the level of DNA, RNA, and protein, allowing investigation of the impact of a retained phosphoglycerate kinase (PGK)-neo cassette located between the mutant beta-major and beta-minor globin genes on expression of these 2 neighboring genes. Although the PGK-neo cassette was expressed at high levels in adult erythroid cells, the abundance of the beta(6I) mRNA was indistinguishable from that of the wild-type counterpart in bone marrow cells. Similarly, the output from the beta-minor globin gene was also normal. Therefore, in this specific location, the retained, transcriptionally active PGK-neo cassette does not disrupt the regulated expression of the adult beta-globin genes. (Blood. 2001;98:65-73)

  13. Molecular cloning and characterization of hemoglobin alpha and beta chains from plateau pika (Ochotona curzoniae) living at high altitude.

    PubMed

    Yingzhong, Yang; Yue, Cao; Guoen, Jin; Zhenzhong, Bai; Lan, Ma; Haixia, Yun; Rili, Ge

    2007-11-15

    Hemoglobin (Hb) plays an important role in oxygen transfer from lung to tissues. Possession of a Hb with high oxygen affinity helps highland animals to adapt to high altitude, has been studied profoundly. Plateau pika (Ochotona curzoniae), a native species living at 3,000-5,000 m above sea level on Qinghai-Tibet Plateau, is a typical hypoxia and low temperature tolerant mammal. To investigate the possible mechanisms of plateau pika Hb in adaptation to high altitude, the complete cDNA and amino acid sequences of plateau pika hemoglobin alpha and beta chains have been described. Compared with human Hb, alterations in important regions can be noted: alpha111 Ala-->Asn, beta35 Tyr-->Phe, beta112 Cys-->Val, beta115 Ala-->Ser, and beta125 Pro-->Gln. Phylogenetic analysis of alpha and beta chains shows that plateau pika is closer to rabbit than to other species. This study provides essential information for elucidating the possible roles of hemoglobin in adaptation to extremely high altitude in plateau pika.

  14. [Cloning of pigeon invariant chain (Ii) gene by RACE].

    PubMed

    Liu, Gang; Zhong, Da-Lian; Liu, Xue-Lan; Yu, Wei-Yi

    2008-01-01

    In order to compare the structure and function of pigeon invariant chain (pIi) gene with other avian's, pIi gene was cloned using a method of RACE (Rapid Amplification of cDNA Ends). Firstly, according to high conservative nucleotide sequence of homologous fragment in avian invariant chain (Ii) gene, a pair of degenerated primer was designed, and a special DNA fragment was gained from pigeon spleen cell RNA by PCR. Then based on the sequence of gained DNA fragment, some new primers were designed, and the 3'terminal and the 5'terminal of pIi gene were cloned by RACE respectively. Finally a complete cDNA of pIi was to extend with newly designed primer by PCR. The product was identified by electrophresis and sequence analysis. The results of sequencing indicate that pIi gene is 1,050 bp in length (GenBank No. AY904337), which includes an open reading frame of 633 bp encoding a precursor protein with 211 amino acid residues. In comparison with the nucleotide sequences of other species' Ii genes, pIi is similar to chicken's, showing an overall identity of 82.8 with chicken and over 52.0 with human and other mammalian animals. In addition, some amino acid residues in Ii molecule manifest extremely conservative among animals, which suggests that they could have an important biological function.

  15. Human T-cell receptor v{beta} gene polymorphism and multiple sclerosis

    SciTech Connect

    Wei, S.; Charmley, P.; Birchfield, R.I.; Concannon, P.

    1995-04-01

    Population-based genetic associations have been reported between RFLPs detected with probes corresponding to the genes encoding the {beta} chain of the T-cell receptor for antigen (RCRB) and a variety of autoimmune disorders. In the case of multiple sclerosis (MS), these studies have localized a putative disease-associated gene to a region of {approximately}110 kb in length, located within the TCRB locus. In the current study, all 14 known TCRBV (variable region) genes within the region of localization were mapped and identified. The nucleotide sequences of these genes were determined in a panel of six MS patients and six healthy controls, who were human-leukocyte antigen and TCRB-RFLP haplotype matched. Nine of the 14 TCRBV genes studied showed evidence of polymorphism. PCR-based assays for each of these polymorphic genes were developed, and allele and genotype frequencies were determined in a panel of DNA samples from 48 MS patients and 60 control individuals. No significant differences in allele, genotype, or phenotype frequencies were observed between the MS patients and controls for any of the 14 TCRBV-gene polymorphisms studied. In light of the extensive linkage disequilibrium across the region studied, the saturating numbers of polymorphisms examined, and the direct sequence analysis of all BV genes in the region, these results suggest that it is unlikely that germ-line polymorphism in the TCRBV locus makes a major contribution to MS susceptibility. The TCRBV coding region-specific markers generated in these studies, as well as the approach of testing for associations with specific functionally relevant polymorphic sites within individual BV genes, should be useful in the evaluation of the many reported disease associations involving the human TCRB region. 22 refs., 1 fig., 3 tabs.

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

    PubMed

    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.

  17. Dopamine beta-hydroxylase gene modulates individuals’ empathic ability

    PubMed Central

    Gong, Pingyuan; Liu, Jinting; Li, She

    2014-01-01

    Dopamine beta-hydroxylase (DBH), an enzyme that converts dopamine to norepinephrine, has broad influences on social functions. In this study, we examined to what extent two polymorphisms (−1021C/T and a 19 bp insertion/deletion) in DBH gene modulate individuals’ empathic perception and response, which were measured, respectively, by reading the mind in the eyes test and the empathic concern subscale of interpersonal reactivity index. Results showed that polymorphism at −1021C/T, but not the 19 bp insertion/deletion, accounts for 2.3% variance of empathic perception and 1.4% variance of empathic response. Individuals with the CC genotype, which is associated with higher DBH activity, manifested greater empathic ability than those with CT/TT genotypes. These findings demonstrate the importance of DBH −1021C/T as a genetic basis of empathy and in predicting individual differences in social and affective processing. PMID:23988761

  18. Molecular dynamics studies of side chain effect on the beta-1,3-D-glucan triple helix in aqueous solution.

    PubMed

    Okobira, Tadashi; Miyoshi, Kentaro; Uezu, Kazuya; Sakurai, Kazuo; Shinkai, Seiji

    2008-03-01

    beta-1,3-D-glucans have been isolated from fungi as right-handed 6(1) triple helices. They are categorized by the side chains bound to the main triple helix through beta-(1-->6)-D-glycosyl linkage. Indeed, since a glucose-based side chain is water soluble, the presence and frequency of glucose-based side chains give rise to significant variation in the physical properties of the glucan family. Curdlan has no side chains and self-assembles to form an water-insoluble triple helical structure, while schizophyllan, which has a 1,6-D-glucose side chain on every third glucose unit along the main chain, is completely water soluble. A thermal fluctuation in the optical rotatory dispersion is observed for the side chain, indicating probable co-operative interaction between the side chains and water molecules. This paper documents molecular dynamics simulations in aqueous solution for three models of the beta-1,3-D-glucan series: curdlan (no side chain), schizophyllan (a beta-(1-->6)-D-glycosyl side-chain at every third position), and a hypothetical triple helix with a side chain at every sixth main-chain glucose unit. A decrease was observed in the helical pitch as the population of the side chain increased. Two types of hydrogen bonding via water molecules, the side chain/main chain and the side chain/side chain hydrogen bonding, play an important role in determination of the triple helix conformation. The formation of a one-dimensional cavity of diameter about 3.5 A was observed in the schizophyllan triple helix, while curdlan showed no such cavity. The side chain/side chain hydrogen bonding in schizophyllan and the hypothetical beta-1,3-D-glucan triple helix could cause the tilt of the main-chain glucose residues to the helix.

  19. Functional polymorphism of each of the two HLA-DR beta chain loci demonstrated with antigen-specific DR3- and DRw52-restricted T cell clones

    PubMed Central

    1988-01-01

    HLA-DR3- and HLA-DRw52-associated functional polymorphism was investigated with selected tetanus toxoid (TT)-specific T cell clones. We have shown earlier that HLA-DR antigens are encoded by two distinct loci, DR beta I and DR beta III. The alloantigenic determinant(s) defined by the serological HLA-DR3 specificity map to the former, while the supratypic HLA-DRw52 determinants map to DR beta III. Furthermore, we have recently recognized by DNA sequencing three alleles of HLA- DRw52 at locus DR beta III, referred to as 52 a, b, and c. Our objective was to correlate the pattern of T cell restriction with the gene products of individual DR beta chain loci and with the three newly described alleles of locus DR beta III. Among the selected T cell clones, 5 reacted exclusively when TT was presented by HLA-DR3+ APCs (TT-DR3-APC). In contrast, two T cell clones were stimulated by TT- DRw52-APC. More specifically, these two T cell clones (Clones 10 and 16) were stimulated by different subsets of TT-DRw52-APC. Clone 16 responded to some DR3 and TT-DRw6-APC, while clone 10 was stimulated by other TT-DR3 and TT-DRw6, and all TT-DR5-APC. This same pattern of DRw52 restriction was found in panel, as well as in family studies. Because this suggested a correlation with the pattern of DRw52 polymorphism observed earlier by DNA sequencing and oligonucleotide hybridization, the APC used in these experiments were typed for the 52 a, b, and c alleles of locus DR beta III by allele-specific oligonucleotide probes. This distribution overlapped exactly with the stimulation pattern defined by the T cell clones. Clone 16 responded to TT-52a-APC, clone 10 to TT-52b-APC, and both clones to a TT-52c-APC. The response of the T cell clones was inhibited differentially by mAbs to DR. Raising TT concentration, or increasing HLA-class II expression with INF-gamma both affected the magnitude of response of the TT- specific clones but did not modify their specificities. These results demonstrate that

  20. Complementary DNA sequences encoding the multimammate rat MHC class II DQ alpha and beta chains and cross-species sequence comparison in rodents.

    PubMed

    de Bellocq, J Goüy; Leirs, H

    2009-09-01

    Sequences of the complete open reading frame (ORF) for rodents major histocompatibility complex (MHC) class II genes are rare. Multimammate rat (Mastomys natalensis) complementary DNA (cDNA) encoding the alpha and beta chains of MHC class II DQ gene was cloned from a rapid amplifications of cDNA Emds (RACE) cDNA library. The ORFs consist of 801 and 771 bp encoding 266 and 256 amino acid residues for DQB and DQA, respectively. The genomic structure of Mana-DQ genes is globally analogous to that described for other rodents except for the insertion of a serine residue in the signal peptide of Mana-DQB, which is unique among known rodents.

  1. Gene expression assay in blood and various tissues using a single-tube real-time reverse transcription-polymerase chain reaction method using an oligodeoxythymidine-immobilized polymerase chain reaction tube.

    PubMed

    Harikai, N; Saito, S; Tanaka, A; Kinoshita, K

    2009-06-01

    A single-tube real-time reverse transcription-polymerase chain reaction (RT-PCR) method has been developed which makes it possible to conduct the entire procedure, from nucleic acid extraction to product detection, in a single PCR tube. In this study, we developed the method using an oligodeoxythymidine-immobilized PCR tube, which enables simple and rapid mRNA extraction and quantification of target genes in blood and other tissues. The beta-actin gene was analyzed from lysates of blood and various tissues using this method. The data showed a good correlation between the plotted threshold cycle values and log(10) of blood and tissue amounts without a reduction in PCR efficiency. Gene expression of interleukin-1beta in blood from lipopolysaccharide (LPS)-stimulated rats and of beta(3)-adrenoceptors in adipose tissue from SHRSP.Z-Lepr (fa)/IzmDmcr (obese SHRSP) rats was also analyzed using the single-tube method, as well as a general real-time RT-PCR method, using RNA purified with a silica membrane column. In both methods, the copy number ratio of interleukin-1beta to beta-actin in LPS-stimulated rats was higher than in control rats, and the ratio of beta(3)-adrenoceptors to beta-actin in obese SHRSP rats was lower than in lean littermates. These results indicate that the single-tube method can provide results equivalent to those from general real-time RTPCR methods in gene expression analysis.

  2. [Nucleotide sequence of genes for alpha- and beta-subunits of luciferase from Photobacterium leiognathi].

    PubMed

    Illarionov, B A; Protopopova, M V; Karginov, V A; Mertvetsov, N P; Gitel'zon, I I

    1988-03-01

    Nucleotide sequence of the Photobacterium leiognathi DNA containing genes of alpha and beta subunits of luciferase has been determined. We also deduced amino acid sequence and molecular mass of luciferase and localized luciferase genes in the sequenced DNA fragment.

  3. Genotyping of Giardia duodenalis from humans and dogs from Mexico using a beta-giardin nested polymerase chain reaction assay.

    PubMed

    Lalle, Marco; Jimenez-Cardosa, Enedina; Cacciò, Simone M; Pozio, Edoardo

    2005-02-01

    Cysts of Giardia duodenalis were collected in Mexico from symptomatic children (n = 9) and from pet dogs (n = 5), and they were directly characterized by nested polymerase chain reaction (PCR) amplification of the beta-giardin gene. Eight isolates of human origin established as in vitro cultures and 2 reference strains, representing assemblages A and B of G. duodenalis, were also analyzed. PCR-restriction fragment length polymorphism showed that all isolates belonged to assemblage A. Sequence analyses indicated that the large majority of isolates were of the A1 genotype; interestingly, 2 human isolates displayed the A3 genotype, which has been previously identified in human isolates from Italy. The presence of cysts of the A1 and A3 genotypes in isolates from pet dogs is consistent with their role as reservoirs for human infection, although further studies are needed to confirm the occurrence of zoonotic transmission. Remarkably, cysts of assemblage B have not been found in any of the Mexican isolates studied to date.

  4. The amino-acid sequence of alpha A- and beta-chains from the major hemoglobin component of American flamingo (Phoenicopterus ruber ruber).

    PubMed

    Godovac-Zimmermann, J; Braunitzer, G

    1984-04-01

    The complete amino-acid sequence of alpha A- and beta-chains from the major hemoglobin component (HbA) of American Flamingo ( Phoenicopterus ruber ruber) is presented. The minor component (HbD) with alpha D-chains was detected in similar amounts (25%) as in chicken and pheasant hemoglobins. The comparison of American Flamingo and Greylag Goose (Anser anser) hemoglobins shows that alpha A-chains differ by 22 exchanges and beta-chains by only 4 exchanges. Two substitutions modify alpha 1 beta 1-contacts. Amino-acid replacements between American Flamingo and other bird hemoglobins are discussed.

  5. Amyloid-beta aggregation: selective inhibition of aggregation in mixtures of amyloid with different chain lengths.

    PubMed Central

    Snyder, S W; Ladror, U S; Wade, W S; Wang, G T; Barrett, L W; Matayoshi, E D; Huffaker, H J; Krafft, G A; Holzman, T F

    1994-01-01

    One of the clinical manifestations of Alzheimer's disease is the deposition of the 39-43 residue amyloid-beta (A beta) peptide in aggregated fibrils in senile plaques. Characterization of the aggregation behavior of A beta is one of the critical issues in understanding the role of A beta in the disease process. Using solution hydrodynamics, A beta was observed to form three types of species in phosphate-buffered saline: insoluble aggregates with sedimentation coefficients of approximately 50,000 S and molecular masses of approximately 10(9) Da, "soluble aggregates" with sedimentation coefficients of approximately 30 S and masses of approximately 10(6) Da, and monomer. When starting from monomer, the aggregation kinetics of A beta 1-40 (A beta 40) and A beta 1-42 (A beta 42), alone and in combination, reveal large differences in the tendency of these peptides to aggregate as a function of pH and other solution conditions. At pH 4.1 and 7.0-7.4, aggregation is significantly slower than at pH 5 and 6. Under all conditions, aggregation of the longer A beta 42 was more rapid than A beta 40. Oxidation of Met-35 to the sulfoxide in A beta 40 enhances the aggregation rate over that of the nonoxidized peptide. Aggregation was found to be dependent upon temperature and to be strongly dependent on peptide concentration and ionic strength, indicating that aggregation is driven by a hydrophobic effect. When A beta 40 and A beta 42 are mixed together, A beta 40 retards the aggregation of A beta 42 in a concentration-dependent manner. Shorter fragments have a decreasing ability to interfere with A beta 42 aggregation. Conversely, the rate of aggregation of A beta 40 can be significantly enhanced by seeding slow aggregating solutions with preformed aggregates of A beta 42. Taken together, the inhibition of A beta 42 aggregation by A beta 40, the seeding of A beta 40 aggregation by A beta 42 aggregates, and the chemical oxidation of A beta 40 suggest that the relative abundance and

  6. Chromosome mapping of the human arrestin (SAG), {beta}-arrestin 2 (ARRB2), and {beta}-adrenergic receptor kinase 2 (ADRBK2) genes

    SciTech Connect

    Calabrese, G.; Sallese, M.; Stornaiuolo, A.

    1994-09-01

    Two types of proteins play a major role in determining homologous desensitization of G-coupled receptors: {beta}-adrenergic receptor kinase ({beta}ARK), which phosphorylates the agonist-occupied receptor and its functional cofactor, {beta}-arrestin. Both {beta}ARK and {beta}-arrestin are members of multigene families. The family of G-protein-coupled receptor kinases includes rhodopsin kinase, {beta}ARK1, {beta}ARK2, IT11-A (GRK4), GRK5, and GRK6. The arrestin/{beta}-arrestin gene family includes arrestin (also known as S-antigen), {beta}-arrestin 1, and {beta}-arrestin 2. Here we report the chromosome mapping of the human genes for arrestin (SAG), {beta}arrestin 2 (ARRB2), and {beta}ARK2 (ADRBK2) by fluorescence in situ hybridization (FISH). FISH results confirmed the assignment of the gene coding for arrestin (SAG) to chromosome 2 and allowed us to refine its localization to band q37. The gene coding for {beta}-arrestin 2 (ARRB2) was mapped to chromosome 17p13 and that coding for {beta}ARK2 (ADRBK2) to chromosome 22q11. 17 refs., 1 fig.

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

    PubMed

    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.

  8. A human high affinity interleukin-5 receptor (IL5R) is composed of an IL5-specific alpha chain and a beta chain shared with the receptor for GM-CSF.

    PubMed

    Tavernier, J; Devos, R; Cornelis, S; Tuypens, T; Van der Heyden, J; Fiers, W; Plaetinck, G

    1991-09-20

    cDNA clones encoding two receptor proteins involved in the binding of human interleukin 5 (hIL5) have been isolated. A first class codes for an IL5-specific chain (hIL5R alpha). The major transcript of this receptor gene, as analyzed in both HL-60 eosinophilic cells and eosinophilic myelocytes grown from cord blood, encodes a secreted form of this receptor. This soluble hIL5R alpha has antagonistic properties. A second component of the hIL5R is found to be identical to the beta chain of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) high affinity receptor. The finding that IL5 and GM-CSF share a receptor subunit provides a molecular basis for the observation that these cytokines can partially interfere with each other's binding and have highly overlapping biological activities on eosinophils.

  9. Interleukin 10 and transforming growth factor beta 1 gene polymorphisms in juvenile idiopathic arthritis.

    PubMed

    Harsini, S; Ziaee, V; Maddah, M; Rezaei, A; Sadr, M; Zoghi, S; Moradinejad, M H; Tahghighi, F; Aghighi, Y; Rezaei, N

    2016-01-01

    The aim of this study is to identify the associations between interleukin 10 (IL-10) and transforming growth factor beta 1 (TGF-β1) gene polymorphisms and individual susceptibility to juvenile idiopathic arthritis (JIA) in a group of Iranian patients. Cytokine genes, including IL-10 and TGF-β1, are known to play important roles in the pathogenesis of JIA. Using polymerase chain reaction with sequence-specific primers method, the frequency of alleles, genotypes and haplotypes of IL-10 (positions -1082, -819, -592) and TGF-β1 (codon 10, codon 25) single-nucleotide polymorphisms (SNPs) were investigated in 55 patients with JIA as a case group and compared with 140 healthy unrelated controls. The G allele was significantly less frequent at TGF-β1 codon 25 in patients with JIA than in the controls (p < 0.01). The frequency of CT genotype at TGF-β1 codon 10 was found to be higher in healthy individuals in comparison with that in patients group (p = 0.04). We observed no differences in the frequency of alleles, genotypes and haplotypes of IL-10 gene between the groups of patients and controls. Considering the low frequency of existence of TGF-β1 G allele at codon 25 as well as TGF-β1 CT genotype at codon 10 in patients with JIA, it seems that these cytokine gene polymorphisms could play role as the protective factors against JIA.

  10. Platelet-derived growth factor (PDGF) B-chain gene expression by activated blood monocytes precedes the expression of the PDGF A-chain gene

    SciTech Connect

    Martinet, Y.; Jaffe, H.A.; Yamauchi, K.; Betsholtz, C.; Westermark, B.; Heldin, C.H.; Crystal, R.G.

    1987-05-01

    When activated, normal human blood monocytes are known to express the c-sis proto-oncogene coding for PDGF B-chain. Since normal human platelet PDGF molecules are dimers of A and B chains and platelets and monocytes are derived from the same marrow precursors, activated blood monocytes were simultaneously evaluated for their expression of PDGF A and B chain genes. Human blood monocytes were purified by adherence, cultured with or without activation by lipopolysaccharide and poly(A)+ RNA evaluated using Northern analysis and /sup 32/P-labeled A-chain and B-chain (human c-sis) probes. Unstimulated blood monocytes did not express either A-chain or B-chain genes. In contrast, activated monocytes expressed a 4.2 kb mRNA B-chain transcript at 4 hr, but the B-chain mRNA levels declined significantly over the next 18 hr. In comparison, activated monocytes expressed very little A-chain mRNA at 4 hr, but at 12 hr 1.9, 2.3, and 2.8 kb transcripts were observed and persisted through 24 hr. Thus, activation of blood monocytes is followed by PDGF B-chain gene expression preceding PDGF A-chain gene expression, suggesting a difference in the regulation of the expression of the genes for these two chains by these cells.

  11. Lambda light chain myeloma with co-migrating paraprotein at beta region on agarose gel electrophoresis: a case report.

    PubMed

    Siti Sarah, M; Nor Aini, U; Nurismah, M I; Hafiza, A; Khalidah, M; Mokhtar, A B; Das, S

    2014-01-01

    Paraproteinemia is one of the diagnostic features of multiple myeloma. A commonly used method is the detection of paraprotein by agarose gel electrophoresis (AGE) followed by by immunofixation electrophoresis (IFE) to confirm monoclonality. Due to their smaller size, immunoglobulin A (IgA) and light chain only paraproteins may appear at the beta or even alpha 2 protein fractions. Here, we discuss a case report of a 47-year-old man who presented with pathological fracture of third thoracic (T3) vertebra. Serum protein electrophoresis (SPE) was initially reported as no paraprotein detected. However, a bone biopsy was reported to show plasma cell proliferation with light chain restriction. A repeat sample for protein electrophoresis together with IFE revealed lambda light chain paraprotein co-migrating at the beta region. The beta band plus paraprotein was quantitated as 4.3 g/L (7.0%), which was within normal limits of the beta protein fraction. Hence, it has to be remembered that if the SPE is negative, it does not necessarily mean that the paraprotein is absent in cases which are highly suspicious.

  12. Regulatory myosin light-chain genes of Caenorhabditis elegans.

    PubMed Central

    Cummins, C; Anderson, P

    1988-01-01

    We have cloned and analyzed the Caenorhabditis elegans regulatory myosin light-chain genes. C. elegans contains two such genes, which we have designated mlc-1 and mlc-2. The two genes are separated by 2.6 kilobases and are divergently transcribed. We determined the complete nucleotide sequences of both mlc-1 and mlc-2. A single, conservative amino acid substitution distinguishes the sequences of the two proteins. The C. elegans proteins are strongly homologous to regulatory myosin light chains of Drosophila melanogaster and vertebrates and weakly homologous to a superfamily of eucaryotic calcium-binding proteins. Both mlc-1 and mlc-2 encode abundant mRNAs. We mapped the 5' termini of these transcripts by using primer extension sequencing of mRNA templates. mlc-1 mRNAs initiate within conserved hexanucleotides at two different positions, located at -28 and -38 relative to the start of translation. The 5' terminus of mlc-2 mRNA is not encoded in the 4.8-kilobase genomic region upstream of mlc-2. Rather, mlc-2 mRNA contains at its 5' end a short, untranslated leader sequence that is identical to the trans-spliced leader sequence of three C. elegans actin genes. Images PMID:3244358

  13. T cell receptor repertoire differences between African Americans and Caucasians associated with polymorphism of the TCRBV3S1 (V{beta}3.1) gene

    SciTech Connect

    De Inocencio, J.; Glass, D.N.; Hirsch, R.

    1995-05-01

    The generation of TCR diversity occurs primarily through rearrangement of germline DNA. Genetic polymorphism of the TCR chains appears to be a rarer mechanism for generating repertoire differences between races. Flow cytometric analysis of the TCR V{beta} repertoire in a population of healthy African Americans (n = 30) and Caucasians (n = 30) revealed a significant difference in the frequency of cells bearing V{beta}3.1, but not V{beta}2, V{beta}5.1, V{beta}5.2-5.3, V{beta}6.7, V{beta}8.1-8.2, V{beta}12.1, V{beta}13.3, or V{beta}19. African Americans had a significantly lower frequency of V{beta}3.1{sup +} cells, in both the CD4{sup +} (2.55 {+-} 0.36% vs 4.85 {+-} 0.43%, p = 0.0001) and the CD8{sup +} (3.03 {+-} 0.54% vs 5.32 {+-} 0.57%, p = 0.004) population than did Caucasians, and this difference was independent of the age of the individuals. Analysis of genomic DNA revealed that the observed differences in frequency of V{beta}3.1{sup +} cells correlated with a recently described polymorphism of the recombination signal sequence of the TCRBV3S1 gene. Allele 1, associated with a lower frequency of V{beta}3.1{sup +} cells, was more commonly present in African Americans (0.68 vs 0.43), whereas allele 2, associated with a higher frequency of V{beta}3.1{sup +} cells, was more commonly present in Caucasians (0.31 vs 0.56). This study demonstrates the potential for TCR repertoire differences, based on genetic polymorphism, between African Americans and Caucasians. 31 refs., 2 figs., 5 tabs.

  14. Ultrasound-mediated interferon {beta} gene transfection inhibits growth of malignant melanoma

    SciTech Connect

    Yamaguchi, Kazuki; Feril, Loreto B.; Tachibana, Katsuro; Takahashi, Akira; Matsuo, Miki; Endo, Hitomi; Harada, Yoshimi; Nakayama, Juichiro

    2011-07-22

    Highlights: {yields} Successful ultrasound-mediated transfection of melanoma (C32) cells with IFN-{beta} genes both in vitro and in vivo. {yields} Ultrasound-mediated IFN-{beta} transfection inhibited proliferation of melanoma cells in vitro. {yields} Ultrasound-mediated IFN-{beta} transfection inhibited melanoma tumor growth in vivo. -- Abstract: We investigated the effects of ultrasound-mediated transfection (sonotransfection) of interferon {beta} (IFN-{beta}) gene on melanoma (C32) both in vitro and in vivo. C32 cells were sonotransfected with IFN-{beta} in vitro. Subcutaneous C32 tumors in mice were sonicated weekly immediately after intra-tumor injection with IFN-{beta} genes mixed with microbubbles. Successful sonotransfection with IFN-{beta} gene in vitro was confirmed by ELISA, which resulted in C32 growth inhibition. In vivo, the growth ratio of tumors transfected with IFN-{beta} gene was significantly lower than the other experimental groups. These results may lead to a new method of treatment against melanoma and other hard-to-treat cancers.

  15. Unusual association of beta 2-microglobulin with certain class I heavy chains of the murine major histocompatibility complex.

    PubMed Central

    Bushkin, Y; Tung, J S; Pinter, A; Michaelson, J; Boyse, E A

    1986-01-01

    Class I products of the major histocompatibility complex (MHC) comprise a heavy chain of about 45 kDa noncovalently linked to a 12-kDa beta 2-microglobulin (beta 2m) light chain encoded on a different chromosome. We find that class I products of some mouse strains include an additional 62-kDa molecule which on the following evidence consists of a heavy chain linked covalently with beta 2m. Production of the 62-kDa protein invariably accorded with the occurrence of cysteine at position 121 of the heavy chain (Kb,Kbm1,Kbm3,Dd, and Ld). Substitution of arginine at position 121 invariably accorded with absence of the 62-kDa protein (Kbm6,Kbm7,Kbm9,Kd, and Db). On the basis of observed production versus nonproduction of the 62-kDa molecule, predictions are made regarding residue 121 in class I products for which this is not yet known; namely, Kk, Ks, and Dk, which produce the 62-kDa molecule, as compared with Kj, Qa-2, and TL, which do not. Reported differences in immunologic reactivity between Kb mutant strains with Arg-121 in place of Cys-121 imply that the occurrence of 62-kDa class I products in mice of Cys-121 genotype has functional consequences. Images PMID:3510435

  16. Cellular distribution and molecular heterogeneity of MAC393 antigen (clusterin, beta-chain) on the surface membrane of bull spermatozoa.

    PubMed

    Howes, E A; Hurst, S; Laslop, A; Jones, R

    1998-07-01

    The distribution and size of a surface membrane antigen identified by a monoclonal antibody (MAC9393) have been examined in testicular and epididymal bovine sperm preparations. Western blots indicated a substantial decrease in molecular mass of the antigen during epididymal maturation from approximately 87 kDa in the testis to approximately 35 kDa in the cauda epididymidis. This was accompanied by a change in its cellular localization from the neck and whole head to the acrosomal region. N-terminal microsequencing identified MAC393 antigen as the beta-chain of clusterin. A polyclonal antiserum to the alpha-chain of clusterin recognized both testicular and epididymal forms and revealed that the heterodimer was present on the sperm tail as well as the acrosome. These findings are explained by the co-existence of dimeric and monomeric pools of clusterin on spermatozoa. The polyclonal antiserum recognizes both testicular and epididymal forms of the heterodimer and although the monoclonal antibody binds to the testicular heterodimer, it only recognizes the beta-chain monomer of epididymal clusterin. These findings support previous observations made on human spermatozoa that two forms of clusterin, the beta-chain monomer and the heterodimer, are present on the surface membrane and in seminal plasma.

  17. Genomic organization of the human {beta}-catenin gene (CTNNB1)

    SciTech Connect

    Nollet, F.; Berx, G.; Molemans, F.; Roy, F. van

    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.

  18. Detection of Clostridium septicum hemolysin gene by polymerase chain reaction.

    PubMed

    Takeuchi, S; Hashizume, N; Kinoshita, T; Kaidoh, T; Tamura, Y

    1997-09-01

    A polymerase chain reaction (PCR) was developed for the detection of the hemolysin (alpha toxin) gene of Clostridium septicum. The PCR primers were designed from the sequence of the hemolysin gene and synthesized. A DNA fragment of 270 bp was amplified from 10 strains of C. septicum, but was not from strains of C. chauvoei, C. perfringens, C. novyi, or C. haemolyticum. When the PCR product was digested with Sau3AI, two DNA fragments of the expected 148 bp and 122 bp were recognized. The lowest detectable threshold of PCR for the hemolysin gene was 3.8 x 10(3) cells/ml. The PCR technique may be useful for rapid detection or identification of C. septicum associated with malignant edema.

  19. Identification of the gene for fly non-muscle myosin heavy chain: Drosophila myosin heavy chains are encoded by a gene family.

    PubMed Central

    Kiehart, D P; Lutz, M S; Chan, D; Ketchum, A S; Laymon, R A; Nguyen, B; Goldstein, L S

    1989-01-01

    In contrast to vertebrate species Drosophila has a single myosin heavy chain gene that apparently encodes all sarcomeric heavy chain polypeptides. Flies also contain a cytoplasmic myosin heavy chain polypeptide that by immunological and peptide mapping criteria is clearly different from the major thoracic muscle isoform. Here, we identify the gene that encodes this cytoplasmic isoform and demonstrate that it is distinct from the muscle myosin heavy chain gene. Thus, fly myosin heavy chains are the products of a gene family. Our data suggest that the contractile function required to power myosin based movement in non-muscle cells requires myosin diversity beyond that available in a single heavy chain gene. In addition, we show, that accumulation of cytoplasmic myosin transcripts is regulated in a developmental stage specific fashion, consistent with a key role for this protein in the movements of early embryogenesis. Images PMID:2498088

  20. Analysis of HLA-DR glycoproteins by DNA-mediated gene transfer. Definition of DR2 beta gene products and antigen presentation to T cell clones from leprosy patients

    PubMed Central

    1988-01-01

    We have used DNA-mediated gene transfer to express HLA class II molecules in mouse L cells for serological, biochemical, and functional analysis. cDNA clones encoding the DR2 beta a and DR2 beta b products of the DR2Dw2 haplotype were subcloned into a mouse Moloney leukemia virus-based expression vector (pJ4) and transfected separately into mouse L cells together with a HLA-DR alpha/pJ4 construct. These transfectants have allowed differential analysis of the two DR2 beta products in a manner normally prohibited by the concomitant expression seen in B cells. Two-dimensional SDS-PAGE analysis of the transfectants defines the more acidic beta chain as the product of the DR2 beta a sequence, and the more basic chain as the product of the DR2 beta b sequence. The LDR2a transfectants present antigen efficiently to M.leprae-specific T cell clones and are capable of presenting synthetic peptide, 65-kD recombinant mycobacterial antigen and M.leprae. Of the DR2Dw2-restricted T cell clones we have tested, all use the DR2 beta a chain as their restriction element. Inhibition studies with mAbs demonstrate the dependence of presentation by the transfectant on class II and CD4, while mAbs against LFA-1, which substantially inhibit presentation by B-lymphoblastoid cell lines, do not inhibit transfectant presentation. PMID:3128633

  1. Identification of Leptospira serovars by RFLP of the RNA polymerase beta subunit gene (rpoB)

    PubMed Central

    Jung, Lenice Roteia Cardoso; Bomfim, Maria Rosa Quaresma; Kroon, Erna Geessien; Nunes, Álvaro Cantini

    2015-01-01

    Leptospires are usually classified by methods based on DNA-DNA hybridization and the conventional cross-agglutination absorption test, which uses polyclonal antibodies against lipopolysaccharides. In this study, the amplification of the rpoB gene, which encodes the beta-subunit of RNA polymerase, was used as an alternative tool to identify Leptospira. DNA extracts from sixty-eight serovars were obtained, and the hypervariable region located between 1990 and 2500-bp in the rpoB gene was amplified by polymerase chain reaction (PCR). The 600-bp amplicons of the rpoB gene were digested with the restriction endonucleases TaqI, Tru1I, Sau3AI and MslI, and the restriction fragments were separated by 6% polyacrylamide gel electrophoresis. Thirty-five fragment patters were obtained from the combined data of restriction fragment length polymorphism (PCR-RFLP) analysis and used to infer the phylogenetic relationships among the Leptospira species and serovars. The species assignments obtained were in full agreement with the established taxonomic classifications. Twenty-two serovars were effectively identified based on differences in their molecular profiles. However, the other 46 serovars remained clustered in groups that included more than one serovar of different species. This study demonstrates the value of RFLP analysis of PCR-amplified rpoB as an initial method for identifying Leptospira species and serovars. PMID:26273261

  2. Polymorphisms in the Estrogen Receptor Beta Gene and the Risk of Unexplained Recurrent Spontaneous Abortion

    PubMed Central

    Mahdavipour, Marzieh; Zarei, Saeed; Fatemi, Ramina; Edalatkhah, Haleh; Heidari-Vala, Hamed; Jeddi-Tehrani, Mahmood; Idali, Farah

    2017-01-01

    Background: Recurrent Spontaneous Abortion (RSA) is caused by multiple genetic and non-genetic factors. Around 50% of the RSA cases have no known etiology and are considered as Unexplained RSA (URSA). Estrogens, via binding to their receptors, play an important role in female reproduction. This study aimed to investigate whether single nucleotide polymorphisms (SNPs; +1082G/A, +1730G/A and rs1256030 C/T) in the estrogen receptor beta (ESR2) gene are associated with susceptibility to URSA in a population of Iranian women. Methods: In this case-control study, the study groups consisted of 240 subjects with a history of URSA and 102 fertile women as controls. Serum levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) were measured on day 2–3 of menstrual cycle. Two functional SNPs, +1082G/A (a silent mutation in exon 5) and +1730G/A (3′ untranslated region of the exon 8), and one intron, rs1256030C/T, in the ESR2 gene were genotyped, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Results: Serum levels of LH were significantly increased in URSA women. No significant differences in distribution of +1082G/A, +1730G/A and rs1256030C/T between URSA and control groups were observed. Conclusion: Our findings suggest that the studied SNPs on ESR2 gene may not be associated with URSA. PMID:28706612

  3. An unconventional myosin heavy chain gene from Drosophila melanogaster.

    PubMed

    Kellerman, K A; Miller, K G

    1992-11-01

    As part of a study of cytoskeletal proteins involved in Drosophila embryonic development, we have undertaken the molecular analysis of a 140-kD ATP-sensitive actin-binding protein (Miller, K. G., C. M. Field, and B. M. Alberts. 1989. J. Cell Biol. 109:2963-2975). Analysis of cDNA clones encoding this protein revealed that it represents a new class of unconventional myosin heavy chains. The amino-terminal two thirds of the protein comprises a head domain that is 29-33% identical (60-65% similar) to other myosin heads, and contains ATP-binding, actin-binding and calmodulin/myosin light chain-binding motifs. The carboxy-terminal tail has no significant similarity to other known myosin tails, but does contain a approximately 100-amino acid region that is predicted to form an alpha-helical coiled-coil. Since the unique gene that encodes this protein maps to the polytene map position 95F, we have named the new gene Drosophila 95F myosin heavy chain (95F MHC). The expression profile of the 95F MHC gene is complex. Examination of multiple cDNAs reveals that transcripts are alternatively spliced and encode at least three protein isoforms; in addition, a fourth isoform is detected on Western blots. Developmental Northern and Western blots show that transcripts and protein are present throughout the life cycle, with peak expression occurring during mid-embryogenesis and adulthood. Immunolocalization in early embryos demonstrates that the protein is primarily located in a punctate pattern throughout the peripheral cytoplasm. Most cells maintain a low level of protein expression throughout embryogenesis, but specific tissues appear to contain more protein. We speculate that the 95F MHC protein isoforms are involved in multiple dynamic processes during Drosophila development.

  4. Amino acid sequence of the alpha- and beta-globin chains of the Erabu sea snake (Laticaudia semifasciata).

    PubMed

    Eguchi, Yukinori; Eguchi, Tomoko

    2003-07-01

    We determined the complete amino acid sequences of the Erabu sea snake (Laticaudia semifasciata) hemoglobin by analyzing the intact globin chains, enzymatically digested fragments, and chemical cleavage fragments to clarify the molecular evolution and phylogenetic classification of the sea snake. The Erabu sea snake has two types of hemoglobin components, Hb-I and Hb-II, which contain different alpha- and beta-chains. This is the second report of the complete primary structure for hemoglobin of snakes. The sequences were compared with those of other reptilian hemoglobins. Amino acids at positions critical for the structure and physiological functions of hemoglobin were loosely conserved. The requirements for binding of ATP and of diphosphoglycerate as allosteric effectors of beta-globins seemed to be fulfilled.

  5. Cloning and sequencing of the gene encoding thermophilic beta-amylase of Clostridium thermosulfurogenes.

    PubMed Central

    Kitamoto, N; Yamagata, H; Kato, T; Tsukagoshi, N; Udaka, S

    1988-01-01

    A gene coding for thermophilic beta-amylase of Clostridium thermosulfurogenes was cloned into Bacillus subtilis, and its nucleotide sequence was determined. The nucleotide sequence suggested that the thermophilic beta-amylase is translated from monocistronic mRNA as a secretory precursor with a signal peptide of 32 amino acid residues. The deduced amino acid sequence of the mature beta-amylase contained 519 residues with a molecular weight of 57,167. The amino acid sequence of the C. thermosulfurogenes beta-amylase showed 54, 32, and 32% homology with those of the Bacillus polymyxa, soybean, and barley beta-amylases, respectively. Twelve well-conserved regions were found among the amino acid sequences of the four beta-amylases. To elucidate the mechanism rendering the C. thermosulfurogenes beta-amylase thermophilic, its amino acid sequence was compared with that of the B. polymyxa beta-amylase. The C. thermosulfurogenes beta-amyulase contained more Cys residues and fewer hydrophilic amino acid residues than the B. polymyxa beta-amylase did. Several regions were found in the amino acid sequence of the C. thermosulfurogenes beta-amylase, where the hydrophobicity was remarkably high as compared with that of the corresponding regions of the B. polymyxa beta-amylase. PMID:2461360

  6. Targeted disruption of the mouse beta1-adrenergic receptor gene: developmental and cardiovascular effects.

    PubMed Central

    Rohrer, D K; Desai, K H; Jasper, J R; Stevens, M E; Regula, D P; Barsh, G S; Bernstein, D; Kobilka, B K

    1996-01-01

    At least three distinct beta-adrenergic receptor (beta-AR) subtypes exist in mammals. These receptors modulate a wide variety of processes, from development and behavior, to cardiac function, metabolism, and smooth muscle tone. To understand the roles that individual beta-AR subtypes play in these processes, we have used the technique of gene targeting to create homozygous beta 1-AR null mutants (beta 1-AR -/-) in mice. The majority of beta 1-AR -/- mice die prenatally, and the penetrance of lethality shows strain dependence. Beta l-AR -/- mice that do survive to adulthood appear normal, but lack the chronotropic and inotropic responses seen in wild-type mice when beta-AR agonists such as isoproterenol are administered. Moreover, this lack of responsiveness is accompanied by markedly reduced stimulation of adenylate cyclase in cardiac membranes from beta 1-AR -/- mice. These findings occur despite persistent cardiac beta 2-AR expression, demonstrating the importance of beta 1-ARs for proper mouse development and cardiac function, while highlighting functional differences between beta-AR subtypes. Images Fig. 1 Fig. 3 PMID:8693001

  7. Dopamine beta-hydroxylase gene modulates individuals' empathic ability.

    PubMed

    Gong, Pingyuan; Liu, Jinting; Li, She; Zhou, Xiaolin

    2014-09-01

    Dopamine beta-hydroxylase (DBH), an enzyme that converts dopamine to norepinephrine, has broad influences on social functions. In this study, we examined to what extent two polymorphisms (-1021C/T and a 19 bp insertion/deletion) in DBH gene modulate individuals' empathic perception and response, which were measured, respectively, by reading the mind in the eyes test and the empathic concern subscale of interpersonal reactivity index. Results showed that polymorphism at -1021C/T, but not the 19 bp insertion/deletion, accounts for 2.3% variance of empathic perception and 1.4% variance of empathic response. Individuals with the CC genotype, which is associated with higher DBH activity, manifested greater empathic ability than those with CT/TT genotypes. These findings demonstrate the importance of DBH -1021C/T as a genetic basis of empathy and in predicting individual differences in social and affective processing. © The Author (2013). Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

  8. Inhibition of spermidine synthase gene expression by transforming growth factor-beta 1 in hepatoma cells.

    PubMed Central

    Nishikawa, Y; Kar, S; Wiest, L; Pegg, A E; Carr, B I

    1997-01-01

    We screened genes responsive to transforming growth factor-beta (TGF-beta 1) protein in a human hepatoma cell line (Hep3B) using a PCR-mediated differential display technique, in order to investigate the mechanisms involved in TGF-beta-induced growth suppression. We found a gene that was down-regulated by TGF-beta 1 to be completely identical in an approx. 620 bp segment to the gene for the enzyme spermidine synthase, which mediates the conversion of putrescine into spermidine. Both spermidine synthase mRNA expression and its enzyme activity were decreased after TGF-beta 1 treatment of Hep3B cells. The inhibition of spermidine synthase gene expression by TGF-beta 1 protein was also observed in other hepatoma cell lines. The expression of genes for other biosynthetic enzymes in polyamine metabolism (ornithine decarboxylase and S-adenosylmethionine decarboxylase) was also inhibited to the same extent as for spermidine synthase, while the gene expression of spermidine/spermine N1-acetyltransferase, a catabolic enzyme, was relatively resistant to TGF-beta 1. Spermine levels in Hep3B cells were decreased by TGF-beta 1 treatment, although the levels of spermidine and putrescine were unchanged, probably due to compensation by remaining spermidine/spermine N1-acetyltransferase activity. Exogenously added spermidine or spermine, but not putrescine, partially antagonized the growth-inhibitor effects of TGF-beta 1 on Hep3B cells. Our data suggest that down-regulation of gene expression of the enzymes involved in polyamine metabolism, including spermidine synthase, may be associated with the mechanism of TGF-beta-induced growth suppression. PMID:9020892

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

    SciTech Connect

    Dudas, Jozsef; Fullar, Alexandra; Bitsche, Mario; Schartinger, Volker; Kovalszky, Ilona; Sprinzl, Georg Mathias; Riechelmann, Herbert

    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

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

    PubMed

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

    1984-04-01

    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 the delta beta 0-thalassemic non-alpha globin fragments were defined providing the basis for cloning the delta beta 0-thalassemic beta-globin gene from the DNA of a heterozygous patient. This gene appears to carry a C----T single mutation causing the appearance of a stop codon at amino acid position 39 of the beta-globin gene. This mutation was previously reported in beta 0-thalassemic patients, in linkage with different haplotypes. We conclude that Sardinian delta beta 0-thalassemia is the result of two separate mutations, the former one (unknown) responsible for persistent expression of gamma-globin genes, the latter for beta 0-thalassemia.

  11. Molecular characterization of an atypical beta-thalassemia caused by a large deletion in the 5' beta-globin gene region.

    PubMed Central

    Popovich, B W; Rosenblatt, D S; Kendall, A G; Nishioka, Y

    1986-01-01

    We describe a Canadian family of Czechoslovakian descent that came to our attention because of an HbA2 percentage approximately twice that of an average case of heterozygous beta-thalassemia. This unique phenotype suggested to us the possibility of a novel genetic mechanism being responsible for their beta-thalassemia. To investigate this possibility, we mapped, cloned, and sequenced the mutant beta-globin allele. This molecular analysis demonstrated the presence of a unique 4,237 base pair (bp) deletion extending from 3.3 kilobases (kb) 5' of the beta-globin mRNA cap site to approximately the middle of beta IVS-2. This truncated beta-globin gene further extends the heterogeneity of mutations known to cause beta-thalassemia and delineates new sequences involved in nonhomologous recombination events in the beta-globin gene region. Images Fig. 2 Fig. 4 Fig. 5 Fig. 6 PMID:3799598

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

    PubMed

    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. [Investigation of beta-lactamase genes and clonal relationship among the extended-spectrum beta-lactamase producing nosocomial Escherichia coli isolates].

    PubMed

    Görgeç, Sündüz; Kuzucu, Çiğdem; Otlu, Barış; Yetkin, Funda; Ersoy, Yasemin

    2015-01-01

    Extended-spectrum beta-lactamase (ESBL) producing microorganisms currently cause a major problem. Among theseCTX-M beta-lactamase producing Escherichia coli has also disseminated worldwide as an important cause of both nosocomial and community-acquired infections. The aims of this study were to determine the prevalence of the beta-lactamase genes, antibiotic susceptibilities and clonal relationships of ESBL-producing nosocomial E.coli isolates. A total of 76 ESBL-producing E.coli strains isolated from urine (n= 26), blood (n= 25) and wound (n= 25) specimens of hospitalized patients identified as nosocomial infection agents according to the CDC criteria between June 2010-June 2011 were included in the study. Antibiotic susceptibilities of the isolates were detected by Kirby-Bauer disc diffusion method according to CLSI recommendations. ESBL production was tested by double disc diffusion method, and cefotaxime/cefotaxime-clavulanic acid E-test strips (AB Biodisk, Sweden) were used for indeterminate results. Presence of TEM, SHV, CTX-M, OXA-2 group, 0XA-10 group, PER, VEB and GES beta-lactamase genes were investigated by polymerase chain reaction (PCR) using specific primers. Pulsed-field gel electrophoresis (PFGE) method was used for the detection of clonal relationships among the strains. Most of the ESBL-producing E.coli strains were isolated from samples of inpatients in intensive care (35%), internal medicine (16%) and general surgery (13%) units. All of the 76 strains were found susceptible to imipenem, meropenem and amikacin; however all were resistant to cefotaxime and ceftriaxone. The susceptibility rates of the isolates to cefoxitin, ertapenem, cefoperazone/sulbactam, piperacillin-tazobactam, gentamicin, ciprofloxacin, cefepime, amoxicillin-clavulanic acid, aztreonam and ceftazidime were 96%, 83%, 63%, 61%, 50%, 41%, 25%, 21%, 20% and 18%, respectively. Among E.coli isolates, the frequency of CTX-M, TEM, OXA-2 group, PER, SHV and OXA-10 group beta

  14. Beta-lactam antibiotic biosynthetic genes have been conserved in clusters in prokaryotes and eukaryotes.

    PubMed Central

    Smith, D J; Burnham, M K; Bull, J H; Hodgson, J E; Ward, J M; Browne, P; Brown, J; Barton, B; Earl, A J; Turner, G

    1990-01-01

    A cosmid clone containing closely linked beta-lactam antibiotic biosynthetic genes was isolated from a gene library of Flavobacterium sp. SC 12,154. The location within the cluster of the DNA thought to contain the gene for delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS), the first step in the beta-lactam antibiotic biosynthetic pathway, was identified by a novel method. This DNA facilitated the isolation, by cross-hybridization, of the corresponding DNA from Streptomyces clavuligerus ATCC 27064, Penicillium chrysogenum Oli13 and Aspergillus nidulans R153. Evidence was obtained which confirmed that the cross-hybridizing sequences contained the ACVS gene. In each case the ACVS gene was found to be closely linked to other beta-lactam biosynthetic genes and constituted part of a gene cluster. Images Fig. 1. Fig. 2. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:2107074

  15. Diverse locations of amino acids in HLA-DR beta chains involved in polymorphic antibody binding epitopes on DR(alpha, beta 1*0101), DR(alpha, beta 1*1101), and DR(alpha,beta 3*0202) molecules.

    PubMed

    Fu, X T; Klohe, E; Alber, C; Yu, W Y; Ferrara, G B; Pistillo, M P; Ballas, M; Karr, R W

    1992-03-01

    In a previous study, we used transfectants expressing hybrid HLA-DR(beta 1*0403)/DR(beta 1*0701) chains to map sequences involved in polymorphic antibody binding epitopes on DR(alpha, beta 1*0403) or DR(alpha, beta 1*0701) molecules. Amino acids 1-40 of the beta 1 domain were found to make the major contributions to most of the antibody binding epitopes studied. To begin to localize sequences that contribute to polymorphic antibody epitopes on DR(alpha,beta 1*0101), DR(alpha,beta 1*1101) and DR(alpha,beta 3*0202) molecules, we used indirect immunofluorescence and flow cytometry to assess the binding of mAb to transfectants expressing hybrid DR(beta 1*0101)/DR(beta 1*1101) or DR(beta 1*1101)/DR(beta 3*0202) chains that divide the DR beta chain into three segments: amino acids 1-40, 41-97, and the beta 2 domain. The results indicate that amino acids 41-97 of the beta 1 domain on DR(beta 1*0101), DR(beta 1*1101), or DR(beta 3*0202) are critical in most of the epitopes, including those recognized by human antibodies MP4 and MP12, and mouse mAb GS88.2, I-LR1, 21r5, and 7.3.19.1, whereas amino acids 1-40 of DR(beta 1*1101) are critical in the epitope recognized by the MCS-7 mAb, and both segments 1-40 and 41-97 of DR(beta 1*1101) are important in the epitopes recognized by the I-LR2 and UL-52 mAbs. Based on these data and comparison of DR beta allelic protein sequences, the residues that may play critical roles in these antibody binding epitopes are predicted.

  16. Somatic generation of hybrid antibody H chain genes in transgenic mice via interchromosomal gene conversion

    PubMed Central

    1994-01-01

    We have constructed lines of mice with transgenes containing an antibody heavy (H) chain variable region (VHDJH) gene and various amounts of natural immunoglobulin (Ig) and plasmid flanking DNA. In these lines, recombination of the transgene and the endogenous Igh locus takes place in B cells, leading to the expression of functional H chains partially encoded by the transgenic VHDJH gene. Here, we demonstrate that the transgenic VHDJH gene, and various amounts of flanking sequence are recombined with Igh locus DNA via interchromosomal gene conversion. The structures of the resulting "hybrid" transgene-Igh H chain loci are consistent with the 3' end of the conversion occurring in regions of sequence identity, and the 5' end taking place between regions of little or no homology. This mode of antibody transgene recombination with the Igh locus is fundamentally different from the previously reported "trans H chain class switching" that results in reciprocal translocations. In contrast, this recombination resembles events previously observed in mammalian tissue culture cells between adjacent homologous chromosomal sequences, or transfected DNA and a homologous chromosomal target. Our data indicate that this recombination takes place at a low frequency, and that the frequency is influenced by both the length and extent of homology between the transgene and the Igh locus, but is not greatly affected by transgene copy number. This recombination pathway provides a novel approach for the subtle alteration of the clonal composition of the mouse B cell compartment in vivo using VH genes with defined structures and functions. PMID:8270869

  17. Digitalis purpurea P5 beta R2, encoding steroid 5 beta-reductase, is a novel defense-related gene involved in cardenolide biosynthesis.

    PubMed

    Pérez-Bermúdez, Pedro; García, Aurelio A Moya; Tuñón, Iñaki; Gavidia, Isabel

    2010-02-01

    The stereospecific 5 beta-reduction of progesterone is a required step for cardiac glycoside biosynthesis in foxglove plants. Recently, we have isolated the gene P5 beta R, and here we investigate the function and regulation of P5 beta R2, a new progesterone 5 beta-reductase gene from Digitalis purpurea. P5 beta R2 cDNA was isolated from a D. purpurea cDNA library and further characterized at the biochemical, structural and physiological levels. Like P5 beta R, P5 beta R2 catalyzes the 5 beta-reduction of the Delta(4) double bond of several steroids and is present in all plant organs. Under stress conditions or on treatment with chemical elicitors, P5 beta R expression does not vary, whereas P5 beta R2 is highly responsive. P5 beta R2 expression is regulated by ethylene and hydrogen peroxide. The correlation between P5 beta R2 expression and cardenolide formation demonstrates the key role of this gene in cardenolide biosynthesis, and therefore in the chemical defense of foxglove plants.

  18. A recombinant single-chain human class II MHC molecule (HLA-DR1) as a covalently linked heterotrimer of alpha chain, beta chain, and antigenic peptide, with immunogenicity in vitro and reduced affinity for bacterial superantigens.

    PubMed

    Zhu, X; Bavari, S; Ulrich, R; Sadegh-Nasseri, S; Ferrone, S; McHugh, L; Mage, M

    1997-08-01

    Major histocompatibility complex (MHC) class II molecules bind to numerous peptides and display these on the cell surface for T cell recognition. In a given immune response, receptors on T cells recognize antigenic peptides that are a minor population of MHC class II-bound peptides. To control which peptides are presented to T cells, it may be desirable to use recombinant MHC molecules with covalently bound antigenic peptides. To study T cell responses to such homogeneous peptide-MHC complexes, we engineered an HLA-DR1 cDNA coding for influenza hemagglutinin, influenza matrix, or HIV p24 gag peptides covalently attached via a peptide spacer to the N terminus of the DR1 beta chain. Co-transfection with DR alpha cDNA into mouse L cells resulted in surface expression of HLA-DR1 molecules that reacted with monoclonal antibodies (mAb) specific for correctly folded HLA-DR epitopes. This suggested that the spacer and peptide did not alter expression or folding of the molecule. We then engineered an additional peptide spacer between the C terminus of a truncated beta chain (without transmembrane or cytoplasmic domains) and the N terminus of full-length DR alpha chain. Transfection of this cDNA into mouse L cells resulted in surface expression of the entire covalently linked heterotrimer of peptide, beta chain, and alpha chain with the expected molecular mass of approximately 66 kDa. These single-chain HLA-DR1 molecules reacted with mAb specific for correctly folded HLA-DR epitopes, and identified one mAb with [MHC + peptide] specificity. Affinity-purified soluble secreted single-chain molecules with truncated alpha chain moved in electrophoresis as compact class II MHC dimers. Cell surface two-chain or single-chain HLA-DR1 molecules with a covalent HA peptide stimulated HLA-DR1-restricted HA-specific T cells. They were immunogenic in vitro for peripheral blood mononuclear cells. The two-chain and single-chain HLA-DR1 molecules with covalent HA peptide had reduced binding

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

    SciTech Connect

    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.

  20. Genomic structure of the human beta-PIX gene and its alteration in gastric cancer.

    PubMed

    Li, Zhong you; Wang, You jie; Song, Jian ping; Kataoka, Hideki; Yoshii, Shigeto; Gao, Chang ming; Wang, Ya ping; Zhou, Jian nong; Ota, Satoshi; Tanaka, Masamitsu; Sugimura, Haruhiko

    2002-03-28

    beta-PIX, a newly identified p21-activated kinase (PAK)-interacting exchange factors (PIX), encodes a guanine nucleotide exchange factor for Rho guanosine triphosphatases. Characterization of beta-PIX gene was performed using the BAC Library method. The beta-PIX gene has 17 exons and an A/T polymorphism at the 32nd base upstream of the intron/exon junction of exon 7. The frequencies of genotypes A/T, A/A and T/T were 23.6% (13/55), 72.7% (40/55) and 3.6% (2/55), respectively; these frequencies are in Hardy-Weinberg equilibrium. Two out of 14 informative tumors (14.3%) were shown to have lost their heterozygosity at this locus, but no mutations in the remaining alleles were detected. In addition, we examined the gene-expression profile in another set of 30 gastric samples, but no significant over-expression of either the beta-PIX gene or the alpha-PIX gene was found. Though the beta-PIX gene has been speculated to potentially have tumor-related biological characteristics, the findings of the present study suggest that the involvement of beta-PIX gene in human gastric carcinogenesis is minimal.

  1. On the road to gene therapy for beta-thalassemia and sickle cell anemia.

    PubMed

    Bank, Arthur

    2008-01-01

    Human globin gene therapy is a potential cure for sickle cell disease and beta-thalassemia (Cooley anemia). A clinical trial of this treatment is currently under way in Paris using lentiglobin vectors.

  2. Induction of cytochrome P450IA1 gene expression in rat epidermis and human keratinocytes by. beta. -napthoflavone and benzanthracene

    SciTech Connect

    Khan, I.U.; Mukhtar, H.; Bickers, D.R.; Haqqi, T.M. )

    1991-03-15

    Cytochrome P450IA1 (P450IA1) plays a major role in the bioactivation of procarcinogens in various tissues including skin. However, factors controlling the expression of P450IA1 gene message in mammalian skin are unknown. In this study, the polymerase chain reaction (PCR) using specific primers was employed to study the expression of P450IA1 mRNA transcripts in rat epidermis and human keratinocytes (HK) treated with {beta}-napthoflavone ({beta}NF) and benzanthracene (BA). Total RNA was extracted from the epidermis of control and inducer-treated 4-day-old and adult Sprague Dawley rats, and from control and inducer-treated HL. cDNAs were synthesized using random primers and reverse transcriptase. PCR products were analyzed on agarose gel and quantitated by densitometry. Inducer treatment of rats and HK resulted in several-fold increases in aryl hydrocarbon hydroxylase (AHH) activity. The level of P450IA1 gene message increased 2-5-fold in treated animals as compared to controls; higher basal level and inducibility in adult than in 4-day-old rats. This induction occurred as early as 4 h after {beta}NF application, reached a maximum at 16 h and returned to basal levels by 36 h. Exposure to {beta}NF and BA resulted in 2-3-fold increase in gene message in HK. Northern blot analysis complemented PCR data. These results indicate that in mammalian skin P450IA1 gene expression is increased by the inducers of epidermal AHH activity.

  3. Identification of Androgen Receptor and Beta-Catenin Target Genes in Prostate and Prostate Cancer

    DTIC Science & Technology

    2013-10-01

    Transdisciplinary Research in Epigenetics and Cancer Journal Clubs and Transdisciplinary Science Meetings, biweekly and monthly 3. To gain expertise...Target Genes in Prostate and Prostate Cancer PRINCIPAL INVESTIGATOR: Laura Lamb CONTRACTING ORGANIZATION: Washington University...TITLE AND SUBTITLE Identification of Androgen Receptor and Beta-Catenin Target Genes in Prostate and Prostate Cancer 5a. CONTRACT NUMBER Genes in

  4. Chromosomal mapping, sequence and transcription analysis of the porcine fertilin beta gene (ADAM2).

    PubMed

    Day, A E; Quilter, C R; Sargent, C A; Mileham, A J

    2003-10-01

    Fertilin beta (ADAM2) forms a part of the heterodimeric surface protein fertilin, found on the plasma membrane of mammalian sperm, and has been implicated in the process of sperm-egg fusion. Analysis of cDNA products obtained from adult porcine testis mRNA has presented a sequence corresponding to 2620 bp of the ADAM2 gene. This sequence contained an open reading frame encoding a 735-amino acid protein and homologous to ADAM2 genes known in other mammalian species. Polymerase chain reaction (PCR) analysis of genomic DNA showed that the 2620 bp of cDNA sequence comprises at least 21 exons and spans approximately 76 kb of genomic DNA, with its size and structure being relatively conserved between mouse, human and pig. Fluorescence in situ hybridization was used to map ADAM2 to chromosome 15 of the pig, using a bacterial artificial chromosome clone from the PigE BAC library. This finding is consistent with comparative mapping experiments performed between pig and human chromosomes. Analysis of nine mRNA samples, by reverse transcriptase-PCR, from different porcine tissues has also suggested that expression of ADAM2 is limited to the testis, a finding that is consistent with other mammalian species.

  5. APOE and A-betaPP Gene Variation in Cortical and Cerebrovascular Amyloid-beta Pathology and Alzheimer’s Disease: A Population-Based Analysis

    PubMed Central

    Peuralinna, Terhi; Tanskanen, Maarit; Mäkelä, Mira; Polvikoski, Tuomo; Paetau, Anders; Kalimo, Hannu; Sulkava, Raimo; Hardy, John; Lai, Shiao-Lin; Arepalli, Sampath; Hernandez, Dena; Traynor, Bryan J.; Singleton, Andrew; Tienari, Pentti J.; Myllykangas, Liisa

    2012-01-01

    Cortical and cerebrovascular amyloid-beta (A-beta) deposition is a hallmark of Alzheimer’s disease (AD), but also occurs in elderly people not affected by dementia. The apolipoprotein E (APOE) epsilon4 is a major genetic modulator of A-beta deposition and AD risk. Variants of the amyloid-beta protein precursor (A-betaPP) gene have been reported to contribute to AD and cerebral amyloid angiopathy (CAA). We analyzed the role of APOE and A-beta PP variants in cortical and cerebrovascular A-beta deposition, and neuropathologically verified AD (based on modified NIA-RI criteria) in a population-based autopsy sample of Finns aged ≥85 years (Vantaa85 + Study; n = 282). Our updated analysis of APOE showed strong associations of the epsilon4 allele with cortical (p = 4.91×10−17) and cerebrovascular (p = 9.87×10−11) A-beta deposition as well as with NIA-RI AD (p = 1.62×10−8). We also analyzed 60 single nucleotide polymorphisms (SNPs) at the A-betaPP locus. In single SNP or haplotype analyses there were no statistically significant A-betaPP locus associations with cortical or cerebrovascular A-beta deposition or with NIA-RI AD. We sequenced the promoter of the A-betaPP gene in 40 subjects with very high A-beta deposition, but none of these subjects had any of the previously reported or novel AD-associated mutations. These results suggest that cortical and cerebrovascular A-beta depositions are useful quantitative traits for genetic studies, as highlighted by the strong associations with the APOE epsilon4 variant. Promoter mutations or common allelic variation in the A-betaPP gene do not have a major contribution to cortical or cerebrovascular A-beta deposition, or very late-onset AD in this Finnish population based study. PMID:21654062

  6. The sizes of the CDR3 hypervariable regions of the murine T-cell receptor beta chains vary as a function of the recombined germ-line segments.

    PubMed Central

    Pannetier, C; Cochet, M; Darche, S; Casrouge, A; Zöller, M; Kourilsky, P

    1993-01-01

    A method using PCR amplification and primer extension with fluorescent oligonucleotides was developed to analyze T-cell repertoires. The sizes of the hypervariable CDR3-like regions of the murine T-cell antigen receptor beta chains were measured for all possible V beta-J beta combinations. This analysis shows that beta chains are distributed into at least 2000 groups, a value that provides a lower limit to their complexity. The CDR3 sizes appear to be dependent on the J beta and especially the V beta segment used and correlates with amino acid sequence motifs in the corresponding CDR1 region. This feature of T-cell receptors is discussed. Images Fig. 3 PMID:8483950

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

    PubMed

    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.

  8. Chain-length dependence of alpha-helix to beta-sheet transition in polylysine: model of protein aggregation studied by temperature-tuned FTIR spectroscopy.

    PubMed

    Dzwolak, Wojciech; Muraki, Takeshi; Kato, Minoru; Taniguchi, Yoshihiro

    2004-03-01

    The chain-length dependence of the alpha-helix to beta-sheet transition in poly(L-lysine) is studied by temperature-tuned FTIR spectroscopy. This study shows that heterogeneous samples of poly(L-lysine), comprising polypeptide chains with various lengths, undergo the alpha-beta transition at an intermediate temperature compared to homogeneous ingredients. This holds true as long as each individual fraction of the polypeptide is capable of adopting an antiparallel beta-sheet structure. The tendency is that the longer chain is, the lower the alpha-beta transition temperature is, which has been linked to the presence of distorted or solvated helices with turns or beta sheets in elongating chains of poly(L-lysine). As such helical structures are apparently conducive to the alpha-beta transition, this draws a comparison to the hypothesis of metastable protein conformational states being a common stage in amyloid-formation pathways. The antiparallel architecture of the beta sheet is likely to reflect the pretransition interhelical interactions in poly(L-lysine). Namely, the chains are arranged in an antiparallel manner because of energetically favored antiparallel pre-assembly of dipolar alpha helices. Copyright 2004 Wiley Periodicals, Inc.

  9. Effects of side-chain orientation on the 13C chemical shifts of antiparallel beta-sheet model peptides.

    PubMed

    Villegas, Myriam E; Vila, Jorge A; Scheraga, Harold A

    2007-02-01

    The dependence of the (13)C chemical shift on side-chain orientation was investigated at the density functional level for a two-strand antiparallel beta-sheet model peptide represented by the amino acid sequence Ac-(Ala)(3)-X-(Ala)(12)-NH(2) where X represents any of the 17 naturally occurring amino acids, i.e., not including alanine, glycine and proline. The dihedral angles adopted for the backbone were taken from, and fixed at, observed experimental values of an antiparallel beta-sheet. We carried out a cluster analysis of the ensembles of conformations generated by considering the side-chain dihedral angles for each residue X as variables, and use them to compute the (13)C chemical shifts at the density functional theory level. It is shown that the adoption of the locally-dense basis set approach for the quantum chemical calculations enabled us to reduce the length of the chemical-shift calculations while maintaining good accuracy of the results. For the 17 naturally occurring amino acids in an antiparallel beta-sheet, there is (i) good agreement between computed and observed (13)C(alpha) and (13)C(beta) chemical shifts, with correlation coefficients of 0.95 and 0.99, respectively; (ii) significant variability of the computed (13)C(alpha) and (13)C(beta) chemical shifts as a function of chi(1) for all amino acid residues except Ser; and (iii) a smaller, although significant, dependence of the computed (13)C(alpha) chemical shifts on chi(xi) (with xi > or = 2) compared to chi(1) for eleven out of seventeen residues. Our results suggest that predicted (13)C(alpha) and (13)C(beta) chemical shifts, based only on backbone (phi,psi) dihedral angles from high-resolution X-ray structure data or from NMR-derived models, may differ significantly from those observed in solution if the dihedral-angle preferences for the side chains are not taken into account.

  10. Identification of a Novel Single Nucleotide Polymorphism in Porcine Beta-Defensin-1 Gene.

    PubMed

    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.

  11. Cloning and expression in Pichia pastoris of a blue mussel (Mytilus edulis) beta-mannanase gene.

    PubMed

    Xu, Bingze; Sellos, Daniel; Janson, Jan-Christer

    2002-03-01

    Using PCR, cloning and sequencing techniques, a 1.1-kb complementary DNA fragment encoding for a beta-mannanase (mannan endo-1,4-beta-mannosidase, EC 3.2.1.78) has been identified in the digestive gland of blue mussel, Mytilus edulis. The cDNA sequence shows significant sequence identity to several beta-mannanases in glycoside hydrolase family 5. The beta-mannanase gene has been isolated and sequenced from gill tissue of blue mussel and contains five introns. The beta-mannanase has been expressed extracellularly in Pichia pastoris using the Saccharomyces cerevisiae alpha-factor signal sequence. The beta-mannanase was produced in a 14-L fermenter with an expression level of 900 mg.L-1. The expression level is strongly affected by the induction temperature. A two-step purification procedure, composed of a combination of immobilized metal ion affinity chromatography and ion exchange chromatography, is required to give a pure beta-mannanase. However, due to post-translational modifications, structural varieties regarding molecular mass and isoelectric point were obtained. The specific activity of the purified recombinant M. edulis beta-mannanase was close to that of the wild-type enzyme. Also pH and temperature optima were the same as for the native protein. In conclusion, P. pastoris is regarded as a suitable host strain for the production of blue mussel beta-mannanase. This is the first time a mollusc beta-mannanase has been characterized at the DNA level.

  12. A Drosophila gene encoding a protein resembling the human beta-amyloid protein precursor.

    PubMed Central

    Rosen, D R; Martin-Morris, L; Luo, L Q; White, K

    1989-01-01

    We have isolated genomic and cDNA clones for a Drosophila gene resembling the human beta-amyloid precursor protein (APP). This gene produces a nervous system-enriched 6.5-kilobase transcript. Sequencing of cDNAs derived from the 6.5-kilobase transcript predicts an 886-amino acid polypeptide. This polypeptide contains a putative transmembrane domain and exhibits strong sequence similarity to cytoplasmic and extracellular regions of the human beta-amyloid precursor protein. There is a high probability that this Drosophila gene corresponds to the essential Drosophila locus vnd, a gene required for embryonic nervous system development. Images PMID:2494667

  13. A Drosophila gene encoding a protein resembling the human. beta. -amyloid protein precursor

    SciTech Connect

    Rosen, D.R.; Martin-Morris, L.; Luo, L.; White, K. )

    1989-04-01

    The authors have isolated genomic and cDNA clones for a Drosophila gene resembling the human {beta}-amyloid precursor protein (APP). This gene produces a nervous system-enriched 6.5-kilobase transcript. Sequencing of cDNAs derived from the 6.5-kilobase transcript predicts an 886-amino acid polypeptide. This polypeptide contains a putative transmembrane domain and exhibits strong sequence similarity to cytoplasmic and extracellular regions of the human {beta}-amyloid precursor protein. There is a high probability that this Drosophila gene corresponds to the essential Drosophila locus vnd, a gene required for embryonic nervous system development.

  14. Modification of the beta 2-adrenergic receptor to engineer a receptor-effector complex for gene therapy.

    PubMed

    Small, K M; Brown, K M; Forbes, S L; Liggett, S B

    2001-08-24

    Depressed G-protein-coupled receptor (GPCR) signaling has been implicated as a component of the pathophysiology of a number of complex diseases including heart failure and asthma, and augmentation or restoration of signaling by various means has been shown to improve organ function. Because some properties of native GPCRs are disadvantageous for ectopic therapeutic expression, we utilized the beta(2)-adrenergic receptor (beta(2)AR) as a scaffold to construct a highly modified therapeutic receptor-effector complex (TREC) suitable for gene therapy. Altogether, 19 modifications were made to the receptor. The ligand-binding site was re-engineered in TM-3 so that a beta-hydroxylmethyl side chain acts as a proton donor for the binding of a novel ligand. In addition, sites critical for agonist-promoted down-regulation in the amino terminus and for phosphorylation by GPCR kinases, and protein kinases A and C, in the third intracellular loop and the carboxyl terminus of the receptor were altered. These modifications of the receptor resulted in depressed agonist-stimulated adenylyl cyclase activity (26.8 +/- 2.1 versus 41.4 +/- 8 pmol/min/mg for wild-type beta(2)AR). This was fully restored by fusing the carboxyl terminus of the modified receptor to G alpha(s) (43.3 +/- 2.7 pmol/min/mg). The fully modified fused receptor was not activated by beta-agonists but rather by a nonbiogenic amine agonist that itself failed to activate the wild-type beta(2)AR. This two-way selectivity thus provides targeted activation based on physiologic status. Furthermore, the TREC did not display tachyphylaxis to prolonged agonist exposure (desensitization was 1 +/- 5% versus 55 +/- 4% for wild-type beta(2)AR). Thus, despite extensive alterations in regions of conformational lability, the beta(2)AR can be tailored to have optimal signaling characteristics for gene therapy. As a general paradigm, TRECs for enhancement of other G-protein signaling appear to be feasible for modification of other

  15. A novel short-chain peptide BmKX from the Chinese scorpion Buthus martensi Karsch, sequencing, gene cloning and structure determination.

    PubMed

    Wang, Chun-guang; Cai, Zheng; Lu, Wuyuan; Wu, Jihui; Xu, Yingqi; Shi, Yunyu; Chi, Cheng-wu

    2005-03-01

    Scorpion venom is a rich source of bioactive peptides. From the venom of Chinese scorpion Buthus martensi Karsch (BmK), a novel short chain peptide BmKX of 31-amino acid residues was purified, and its amino acid sequence and gene structure were determined. The gene of BmKX was composed of two exons interrupted by an 86-bp intron at the codon-7 upstream of the mature peptide. Although its gene structure is similar to those of other known scorpion toxins, its amino acid sequence, especially the cysteine framework, is different from those of all other known subfamilies of short-chain scorpion toxins. The solution structure of BmKX, determined with two-dimensional NMR spectroscopy, shows that BmKX also forms a typical cysteine-stabilized alpha/beta scaffold adopted by most short-chain scorpion toxins, consisting of a short 3(10)-helix and a two-stranded antiparallel beta-sheet, and the short N-terminal segment forms a pseudo-strand of the beta-sheet. However, the orientation between the helix and the beta-sheet is significantly different from the others, which might be the reason for its unique but still unclear physiological function.

  16. The C-terminus of the {gamma}2 chain but not of the {beta}3 chain of laminin-332 is indirectly but indispensably necessary for integrin-mediated cell reactions

    SciTech Connect

    Navdaev, Alexei; Heitmann, Vanessa; Santana Evangelista, Karla de; Moergelin, Matthias; Wegener, Joachim; Eble, Johannes A.

    2008-02-01

    Using a recombinant mini-laminin-332, we showed that truncation of the three C-terminal amino acids of the {gamma}2 chain, but not of the C-terminal amino acid of the {beta}3 chain, completely abolished {alpha}3{beta}1 integrin binding and its cellular functions, such as attachment and spreading. However, a synthetic peptide mimicking the {gamma}2 chain C-terminus did not interfere with {alpha}3{beta}1 integrin binding or cell adhesion and spreading on laminin-332 as measured by protein interaction assays and electric cell-substrate impedance sensing. Nor was the soluble peptide able to restore the loss of integrin-mediated cell adhesiveness to mini-laminin-332 after deletion of the {gamma}2 chain C-terminus. These findings spoke against the hypothesis that the {gamma}2 chain C-terminus of laminin-332 is a part of the {alpha}3{beta}1 integrin interaction site. In addition, structural studies with electron microscopy showed that truncation of the {gamma}2 chain C-terminus opened up the compact supradomain structure of LG1-3 domains. Thus, by inducing or stabilizing an integrin binding-competent conformation or array of the LG1-3 domains, the {gamma}2 chain C-terminus plays an indirect but essential role in laminin-332 recognition by {alpha}3{beta}1 integrin and, hence, its cellular functions.

  17. The exon-intron organization of the human erythroid [beta]-spectrin gene

    SciTech Connect

    Amin, K.M.; Forget, B.G. ); Scarpa, A.L.; Curtis, P.J. ); Winkelmann, J.C. )

    1993-10-01

    The human erythrocyte [beta]-spectrin gene DNA has been cloned from overlapping human genomic phage and cosmid recombinants. The entire erythroid [beta]-spectrin mRNA is encoded by 32 exons that range in size from 49 to 871 bases. The exon/intron junctions have been identified and the exons mapped. There is no correlation between intron positions and the repeat units of 106 amino acids within domain II of the [beta]-spectrin gene. The scatter of the introns over the 17 repeats argues against the 106-amino-acid unit representing a minigene that underwent repeated duplication resulting in the present [beta]-spectrin gene. In fact, the two largest exons, exon 14 (871 bp) and 16 (757 bp), extend over 4 and 3 repeat units of 106 amino acids, respectively, while repeat [beta]10 is encoded by 4 exons. No single position of an intron in the [beta]-spectrin gene is conserved between any of the 17 [beta]-spectrin and 22 [alpha]-spectrin repeat units. The nucleotide sequences of the exon/intron boundaries conform to the consensus splice site sequences except for exon 20, whose 5[prime] donor splice-site sequence begins with GC. The [beta]-spectrin isoform present in the human brain, the skeletal muscle, and the cardiac muscle is an alternatively spliced product of the erythroid [beta]-spectrin gene. This splice site is located within the coding sequences of exon 32 and its utilization in nonerythroid tissues leads to the use of 4 additional downstream exons with a size range of 44 to 530 bp. 55 refs., 3 figs., 3 tabs.

  18. Comparative Studies of Vertebrate Beta Integrin Genes and Proteins: Ancient Genes in Vertebrate Evolution

    PubMed Central

    Holmes, Roger S.; Rout, Ujjwal K.

    2011-01-01

    Intregins are heterodimeric α- and β-subunit containing membrane receptor proteins which serve various cell adhesion roles in tissue repair, hemostasis, immune response, embryogenesis and metastasis. At least 18 α- (ITA or ITGA) and 8 β-integrin subunits (ITB or ITGB) are encoded on mammalian genomes. Comparative ITB amino acid sequences and protein structures and ITB gene locations were examined using data from several vertebrate genome projects. Vertebrate ITB genes usually contained 13–16 coding exons and encoded protein subunits with ∼800 amino acids, whereas vertebrate ITB4 genes contained 36-39 coding exons and encoded larger proteins with ∼1800 amino acids. The ITB sequences exhibited several conserved domains including signal peptide, extracellular β-integrin, β-tail domain and integrin β-cytoplasmic domains. Sequence alignments of the integrin β-cytoplasmic domains revealed highly conserved regions possibly for performing essential functions and its maintenance during vertebrate evolution. With the exception of the human ITB8 sequence, the other ITB sequences shared a predicted 19 residue α-helix for this region. Potential sites for regulating human ITB gene expression were identified which included CpG islands, transcription factor binding sites and microRNA binding sites within the 3′-UTR of human ITB genes. Phylogenetic analyses examined the relationships of vertebrate beta-integrin genes which were consistent with four major groups: 1: ITB1, ITB2, ITB7; 2: ITB3, ITB5, ITB6; 3: ITB4; and 4: ITB8 and a common evolutionary origin from an ancestral gene, prior to the appearance of fish during vertebrate evolution. The phylogenetic analyses revealed that ITB4 is the most likely primordial form of the vertebrate β integrin subunit encoding genes, that is the only β subunit expressed as a constituent of the sole integrin receptor ‘α6β4’ in the hemidesmosomes of unicellular organisms. PMID:24970121

  19. Cardiac beta-myosin heavy chain defects in two families with non-compaction cardiomyopathy: linking non-compaction to hypertrophic, restrictive, and dilated cardiomyopathies.

    PubMed

    Hoedemaekers, Yvonne M; Caliskan, Kadir; Majoor-Krakauer, Danielle; van de Laar, Ingrid; Michels, Michelle; Witsenburg, Maarten; ten Cate, Folkert J; Simoons, Maarten L; Dooijes, Dennis

    2007-11-01

    Cardiomyopathies are classified according to distinct morphological characteristics. They occur relatively frequent and are an important cause of mortality and morbidity. Isolated ventricular non-compaction or non-compaction cardiomyopathy (NCCM) is characterized by an excessively thickened endocardial layer with deep intertrabecular recesses, reminiscent of the myocardium during early embryogenesis. Aims Autosomal-dominant as well as X-linked inheritance for NCCM has been described and several loci have been associated with the disease. Nevertheless, a major genetic cause for familial NCCM remains to be identified. Methods and Results We describe, in two separate autosomal-dominant NCCM families, the identification of mutations in the sarcomeric cardiac beta-myosin heavy chain gene (MYH7), known to be associated with hypertrophic cardiomyopathy (HCM), restricted cardiomyopathy (RCM), and dilated cardiomyopathy (DCM). Conclusion These results confirm the genetic heterogeneity of NCCM and suggest that the molecular classification of cardiomyopathies includes an MYH7-associated spectrum of NCCM with HCM, RCM, and DCM.

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

    PubMed

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

    NASA Astrophysics Data System (ADS)

    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 < 0.05) higher during the thermal stress. Pearson correlation coefficient analysis revealed that the expression of ATPase Β1, ATPase B2, and ATPase B3 is highly correlated ( 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.

  2. Evidence for a physical association between the Shc-PTB domain and the beta c chain of the granulocyte-macrophage colony-stimulating factor receptor.

    PubMed

    Pratt, J C; Weiss, M; Sieff, C A; Shoelson, S E; Burakoff, S J; Ravichandran, K S

    1996-05-24

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates the growth and function of several myeloid cell types at different stages of maturation. The effects of GM-CSF are mediated through a high affinity receptor that is composed of two chains: a unique, ligand-specific alpha chain and a beta common chain (beta c) that is also a component of the receptors for interleukin 3 (IL-3) and IL-5. Beta c plays an essential role in the transduction of extra cellular signals to the nucleus through its recruitment of secondary messengers. Several downstream signaling events induced by GM-CSF stimulation have been described, including activation of tyrosine kinases and tyrosine phosphorylation of cellular proteins (including beta c) and activation of the Ras/mitogen-activated protein kinase and the JAK/STAT pathways. A region within the beta c cytoplasmic tail (amino acids 517-763) has been reported to be necessary for tyrosine phosphorylation of the adapter protein, Shc, and for the subsequent GM-CSF-induced activation of Ras. In this paper, we describe a physical association between the tyrosine phosphorylated GM-CSF receptor (GMR)-beta c chain and Shc in vivo. Using a series of cytoplasmic truncation mutants of beta c and various mutant Shc proteins, we demonstrate that the N-terminal phosphotyrosine-binding (PTB) domain of Shc binds to a short region of beta c (amino acids 549-656) that contains Tyr577. Addition of a specific phosphopeptide encoding amino acids surrounding this tyrosine inhibited the interaction between beta c and shc. Moreover, mutation of a key residue within the phosphotyrosine binding pocket of the Shc-PTB domain abrogated its association with beta c. These observations provide an explanation for the previously described requirement for Tyr577 of beta c for GM-CSF-induced tyrosine phosphorylation of Shc and have implications for Ras activation through the GM-CSF, IL-3, and IL-5 receptors.

  3. Growth inhibition of human pancreatic cancer cells by human interferon-beta gene combined with gemcitabine.

    PubMed

    Endou, Masato; Mizuno, Masaaki; Nagata, Takuya; Tsukada, Kazuhiro; Nakahara, Norimoto; Tsuno, Takaya; Osawa, Hirokatsu; Kuno, Tomohiko; Fujita, Mitsugu; Hatano, Manabu; Yoshida, Jun

    2005-02-01

    We examined the anti-tumor effect of cationic multilamellar liposome containing human IFN-beta (huIFN-beta) gene against cultured human pancreatic cancer cells. We also evaluated the combined effect of huIFN-beta gene entrapped in liposomes and gemcitabine. Furthermore, we examined the anti-tumor mechanisms of the therapy, with emphasis on the Ras-related signal pathway. Three human pancreatic cancer cell lines (AsPc-1, MIAPaCa-2, and PANC-1) were used in this study. The growth inhibition together with the therapy were evaluated by WST-1 assay; the production of huIFN-beta protein was measured by ELISA; the cell cycle and apoptosis were analyzed using a FACScan flow cytometer; the protein levels of Son of sevenless (SOS-1) and Ras-GAP were measured by Western blotting; and the activation of Ras-GTP was evaluated by the immunoprecipitation method. As a result, we found that huIFN-beta gene entrapped in liposomes demonstrated a strong anti-tumor effect against human pancreatic cancer cells. The treatment that combined huIFN-beta gene entrapped in liposomes and gemcitabine was more effective than each treatment alone. Although gemcitabine remarkably reduced the level of SOS-1, the above combined therapy reduced the level of SOS-1 even more significantly. Both huIFN-beta gene entrapped in liposomes and the com-bination of huIFN-beta gene entrapped in liposomes and gemcitabine increased the level of Ras-GAP, and decreased the activity of Ras-GTP. These results suggest that this combination therapy can induce strong anti-tumor activity against human pancreatic cancer cells through the regulation of the Ras-related signal pathway.

  4. Translocation of peptides through microsomal membranes is a rapid process and promotes assembly of HLA-B27 heavy chain and beta 2- microglobulin translated in vitro

    PubMed Central

    1991-01-01

    We have translated major histocompatibility complex (MHC) class I heavy chains and human beta 2-microglobulin in vitro in the presence of microsomal membranes and a peptide from the nucleoprotein of influenza A. This peptide stimulates assembly of HLA-B27 heavy chain and beta 2- microglobulin about fivefold. By modifying this peptide to contain biotin at its amino terminus, we could precipitate HLA-B27 heavy chains with immobilized streptavidin, thereby directly demonstrating class I heavy chain-peptide association under close to physiological conditions. The biotin-modified peptide stimulates assembly to the same extent as the unmodified peptide. Both peptides bind to the same site on the HLA-B27 molecule. Immediately after synthesis of the HLA-B27 heavy chain has been completed, it assembles with beta 2-microglobulin and peptide. These interactions occur in the lumen of the microsomes (endoplasmic reticulum), demonstrating that the peptide must cross the microsomal membrane in order to promote assembly. The transfer of peptide across the microsomal membrane is a rapid process, as peptide binding to heavy chain-beta 2-microglobulin complexes is observed in less than 1 min after addition of peptide. By using microsomes deficient of beta 2-microglobulin (from Daudi cells), we find a strict requirement of beta 2-microglobulin for detection of peptide interaction with the MHC class I heavy chain. Furthermore, we show that heavy chain interaction with beta 2-microglobulin is likely to precede peptide binding. Biotin-modified peptides are likely to become a valuable tool in studying MHC antigen interaction and assembly. PMID:1955465

  5. Reference genes for quantitative real-time polymerase chain reaction studies in soybean plants under hypoxic conditions.

    PubMed

    Nakayama, T J; Rodrigues, F A; Neumaier, N; Marcelino-Guimarães, F C; Farias, J R B; de Oliveira, M C N; Borém, A; de Oliveira, A C B; Emygdio, B M; Nepomuceno, A L

    2014-02-13

    Quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful tool used to measure gene expression. However, because of its high sensitivity, the method is strongly influenced by the quality and concentration of the template cDNA and by the amplification efficiency. Relative quantification is an effective strategy for correcting random and systematic errors by using the expression level of reference gene(s) to normalize the expression level of the genes of interest. To identify soybean reference genes for use in studies of flooding stress, we compared 5 candidate reference genes (CRGs) with the NormFinder and GeNorm programs to select the best internal control. The expression stability of the CRGs was evaluated in root tissues from soybean plants subjected to hypoxic conditions. Elongation factor 1-beta and actin-11 were identified as the most appropriate genes for RT-qPCR normalization by both the NormFinder and GeNorm analyses. The expression profiles of the genes for alcohol dehydrogenase 1, sucrose synthase 4, and ascorbate peroxidase 2 were analyzed by comparing different normalizing combinations (including no normalization) of the selected reference genes. Here, we have identified potential genes for use as references for RT-qPCR normalization in experiments with soybean roots growing in O2-depleted environments, such as flooding-stressed plants.

  6. Association of polymorphisms in the beta2-adrenoreceptor gene with higher levels of parasitic infection.

    PubMed

    Ramsay, C E; Hayden, C M; Tiller, K J; Burton, P R; Hagel, I; Palenque, M; Lynch, N R; Goldblatt, J; LeSouëf, P N

    1999-03-01

    The diminishing incidence of parasitic infection in westernised societies has been suggested to result in an increased prevalance of asthma. Asthma is a polygenic disease and genome screens have shown that genes on chromosome 5q31-33 are strongly linked to the disease. The gene for the beta2-adrenoreceptor is located in this region and two polymorphisms have been identified that result in amino acid changes at positions 16 (ArgGly) and 27 (GlnGlu). To determine whether these polymorphisms influence asthma and parasitic infection, a genotype/phenotype study has been performed on a cohort of 126 children from Coche Island in Venezuela. There is a high incidence of asthma on the island and intestinal helminthiasis is endemic. Genotyping for both polymorphisms was carried out by using the polymerase chain reaction and allele-specific oligonucleotide hybridisation. Genotype frequencies in this cohort were consistent with other studies and both polymorphisms were in significant linkage disequilibrium. Individuals who were homozygous for Arg16 had significantly higher levels of specific IgE to Ascaris lumbricoides (P=0.002), significantly higher A. lumbricoides egg counts (P<0.001) and significantly larger wheal sizes following skin-prick testing with A. lumbricoides allergen (P=0.008). There was no association between either polymorphism and total serum IgE or asthma in this population. A combination of mast cell degranulation and the lung migratory phase of A. lumbricoides larvae may result in bronchoconstriction in infected individuals. These results suggest that the Gly 16 allele confers resistance to high levels of parasitic infection in this population. An alternative explanation for the association is that it may be the result of linkage disequilibrium with other genes in the chromosome 5q31-33 region.

  7. The human ATP synthase beta subunit gene: sequence analysis, chromosome assignment, and differential expression.

    PubMed

    Neckelmann, N; Warner, C K; Chung, A; Kudoh, J; Minoshima, S; Fukuyama, R; Maekawa, M; Shimizu, Y; Shimizu, N; Liu, J D

    1989-11-01

    In humans, the functional F0F1-ATP synthase beta subunit gene is located on chromosome 12 in the p13----qter region. Other partially homologous sequences have been detected on chromosomes 2 and 17. The bona fide beta subunit gene has 10 exons encoding a leader peptide of 49 amino acids and a mature protein of 480 amino acids. Thirteen Alu family DNA repeats are found upstream from the gene and in four introns. The gene has four "CCAAT" sequences upstream and in close proximity to the transcriptional initiation site. A 13-bp motif is found in the 5' nontranscribed region of both the beta subunit gene and an ADP/ATP translocator gene that is expressed in high levels in cardiac and skeletal muscle. Analysis of the beta subunit mRNA levels reveals marked differences among tissues. The highest levels are found in heart, lower levels in skeletal muscle, and the lowest levels in liver and kidney. These findings suggest that the tissue-specific levels of ATP synthase beta subunit mRNA may be generated through transcriptional control.

  8. Expression and nucleotide sequence of the Clostridium acetobutylicum beta-galactosidase gene cloned in Escherichia coli.

    PubMed Central

    Hancock, K R; Rockman, E; Young, C A; Pearce, L; Maddox, I S; Scott, D B

    1991-01-01

    A gene library for Clostridium acetobutylicum NCIB 2951 was constructed in the broad-host-range cosmid pLAFR1, and cosmids containing the beta-galactosidase gene were isolated by direct selection for enzyme activity on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside) plates after conjugal transfer of the library to a lac deletion derivative of Escherichia coli. Analysis of various pSUP202 subclones of the lac cosmids on X-Gal plates localized the beta-galactosidase gene to a 5.1-kb EcoRI fragment. Expression of the Clostridium beta-galactosidase gene in E. coli was not subject to glucose repression. By using transposon Tn5 mutagenesis, two gene loci, cbgA (locus I) and cbgR (locus II), were identified as necessary for beta-galactosidase expression in E. coli. DNA sequence analysis of the entire 5.1-kb fragment identified open reading frames of 2,691 and 303 bp, corresponding to locus I and locus II, respectively, and in addition a third truncated open reading frame of 825 bp. The predicted gene product of locus I, CbgA (molecular size, 105 kDa), showed extensive amino acid sequence homology with E. coli LacZ, E. coli EbgA, and Klebsiella pneumoniae LacZ and was in agreement with the size of a polypeptide synthesized in maxicells containing the cloned 5.1-kb fragment. The predicted gene product of locus II, CbgR (molecular size, 11 kDa) shares no significant homology with any other sequence in the current DNA and protein sequence data bases, but Tn5 insertions in this gene prevent the synthesis of CbgA. Complementation experiments indicate that the gene product of cbgR is required in cis with cbgA for expression of beta-galactosidase in E. coli. Images PMID:1850729

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

    SciTech Connect

    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.

  10. Regulatory Divergence among Beta-Keratin Genes during Bird Evolution.

    PubMed

    Bhattacharjee, Maloyjo Joyraj; Yu, Chun-Ping; Lin, Jinn-Jy; Ng, Chen Siang; Wang, Tzi-Yuan; Lin, Hsin-Hung; Li, Wen-Hsiung

    2016-11-01

    Feathers, which are mainly composed of α- and β-keratins, are highly diversified, largely owing to duplication and diversification of β-keratin genes during bird evolution. However, little is known about the regulatory changes that contributed to the expressional diversification of β-keratin genes. To address this issue, we studied transcriptomes from five different parts of chicken contour and flight feathers. From these transcriptomes we inferred β-keratin enriched co-expression modules of genes and predicted transcription factors (TFs) of β-keratin genes. In total, we predicted 262 TF-target gene relationships in which 56 TFs regulate 91 β-keratin genes; we validated 14 of them by in vitro tests. A dual criterion of TF enrichment and "TF-target gene" expression correlation identified 26 TFs as the major regulators of β-keratin genes. According to our predictions, the ancestral scale and claw β-keratin genes have common and unique regulators, whereas most feather β-keratin genes show chromosome-wise regulation, distinct from scale and claw β-keratin genes. Thus, after expansion from the β-keratin gene on Chr7 to other chromosomes, which still shares a TF with scale and claw β-keratin genes, most feather β-keratin genes have recruited distinct or chromosome-specific regulators. Moreover, our data showed correlated gene expression profiles, positive or negative, between predicted TFs and their target genes over the five studied feather regions. Therefore, regulatory divergences among feather β-keratin genes have contributed to structural differences among different parts of feathers. Our study sheds light on how feather β-keratin genes have diverged in regulation from scale and claw β-keratin genes and among themselves. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  11. Beta-sheet side chain polymers synthesized by atom-transfer radical polymerization.

    PubMed

    Ayres, Lee; Adams, P Hans H M; Löwik, Dennis W P M; van Hest, Jan C M

    2005-01-01

    Silks are a widely studied class of naturally occurring structural proteins. Dragline spider silk, in particular, is considered to be nature's high-performance material due to its remarkable combination of strength and toughness. These mechanical properties stem from the protein secondary structure, a combination of well-defined beta-sheets in a less well-defined glycine-rich matrix. The translation of this structure into a synthetic polymer was the aim of this investigation. To achieve this, a peptide-based monomer containing the sequence alanine-glycine-alanine-glycine, a well-known beta-sheet-forming sequence found in silk, was synthesized. Using atom-transfer radical polymerization and a bifunctional initiator, a well-defined peptide-based polymer was prepared. This was then used as the macroinitiator for the polymerization of methyl methacrylate. The resulting well-defined triblock copolymer was analyzed using IR spectroscopy, which clearly showed beta-sheet secondary structure had been introduced.

  12. Laminins containing the beta2 chain modulate the precise organization of CNS synapses.

    PubMed

    Egles, Christophe; Claudepierre, Thomas; Manglapus, Mary K; Champliaud, Marie-France; Brunken, William J; Hunter, Dale D

    2007-03-01

    Synapses are formed and stabilized by concerted interactions of pre-, intra-, and post-synaptic components; however, the precise nature of the intrasynaptic components in the CNS remains obscure. Potential intrasynaptic components include extracellular matrix molecules such as laminins; here, we isolate beta2-containing laminins, including perhaps laminins 13 (alpha3beta2gamma3) and 14 (alpha4beta2gamma3), from CNS synaptosomes suggesting a role for these molecules in synaptic organization. Indeed, hippocampal synapses that form in vivo in the absence of these laminins are malformed at the ultrastructural level and this malformation is replicated in synapses formed in vitro, where laminins are provided largely by the post-synaptic neuron. This recapitulation of the in vivo function of laminins in vitro suggests that the malformations are a direct consequence of the removal of laminins from the synapse. Together, these results support a role for neuronal laminins in the structural integrity of central synapses.

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

    PubMed

    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

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

    PubMed

    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.

  15. Synthesis and ocular antihypertensive activity of new imidazolidine derivatives containing a beta-blocking side chain.

    PubMed

    Huber, D; Ehrhardt, J D; Decker, N; Himber, J; Andermann, G; Leclerc, G

    1991-11-01

    The syntheses of new phenylimidazolidine derivatives (3-6)1 containing a propanolamine oxime or an oxypropanolamine moiety attached either to the aromatic or to the imidazolidine ring are described. These compounds were evaluated for potential ocular antihypertensive activity in alpha-chymotrypsin-induced ocular hypertension in rabbits. These compounds represent a unique series of effective ocular antihypertensive agents that despite possessing structural characteristics of beta-blockers and of imidazolidine derivatives, exhibit weak alpha- and beta-adrenergic agonist and antagonist activities. These findings may be of significant therapeutic importance in the medical management of glaucoma.

  16. Cloning and sequence determination of a human rheumatoid factor light-chain gene.

    PubMed Central

    Jirik, F R; Sorge, J; Fong, S; Heitzmann, J G; Curd, J G; Chen, P P; Goldfien, R; Carson, D A

    1986-01-01

    The contribution of germ-line variable regions to autoantibody formation in humans is poorly understood. To study the gene structure of a human autoantibody, chronic lymphatic leukemia (CLL) cells from a patient with an IgM anti-IgG (rheumatoid factor, RF) paraprotein were utilized. The rearranged immunoglobulin gene encoding the kappa light chain for the RF was cloned, and the nucleic acid sequence of its variable region was determined. As demonstrated by Southern blot analysis using a kappa joining-region probe, the CLL cells, stable CLL-WIL2-729-HF2 RF-secreting hybridomas, and the cloned light-chain gene all had an identical restriction fragment containing the rearranged light-chain gene. The CLL RF light chains reacted weakly with an antipeptide antibody against a primary structure-dependent idiotype present on the light chains of the majority of IgM RF paraproteins. The nucleotide and predicted amino acid sequences of the CLL light-chain gene place it in the kappa III variable-region subgroup, and a comparison to known RF paraproteins reveals marked homology to the light-chain amino acid sequence of the IgM RF paraprotein Pom. Both Pom and the CLL light chain appear to identify a second kappa III gene or gene group that is able to encode RF paraprotein light chains. Images PMID:3083417

  17. Genes involved in long-chain alkene biosynthesis in Micrococcus luteus.

    PubMed

    Beller, Harry R; Goh, Ee-Been; Keasling, Jay D

    2010-02-01

    Aliphatic hydrocarbons are highly appealing targets for advanced cellulosic biofuels, as they are already predominant components of petroleum-based gasoline and diesel fuels. We have studied alkene biosynthesis in Micrococcus luteus ATCC 4698, a close relative of Sarcina lutea (now Kocuria rhizophila), which 4 decades ago was reported to biosynthesize iso- and anteiso-branched, long-chain alkenes. The underlying biochemistry and genetics of alkene biosynthesis were not elucidated in those studies. We show here that heterologous expression of a three-gene cluster from M. luteus (Mlut_13230-13250) in a fatty acid-overproducing Escherichia coli strain resulted in production of long-chain alkenes, predominantly 27:3 and 29:3 (no. carbon atoms: no. C=C bonds). Heterologous expression of Mlut_13230 (oleA) alone produced no long-chain alkenes but unsaturated aliphatic monoketones, predominantly 27:2, and in vitro studies with the purified Mlut_13230 protein and tetradecanoyl-coenzyme A (CoA) produced the same C(27) monoketone. Gas chromatography-time of flight mass spectrometry confirmed the elemental composition of all detected long-chain alkenes and monoketones (putative intermediates of alkene biosynthesis). Negative controls demonstrated that the M. luteus genes were responsible for production of these metabolites. Studies with wild-type M. luteus showed that the transcript copy number of Mlut_13230-13250 and the concentrations of 29:1 alkene isomers (the dominant alkenes produced by this strain) generally corresponded with bacterial population over time. We propose a metabolic pathway for alkene biosynthesis starting with acyl-CoA (or-ACP [acyl carrier protein]) thioesters and involving decarboxylative Claisen condensation as a key step, which we believe is catalyzed by OleA. Such activity is consistent with our data and with the homology (including the conserved Cys-His-Asn catalytic triad) of Mlut_13230 (OleA) to FabH (beta-ketoacyl-ACP synthase III), which

  18. Critical contribution of beta chain residue 57 in peptide binding ability of both HLA-DR and -DQ molecules.

    PubMed Central

    Nepom, B S; Nepom, G T; Coleman, M; Kwok, W W

    1996-01-01

    Position 57 in the beta chain of HLA class II molecules maintains an Asp/non-Asp dimorphism that has been conserved through evolution and is implicated in susceptibility to some autoimmune diseases. The latter effect may be due to the influence of this residue on the ability of class II alleles to bind specific pathogenic peptides. We utilized highly homologous pairs of both DR and DQ alleles that varied at residue 57 to investigate the impact of this dimorphism on binding of model peptides. Using a direct binding assay of biotinylated peptides on whole cells expressing the desired alleles, we report several peptides that bind differentially to the allele pairs depending on the presence or absence of Asp at position 57. Peptides with negatively charged residues at anchor position 9 bind well to alleles not containing Asp at position 57 in the beta chain but cannot bind well to homologous Asp-positive alleles. By changing the peptides at the single residue predicted to interact with this position 57, we demonstrate a drastically altered or reversed pattern of binding. Ala analog peptides confirm these interactions and identify a limited set of interaction sites between the bound peptides and the class II molecules. Clarification of the impact of specific class II polymorphisms on generating unique allele-specific peptide binding "repertoires" will aid in our understanding of the development of specific immune responses and HLA-associated diseases. PMID:8692969

  19. Identification alpha-2-HS-glycoprotein precursor and tubulin beta chain as serology diagnosis biomarker of colorectal cancer

    PubMed Central

    2014-01-01

    Background Colorectal cancer (CRC) remains a major worldwide cause of cancer-related morbidity and mortality largely due to the insidious onset of the disease. The current clinical procedures utilized for disease diagnosis are invasive, unpleasant, and inconvenient. Hence, the need for simple blood tests that could be used for the early detection is crucial for its ultimate control and prevention. Methods The present work is a case–control study focused on proteomic analysis of serum of healthy volunteers and CRC patients by the ClinProt profiling technology based on mass spectrometry. This approach allowed to identifying a pattern of proteins/peptides able to differentiate the studied populations. Moreover, some of peptides differentially expressed in the serum of patients as compared to healthy volunteers were identified by LTQ Orbitrap XL. Results A Quick Classifier Algorithm was used to construct the peptidome patterns (m/z 1208, 1467, 1505, 1618, 1656 and 4215) for the identification of CRC from healthy volunteers with accuracy close to 100% (>CEA, P < 0.05). Peaks at m/z 1505 and 1618 were identified as alpha-2-HS-glycoprotein precursor and tubulin beta chain, respectively. Conclusions Alpha-2-HS-glycoprotein precursor and tubulin beta chain could be involved in the pathogenesis of CRC and perform as potential serology diagnosis biomarker. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4796578761089186. PMID:24618180

  20. Methionine adenosyltransferase II beta subunit gene expression provides a proliferative advantage in human hepatoma.

    PubMed

    Martínez-Chantar, Maria L; García-Trevijano, Elena R; Latasa, M Ujue; Martín-Duce, Antonio; Fortes, Puri; Caballería, Juan; Avila, Matías A; Mato, José M

    2003-04-01

    Of the 2 genes (MAT1A, MAT2A) encoding methionine adenosyltransferase, the enzyme that synthesizes S-adenosylmethionine, MAT1A, is expressed in liver, whereas MAT2A is expressed in extrahepatic tissues. In liver, MAT2A expression associates with growth, dedifferentiation, and cancer. Here, we identified the beta subunit as a regulator of proliferation in human hepatoma cell lines. The beta subunit has been cloned and shown to lower the K(m) of methionine adenosyltransferase II alpha2 (the MAT2A product) for methionine and to render the enzyme more susceptible to S-adenosylmethionine inhibition. Methionine adenosyltransferase II alpha2 and beta subunit expression was analyzed in human and rat liver and hepatoma cell lines and their interaction studied in HuH7 cells. beta Subunit expression was up- and down-regulated in human hepatoma cell lines and the effect on DNA synthesis determined. We found that beta subunit is expressed in rat extrahepatic tissues but not in normal liver. In human liver, beta subunit expression associates with cirrhosis and hepatoma. beta Subunit is expressed in most (HepG2, PLC, and Hep3B) but not all (HuH7) hepatoma cell lines. Transfection of beta subunit reduced S-adenosylmethionine content and stimulated DNA synthesis in HuH7 cells, whereas down-regulation of beta subunit expression diminished DNA synthesis in HepG2. The interaction between methionine adenosyltransferase II alpha2 and beta subunit was demonstrated in HuH7 cells. Our findings indicate that beta subunit associates with cirrhosis and cancer providing a proliferative advantage in hepatoma cells through its interaction with methionine adenosyltransferase II alpha2 and down-regulation of S-adenosylmethionine levels.

  1. Cloning and characterization of the human beta2-glycoprotein I (beta2-GPI) gene promoter: roles of the atypical TATA box and hepatic nuclear factor-1alpha in regulating beta2-GPI promoter activity.

    PubMed Central

    Wang, Hsueh-Hsiao; Chiang, An-Na

    2004-01-01

    Beta2-glycoprotein I (beta2-GPI) is a plasma glycoprotein primarily synthesized in the liver. The interindividual variability of beta2-GPI expression in subjects with various metabolic syndromes and disease states suggests that it may have clinical importance. However, the regulation of beta2-GPI gene expression has not been clarified. To gain more insight into the control of beta2-GPI gene expression, we cloned the 4.1-kb 5'-flanking region and characterized the proximal promoter of the beta2- GPI gene in this study. Cis -acting elements required for beta2-GPI promoter activity were identified with transient transfection assays in the hepatoma cell lines HepG2 and Huh7 and in non-hepatic HeLa cells. Serial deletion analyses of the beta2-GPI 5'-flanking sequence revealed that the region from -197 to +7 had strong promoter activity in hepatoma cells but not in HeLa cells. Truncation and site-directed mutagenesis of putative cis -elements within this region showing an atypical TATA box and a HNF-1 (hepatic nuclear factor-1) element were both essential for the beta2-GPI promoter activity. Subsequent gel mobility shift assays confirmed the interaction of HNF-1alpha with the HNF-1 site residing downstream of the TATA box. Co-transfection of beta2-GPI promoter-luciferase vector with HNF-1alpha expression vector in Huh7 and HNF-1-deficient HeLa cells demonstrated the transactivation effect of HNF-1alpha on beta2-GPI promoter activity. In addition, overexpression of HNF-1alpha enhanced the endogenous beta2-GPI expression. These results suggest that the atypical TATA box and HNF-1 cis-element are critical for beta2-GPI transcription and HNF-1alpha may play an important role in cell-specific regulation of beta2-GPI gene expression. PMID:14984368

  2. The regulated expression of beta-globin genes introduced into mouse erythroleukemia cells.

    PubMed

    Chao, M V; Mellon, P; Charnay, P; Maniatis, T; Axel, R

    1983-02-01

    We have introduced a hybrid mouse-human beta-globin gene as well as the intact human beta-globin gene into murine erythroleukemia (MEL) cells and have demonstrated that these genes are appropriately regulated during differentiation of the MEL cell in culture. The addition of chemical inducers to cotransformed cells results in a 5 to 50 fold increase in the level of mRNA transcribed from the exogenous globin gene. S1 nuclease and primer extension analyses demonstrate that these mRNAs initiate and terminate correctly. Nuclear transcription experiments indicate that induction of hybrid mRNA results at least in part from the increase in the rate of globin gene transcription. Furthermore, the induction appears to be specific for globin genes within an erythroid cell. These results permit the study of expression of the globin gene during erythroid differentiation and suggest that the specific induction of the globin gene is an inherent property of DNA sequences within or flanking the beta-globin genes. Moreover, the fact that the human and hybrid globin genes are both inducible in MEL cells suggests that these regulatory sequences are conserved between mouse and human cells.

  3. Stalk region of beta-chain enhances the coreceptor function of CD8.

    PubMed

    Wong, Jenny S; Wang, Xiaosong; Witte, Torsten; Nie, Linghu; Carvou, Nicolas; Kern, Petra; Chang, Hsiu-Ching

    2003-07-15

    CD8 glycoproteins are expressed as either alphaalpha homodimers or alphabeta heterodimers on the surface of T cells. CD8alphabeta is a more efficient coreceptor than the CD8alphaalpha for peptide Ag recognition by TCR. Each CD8 subunit is composed of four structural domains, namely, Ig-like domain, stalk region, transmembrane region, and cytoplasmic domain. In an attempt to understand why CD8alphabeta is a better coreceptor than CD8alphaalpha, we engineered, expressed, and functionally tested a chimeric CD8alpha protein whose stalk region is replaced with that of CD8beta. We found that the beta stalk region enhances the coreceptor function of chimeric CD8alphaalpha to a level similar to that of CD8alphabeta. Surprisingly, the beta stalk region also restored functional activity to an inactive CD8alpha variant, carrying an Ala mutation at Arg(8) (R8A), to a level similar to that of wild-type CD8alphabeta. Using the R8A variant of CD8alpha, a panel of anti-CD8alpha Abs, and three MHC class I (MHCI) variants differing in key residues known to be involved in CD8alpha interaction, we show that the introduction of the CD8beta stalk leads to a different topology of the CD8alpha-MHCI complex without altering the overall structure of the Ig-like domain of CD8alpha or causing the MHCI to employ different residues to interact with the CD8alpha Ig domain. Our results show that the stalk region of CD8beta is capable of fine-tuning the coreceptor function of CD8 proteins as a coreceptor, possibly due to its distinct protein structure, smaller physical size and the unique glycan adducts associated with this region.

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

    PubMed

    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.

  5. Genomic organization and sequences of immunoglobulin light chain genes in a primitive vertebrate suggest coevolution of immunoglobulin gene organization.

    PubMed Central

    Shamblott, M J; Litman, G W

    1989-01-01

    The genomic organization and sequence of immunoglobulin light chain genes in Heterodontus francisci (horned shark), a phylogenetically primitive vertebrate, have been characterized. Light chain variable (VL) and joining (JI) segments are separated by 380 nucleotides and together with the single constant region exon (CI), occupy less than 2.7 kb, the closest linkage described thus far for a rearranging gene system. The VL segment is flanked by a characteristic recombination signal sequence possessing a 12 nucleotide spacer; the recombination signal sequence flanking the JL segment is 23 nucleotides. The VL genes, unlike heavy chain genes, possess a typical upstream regulatory octamer as well as conserved enhancer core sequences in the intervening sequence separating JL and CL. Restriction mapping and genomic Southern blotting are consistent with the presence of multiple light chain gene clusters. There appear to be considerably fewer light than heavy chain genes. Heavy and light chain clusters show no evidence of genomic linkage using field inversion gel electrophoresis. The findings of major differences in the organization and functional rearrangement properties of immunoglobulin genes in species representing different levels of vertebrate evolution, but consistent similarity in the organization of heavy and light chain genes within a species, suggests that these systems may be coevolving. Images PMID:2511000

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

    PubMed

    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

  7. Genomic organization and characterization of a three-gene rat adult beta-globin haplotype.

    PubMed

    Au, D M; Wong, W M; Tam, J W; Cheng, L Y; Lam, V M

    1995-11-20

    The isolation and detailed characterization of a three-beta-globin gene (GloB) haplotype in the Sprague-Dawley (S-D) rat is described. An enriched library, lambda SDHelib, was screened with a human GloB probe, humbg44, and from which a beta minor gene, Rathbbz, was isolated, sequenced and characterized. A S-D rat GloB-specific probe, Ratbgze12, derived from the Rathbbz gene, was then used to screen a S-D rat genomic library, lambda SDglib. The clone T1510 was isolated and identified to include the entire Rathbbz gene and part of another GloB gene, Rathbby, which was 5' upstream from Rathbbz. Chromosomal walking upstream using the riboprobe, rnaT71, led to the isolation of an overlapping clone, Ta49, which was shown to include two full-length GloB genes; the most 5' was Rathbbx followed by Rathbby. Sequence data suggests that Rathbbx is a beta major gene, whereas Rathbby is a hybrid gene of Rathbbx and Rathbbz. Genomic hybridization confirmed this particular three-gene haplotype in the S-D rat. This haplotype, a1, may be the prototype of the GloB cluster in rat.

  8. Identification and characterization of a novel repressor of beta-interferon gene expression.

    PubMed

    Keller, A D; Maniatis, T

    1991-05-01

    We have identified and characterized a novel repressor of human beta-interferon (beta-IFN) gene expression. This protein, designated PRDI-BF1, binds specifically to the PRDI element of the beta-IFN gene promoter and is distinct from previously reported proteins that bind to this sequence. PRDI-BF1 is an 88-kD protein containing five zinc-finger motifs. Cotransfection experiments in cultured mammalian cells revealed that PRDI-BF1 is a potent repressor of PRDI-dependent transcription. PRDI-BF1 blocks virus induction of the intact beta-IFN gene promoter and of synthetic promoters containing multiple PRDI sites. PRDI-BF1 can also block the SV40 enhancer when PRDI sites are located between the enhancer and the promoter. This repression is highly dependent on the location of the PRDI sites, however, indicating that PRDI-BF1 cannot act at a distance. On the basis of the properties of PRDI-BF1 and the observation that PRDI-BF1 mRNA accumulation is virus inducible, we propose that PRDI-BF1 may act as a postinduction repressor of the beta-IFN gene by displacing positive regulatory proteins from the PRDI site of the promoter.

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

    PubMed

    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.

  10. Major histocompatibility complex gene product expression on pancreatic beta cells in acutely diabetic BB rats.

    PubMed Central

    Issa-Chergui, B.; Yale, J. F.; Vigeant, C.; Seemayer, T. A.

    1988-01-01

    Type I diabetes mellitus was induced in young, diabetes-prone BB rats by the passive transfer of concanavalin A-activated T lymphocytes from the spleens of acutely diabetic BB rats. The pancreas of the recipients was examined 1-2 days after the onset of glycosuria by immunocytochemistry by means of monoclonal antibodies for determining whether 1) Class I and/or II major histocompatibility gene complex (MHC) products were expressed on beta cells and 2) the mononuclear cell infiltrates were represented by T cells. Marked expression of Class I MHC gene products was evident on beta cells. In contrast, Class II MHC gene products were not identified on normal-appearing beta cells. Dendritic cells dispersed throughout the acinar and interstitial pancreas were markedly increased in number. The mononuclear cell infiltrate contained few cells (1-15%) recognized by a pan-T cell marker. Although it is possible that this passive transfer model might differ considerably from the spontaneously occurring diabetic state in the rat, this study suggests that 1) Class I, rather than Class II, MHC gene expression may be pivotal to beta-cell injury in diabetic rats, and 2) non-T cells may constitute an effector cell population central to beta-cell necrosis in Type I diabetes mellitus. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:3276208

  11. The chicken transforming growth factor-beta 3 gene: genomic structure, transcriptional analysis, and chromosomal location.

    PubMed

    Burt, D W; Dey, B R; Paton, I R; Morrice, D R; Law, A S

    1995-02-01

    In this paper, we report the isolation, characterization, and mapping of the chicken transforming growth factor-beta 3 (TGF-beta 3) gene. The gene contains seven exons and six introns spanning 16-kb of the chicken genome. A comparison of the 5'-flanking regions of human and chicken TGF-beta 3 genes reveals two regions of sequence conservation. The first contains ATF/CRE and TBP/TATA sequence motifs within an 87-bp region. The second is a 162-bp region with no known sequence motifs. Identification of transcription start sites using chicken RNA isolated from various embryonic and adult tissues reveals two sites of initiation, P1 and P2, which map to these two conserved regions. Comparison of 3'-flanking regions of chicken and mammalian TGF-beta 3 genes also revealed conserved sequences. The most significant homologies were found in the 3'-most end of the transcribed region. DNA sequence analysis of chicken TGF-beta 3 cDNAs isolated by 3'-RACE revealed multiple polyadenylation sites unusually distant from a poly(A) signal motif. A Msc I restriction fragment length polymorphism (RFLP) marker was used to map the TGFB3 locus to linkage group E7 on the East Lansing reference backcross. Linkage to the TH locus showed that the TGFB3 locus was physically located on chicken chromosome 5.

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

    PubMed

    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.

  13. Neofunctionalization of Chromoplast Specific Lycopene Beta Cyclase Gene (CYC-B) in Tomato Clade

    PubMed Central

    Mohan, Vijee; Pandey, Arun; Sreelakshmi, Yellamaraju; Sharma, Rameshwar

    2016-01-01

    The ancestor of tomato underwent whole genome triplication ca. 71 Myr ago followed by widespread gene loss. However, few of the triplicated genes are retained in modern day tomato including lycopene beta cyclase that mediates conversion of lycopene to β-carotene. The fruit specific β-carotene formation is mediated by a chromoplast-specific paralog of lycopene beta cyclase (CYC-B) gene. Presently limited information is available about how the variations in CYC-B gene contributed to its neofunctionalization. CYC-B gene in tomato clade contained several SNPs and In-Dels in the coding sequence (33 haplotypes) and promoter region (44 haplotypes). The CYC-B gene coding sequence in tomato appeared to undergo purifying selection. The transit peptide sequence of CYC-B protein was predicted to have a stronger plastid targeting signal than its chloroplast specific paralog indicating a possible neofunctionalization. In promoter of two Bog (Beta old gold) mutants, a NUPT (nuclear plastid) DNA fragment of 256 bp, likely derived from a S. chilense accession, was present. In transient expression assay, this promoter was more efficient than the “Beta type” promoter. CARGATCONSENSUS box sequences are required for the binding of the MADS-box regulatory protein RIPENING INHIBITOR (RIN). The loss of CARGATCONSENSUS box sequence from CYC-B promoter in tomato may be related to attenuation of its efficiency to promote higher accumulation of β-carotene than lycopene during fruit ripening. PMID:27070417

  14. Immunoglobulin heavy and light chains and T-cell receptor beta and gamma chains PCR assessment on cytological samples. A study comparing FTA cards and cryopreserved lymph node fine-needle cytology.

    PubMed

    Peluso, A L; Cozzolino, I; Bottiglieri, A; Lucchese, L; Di Crescenzo, R M; Langella, M; Selleri, C; Zeppa, P

    2017-06-01

    To evaluate and compare the DNA yield and quality extracted from lymph node fine needle cytology (FNC) samples stored on FTA cards to those cryopreserved, and to assess the immunoglobulin heavy and light chains (IGHK) and T-Cell receptor beta and gamma chains (TCRBG) PCR tests. DNA extractions were performed on FNC of 80 non-Hodgkin lymphomas (NHL), four myelomas and 56 benign reactive hyperplasias (BRH) cryopreserved and stored on FTA cards. The JAK2 gene was amplified to assess the DNA integrity and the IGHK/TCRBG clonality status was tested. IGHK monoclonality was found in 99% of B-cell NHL and 100% of myeloma. TCRBG monoclonality was found in 100% of T-cell NHL. TCRBG polyclonality was detected in 97% of B-cell NHL, 100% of myeloma and 96% of BRH. IGHK/TCRBG PCR data were confirmed by histological and/or follow-up controls. No differences were found in the DNA quality between cryopreservation and FTA cards storage methods. IGHK/TCRBG PCR of the lymphoproliferative process on FTA cards is comparable to those cryopreserved. FTA cards can be used to store lymph node FNC for further molecular investigations. © 2016 John Wiley & Sons Ltd.

  15. Sequence heterogeneity, multiplicity, and genomic organization of alpha- and beta-tubulin genes in sea urchins.

    PubMed Central

    Alexandraki, D; Ruderman, J V

    1981-01-01

    We analyzed the multiplicity, heterogeneity, and organization of the genes encoding the alpha and beta tubulins in the sea urchin Lytechinus pictus by using cloned complementary deoxyribonucleic acid (cDNA) and genomic tubulin sequences. cDNA clones were constructed by using immature spermatogenic testis polyadenylic acid-containing ribonucleic acid as a template. alpha- and beta-tubulin clones were identified by hybrid selection and in vitro translation of the corresponding messenger ribonucleic acids, followed by immunoprecipitation and two-dimensional gel electrophoresis of the translation products. The alpha cDNA clone contains a sequence that encodes the 48 C-terminal amino acids of alpha tubulin and 104 base pairs of the 3' nontranslated portion of the messenger ribonucleic acid. The beta cDNA insertion contains the coding sequence for the 100-C terminal amino acids of beta tubulin and 83 pairs of the 3' noncoding sequence. Hybrid selections performed at different criteria demonstrated the presence of several heterogeneous, closely related tubulin messenger ribonucleic acids, suggesting the existence of heterogeneous alpha- and beta-tubulin genes. Hybridization analyses indicated that there are at least 9 to 13 sequences for each of the two tubulin gene families per haploid genome. Hybridization of the cDNA probes to both total genomic DNA and cloned germline DNA fragments gave no evidence for close physical linkage of alpha-tubulin genes with beta-tubulin genes at the DNA level. In contrast, these experiments indicated that some genes within the same family are clustered. Images PMID:6287219

  16. Sequence heterogeneity, multiplicity, and genomic organization of. cap alpha. - and. beta. -tubulin genes in Sea Urchins

    SciTech Connect

    Alexandraki, D.; Ruderman, J.V.

    1981-12-01

    The authors analyzed the multiplicity, heterogeneity, and organization of the genes encoding the ..cap alpha.. and ..beta.. tubulins in the sea urchin Lytechinus pictus by using cloned complementary deoxyribonucleic acid (cDNA) and genomic tubulin sequences. cDNA clones were constructed by using immature spermatogenic testis polyadenylic acid-containing ribonucleic acid as a template. ..cap alpha.. and ..beta..-tubulin clones were identified by hybrid selection and in vitro translation of the corresponding messenger ribonucleic acids, followed by immunoprecipitation and two-dimensional gel electrophoresis of the translation products. The ..cap alpha.. cDNA clone contains a sequence that encodes the 48 C-terminal amino acids of ..cap alpha.. tubulin and 104 base pairs of the 3' nontranslated portion of the messenger ribonucleic acid. The ..beta.. cDNA insertion contains the coding sequence for the 100 C-terminal amino acids of ..beta.. tubulin and 83 base pairs of the 3' noncoding sequence. Hybrid selections performed at different criteria demonstrated the presence of several heterogeneous, closely related tubulin messenger ribonucleic acids, suggesting the existence of heterogeneous ..cap alpha..- and ..beta..-tubulin genes. Hybridization analyses indicated that there are at least 9 to 13 sequences for each of the two tubulin gene families per haploid genome. Hybridization of the cDNA probes to both total genomic DNA and cloned germline DNA fragments gave no evidence for close physical linkage of ..cap alpha..-tubulin genes with ..beta..-tubulin genes at the DNA level. In contrast, these experiments indicated that some genes within the same family are clustered.

  17. Evolution and molecular characterization of a beta-globin gene from the Australian Echidna Tachyglossus aculeatus (Monotremata).

    PubMed

    Lee, M H; Shroff, R; Cooper, S J; Hope, R

    1999-07-01

    Coinciding with a period in evolution when monotremes, marsupials, and eutherians diverged from a common ancestor, a proto-beta-globin gene duplicated, producing the progenitors of mammalian embryonic and adult beta-like globin genes. To determine whether monotremes contain orthologues of these genes and to further investigate the evolutionary relationships of monotremes, marsupials, and eutherians, we have determined the complete DNA sequence of an echidna (Tachyglossus aculeatus) beta-like globin gene. Conceptual translation of the gene and sequence comparisons with eutherian and marsupial beta-like globin genes and echidna adult beta-globin indicate that the gene is adult expressed. Phylogenetic analyses do not clearly resolve the branching pattern of mammalian beta-like globin gene lineages and it is therefore uncertain whether monotremes have orthologues of the embryonic beta-like globin genes of marsupials and eutherians. Four models are proposed that provide a framework for interpreting further studies on the evolution of beta-like globin genes in the context of the evolution of monotremes, marsupials, and eutherians.

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

    PubMed

    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.

  19. Five variants of the beta-globin gene without clinical or hematological effects: Hb Maryland [beta 47(CD6)Asp-->His], Hb Kent [beta 37(C3)Trp-->Cys], Hb Visayan [beta 136(H14)Gly-->Cys], Hb Cutlerville [beta 138(H16)Ala-->Val] and Hb Hornchurch [beta 43(CD2)Glu-->Lys].

    PubMed

    Hoyer, James D; Kaur, Prabhjot; Kozak, Elizabeth A; Lum, Sophia; Alter, David N

    2008-01-01

    We report on five hemoglobin (Hb) beta chain variants that were initially identified either by electrophoretic, chromatographic or isoelectric focusing (IEF) methods. These variants do not appear to be associated with clinical or hematological abnormalities. All variants were confirmed by DNA sequence analysis.

  20. Conformation of methyl beta-lactoside bound to the ricin B-chain: Interpretation of transferred nuclear Overhauser effects facilitated by spin simulation and selective deuteration

    SciTech Connect

    Bevilacqua, V.L.; Thomson, D.S.; Prestegard, J.H. )

    1990-06-12

    Spin simulation and selective deuteration have been used to aid in the interpretation of 1D transferred nuclear Overhauser effect (TRNOE) NMR experiments on ricin B-chain/ligand systems. Application of these methods has revealed a change in the conformation of deuterated methyl beta-lactoside upon binding to the ricin B-chain which results in a slight change in glycosidic torsional angels which appear to dominate in the solution conformation. The combination of simulation and experiment also shows an important sensitivity of TRNOE magnitudes to dissociation rate constants and available spin-diffusion pathways for the ricin B-chain/ligand systems under study. The sensitivity to dissociation rates allows determination of rate constants for methyl beta-lactoside and methyl beta-galactoside of 50 and 300 s-1, respectively.

  1. Myosin Heavy Chain Gene Expression in Developing Neonatal Skeletal Muscle: Involvement of the Nerve, Gravity, and Thyroid State

    NASA Technical Reports Server (NTRS)

    Baldwin, K. M.; Adams, G.; Haddad, F.; Zeng, M.; Qin, A.; Qin, L.; McCue, S.; Bodell, P.

    1999-01-01

    The myosin heavy chain (MHC) gene family encodes at least six MHC proteins (herein designated as neonatal, embryonic, slow type I (beta), and fast IIa, IIx, and IIb) that are expressed in skeletal muscle in a muscle-specific and developmentally-regulated fashion. At birth, both antigravity (e.g. soleus) and locomotor (e.g., plantaris) skeletal muscles are undifferentiated relative to the adult MHC phenotype such that the neonatal and embryonic MHC isoforms account for 80 - 90% of the MHC pool in a fast locomotor muscle; whereas, the embryonic and slow, type I isoforms account for approx. 90% of the pool in a typical antigravity muscle. The goal of this study was to investigate the role of an intact nerve, gravity and thyroid hormone (T3), as well as certain interactions of these interventions, on MHC gene expression in developing neonatal skeletal muscles of rodents.

  2. Myosin Heavy Chain Gene Expression in Developing Neonatal Skeletal Muscle: Involvement of the Nerve, Gravity, and Thyroid State

    NASA Technical Reports Server (NTRS)

    Baldwin, K. M.; Adams, G.; Haddad, F.; Zeng, M.; Qin, A.; Qin, L.; McCue, S.; Bodell, P.

    1999-01-01

    The myosin heavy chain (MHC) gene family encodes at least six MHC proteins (herein designated as neonatal, embryonic, slow type I (beta), and fast IIa, IIx, and IIb) that are expressed in skeletal muscle in a muscle-specific and developmentally-regulated fashion. At birth, both antigravity (e.g. soleus) and locomotor (e.g., plantaris) skeletal muscles are undifferentiated relative to the adult MHC phenotype such that the neonatal and embryonic MHC isoforms account for 80 - 90% of the MHC pool in a fast locomotor muscle; whereas, the embryonic and slow, type I isoforms account for approx. 90% of the pool in a typical antigravity muscle. The goal of this study was to investigate the role of an intact nerve, gravity and thyroid hormone (T3), as well as certain interactions of these interventions, on MHC gene expression in developing neonatal skeletal muscles of rodents.

  3. 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.

    PubMed

    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.

  4. The genomic fingerprinting of the coding region of the beta-tubulin gene in Leishmania identification.

    PubMed

    Luis, L; Ramírez, A; Aguilar, C M; Eresh, S; Barker, D C; Mendoza-León, A

    1998-06-01

    We have demonstrated the polymorphism of the beta-tubulin gene region in Leishmania and its value in the identification of the parasite. In this work we have shown that the coding region of the gene has sufficient variation to accurately discriminate these parasites at the subgenus level. Nevertheless, intrasubgenus diversity, for particular restriction enzymes, was found in New World Leishmania belonging to the Leishmania subgenus. For instance, differences were found between mexicana and amazonensis strains. A unique pattern at the species level was found in particular species of both subgenera, e.g. L. (L.) major strain P and L. (L.) tropica belonging to the Leishmania subgenus, and L. (V.) panamensis strain LS94 from the Viannia subgenus. Particular endonucleases are diagnostic in Leishmania species discrimination as in the case of PvuII for the mexicana and amazonensis. This variation evidenced in the beta-tubulin gene region of Leishmania also occurred in other Kinetoplastida e.g. Trypanosoma cruzi, Leptomonas spp. and Crithidia spp. Moreover, these organisms showed a different genomic fingerprinting for the beta-tubulin gene among them and also Leishmania. Thus, the polymorphism of the coding region of the beta-tubulin gene can be used as a molecular marker for the identification of Leishmania.

  5. Nitric oxide stimulates insulin gene transcription in pancreatic {beta}-cells

    SciTech Connect

    Campbell, S.C. . E-mail: s.c.campbell@ncl.ac.uk; Richardson, H.; Ferris, W.F.; Butler, C.S.; Macfarlane, W.M.

    2007-02-23

    Recent studies have identified a positive role for nitric oxide (NO) in the regulation of pancreatic {beta}-cell function. The aim of this study was to determine the effects of short-term exposure to NO on {beta}-cell gene expression and the activity of the transcription factor PDX-1. NO stimulated the activity of the insulin gene promoter in Min6 {beta}-cells and endogenous insulin mRNA levels in both Min6 and isolated islets of Langerhans. Addition of wortmannin prior to NO stimulation blocked the observed increases in insulin gene promoter activity. Although NO addition stimulated the phosphorylation of p38, inhibition by SB203580 did not block the effect of NO on the insulin gene promoter. NO addition also stimulated both the nuclear accumulation and the DNA binding activity of PDX-1. This study has shown that over 24 h, NO stimulates insulin gene expression, PI-3-kinase activity and the activity of the critical {beta}-cell transcription factor PDX-1.

  6. The maize auxotrophic mutant orange pericarp is defective in duplicate genes for tryptophan synthase beta.

    PubMed Central

    Wright, A D; Moehlenkamp, C A; Perrot, G H; Neuffer, M G; Cone, K C

    1992-01-01

    orange pericarp (orp) is a seedling lethal mutant of maize caused by mutations in the duplicate unlinked recessive loci orp1 and orp2. Mutant seedlings accumulate two tryptophan precursors, anthranilate and indole, suggesting a block in tryptophan biosynthesis. Results from feeding studies and enzyme assays indicate that the orp mutant is defective in tryptophan synthase beta activity. Thus, orp is one of only a few amino acid auxotrophic mutants to be characterized in plants. Two genes encoding tryptophan synthase beta were isolated from maize and sequenced. Both genes encode polypeptides with high homology to tryptophan synthase beta enzymes from other organisms. The cloned genes were mapped by restriction fragment length polymorphism analysis to approximately the same chromosomal locations as the genetically mapped factors orp1 and orp2. RNA analysis indicates that both genes are expressed in all tissues examined from normal plants. Together, the biochemical, genetic, and molecular data verify the identity of orp1 and orp2 as duplicate structural genes for the beta subunit of tryptophan synthase. PMID:1356534

  7. The maize auxotrophic mutant orange pericarp is defective in duplicate genes for tryptophan synthase beta.

    PubMed

    Wright, A D; Moehlenkamp, C A; Perrot, G H; Neuffer, M G; Cone, K C

    1992-06-01

    orange pericarp (orp) is a seedling lethal mutant of maize caused by mutations in the duplicate unlinked recessive loci orp1 and orp2. Mutant seedlings accumulate two tryptophan precursors, anthranilate and indole, suggesting a block in tryptophan biosynthesis. Results from feeding studies and enzyme assays indicate that the orp mutant is defective in tryptophan synthase beta activity. Thus, orp is one of only a few amino acid auxotrophic mutants to be characterized in plants. Two genes encoding tryptophan synthase beta were isolated from maize and sequenced. Both genes encode polypeptides with high homology to tryptophan synthase beta enzymes from other organisms. The cloned genes were mapped by restriction fragment length polymorphism analysis to approximately the same chromosomal locations as the genetically mapped factors orp1 and orp2. RNA analysis indicates that both genes are expressed in all tissues examined from normal plants. Together, the biochemical, genetic, and molecular data verify the identity of orp1 and orp2 as duplicate structural genes for the beta subunit of tryptophan synthase.

  8. Development of real-time PCR method for the detection and the quantification of a new endogenous reference gene in sugar beet "Beta vulgaris L.": GMO application.

    PubMed

    Chaouachi, Maher; Alaya, Akram; Ali, Imen Ben Haj; Hafsa, Ahmed Ben; Nabi, Nesrine; Bérard, Aurélie; Romaniuk, Marcel; Skhiri, Fethia; Saïd, Khaled

    2013-01-01

    KEY MESSAGE : Here, we describe a new developed quantitative real-time PCR method for the detection and quantification of a new specific endogenous reference gene used in GMO analysis. The key requirement of this study was the identification of a new reference gene used for the differentiation of the four genomic sections of the sugar beet (Beta vulgaris L.) (Beta, Corrollinae, Nanae and Procumbentes) suitable for quantification of genetically modified sugar beet. A specific qualitative polymerase chain reaction (PCR) assay was designed to detect the sugar beet amplifying a region of the adenylate transporter (ant) gene only from the species of the genomic section I of the genus Beta (cultivated and wild relatives) and showing negative PCR results for 7 species of the 3 other sections, 8 related species and 20 non-sugar beet plants. The sensitivity of the assay was 15 haploid genome copies (HGC). A quantitative real-time polymerase chain reaction (QRT-PCR) assay was also performed, having high linearity (R (2) > 0.994) over sugar beet standard concentrations ranging from 20,000 to 10 HGC of the sugar beet DNA per PCR. The QRT-PCR assay described in this study was specific and more sensitive for sugar beet quantification compared to the validated test previously reported in the European Reference Laboratory. This assay is suitable for GMO quantification in routine analysis from a wide variety of matrices.

  9. Lampreys have a single gene cluster for the fast skeletal myosin heavy chain gene family.

    PubMed

    Ikeda, Daisuke; Ono, Yosuke; Hirano, Shigeki; Kan-no, Nobuhiro; Watabe, Shugo

    2013-01-01

    Muscle tissues contain the most classic sarcomeric myosin, called myosin II, which consists of 2 heavy chains (MYHs) and 4 light chains. In the case of humans (tetrapod), a total of 6 fast skeletal-type MYH genes (MYHs) are clustered on a single chromosome. In contrast, torafugu (teleost) contains at least 13 fast skeletal MYHs, which are distributed in 5 genomic regions; the MYHs are clustered in 3 of these regions. In the present study, the evolutionary relationship among fast skeletal MYHs is elucidated by comparing the MYHs of teleosts and tetrapods with those of cyclostome lampreys, one of two groups of extant jawless vertebrates (agnathans). We found that lampreys contain at least 3 fast skeletal MYHs, which are clustered in a head-to-tail manner in a single genomic region. Although there was apparent synteny in the corresponding MYH cluster regions between lampreys and tetrapods, phylogenetic analysis indicated that lamprey and tetrapod MYHs have independently duplicated and diversified. Subsequent transgenic approaches showed that the 5'-flanking sequences of Japanese lamprey fast skeletal MYHs function as a regulatory sequence to drive specific reporter gene expression in the fast skeletal muscle of zebrafish embryos. Although zebrafish MYH promoters showed apparent activity to direct reporter gene expression in myogenic cells derived from mice, promoters from Japanese lamprey MYHs had no activity. These results suggest that the muscle-specific regulatory mechanisms are partially conserved between teleosts and tetrapods but not between cyclostomes and tetrapods, despite the conserved synteny.

  10. Lampreys Have a Single Gene Cluster for the Fast Skeletal Myosin Heavy Chain Gene Family

    PubMed Central

    Ikeda, Daisuke; Ono, Yosuke; Hirano, Shigeki; Kan-no, Nobuhiro; Watabe, Shugo

    2013-01-01

    Muscle tissues contain the most classic sarcomeric myosin, called myosin II, which consists of 2 heavy chains (MYHs) and 4 light chains. In the case of humans (tetrapod), a total of 6 fast skeletal-type MYH genes (MYHs) are clustered on a single chromosome. In contrast, torafugu (teleost) contains at least 13 fast skeletal MYHs, which are distributed in 5 genomic regions; the MYHs are clustered in 3 of these regions. In the present study, the evolutionary relationship among fast skeletal MYHs is elucidated by comparing the MYHs of teleosts and tetrapods with those of cyclostome lampreys, one of two groups of extant jawless vertebrates (agnathans). We found that lampreys contain at least 3 fast skeletal MYHs, which are clustered in a head-to-tail manner in a single genomic region. Although there was apparent synteny in the corresponding MYH cluster regions between lampreys and tetrapods, phylogenetic analysis indicated that lamprey and tetrapod MYHs have independently duplicated and diversified. Subsequent transgenic approaches showed that the 5′-flanking sequences of Japanese lamprey fast skeletal MYHs function as a regulatory sequence to drive specific reporter gene expression in the fast skeletal muscle of zebrafish embryos. Although zebrafish MYH promoters showed apparent activity to direct reporter gene expression in myogenic cells derived from mice, promoters from Japanese lamprey MYHs had no activity. These results suggest that the muscle-specific regulatory mechanisms are partially conserved between teleosts and tetrapods but not between cyclostomes and tetrapods, despite the conserved synteny. PMID:24376886

  11. Light controls phospholipase A2alpha and beta gene expression in Citrus sinensis.

    PubMed

    Liao, Hui-Ling; Burns, Jacqueline K

    2010-05-01

    The low-molecular weight secretory phospholipase A2alpha (CssPLA2alpha) and beta (CsPLA2beta) cloned in this study exhibited diurnal rhythmicity in leaf tissue of Citrus sinensis. Only CssPLA2alpha displayed distinct diurnal patterns in fruit tissues. CssPLA2alpha and CsPLA2beta diurnal expression exhibited periods of approximately 24 h; CssPLA2alpha amplitude averaged 990-fold in the leaf blades from field-grown trees, whereas CsPLA2beta amplitude averaged 6.4-fold. Diurnal oscillation of CssPLA2alpha and CsPLA2beta gene expression in the growth chamber experiments was markedly dampened 24 h after transfer to continuous light or dark conditions. CssPLA2alpha and CsPLA2beta expressions were redundantly mediated by blue, green, red and red/far-red light, but blue light was a major factor affecting CssPLA2alpha and CsPLA2beta expression. Total and low molecular weight CsPLA2 enzyme activity closely followed diurnal changes in CssPLA2alpha transcript expression in leaf blades of seedlings treated with low intensity blue light (24 micromol m(-2) s(-1)). Compared with CssPLA2alpha basal expression, CsPLA2beta expression was at least 10-fold higher. Diurnal fluctuation and light regulation of PLA2 gene expression and enzyme activity in citrus leaf and fruit tissues suggests that accompanying diurnal changes in lipophilic second messengers participate in the regulation of physiological processes associated with phospholipase A2 action.

  12. Contribution of peroxisomal beta-oxidation system to the chain-shortening of N-(alpha-methylbenzyl)azelaamic acid in rat liver.

    PubMed

    Suzuki, H; Mori, K; Yamada, J; Suga, T

    1990-06-15

    Hepaptic peroxisomal and mitochondrial beta-oxidation of N-(alpha-methylbenzyl)azelaamic acid (C9), which is a possible metabolic intermediate of Melinamide, a potent hypocholesterolemic drug, were investigated. Isolated hepatocytes generated H2O2 when incubated with C9, indicating that C9 served as the substrate for peroxisomal beta-oxidation. Also with isolated peroxisomes a significant activity of peroxisomal beta-oxidation for C9-CoA measured by following cyanide-insensitive NAD reduction was observed, when the chain-shortened products such as C7 and C5 were detected from the incubation mixture of C9-CoA, and so NADH, acetyl-CoA and C2 units split off from C9-CoA were produced in stoichiometric amounts. In contrast, the mitochondrial beta-oxidation for C9 measured by following ketone body production and antimycin A-sensitive O2 consumption was not detectable, indicating that C9 is not metabolized by mitochondrial beta-oxidation. Comparative study of beta-oxidation capacities in peroxisomes and mitochondria indicate that the beta-oxidation of C9 occurs exclusively in peroxisomes. Also, the formation activity of C2 units liberated from C9 in intact hepatocytes reflects the peroxisomal beta-oxidation activity of liver homogenates with a highly close correlation. Therefore, it is concluded that C9 can be an excellent substrate for estimating peroxisomal beta-oxidation activity in intact cells.

  13. Transforming growth factor-beta receptor requirements for the induction of the endothelin-1 gene.

    PubMed

    Castañares, Cristina; Redondo-Horcajo, Mariano; Magan-Marchal, Noemi; Lamas, Santiago; Rodriguez-Pascual, Fernando

    2006-06-01

    Expression of the endothelin (ET)-1 gene is subject to complex regulation by numerous factors, among which the cytokine transforming growth factor-beta (TGF-beta) is one of the most important. TGF-beta action is based on the activation of the Smad signaling pathway. Smad proteins activate transcription of the gene by cooperation with activator protein-1 (AP-1) at specific sites on the ET-1 promoter. Smad signaling pathway is initiated by binding of the cytokine to a heteromeric complex of type I and type II receptors. Signal is then propagated to the nucleus by specific members of the Smad family. Most cell types contain a type I receptor known as ALK5. However, endothelial cells are unique because they coexpress an additional type I receptor named ALK1. These forms do not constitute redundant receptors with the same function, but they actually activate different Smad-mediated expression programs that lead to specific endothelial phenotypes. TGF-beta/ALK5/Smad3 pathway is associated to a mature endothelium because it leads to inhibition of cell migration/proliferation. Conversely, TGF-beta/ALK1/Smad5 activates both processes and is more related to the angiogenic state. We have analyzed the TGF-beta receptor subtype requirements for the activation of the ET-1 gene. For that purpose, we have overexpressed type I receptor and Smad isoforms in endothelial cells and analyzed the effect on ET-1 expression. Our experiments indicate that TGF-beta induces ET-1 expression preferentially through the activation of the ALK5/Smad3 pathway and, therefore, the expression of the vaso-constrictor may be associated to a quiescent and mature endothelial phenotype.

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

    PubMed

    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.

  15. A new compound heterozygous frameshift mutation in the type II 3{beta}-hydroxysteroid dehydrogenase 3{beta}-HSD gene causes salt-wasting 3{beta}-HSD deficiency congenital adrenal hyperplasia

    SciTech Connect

    Zhang, L.; Sakkal-Alkaddour, S.; Chang, Ying T.; Yang, Xiaojiang; Songya Pang

    1996-01-01

    We report a new compound heterozygous frameshift mutation in the type II 3{Beta}-hydroxysteroid dehydrogenase (3{beta}-HSD) gene in a Pakistanian female child with the salt-wasting form of 3{Beta}-HSD deficiency congenital adrenal hyperplasia. The etiology for her congenital adrenal hyperplasia was not defined. Although the family history suggested possible 3{beta}-HSd deficiency disorder, suppressed adrenal function caused by excess glucocorticoid therapy in this child at 7 yr of age did not allow hormonal diagnosis. To confirm 3{beta}-HSD deficiency, we sequenced the type II 3{beta}-HSD gene in the patient, her family, and the parents of her deceased paternal cousins. The type II 3{beta}-HSD gene region of a putative promotor, exons I, II, III, and IV, and exon-intron boundaries were amplified by PCR and sequenced in all subjects. The DNA sequence of the child revealed a single nucleotide deletion at codon 318 [ACA(Thr){r_arrow}AA] in exon IV in one allele, and two nucleotide deletions at codon 273 [AAA(Lys){r_arrow}A] in exon IV in the other allele. The remaining gene sequences were normal. The codon 318 mutation was found in one allele from the father, brother, and parents of the deceased paternal cousins. The codon 273 mutation was found in one allele of the mother and a sister. These findings confirmed inherited 3{beta}-HSD deficiency in the child caused by the compound heterozygous type II 3{beta}-HSD gene mutation. Both codons at codons 279 and 367, respectively, are predicted to result in an altered and truncated type II 3{beta}-HSD protein, thereby causing salt-wasting 3{beta}-HSD deficiency in the patient. 21 refs., 2 figs., 1 tab.

  16. Imputing Variants in HLA-DR Beta Genes Reveals That HLA-DRB1 Is Solely Associated with Rheumatoid Arthritis and Systemic Lupus Erythematosus

    PubMed Central

    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

  17. Polymorphism of the complement C8A and -B genes in two families with C8 beta deficiency and neisserial infections.

    PubMed

    Barba, G M; Kaufmann, T J; Schneider, P M; Rittner, C; Brai, M

    1994-07-01

    Serum samples from members of two Italian families with complement C8 beta deficiency were studied by SDS-PAGE under nonreducing conditions and by IEF. The proband of family I had suffered from two episodes of purulent meningitis and two of her uncles had suffered from only one episode, while the proband of family II had suffered from three different episodes. In contrast to previous findings, where C8 beta deficiency was cosegregating with C8A (alpha-gamma) allotype A, the proband of family II had the C8A allotype B. In addition, in one of her sons a novel variant of the C8 beta chain was detected. Studies at the DNA level in family I, using a recently described PCR system, demonstrate the presence of a C-T exchange, which creates a stop codon in exon 9 of the C8B gene, as cause for the C8 beta deficiency. The mutated allele does not carry the TaqI polymorphic restriction site located in intron 11 of the C8B gene. These findings provide evidence for multiple recombination events within the genetic region of the defective C8B gene.

  18. Platypus globin genes and flanking loci suggest a new insertional model for beta-globin evolution in birds and mammals.

    PubMed

    Patel, Vidushi S; Cooper, Steven J B; Deakin, Janine E; Fulton, Bob; Graves, Tina; Warren, Wesley C; Wilson, Richard K; Graves, Jennifer A M

    2008-07-25

    Vertebrate alpha (alpha)- and beta (beta)-globin gene families exemplify the way in which genomes evolve to produce functional complexity. From tandem duplication of a single globin locus, the alpha- and beta-globin clusters expanded, and then were separated onto different chromosomes. The previous finding of a fossil beta-globin gene (omega) in the marsupial alpha-cluster, however, suggested that duplication of the alpha-beta cluster onto two chromosomes, followed by lineage-specific gene loss and duplication, produced paralogous alpha- and beta-globin clusters in birds and mammals. Here we analyse genomic data from an egg-laying monotreme mammal, the platypus (Ornithorhynchus anatinus), to explore haemoglobin evolution at the stem of the mammalian radiation. The platypus alpha-globin cluster (chromosome 21) contains embryonic and adult alpha- globin genes, a beta-like omega-globin gene, and the GBY globin gene with homology to cytoglobin, arranged as 5'-zeta-zeta'-alphaD-alpha3-alpha2-alpha1-omega-GBY-3'. The platypus beta-globin cluster (chromosome 2) contains single embryonic and adult globin genes arranged as 5'-epsilon-beta-3'. Surprisingly, all of these globin genes were expressed in some adult tissues. Comparison of flanking sequences revealed that all jawed vertebrate alpha-globin clusters are flanked by MPG-C16orf35 and LUC7L, whereas all bird and mammal beta-globin clusters are embedded in olfactory genes. Thus, the mammalian alpha- and beta-globin clusters are orthologous to the bird alpha- and beta-globin clusters respectively. We propose that alpha- and beta-globin clusters evolved from an ancient MPG-C16orf35-alpha-beta-GBY-LUC7L arrangement 410 million years ago. A copy of the original beta (represented by omega in marsupials and monotremes) was inserted into an array of olfactory genes before the amniote radiation (>315 million years ago), then duplicated and diverged to form orthologous clusters of beta-globin genes with different expression

  19. Involvement of class II beta-chain amino acid residues 85 and 86 in T-cell allorecognition.

    PubMed

    Eckels, D D; Geiger, M J; Sell, T W; Gorski, J A

    1990-03-01

    Alloreactive T-cell clones were derived by limiting dilution following priming to allogeneic cells bearing HLA-DR1 alloantigens. Clonal specificities were determined by extensive testing on a panel of allogeneic lymphoblastoid cell lines and by blocking studies with monoclonal antibodies specific for HLA-DR, -DQ, and -DP class II molecules. Out of nine DR1-positive cell lines, three failed to stimulate a subset of the T-cell clones in conventional proliferation assays. Proliferation by all of the clones was blocked by anti-DR antibodies, not by anti-DQ or anti-DP, which was consistent with the conclusion that the HLA-DR molecule was recognized. This DR1-associated polymorphism has been identified as Dw20 by the Tenth International Histocompatibility Workshop. The molecular basis for this altered recognition of the DR1 molecule was determined by allele-specific oligonucleotide hybridization and by DNA sequencing studies. The first, second, and third hypervariable regions of all nine DR1-positive cell lines were identical. Valine and glycine were found at positions 85 and 86 of the DR1 beta 1 chain in DR1 molecules from six of the nine lymphoblastoid cell lines, whereas alanine and valine were found in the three variant (Dw20) DR1-positive cells. By analogy with class I structure, residues 85 and 86 would be located at the extreme C-terminal end of the beta-chain alpha helix. Together or separately, these amino acid differences may define a T-cell recognition element on the DR1 molecule serving to contact allospecific T-cell receptors. Alternatively, if allorecognition involves recognition of a self peptide complexed with an allogeneic MHC molecule, then it is possible that the differences T cells recognize on DR1 class II proteins arise from peptide-specific interactions with residues 85 and 86.

  20. An Electrophoretically Silent Polymorphism for the Beta Chains of Rabbit Hemoglobin and Associated Polyribosome Patterns

    PubMed Central

    Garrick, Michael D.; Bricker, J.; Garrick, Laura M.

    1974-01-01

    The β chain of rabbit (Oryctolagus caniculus) hemoglobin has previously been reported to contain a single residue of isoleucine at β112. We have detected other rabbits with either zero isoleucyl residues or half a residue per β chain. This character is polymorphic and inherited as a simple mendelian autosomal codominant.—Normally the modal number of ribosomes per polyribosome is 4 to 6 in reticulocyte lysates; but incubation of rabbit reticulocytes prior to lysis with L-o-methylthreonine (OMT), an isostere of isoleucine, leads to a bimodal distribution in lysates with 2–3 and 8–12 ribosomes as modes. This alteration has been attributed to ribosomal traffic jams caused by starvation for ile-tRNA at mRNA codons corresponding to the locations of isoleucyl residues at positions α10, α17, α55 and β112. We have confirmed this interpretation by incubating OMT with reticulocytes from rabbits with integral, half integral and nil values for isoleucyl residues per β chain to show that formation of the larger clusters of polyribosomes requires that β112 = ile. PMID:4818267

  1. Prevalence of TEM, SHV, and CTX-M Beta-Lactamase genes in the urinary isolates of a tertiary care hospital

    PubMed Central

    Bajpai, Trupti; Pandey, M.; Varma, M.; Bhatambare, G. S.

    2017-01-01

    Introduction: Extended-spectrum beta-lactamases (ESBLs) are the major cause of resistance to beta-lactam antibiotics such as penicillins, cephalosporins, and monobactams. They are derived from the narrow-spectrum beta-lactamases (TEM-1, TEM-2, or SHV-1) by mutations that alter the amino acid configuration around the enzyme active site. Aim: To determine the prevalence of ESBL (blaTEM, blaCTX-M, and blaSHV) genes among the members of Enterobacteriaceae. Methodology: The present prospective study was carried out from January 2015 to June 2015 in the Department of Microbiology and Molecular Medicine of a Teaching Tertiary Care Hospital. A total of 526 urine samples were studied. Seventy-eight isolates were subjected to polymerase chain reaction for detection of ESBL genes. Results: In our study, ESBL genes were detected among 18 (45%) phenotypically confirmed ESBL producers and 20 (52.5%) phenotypically confirmed non-ESBL producers. The gene that predominated was blaTEM (48.7%), followed by blaCTX-M (7.6%) and blaSHV (5.1%). Conclusion: Definitive identification of ESBL genes is only possible by molecular detection methods. Phenotypic tests need to be evaluated periodically as their performance may change with the introduction of new enzymes. PMID:28182026

  2. Pest protection conferred by A Beta vulgaris serine proteinase inhibitor gene

    USDA-ARS?s Scientific Manuscript database

    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. Indep...

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

    ERIC Educational Resources Information Center

    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.

  4. Direct cellobiose production from cellulose using sextuple beta-glucosidase gene deletion Neurospora crassa mutants

    USDA-ARS?s Scientific Manuscript database

    Direct cellobiose production from cellulose by a genetically modified fungus—Neurospora crassa, was explored in this study. A library of N. crassa sextuple beta-glucosidase (bgl) gene deletion strains was constructed. Various concentrations of cellobiose were detected in the culture broth of the N. ...

  5. Characterization and expression of the beta-N-acetylglucosaminidase gene family of Tribolium castaneum

    USDA-ARS?s Scientific Manuscript database

    Enzymes belonging to the Beta-N-acetylglucosaminidase (NAG) family cleave chitin oligosaccharides produced by the action of chitinases on chitin into the constituent N-acetylglucosamine monomer. Four genes encoding putative NAGs in the red flour beetle, Tribolium castaneum, namely TcNAG1, TcFDL, Tc...

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

    ERIC Educational Resources Information Center

    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.

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

    SciTech Connect

    Rogers, Z.R.; Powars, D.R.; Williams, W.D. ); Kinney, T.R. ); Schroeder, W.A. )

    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.

  8. Sequence 274-368 in the beta 3-subunit of the integrin alpha IIb beta 3 provides a ligand recognition and binding domain for the gamma-chain of fibrinogen that is independent of platelet activation.

    PubMed

    Alemany, M; Concord, E; Garin, J; Vinçon, M; Giles, A; Marguerie, G; Gulino, D

    1996-01-15

    Several bacterial-expressed recombinant fragments encompassing the extracellular part of the beta 3 subunit of the integrin alpha IIb beta 3 were shown to recognize and bind soluble and immobilized forms of fibrinogen. Two of them, designated as rIII-11 (beta 3 274-368) and rIII-13 (beta 3 274-403), did not contain the established RGD-ligand binding sequence. In fact, they interacted, in a Ca(2+)-independent manner, with the C-terminal part of the fibrinogen gamma chain. Both beta 3 fragments blocked the participation of fibrinogen in the induction of platelet aggregation induced by adenosine diphosphate. Fragment rIII-13 was recognized by the anti-beta 3 monoclonal antibody B2A. This antibody, which possesses an epitope exposed on both resting and activated platelets, inhibited fibrinogen binding as well as platelet adhesion and aggregation. In conclusion, the results demonstrated that the 274-368 sequence of the beta 3 subunit of integrin alpha IIb beta 3 constitutes a fibrinogen ligand binding domain, distinct from the RGD-binding site, that is required for both platelet adhesion and aggregation.

  9. Chromosome 3p loss of heterozygosity and mutation analysis of the FHIT and beta-cat genes in squamous cell carcinoma of the head and neck.

    PubMed Central

    González, M V; Pello, M F; Ablanedo, P; Suárez, C; Alvarez, V; Coto, E

    1998-01-01

    AIMS: To study the loss of heterozygosity at the short arm of chromosome 3 in primary tumours from patients with squamous cell carcinoma of the head and neck; to determine whether the FHIT gene, mapped to 3p14.2 and the CTNNB1 (beta-cat) gene, mapped to 3p21, are deleted or mutated in these tumours. METHODS: DNA was extracted from fresh tumours. Loss of heterozygosity was assessed by microsatellite analysis of the following markers: D3S1283 and D3S1286 (3p24), D3S966 (3p21), and D3S1300 (3P14.2). Homozygous deletion was determined by radioactive multiplex polymerase chain reaction of exons 5 and 6 of the FHIT gene. The presence of mutations in FHIT exon 5 and beta-cat exon 3 was studied by single strand conformation polymorphism. RESULTS: 50% of informative cases (25/50) showed loss of heterozygosity for at least one of the 3p markers. 3p21 was the region with the highest rate of allelic deletion (63%). No point mutation was found in FHIT exon 5 or beta-cat exon 3. No case showed homozygous deletion for the FHIT (exons 5 and 6) or the beta-cat exon 3. CONCLUSIONS: The short arm of chromosome 3 is often deleted in the head and neck squamous cell carcinomas. In the remaining alleles of the FHIT or beta-cat genes, no evidence was found for point mutations or deletions, documented in other common carcinomas. Inactivation could occur by different mechanisms such as methylation, or other genes (not studied here) could be target of allelic losses in squamous cell carcinoma of the head and neck. Images PMID:9797729

  10. Retroviral-mediated transfer and expression of human. beta. -globin genes in cultured murine and human erythroid cells

    SciTech Connect

    Weber-Benarous, A.; Cone, R.D.; London, I.M.; Mulligan, R.C.

    1988-05-05

    The authors cloned human ..beta..-globin DNA sequences from a genomic library prepared from DNA isolated from the human leukemia cell line K562 and have used the retroviral vector pZip-NeoSV(X)1 to introduce a 3.0-kilobase segment encompassing the globin gene into mouse erythroleukemia cells. Whereas the endogenous K562 ..beta..-globin gene is repressed in K562 cells, when introduced into mouse erythroleukemia cells by retroviral-mediated gene transfer, the ..beta..-globin gene from K562 cells was transcribed and induced 5-20-fold after treatment of the cells with dimethyl sulfoxide. The transcripts were correctly initiated, and expression and regulation of the K562 gene were identical to the expression of a normal human ..beta..-globin gene transferred into mouse erythroleukemia cells in the same way. They have also introduced the normal human ..beta..-globin gene into K562 cells using the same retrovirus vector. SP6 analysis of the RNA isolated from the transduced cells showed that the normal ..beta..-globin gene was transcribed at a moderately high level, before or after treatment with hemin. Based on these data, they suggest that the lack of expression of the endogenous ..beta..-globin gene in K562 cells does not result from an alteration in the gene itself and may not result from a lack of factor(s) necessary for ..beta..-lobin gene transcription. Retroviral-mediated transfer of the human ..beta..-globin gene may, however, uniquely influence expression of the gene K562 cells.

  11. Inhibition of serine palmitoyltransferase in vitro and long-chain base biosynthesis in intact Chinese hamster ovary cells by. beta. -chloroalanine

    SciTech Connect

    Medlock, K.A.; Merrill, A.H. Jr.

    1988-09-06

    The effects of ..beta..-chloroalanine (..beta..-Cl-alanine) on the serine palmitoyltransferase activity and the de novo biosynthesis of sphinganine and sphingenine were investigated in vitro with rat liver microsomes and in vivo with intact Chinese hamster ovary (CHO) cells. The inhibition in vitro was rapid, irreversible, and concentration and time dependent and apparently involved the active site because inactivation only occurred with ..beta..-Cl-L-alanine and was blocked by L-serine. These are characteristics of mechanism-based (suicide) inhibition. Serine palmitoyltransferase (SPT) was also inhibited when intact CHO cells were incubated with ..beta..-Cl-alanine and this treatment inhibited (/sup 14/C)serine incorporation into long-chain bases by intact cells. The concentration dependence of the loss of SPT activity and of long-chain base synthesis was identical. The effects of ..beta..-Cl-alanine appeared to occur with little perturbation of other cell functions: the cells exhibited no loss in cell viability, (/sup 14/C)serine uptake was not blocked, total lipid biosynthesis from (/sup 14/C)acetic acid was not decreased (nor was the appearance of radiolabel in cholesterol and phosphatidylcholine), and (/sup 3/H)thymidine incorporation into DNA was not affected. There appeared to be little effect on protein synthesis based on the incorporation of (/sup 3/H)leucine, which was only decreased by 14%. Although ..beta..-Cl-L-alanine is known to inhibit other pyridoxal 5'-phosphate dependent enzymes, alanine and aspartate transaminases were not inhibited under these conditions. These results establish the close association between the activity of serine palmitoyltransferase and the cellular rate of long-chain base formation and indicate that ..beta..-Cl-alanine and other mechanism-based inhibitors might be useful to study alterations in cellular long-chain base synthesis.

  12. Promoter region of the human platelet-derived growth factor A-chain gene.

    PubMed Central

    Takimoto, Y; Wang, Z Y; Kobler, K; Deuel, T F

    1991-01-01

    The platelet-derived growth factor (PDGF) A- and B-chain genes are widely expressed in mammalian tissues and their homodimeric gene products appear to regulate the autocrine growth of both normal and transformed cells. In this study, we analyzed the 5' flanking sequences of the human PDGF A-chain gene to seek elements important to regulating its transcription. The promoter region was exceptionally G + C-rich and contained a "TATA box" but no "CAAT box." The transcription start site was identified 845 base pairs 5' to the translation initiation site by S1 nuclease mapping and by primer extension. Both in vitro transcription and transient expression of the chloramphenicol acetyltransferase gene linked to the PDGF A-chain 5' flanking sequences established that the putative promoter region was active, and RNase H mapping established that the three characteristic mRNAs (1.9, 2.3, and 2.8 kilobases) used the same transcription start site, which was used in normal endothelial cells and in two human tumor cell lines that express high levels of A-chain transcripts. The results established an exceptionally G + C-rich promoter region and a single transcription start site active for each of the three mRNAs of the PDGF A-chain gene. DNA sites of potential importance in mediating the activation of the PDGF A-chain gene in normal cells and in transformed cell lines expressing high levels of PDGF A chain were identified. Images PMID:1848007

  13. Promoter region of the human platelet-derived growth factor A-chain gene

    SciTech Connect

    Takimoto, Yasuo; Wang, Zhao Yi; Kobler, K.; Deuel, T.F. )

    1991-03-01

    The platelet-derived growth factor (PDGF) A- and B-chain genes are widely expressed in mammalian tissues and their homodimeric gene products appear to regulate the autocrine growth of both normal and transformed cells. In this study, we analyzed the 5{prime} flanking sequences of the human PDGF A-chain gene to seek elements important to regulating its transcription. The promoter reigon was exceptionally G + C-rich and contained a TATA box but no CAAT box. The transcription start site was identified 845 base pairs 5{prime} to the translation initiation site by S1 nuclease mapping and by primer extension. Both in vitro transcription and transient expression of the chloramphenicol acetyltransferase gene linked to the PDGF A-chain 5{prime} flanking sequences established that the putative promoter region was active, and RNase H mapping established that the three characteristic mRNAs used the same transcription start site, which was used in normal endothelial cells and in two human tumor cell lines that express high levels of A-chain transcripts. The results extablished an exceptionally G + C-rich promoter region and a single transcription start site active for each of the three mRNAs of the PDGF A-chain gene. DNA sites of potential importance in mediating the activation of the PDGF A-chain gene in normal cells and in transformed cell lines expressing high levels of PDGF A-chain were identified.

  14. [Alkaline-adapted beta-mannanase of Bacillus pumilus: gene heterologous expression and enzyme characterization].

    PubMed

    Tang, Jiajie; Guo, Su; Wang, Wei; Wei, Wei; Wei, Dongzhi

    2015-11-04

    We expressed a novel alkaline-adapted beta-mannanase gene and characterized the enzyme for potential industrial applications. We obtained a mannanase gene (named man(B)) from Bacillus pumilus Nsic2 and expressed the gene man(B) in Escherichia coli and Bacillus subtilis. Furthermore, we characterized the enzyme. The gene man(B) had an open reading frame of 1104 bp that encoded a polypeptide of 367-amino-acid beta-mannanase (Man(B)). The protein sequence showed the highest identity with the beta-mannanase from B. pumilus CCAM080065. We expressed the gene man(B) in E. coli BL21 (DE3) with the enzyme activity of 11021.3 U/mL. Compared with other mannanases, Man(B) showed higher stability under alkaline conditions and was stable at pH6.0 -9.0. The specific activity of purified Man(B) was 4191 ± 107 U/mg. The K(m) and V(max) values of purified Man(B) were 35.7 mg/mL and 14.9 μmol/(mL x min), respectively. Meanwhile, we achieved recombinant protein secretion expression in B. subtilis WB800N. We achieved heterologous expression of the gene man(B) and characterized its enzyme. The alkaline-adapted Man(B) showed potential value in industrial applications due to its pH stability.

  15. Increased Stability and DNA Site Discrimination of Single Chain Variants of the Dimeric beta-Barrel DNA Binding Domain of the Human Papillomavirus E2 Transcriptional Regulator

    SciTech Connect

    Dellarole,M.; Sanchez, I.; Freire, E.; de Prat-Gay, G.

    2007-01-01

    Human papillomavirus infects millions of people worldwide and is a causal agent of cervical cancer in women. The HPV E2 protein controls the expression of all viral genes through binding of its dimeric C-terminal domain (E2C) to its target DNA site. We engineered monomeric versions of the HPV16 E2C, in order to probe the link of the dimeric {beta}-barrel fold to stability, dimerization, and DNA binding. Two single-chain variants, with 6 and 12 residue linkers (scE2C-6 and scE2C-12), were purified and characterized. Spectroscopy and crystallography show that the native structure is unperturbed in scE2C-12. The single chain variants are stabilized with respect to E2C, with effective concentrations of 0.6 to 6 mM. The early folding events of the E2C dimer and scE2C-12 are very similar and include formation of a compact species in the submillisecond time scale and a non-native monomeric intermediate with a half-life of 25 ms. However, monomerization changes the unfolding mechanism of the linked species from two-state to three-state, with a high-energy intermediate. Binding to the specific target site is up to 5-fold tighter in the single chain variants. Nonspecific DNA binding is up to 7-fold weaker in the single chain variants, leading to an overall 10-fold increased site discrimination capacity, the largest described so far for linked DNA binding domains. Titration calorimetric binding analysis, however, shows almost identical behavior for dimer and single-chain species, suggesting very subtle changes behind the increased specificity. Global analysis of the mechanisms probed suggests that the dynamics of the E2C domain, rather than the structure, are responsible for the differential properties. Thus, the plastic and dimeric nature of the domain did not evolve for a maximum affinity, specificity, and stability of the quaternary structure, likely because of regulatory reasons and for roles other than DNA binding played by partly folded dimeric or monomeric conformers.

  16. [Aspirin-PEI-beta-CyD as a novel non-viral vector for gene transfer].

    PubMed

    Wang, Zhong-Ren; Chen, Dan; Zhou, Jun; Tang, Gu-Ping

    2009-01-01

    To develop a novel non-viral gene delivery vector based on PEI-beta-CyD as backbone modified with aspirin, and to identify its physicochemical characters. 1, 1-carbonyldiimidazole (CDI) was used to bind aspirin onto PEI-beta-CyD to form PEI-beta-CyD-ASP. (1)H-NMR, FT-IR, UV and XRD were used to confirm the polymer structure. The ability of condensation was demonstrated by gel retardation assay. MTT assay was used to test the cell viability in B16, Hela and A293 cell lines. Transfection efficiency of the polymer was tested in B16 cells. The structure of PEI-beta-CyD-ASP was confirmed by (1)H-NMR, FT-IR, UV and XRD, which efficiently condensed plasmid DNA at the N/P ratio of 4. The copolymer showed low cytotoxicity and high transfection efficiency in B16 cells. The synthesized aspirin-PEI-beta-CyD might be a potential gene delivery vector.

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

    PubMed

    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.

  18. Short-chain fatty acids and poly-beta-hydroxyalkanoates: (New) Biocontrol agents for a sustainable animal production.

    PubMed

    Defoirdt, Tom; Boon, Nico; Sorgeloos, Patrick; Verstraete, Willy; Bossier, Peter

    2009-01-01

    Because of the risk of antibiotic resistance development, there is a growing awareness that antibiotics should be used more carefully in animal production. However, a decreased use of antibiotics could result in a higher frequency of pathogenic bacteria, which in its turn could lead to a higher incidence of infections. Short-chain fatty acids (SCFAs) have long been known to exhibit bacteriostatic activity. These compounds also specifically downregulate virulence factor expression and positively influence the gastrointestinal health of the host. As a consequence, there is currently considerable interest in SCFAs as biocontrol agents in animal production. Polyhydroxyalkanoates (PHAs) are polymers of beta-hydroxy short-chain fatty acids. Currently, PHAs are applied as replacements for synthetic polymers. These biopolymers can be depolymerised by many different microorganisms that produce extracellular PHA depolymerases. Interestingly, different studies provided some evidence that PHAs can also be degraded upon passage through the gastrointestinal tract of animals and consequently, adding these compounds to the feed might result in biocontrol effects similar to those described for SCFAs.

  19. Amyloid-beta leads to impaired cellular respiration, energy production and mitochondrial electron chain complex activities in human neuroblastoma cells.

    PubMed

    Rhein, V; Baysang, G; Rao, S; Meier, F; Bonert, A; Müller-Spahn, F; Eckert, A

    2009-09-01

    Evidence suggests that amyloid-beta (Abeta) protein is a key factor in the pathogenesis of Alzheimer's disease (AD) and it has been recently proposed that mitochondria are involved in the biochemical pathway by which Abeta can lead to neuronal dysfunction. Here we investigated the specific effects of Abeta on mitochondrial function under physiological conditions. Mitochondrial respiratory functions and energy metabolism were analyzed in control and in human wild-type amyloid precursor protein (APP) stably transfected human neuroblastoma cells (SH-SY5Y). Mitochondrial respiratory capacity of mitochondrial electron transport chain (ETC) in vital cells was measured with a high-resolution respirometry system (Oxygraph-2k). In addition, we determined the individual activities of mitochondrial complexes I-IV that compose ETC and ATP cellular levels. While the activities of complexes I and II did not change between cell types, complex IV activity was significantly reduced in APP cells. In contrast, activity of complex III was significantly enhanced in APP cells, as compensatory response in order to balance the defect of complex IV. However, this compensatory mechanism could not prevent the strong impairment of total respiration in vital APP cells. As a result, the respiratory control ratio (state3/state4) together with ATP production decreased in the APP cells in comparison with the control cells. Chronic exposure to soluble Abeta protein may result in an impairment of energy homeostasis due to a decreased respiratory capacity of mitochondrial electron transport chain which, in turn, may accelerate neurons demise.

  20. Implication of activin E in glucose metabolism: transcriptional regulation of the inhibin/activin betaE subunit gene in the liver.

    PubMed

    Hashimoto, Osamu; Sekiyama, Kazunari; Matsuo, Tsuyoshi; Hasegawa, Yoshihisa

    2009-09-23

    Activin E is a newly identified member of the transforming growth factor-beta superfamily. To assess the role of activin E in glucose/energy metabolism, we investigated the transcriptional regulation of activin E in the liver. Northern blotting, Western blotting, quantitative real-time polymerase chain reaction (PCR), reporter assays and chromatin immunoprecipitation assays were used in this study. Insulin up-regulated activin E expression at the mRNA and protein level in HepG2 cells. Reporter assays revealed that the putative, functional, promoter sequence of human activin E gene was responsible for the effect of insulin. Mutational analysis of the promoter revealed that CCAAT/enhancer-binding proteins (C/EBPs) play a key role in regulating activin E expression and in the stimulatory effect of insulin on activin E transcription. Chromatin immunoprecipitation assays revealed that the C/EBPs can bind to the activin E promoter in HepG2 cells. The expression of activin E mRNA was up-regulated in the liver of diet-induced obese mice. These observations suggest that activin E plays a pathophysiological role in glucose metabolism.

  1. Stimulator of IFN gene is critical for induction of IFN-beta during Chlamydia muridarum infection.

    PubMed

    Prantner, Daniel; Darville, Toni; Nagarajan, Uma M

    2010-03-01

    Type I IFN signaling has recently been shown to be detrimental to the host during infection with Chlamydia muridarum in both mouse lung and female genital tract. However, the pattern recognition receptor and the signaling pathways involved in chlamydial-induced IFN-beta are unclear. Previous studies have demonstrated no role for TLR4 and a partial role for MyD88 in chlamydial-induced IFN-beta. In this study, we demonstrate that mouse macrophages lacking TLR3, TRIF, TLR7, or TLR9 individually or both TLR4 and MyD88, still induce IFN-beta equivalent to wild type controls, leading to the hypothesis that TLR-independent cytosolic pathogen receptor pathways are crucial for this response. Silencing nucleotide-binding oligomerization domain 1 in HeLa cells partially decreased chlamydial-induced IFN-beta. Independently, small interfering RNA-mediated knockdown of the stimulator of IFN gene (STING) protein in HeLa cells and mouse oviduct epithelial cells significantly decreased IFN-beta mRNA expression, suggesting a critical role for STING in chlamydial-induced IFN-beta induction. Conversely, silencing of mitochondria-associated antiviral signaling proteins and the Rig-I-like receptors, RIG-I, and melanoma differentiation associated protein 5, had no effect. In addition, induction of IFN-beta depended on the downstream transcription IFN regulatory factor 3, and on activation of NF-kappaB and MAPK p38. Finally, STING, an endoplasmic reticulum-resident protein, was found to localize in close proximity to the chlamydial inclusion membrane during infection. These results indicate that C. muridarum induces IFN-beta via stimulation of nucleotide-binding oligomerization domain 1 pathway, and TLR- and Rig-I-like receptor-independent pathways that require STING, culminating in activation of IFN regulatory factor 3, NF-kappaB, and p38 MAPK.

  2. Further characterization of the thrombasthenia-related idiotype OG. Antiidiotype defines a novel epitope(s) shared by fibrinogen B beta chain, vitronectin, and von Willebrand factor and required for binding to beta 3

    PubMed Central

    1994-01-01

    A patient (OG) with Glanzmann thrombasthenia became refractory to platelet transfusion after the production of an immunoglobulin G (IgG) isoantibody (Ab1) specific for the integrin subunit beta 3. To determine the frequency at which the OG idiotype is found in the general population and in immune-mediated disease states, we developed a rabbit polyclonal antibody (Ab2) specific for affinity-purified OG anti-beta 3 Fab. The binding of Ab2 to Ab1 is inhibited by purified alpha IIb beta 3. Ab2 als binds to IgG specific for alpha IIb beta 3 obtained from one nonrelated Glanzmann thrombasthenia patient ES who has developed isoantibodies of similar specificity. On the other hand, Ab2 does not recognize alpha IIb beta 3-specific antibodies produced by two Glanzmann thrombasthenia patients, AF and LUC, who have developed isoantibodies with specificities distinct from that of the OG isoantibody. Moreover, Ab2 does not recognize alpha IIb beta 3-specific antibodies developed by three representative patients with (autoimmune) thrombocytopenic purpura or six representative patients with alloimmune thrombocytopenias, nor does it bind to IgG from any of 13 nonimmunized individuals. We have found that Ab2 also binds to selected protein ligands of alpha IIb beta 3 namely, fibrinogen, vitronectin, and von Willebrand factor, but not to other protein ligands or control proteins, such a fibronectin, type I collagen, and albumin. The epitope(s) recognized by Ab2 on each adhesive protein are either very similar or identical since each protein can inhibit the binding of Ab2 to any of the other proteins. The epitope on fibrinogen recognized by Ab2 resides in the B beta chain, and is likely contained within the first 42 amino acids from the NH2 terminus. Since OG IgG inhibits fibrinogen binding to alpha IIb beta 3, the specificity of the OG idiotype defines a novel binding motif for the integrin alpha IIb beta 3 that is shared by fibrinogen, vitronectin, and von Willebrand factor, but

  3. The genes and mRNA coding for the heavy chains of chick embryonic skeletal myosin.

    PubMed

    Patrinou-Georgoulas, M; John, H A

    1977-10-01

    A size class of polysomes was isolated from chick embryonic leg skeletal muscle which synthesized almost exclusively a polypeptide chain with a molecular weight identical to the myosin heavy chain. The mRNA purified from these polysomes was shown to synthesize the 200,000 dalton polypeptide in the wheat germ cell-free translation system. At least 90% of the polypeptide had properties similar to the myosin heavy chain. Isoelectric focusing indicated that the myosin heavy chain synthesized in vitro contained two chains in equal amounts, as did purified embryonic leg skeletal muscle myosin. The kinetics of hybridization of the complementary DNA with an excess of the myosin heavy chain mRNA (MHC mRNA) indicated the presence of two different mRNA sequences. Reassociation of the cDNA to an excess of the DNA of the genome suggest that there is little, if any, reiteration of the myosin heavy chain genes.

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

    PubMed

    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 < 5%, Group B: HbF between 5 and 15%, and Group C: 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.

  5. Molecular characteristics of the alpha- and beta-tubulin genes of Nosema philosamiae.

    PubMed

    Zhu, Feng; Shen, Zhongyuan; Xu, Li; Guo, Xijie

    2013-11-01

    Microsporidia are intracellular parasites of insects and other higher eukaryotes. The microsporidian Nosema philosamiae Talukdar, 1961 was isolated from the eri silkworm, Philosamia cynthia ricini Grote. In the present study, alpha- and beta-tubulin genes from N. philosamiae were characterized. The identity analysis of nucleotide and amino acid sequences indicated high similarity with species of Nosema Nägeli, 1857 sensu lato (nucleotide sequences, > or = 96.0%; amino acid sequences, > or = 99.0%). However, the tubulin genes of N. philosamiae share low sequence similarity with that of N. ceranae Fries, Feng, da Silva, Slemenda et Pieniazek, 1996 (strain BRL01) and a Nosema/Vairimorpha species. Phylogenies based on alpha-, beta- and combined alpha- plus beta-tubulin gene sequences showed that N. philosamiae, along with the true Nosema species, forms a separate clade with a high bootstrap value, with N. ceranae BRL01 forming a clade of its own. The results indicated that the alpha- and beta-tubulin sequences may be useful as a diagnostic tool to discriminate the true Nosema group from the Nosema/Vairimorpha group.

  6. Bifunctional peptides derived from homologous loop regions in the laminin alpha chain LG4 modules interact with both alpha 2 beta 1 integrin and syndecan-2.

    PubMed

    Yokoyama, Fumiharu; Suzuki, Nobuharu; Kadoya, Yuichi; Utani, Atsushi; Nakatsuka, Hiroko; Nishi, Norio; Haruki, Masahiro; Kleinman, Hynda K; Nomizu, Motoyoshi

    2005-07-19

    Laminin alpha chains show diverse biological functions in a chain-specific fashion. The laminin G-like modules (LG modules) of the laminin alpha chains consist of a 14-stranded beta-sheet sandwich structure with biologically active sequences found in the connecting loops. Previously, we reported that connecting loop regions between beta-strands E and F in the mouse laminin alpha chain LG4 modules exhibited chain-specific activities. In this study, we focus on the homologous loop regions in human laminin alpha chain LG4 modules using five synthetic peptides (hEF-1-hEF-5). These homologous peptides induced chain-specific cellular responses in various cell types. Next, to examine the dual-receptor recognition model, we synthesized chimeras (cEF13A-cEF13E) derived from peptides hEF-1 and hEF-3. All of the chimeric peptides promoted fibroblast attachment as well as the parental peptides. Attachment of fibroblasts to cEF13A and cEF13B was inhibited by anti-integrin alpha2 and beta1 antibodies and by heparin, while cell adhesion to cEF13C, cEF13D, and cEF13E was blocked only by heparin. Actin organization of fibroblasts on cEF13C was not different from that on hEF-3, but cEF13B induced membrane ruffling at the tips of the actin stress fibers. These results suggest that cEF13B had bifunctional effects on cellular behaviors through alpha2beta1 integrin and heparin/heparan sulfate proteoglycan. We conclude that the approach utilizing chimeric peptides is useful for examining cellular mechanisms in dual-receptor systems.

  7. Expression of the Escherichia coli beta-glucuronidase gene in Pseudocercosporella herpotrichoides.

    PubMed Central

    Bunkers, G J

    1991-01-01

    The plant-pathogenic fungus Pseudocercosporella herpotrichoides has been successfully transformed by using two different positive selection systems in combination with the Escherichia coli gusA gene. The selectable markers used in this study were the hygromycin B phosphotransferase gene (hph) from E. coli and the gene (bml) for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa. A lower transformation rate was obtained with the bml system than with the hph system. Conversely, cotransformation frequencies, as determined with medium plates containing the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid, were higher with bml than with hph as the selectable marker. The hygromycin-resistant transformants were mitotically stable, and both the selectable gene and gusA were maintained through conidiation. The vector DNA was integrated into the genome, and the number and sites of insertion varied among transformants. Enzyme assays of mycelial extracts showed that beta-glucuronidase activity was highest in transformants with a high gusA copy number. Expression of gusA during growth of the fungus on plants was easily detectable and did not affect pathogenicity. These results form the basis for construction of a versatile and sensitive reporter gene system for P. herpotrichoides. Images PMID:1746951

  8. Specific transcription and RNA splicing defects in five cloned beta-thalassaemia genes.

    PubMed

    Treisman, R; Orkin, S H; Maniatis, T

    1983-04-14

    Transcriptional analysis of five different cloned beta-thalassaemia genes introduced into cultured mammalian cells revealed specific defects in transcription and RNA splicing. A single base change 87 base pairs to the 5' side of the mRNA cap site significantly lowers the level of transcription and therefore appears to represent a promoter mutation. Three genes contain different single base changes in the first intervening sequence (IVS) 5' splice site. One mutation, at IVS1 position 1, inactivates the splice site completely; the other two, at IVS1 positions 5 and 6, reduce its activity. Each mutation activates the same three cryptic splice sites. The fifth gene contains a single base change within IVS2 at position 745, which results in the formation of abnormal beta-globin RNA that contains an extra exon.

  9. Regulated expression of a complete human beta-globin gene encoded by a transmissible retrovirus vector.

    PubMed Central

    Cone, R D; Weber-Benarous, A; Baorto, D; Mulligan, R C

    1987-01-01

    We introduced a human beta-globin gene into murine erythroleukemia (MEL) cells by infection with recombinant retroviruses containing the complete genomic globin sequence. The beta-globin gene was correctly regulated during differentiation, steady-state mRNA levels being induced 5- to 30-fold after treatment of the cells with the chemical inducer dimethyl sulfoxide. Studies using vectors which yield integrated proviruses lacking transcriptional enhancer sequences indicated that neither retroviral transcription nor the retroviral enhancer sequences themselves had any obvious effect on expression of the globin gene. Viral RNA expression also appeared inducible, being considerably depressed in uninduced MEL cells but approaching normal wild-type levels after dimethyl sulfoxide treatment. We provide data which suggest that the control point for both repression and subsequent activation of virus expression in MEL cells lies in the viral enhancer element. Images PMID:3029570

  10. Rapid Evolution of Beta-Keratin Genes Contribute to Phenotypic Differences That Distinguish Turtles and Birds from Other Reptiles

    PubMed Central

    Li, Yang I.; Kong, Lesheng; Ponting, Chris P.; Haerty, Wilfried

    2013-01-01

    Sequencing of vertebrate genomes permits changes in distinct protein families, including gene gains and losses, to be ascribed to lineage-specific phenotypes. A prominent example of this is the large-scale duplication of beta-keratin genes in the ancestors of birds, which was crucial to the subsequent evolution of their beaks, claws, and feathers. Evidence suggests that the shell of Pseudomys nelsoni contains at least 16 beta-keratins proteins, but it is unknown whether this is a complete set and whether their corresponding genes are orthologous to avian beak, claw, or feather beta-keratin genes. To address these issues and to better understand the evolution of the turtle shell at a molecular level, we surveyed the diversity of beta-keratin genes from the genome assemblies of three turtles, Chrysemys picta, Pelodiscus sinensis, and Chelonia mydas, which together represent over 160 Myr of chelonian evolution. For these three turtles, we found 200 beta-keratins, which indicate that, as for birds, a large expansion of beta-keratin genes in turtles occurred concomitantly with the evolution of a unique phenotype, namely, their plastron and carapace. Phylogenetic reconstruction of beta-keratin gene evolution suggests that separate waves of gene duplication within a single genomic location gave rise to scales, claws, and feathers in birds, and independently the scutes of the shell in turtles. PMID:23576313

  11. Rapid evolution of Beta-keratin genes contribute to phenotypic differences that distinguish turtles and birds from other reptiles.

    PubMed

    Li, Yang I; Kong, Lesheng; Ponting, Chris P; Haerty, Wilfried

    2013-01-01

    Sequencing of vertebrate genomes permits changes in distinct protein families, including gene gains and losses, to be ascribed to lineage-specific phenotypes. A prominent example of this is the large-scale duplication of beta-keratin genes in the ancestors of birds, which was crucial to the subsequent evolution of their beaks, claws, and feathers. Evidence suggests that the shell of Pseudomys nelsoni contains at least 16 beta-keratins proteins, but it is unknown whether this is a complete set and whether their corresponding genes are orthologous to avian beak, claw, or feather beta-keratin genes. To address these issues and to better understand the evolution of the turtle shell at a molecular level, we surveyed the diversity of beta-keratin genes from the genome assemblies of three turtles, Chrysemys picta, Pelodiscus sinensis, and Chelonia mydas, which together represent over 160 Myr of chelonian evolution. For these three turtles, we found 200 beta-keratins, which indicate that, as for birds, a large expansion of beta-keratin genes in turtles occurred concomitantly with the evolution of a unique phenotype, namely, their plastron and carapace. Phylogenetic reconstruction of beta-keratin gene evolution suggests that separate waves of gene duplication within a single genomic location gave rise to scales, claws, and feathers in birds, and independently the scutes of the shell in turtles.

  12. Transfer of nonselectable genes into mouse teratocarcinoma cells and transcription of the transferred human. beta. -globin gene

    SciTech Connect

    Wagner, E.F.; Mintz, B.

    1982-02-01

    Teratocarcinoma (TCC) stem cells can function as vehicles for the introduction of specific recombinant genes into mice. Because most genes do not code for a selectable marker, the authors investigated the transformation efficiency of vectors with a linked selectable gene. In one series, TCC cells first selected for thymidine kinase deficiency were treated with DNA from the plasmid vector PtkH..beta..1 containing the human genomic ..beta..-globin gene and the thymidine kinase gene of herpes simplex virus. A high transformation frequency was obtained after selection in hypoxanthine-aminopterin-thymidine medium. Hybridization tests revealed that the majority of transformants had intact copies of the human gene among three to six total copies per cell. These were associated with cellular DNA sequences as judged from the presence of additional new restriction fragments and from stability of the sequences in tumors produced by injecting the cells subcutaneously. Total polyadenylate-containing RNA from cell cultures of two out of four transformants examined showed hybridization to the human gene probe: one RNA species resembled mature human ..beta..-globin mRNA transcripts; the others were of larger size. In differentiating tumors, various tissues, including hematopoietic cells of TCC provenance could be found. In a second model set of experiments, wild-type TCC cells were used to test a dominant-selection scheme with pSV-gpt vectors. Numerous transformants were isolated, and their transfected DNA was apparently stably integrated. Thus, any gene of choice can be transferred into TCC stem cells even without mutagenesis of the cells, and selected cell clones can be characterized. Cells of interest may then be introduced into early embryos to produce new mouse strains with predetermined genetic changes.

  13. [Cloning of genes coding for 3-alpha-hydroxysteroid dehydrogenase and for (3-17)-beta-hydroxysteroid dehydrogenase from Pseudomonas testosteroni].

    PubMed

    Floch, H H; Abalain, J H; Di Stefano, S; Carré, J L; Abalain-Colloc, M L

    1995-01-01

    A genomic library of Pseudomonas testosteroi total DNA constructed from SauIIIA digests ligated to a lambda gt11 vector was probed with different polyclonal antibodies raised against purified 3 alpha-HSD and (3 beta-17 beta)-HSD. Two different clones reacting with one antibody were selected. The clone reacting with (3-17)beta-HSD antibody contained a 2,661-base pair insert and was found to contained an open reading frame of 765 base pair that corresponds to a protein of 254 amino-acid residues. A 1,492-base pair was inserte in pBR 322 plasmid vector; the recombinant bacterie over expressed the (3-17)beta-HSD gene. The clone reacting with 3 alpha-HSD antibody contained a 1746 base pair insert which contained an open reading frame of 696 base pairs that corresponds to a protein of 231 amino-acid residues. A search for homologous proteins was performed. Distant similarities were found between (3-17)beta-HSD and members of the short-chain alcool dehydrogenase (SCAD) family but no similarity was observed between 3 alpha-HSD and proteins of this family.

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

    PubMed

    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.

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

    PubMed

    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.

  16. Dominant-negative mutation in the beta2 and beta6 proteasome subunit genes affect alternative cell fate decisions in the Drosophila sense organ lineage.

    PubMed

    Schweisguth, F

    1999-09-28

    In Drosophila, dominant-negative mutations in the beta2 and beta6 proteasome catalytic subunit genes have been identified as dominant temperature-sensitive (DTS) mutations. At restrictive temperature, beta2 and beta6 DTS mutations confer lethality at the pupal stage. I investigate here the role of proteasome activity in regulating cell fate decisions in the sense organ lineage at the early pupal stage. Temperature-shift experiments in beta2 and beta6 DTS mutant pupae occasionally resulted in external sense organs with two sockets and no shaft. This double-socket phenotype was strongly enhanced in conditions in which Notch signaling was up-regulated. Furthermore, conditional overexpression of the beta6 dominant-negative mutant subunit led to shaft-to-socket and to neuron-to-sheath cell fate transformations, which are both usually associated with increased Notch signaling activity. Finally, expression of the beta6 dominant-negative mutant subunit led to the stabilization of an ectopically expressed nuclear form of Notch in imaginal wing discs. This study demonstrates that mutations affecting two distinct proteasome catalytic subunits affect two alternative cell fate decisions and enhance Notch signaling activity in the sense organ lineage. These findings raise the possibility that the proteasome targets an active form of the Notch receptor for degradation in Drosophila.

  17. Limited T cell receptor beta-chain usage in the sperm whale myoglobin 110-121/E alpha dA beta d response by H-2d congenic mouse strains.

    PubMed

    Sellins, K S; Danska, J S; Paragas, V; Fathman, C G

    1992-10-01

    The specificity and TCR gene usage of a panel of sperm whale myoglobin (SpWMb)-reactive T cell clones from DBA/2 mice have previously been characterized, to study structure-function relationships between components of the ternary complex consisting of Ag, TCR, and MHC class II molecules, whose interaction leads to Th cell activation. These DBA/2 clones were specific for epitopes within the residue 110 to 121 region of SpWMb, in the context of the mixed isotype molecule E alpha dA beta d, and expressed the TCR V beta 8.2 gene element. SpWMb-specific T cell hybridomas from the H-2d-congenic B10.D2 mouse strain, which differs from the DBA/2 strain only in the non-MHC background, were generated and compared with the T cell hybridomas from DBA/2 mice, in order to investigate the influence of non-MHC genes on the specificity of the T cell response to the 110-121 epitope. V beta usage by these hybridomas was very homogeneous; three of three DBA/2 and eight of nine B10.D2 hybridomas specific for the 110-121 epitope, in the context of the mixed isotype molecule E alpha dA beta d, expressed the V beta 8.2 gene product. Nucleotide and amino acid sequences of D beta, J beta, and N regions were also similar. One 110-121/E alpha dA beta d-specific B10.D2 hybridoma used V beta 7, a V beta that is clonally deleted in DBA/2 mice. These experiments suggest that a similar set of TCR beta genes are used to respond to a given epitope, regardless of non-MHC background, and they support the hypothesis that, despite great variability between individuals in their non-MHC background genes, human HLA-associated diseases might result from the formation of a particular ternary complex consisting of a shared MHC molecule, a common "disease-associated" epitope, and a shared TCR.

  18. Nonrandom association of polymorphic restriction sites in the beta-globin gene cluster.

    PubMed

    Antonarakis, S E; Boehm, C D; Giardina, P J; Kazazian, H H

    1982-01-01

    By using probes for epsilon-, Psibeta(1)-, and beta-globin genes, we found four additional polymorphic restriction sites that have frequencies >0.1 in persons of Mediterranean area origin, Asian Indians, and American Blacks. Three of these (HincII sites) and the two previously described polymorphic HindIII sites [one in intervening sequence (IVS) II of each gamma-globin gene] are distributed over 32 kilobases (kb) of DNA located 5' to the delta-globin gene. This region of DNA comprises two-thirds of the beta-globin gene cluster. Since each of these five polymorphic sites can be present (+) or absent (-), in theory there exist 32 possible combinations of sites (haplotypes). However, in Italians, Greeks, Indians, and Turks, 3 of the 32 haplotypes, (+----), (-+-++), and (-++-+), account for 92% of 89 beta(A) chromosomes examined. The observed frequencies for these haplotypes are 0.64, 0.15, and 0.13 in the populations studied, in contrast to expected frequencies (based on the observed gene frequencies at each of the five sites) of 0.20, 0.006, and 0.005, respectively. In American Blacks, a fourth haplotype, (----+), which is rare in non-Black populations, has a frequency of 0.37 in contrast to its expected frequency of 0.05. These results suggest a nonrandom association of DNA sequences over 32 kb 5' to the delta-globin gene in all populations studied. Two other polymorphic sites 3' to the delta gene (the newly discovered Ava II site in IVS II of the beta-globin gene and the BamHI site 3' to it) are nonrandomly associated with each other but randomly distributed with respect to the above haplotypes. This suggests that randomization of sequences has occurred within 12 kb of DNA between these two nonrandomly associated sequence clusters. Nonrandom association of polymorphic restriction sites has practical consequences in that it limits the usefulness of these additional HincII sites for prenatal diagnosis of hemoglobinopathies by linkage analysis. These sites provide

  19. Structure, chromosome location, and expression of the human. gamma. -actin gene: Differential evolution, location, and expression of the cytoskeletal BETA- and. gamma. -actin genes

    SciTech Connect

    Erba, H.P.; Eddy, R.; Shows, T.; Kedes, L.; Gunning, P.

    1988-04-01

    The accumulation of the cytoskeletal ..beta..-and ..gamma..-actin mRNAs was determined in a variety of mouse tissues and organs. The ..beta..-iosform is always expressed in excess of the ..gamma..-isoform. However, the molar ratio of ..beta..- to ..gamma..-actin mRNA varies from 1.7 in kidney and testis to 12 in sarcomeric muscle to 114 in liver. The authors conclude that, whereas the cytoskeletal ..beta..- and ..gamma..-actins are truly coexpressed, their mRNA levels are subject to differential regulation between different cell types. The human ..gamma..-actin gene has been cloned and sequenced, and its chromosome location has been determined. The gene is located on human chromosome 17, unlike ..beta..-actin which is on chromosome 7. Thus, if these genes are also unlinked in the mouse, the coexpression of the ..beta..- and ..gamma..-actin genes in rodent tissues cannot be determined by gene linkage. Comparison of the human ..beta..- and ..gamma..-actin genes reveals that noncoding sequences in the 5'-flanking region and in intron III have been conserved since the duplication that gave rise to these two genes. In contrast, there are sequences in intron III and the 3'-untranslated region which are not present in the ..beta..-actin gene but are conserved between the human ..gamma..-actin and the Xenopus borealis type 1 actin genes. Such conserved noncoding sequences may contribute to the coexpression of ..beta..- and ..gamma..-actin or to the unique regulation and function of the ..gamma..-actin gene. Finally, the authors demonstrate that the human ..gamma..-actin gene is expressed after introduction into mouse L cells and C2 myoblasts and that, upon fusion of C2 cells to form myotubes, the human ..gamma..-actin gene is appropriately regulated.

  20. The Polymorphisms of Ser49Gly and Gly389Arg in Beta-1-Adrenergic Receptor Gene in Major Depression

    PubMed Central

    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. PMID:28360691

  1. A novel transgenic mouse model produced from lentiviral germline integration for the study of beta-thalassemia gene therapy.

    PubMed

    Li, Wei; Xie, Shuyang; Guo, Xinbing; Gong, Xiuli; Wang, Shu; Lin, Dan; Zhang, Jingzhi; Ren, Zhaorui; Huang, Shuzhen; Zeng, Fanyi; Zeng, Yitao

    2008-03-01

    beta-thalassemia is one of the most common genetic diseases in the world and requires extensive therapy. Lentiviral-mediated gene therapy has been successfully exploited in the treatment of beta-thalassemia and showed promise in clinical application. Using a human beta-globin transgenic mouse line in a beta-thalassemia diseased model generated with a lentiviral-mediated approach, we investigate the stable therapeutic effect on a common thalassemia syndrome. Human beta-globin gene lentiviral vector was constr ucted, followed by subzonal microinjection into single-cell embryos of beta(IVS-2-654)-thalassemia mice to generate a transgenic line. Human beta-globin gene expression was examined with RT-PCR, Western-blotting and ELISA. The hematologic parameters and tissue pathology were investigated over time in founder mice and their off-spring. Transgenic mice with stable expression of the lentivirus carrying human beta-globin gene were obtained. A marked improvement in red blood cell indices and a dramatic reduction in red blood cell anisocytosis, poikilocytosis and target cells were observed. Nucleated cell proportion was greatly decreased in bone marrow, and splenomegaly with extramedullary hematopoiesis was ameliorated. Iron deposition in liver was also reduced. There was a two-fold increase in the survival rate of the beta(IVS-2-654) mice carrying human beta-globin transgene. Significantly, the germline integration of the lentiviral construct was obtained and stable hematologic phenotype correction was observed over the next two generations of the transgenic mice. The generation of human beta-globin transgenic mice in a beta(IVS-2-654)-thalassemia mouse mediated with lentiviral vectors provides a useful model and offers an attractive means to investigate the transgenic stable therapeutic effect in beta-thalassemia.

  2. beta-Glucuronidase is an optimal normalization control gene for molecular monitoring of chronic myelogenous leukemia.

    PubMed

    Lee, Joong Won; Chen, Qiaofang; Knowles, Daniel M; Cesarman, Ethel; Wang, Y Lynn

    2006-07-01

    Quantitative monitoring of breakpoint cluster region (BCR)-Abelson kinase (ABL) transcripts has become indispensable in the clinical care of patients with chronic myelogenous leukemia. Because quantity and quality of RNA in clinical samples are highly variable, a suitable internal normalization control is required for accurate BCR-ABL quantification. However, few studies have examined suitability of the control genes using criteria relevant to residual disease testing. In this study, we evaluated a number of control genes with the application of several novel criteria, including control gene performance on serial patient sample testing and in a residual disease model. We also examined expression of the control genes in BCR-ABL-positive K562 cells in response to Gleevec treatment. We found that beta-glucuronidase is the best control gene among those studied. Importantly, ABL, a widely used control gene, generates misleading BCR-ABL changes that potentially affect the clinical management of chronic myelogenous leukemia patients.

  3. Cloning and expression in Saccharomyces cerevisiae of a Trichoderma reesei beta-mannanase gene containing a cellulose binding domain.

    PubMed Central

    Stålbrand, H; Saloheimo, A; Vehmaanperä, J; Henrissat, B; Penttilä, M

    1995-01-01

    beta-Mannanase (endo-1,4-beta-mannanase; mannan endo-1,4-beta-mannosidase; EC 3.2.1.78) catalyzes endo-wise hydrolysis of the backbone of mannan and heteromannans, including hemicellulose polysaccharides, which are among the major components of plant cell walls. The gene man1, which encodes beta-mannanase, of the filamentous fungus Trichoderma reesei was isolated from an expression library by using antiserum raised towards the earlier-purified beta-mannanase protein. The deduced beta-mannanase consists of 410 amino acids. On the basis of hydrophobic cluster analysis, the beta-mannanase was assigned to family 5 of glycosyl hydrolases (cellulase family A). The C terminus of the beta-mannanase has strong amino acid sequence similarity to the cellulose binding domains of fungal cellulases and is preceded by a serine-, threonine-, and proline-rich region. Consequently, the beta-mannanase is probably organized similarly to the T. reesei cellulases, having a catalytic core domain separated from the substrate-binding domain by an O-glycosylated linker. Active beta-mannanase was expressed and secreted by using the yeast Saccharomyces cerevisiae as the host. The results indicate that the man1 gene encodes the two beta-mannanases with different isoelectric points (pIs 4.6 and 5.4) purified earlier from T. reesei. PMID:7793911

  4. Comparison of the canine and human acid {beta}-galactosidase gene

    SciTech Connect

    Ahern-Rindell, A.J.; Kretz, K.A.; O`Brien, J.S.

    1996-05-17

    Several canine cDNA libraries were screened with human {beta}-galactosidase cDNA as probe. Seven positive clones were isolated and sequenced yielding a partial (2060 bp) canine {beta}-galactosidase cDNA with 86% identity to the human {beta}-galactosidase cDNA. Preliminary analysis of a canine genomic library indicated conservation of exon number and size. Analysis by Northern blotting disclosed a single mRNA of 2.4 kb in fibroblasts and liver from normal dogs and dogs affected with GM1 gangliosidosis. Although incomplete, these results indicate canine GM1 gangliosidosis is a suitable animal model of the human disease and should further efforts to devise a gene therapy strategy for its treatment. 20 refs., 2 figs., 1 tab.

  5. Regulation of the beta-hydroxyacyl ACP dehydratase gene of Picea mariana by alternative splicing.

    PubMed

    Tai, Helen H; Williams, Martin; Iyengar, Abhinav; Yeates, Jessica; Beardmore, Tannis

    2007-01-01

    The gene for beta-hydroxyacyl ACP dehydratase, a de novo fatty acid biosynthetic enzyme, was cloned from Picea mariana (black spruce) and consists of five exons and four introns. The first intron of the beta-hydroxyacyl ACP dehydratase mRNA is alternatively spliced. Retention of intron 1 in splice variants results in truncation of the beta-hydroxyacyl ACP dehydratase ORF at a premature termination codon. In addition, splicing of intron 1 was found to be associated with cold temperature. mRNAs retaining intron 1 increase with seed imbibition at 22 degrees C but not 4 degrees C, whereas, splicing of intron 1 increases in winter weeks with temperatures below freezing. These results provide evidence that alternative splicing may also contribute to regulation of lipid biosynthesis in Picea mariana.

  6. Hemoglobins of reptiles. The primary structures of the alpha I- and beta I-chains of common iguana (Iguana iguana) hemoglobin.

    PubMed

    Rücknagel, K P; Braunitzer, G; Wiesner, H

    1988-10-01

    The primary structures of alpha I- and beta I-chains from the hemoglobins of the Common Iguana (Iguana iguana) are presented. The globin chains were separated on CM-cellulose in 8 M urea buffer. The amino-acid sequences were established by automatic Edman degradation of the native chains, the tryptic peptides and a peptide obtained by cyanogen bromide cleavage. The sequences are compared with human hemoglobin. Amino-acid replacements at positions critical for structure and function of the hemoglobin are discussed. The requirements for binding of ATP and also of DPG as allosteric effectors at the beta-chains seem to be fulfilled. Comparison of the alpha-chains with those of the Viper (Vipera aspis) shows 66 amino-acid substitutions. This number is in the same order of magnitude as the ones found by comparison with alpha-chains of crocodiles and mammals as well as with alpha A-chains of a turtle and birds. This result points towards a period of independent evolution of the reptile lines leading to the Common Iguana on one hand and to the Viper on the other. This time span is comparable to the one separating mammals from reptiles.

  7. The heavy chain of neonatal Fc receptor for IgG is sequestered in endoplasmic reticulum by forming oligomers in the absence of beta2-microglobulin association.

    PubMed Central

    Zhu, Xiaoping; Peng, Junmin; Raychowdhury, Raktima; Nakajima, Atsushi; Lencer, Wayne I; Blumberg, Richard S

    2002-01-01

    The heavy chain (HC) of the neonatal Fc receptor (FcRn) for IgG is non-convalently associated with beta(2)-microglobulin (beta(2)m). In beta(2)m(-/-) mice, FcRn functions are greatly impaired. We sought to determine how FcRn HC, particularly its structure and biogenesis, is affected by the absence of beta(2)m. Human FcRn HC, expressed from the beta(2)m-null cell line FO-1(FcRn), was present as a monomeric 45-kDa protein under reducing conditions but primarily as a 92-kDa oligomeric protein under non-reducing conditions. Two-dimensional electrophoresis and MS analysis showed that the 92-kDa protein was a dimer of the 45-kDa HC. Immunostaining showed that FcRn HC in FO-1(FcRn) was co-localized with the endoplasmic reticulum (ER) protein Bip/GRP78 but not with an endosome protein, EEA1. In contrast, FcRn HC in FO-1(FcRn+beta2m) was detected in both the ER and endosome. The dimeric HC in FcRn oligomers was free of beta(2)m association in FO-1(FcRn+beta2m). Mutation of non-paired cysteine residues at positions 48 and 251 within the human FcRn cDNA failed to eliminate the oligomers. The FcRn HC oligomers could be reduced by reconstitution of FO-1(FcRn) with beta(2)m or by balanced expression of FcRn HC with beta(2)m, or beta(2)m fused with a KDEL retention sequence. Similarly, the majority of FcRn HC isolated from neonatal beta(2)m(-/-) mice was in a dimeric form under non-reducing conditions. The amount of FcRn HC was significantly decreased in beta(2)m(-/-) mice and FO-1(FcRn). Furthermore, beta(2)m-free FcRn HC was sensitive to endoglycosidase digestion. These results indicate that FcRn HC alone can form disulphide-bonded oligomers in the ER, which may represent a misfolded protein. The beta(2)m association with FcRn HC is critical for correct folding of FcRn and exiting the ER for routing to endosomes and the cell surface. PMID:12162790

  8. Sequence and diversity of rabbit T-cell receptor gamma chain genes

    SciTech Connect

    Isono, T.; Kim, C.J.; Seto, A.

    1995-03-01

    The nucleotide sequences of one constant (C), six variable (V), and two joining (J) gene segments coding for the rabbit T-cell receptor gamma chain (Tcrg) were determined by directly sequencing fragments amplified by the cassette-ligation mediated polymerase chain reaction. The Tcrg-C gene segment did not encode a cysteine residue for connection to the Tcr delta chain in the connecting region, and two variant forms of the Tcrg-C gene segment were generated by alternative splicing, like the human Tcrg-C2 gene. Five of six rabbit Tcrg-V gene segments belonged to the same family and displayed similarity to five productive human Tcrg-V1 family genes as well as the mouse Tcrg-V5 gene. The remaining rabbit Tcrg-V gene segment displayed similarity to the human Tcrg-V3 gene. Both rabbit Tcrg-J gene segments displayed similarity to the human Tcrg-J2.1 and 2.3, respectively. These findings suggested that the genomic organization of rabbit Tcrg genes is more similar to that of human than of mouse Tcrg genes. 18 refs., 4 figs., 1 tab.

  9. Two mutations in the transforming growth factor beta-induced gene associated with familial Lattice corneal dystrophy

    PubMed Central

    Cao, Wen-Ping; Yuan, Hai-Gang; Liu, Ping; Li, Xue; Hu, Qi

    2017-01-01

    AIM To report a phenotypic variant pedigree of lattice corneal dystrophy (LCD) associated with two mutations, R124C and A546D, in the transforming growth factor beta-induced gene (TGFBI). METHODS A detailed ocular examination was taken for all participants of a LCD family. Peripheral blood leukocytes from each participant were extracted to obtain the DNA. Polymerase chain reaction (PCR) of all seventeen exons of TGFBI gene was performed. The products were sequenced and analyzed. Histological examination was carried out after a penetrating keratoplasty from the right eye of proband. RESULTS Genetic analysis showed that the proband and all 6 affected individuals harbored both a heterozygous CGC to TGC mutation at codon 124 and a heterozygous GCC to GAC mutation at codon 546 of TGFBI. None of the 100 control subjects and unaffected family members was positive for these two mutations. Ocular examination displayed multiple refractile lattice-like opacities in anterior stroma of the central cornea and small granular deposits in the peripheral cornea. The deposits were stained positively with Congo red indicating be amyloid in nature and situated mainly in the anterior and middle stroma. CONCLUSION We observed a novel LCD family which carried two pathogenic mutations (R124C and A546D) in the TGFBI gene. The phenotypic features were apparently different from those associated with corresponding single mutations. The result reveals that although the definite mutation is the most important genetic cause of the disease, some different modifier alleles may influence the phenotype.

  10. MHC evolution in three salmonid species: a comparison between class II alpha and beta genes.

    PubMed

    Gómez, Daniela; Conejeros, Pablo; Marshall, Sergio H; Consuegra, Sofia

    2010-08-01

    The genes of the major histocompatibility complex (MHC) are amongst the most variable in vertebrates and represent some of the best candidates to study processes of adaptive evolution. However, despite the number of studies available, most of the information on the structure and function of these genes come from studies in mammals and birds in which the MHC class I and II genes are tightly linked and class II alpha exhibits low variability in many cases. Teleost fishes are among the most primitive vertebrates with MHC and represent good organisms for the study of MHC evolution because their class I and class II loci are not physically linked, allowing for independent evolution of both classes of genes. We have compared the diversity and molecular mechanisms of evolution of classical MH class II alpha and class II beta loci in farm populations of three salmonid species: Oncorhynchus kisutch, Oncorhynchus mykiss and Salmo salar. We found single classical class II loci and high polymorphism at both class II alpha and beta genes in the three species. Mechanisms of evolution were common for both class II genes, with recombination and point mutation involved in generating diversity and positive selection acting on the peptide-binding residues. These results suggest that the maintenance of variability at the class IIalpha gene could be a mechanism to increase diversity in the MHC class II in salmonids in order to compensate for the expression of one single classical locus and to respond to a wider array of parasites.

  11. Nested polymerase chain reaction amplification and sequencing analysis of the light-chain and heavy-chain variable regions in the influenza A H1N1 virus hemagglutinin monoclonal antibody gene.

    PubMed

    Li, H J; Guo, C Y; Sun, J Y; Sun, L J; Zhao, P H; Hu, L; Li, Y; Hu, J

    2014-06-11

    The nested polymerase chain reaction (PCR) method was used for the amplification of the influenza A H1N1 virus hemagglutinin monoclonal antibody light-chain and heavy-chain genes. Sequence analysis of the obtained genes was then used to identify common cloning methods of the mouse immunoglobulin-kappa (Igκ) light-chain and heavy-chain variable gene regions. Twenty-two pairs of amplification primers for the mouse Igκ light-chain and heavy-chain variable gene regions were designed, and 6 mouse anti-human H1N1 influenza virus hemagglutinin monoclonal antibody light-chain and heavy-chain variable gene regions were cloned and sequenced. Comparative analysis was conducted between our results and the mouse Ig sequences published in the National Center of Biotechnology Information (NCBI). The nested PCR method effectively avoided cloning the pseudogenes of the monoclonal antibody, and the amino acid sequence obtained was consistent with the characteristics of the mouse Ig variable region. A general method of cloning the mouse Ig light-chain and heavy-chain variable gene regions was established, which provides a basis for further cloning of mouse monoclonal antibody variable gene regions. This study also provides data for further studies of H1N1 influenza virus hemagglutinin antibody binding sites.

  12. A rapid single-tube multiplex polymerase chain reaction assay for the seven most prevalent alpha-thalassemia deletions and alphaalphaalpha(anti 3.7) alpha-globin gene triplication.

    PubMed

    de Mare, Arjan; Groeneger, Antoinette Heijs-Oude; Schuurman, Sander; van den Bergh, Frank A T J M; Slomp, Jennichjen

    2010-01-01

    alpha-Globin gene triplications may exacerbate the alpha chain and beta chain imbalance in beta-thalassemia (beta-thal) and may compensate for the effect of alpha-globin gene deletion in alpha-thal. Identification of an alpha-globin gene triplication is, therefore, valuable in predicting the clinical phenotype of the thalassemias. To be able to detect alpha-globin gene triplications, we have modified an existing multiplex polymerase chain reaction (PCR) assay for the seven most prevalent alpha-globin gene deletions by incorporating two triplication-specific primers and concurrently substituting one of the original primers by a newly designed primer. This modified multiplex PCR assay was evaluated by performing the assay on archival DNA samples and on peripheral blood samples from 163 suspected thalassemia cases. It was found to function properly. Our assay thereby represents the first multiplex PCR assay that can detect both the seven most prevalent alpha-globin gene deletions and the alphaalphaalpha(anti 3.7) alpha-globin gene triplication in a single-tube reaction.

  13. Expression of Caveolin-1 reduces cellular responses to TGF-{beta}1 through down-regulating the expression of TGF-{beta} type II receptor gene in NIH3T3 fibroblast cells

    SciTech Connect

    Lee, Eun Kyung; Lee, Youn Sook; Han, In-Oc; Park, Seok Hee . E-mail: parks@skku.edu

    2007-07-27

    Transcriptional repression of Transforming Growth Factor-{beta} type II receptor (T{beta}RII) gene has been proposed to be one of the major mechanisms leading to TGF-{beta} resistance. In this study, we demonstrate that expression of Caveolin-1 (Cav-1) gene in NIH3T3 fibroblast cells down-regulates the expression of T{beta}RII gene in the transcriptional level, eventually resulting in the decreased responses to TGF-{beta}. The reduced expression of T{beta}RII gene by Cav-1 appeared to be due to the changes of the sequence-specific DNA binding proteins to either Positive Regulatory Element 1 (PRE1) or PRE2 of the T{beta}RII promoter. In addition, Cav-1 expression inhibited TGF-{beta}-mediated cellular proliferation and Plasminogen Activator Inhibitor (PAI)-1 gene expression as well as TGF-{beta}-induced luciferase activity. Furthermore, the inhibition of endogeneous Cav-1 by small interfering RNA increased the expression of T{beta}RII gene. These findings strongly suggest that expression of Cav-1 leads to the decreased cellular responsiveness to TGF-{beta} through down-regulating T{beta}RII gene expression.

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

    NASA Technical Reports Server (NTRS)

    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.

  15. A pseudodeficiency allele (D152N) of the human beta-glucuronidase gene.

    PubMed

    Vervoort, R; Islam, M R; Sly, W; Chabas, A; Wevers, R; de Jong, J; Liebaers, I; Lissens, W

    1995-10-01

    We present evidence that a 480G-->A transition in the coding region of the beta-glucuronidase gene, which results in an aspartic-acid-to-asparagine substitution at amino acid position 152 (D152N), produces a pseudodeficiency allele (GUSBp) that leads to greatly reduced levels of beta-glucuronidase activity without apparent deleterious consequences. The 480G-->A mutation was found initially in the pseudodeficient mother of a child with mucopolysaccharidosis VII (MPSVII), but it was not on her disease-causing allele, which carried the L176F mutation. The 480G-->A change was also present in an unrelated individual with another MPSVII allele who had unusually low beta-glucuronidase activity, but whose clinical symptoms were probably unrelated to beta-glucuronidase deficiency. This individual also had an R357X mutation, probably on his second allele. We screened 100 unrelated normal individuals for the 480G-->A mutation with a PCR method and detected one carrier. Reduced beta-glucuronidase activity following transfection of COS cells with the D152N cDNA supported the causal relationship between the D152N allele and pseudodeficiency. The mutation reduced the fraction of expressed enzyme that was secreted. Pulse-chase experiments indicated that the reduced activity in COS cells was due to accelerated intracellular turnover of the D152N enzyme. They also suggested that a potential glycosylation site created by the mutation is utilized in approximately 50% of the enzyme expressed.

  16. Localisation of Neuregulin 1-{beta}3 to different sub-nuclear structures alters gene expression

    SciTech Connect

    Wang, Ming; Trim, Carol M.; Gullick, William J.

    2011-02-15

    Neuregulins are growth factors that signal via the ErbB3 and ErbB4 receptors. Here we show using immunohistochemistry that they are often expressed in the nucleus of a range of tumour types including soft tissue and breast. The Neuregulin 1 type I-{beta}3 (NRG1-{beta}3) isoform localises to two sub-nuclear compartments in animal cells, nucleoli and spliceosomes. We used NRG1-{beta}3 tagged with photoactivatable GFP and demonstrated that this re-localised from nucleoli to spliceosomes over 90 min. Tyrosine kinase activity was not required for retaining the NRG1-{beta}3 within the nucleus. Mutation of the lysines 14 and 16 or 15 and 16 together prevented nucleolar uptake while four positively charged residues were identified which were required for spliceosome uptake. Molecular modelling suggests that three of these may form a binding site. We showed using a kinome array that NRG1-{beta}3 and a mutant exclusively localising to spliceosomes increased phosphorylation and/or expression of the HER4 and HER2 receptors. Using a transcriptomic analysis the same two constructs induced expression of several messenger RNAs and we confirmed the increased expression at the protein level of the most highly induced, Heat Shock Protein 70B'. These results suggest that Neuregulin activates receptor signalling in spliceosomes leading to altered gene expression.

  17. Behavioral responses to odorants in drosophila require nervous system expression of the beta integrin gene myospheroid.

    PubMed

    Bhandari, Poonam; Gargano, Julia Warner; Goddeeris, Matthew M; Grotewiel, Michael S

    2006-09-01

    Integrins are cell adhesion molecules that mediate numerous developmental processes in addition to a variety of acute physiological events. Two reports implicate a Drosophila beta integrin, betaPS, in olfactory behavior. To further investigate the role of integrins in Drosophila olfaction, we used Gal4-driven expression of RNA interference (RNAi) transgenes to knock down expression of myospheroid (mys), the gene that encodes betaPS. Expression of mys-RNAi transgenes in the wing reduced betaPS immunostaining and produced morphological defects associated with loss-of-function mutations in mys, demonstrating that this strategy knocked down mys function. Expression of mys-RNAi transgenes in the antennae, antennal lobes, and mushroom bodies via two Gal4 lines, H24 and MT14, disrupted olfactory behavior but did not alter locomotor abilities or central nervous system structure. Olfactory behavior was normal in flies that expressed mys-RNAi transgenes via other Gal4 lines that specifically targeted the antennae, the projection neurons, the mushroom bodies, bitter and sweet gustatory neurons, or Pox neuro neurons. Our studies confirm that mys is important for the development or function of the Drosophila olfactory system. Additionally, our studies demonstrate that mys is required for normal behavioral responses to both aversive and attractive odorants. Our results are consistent with a model in which betaPS mediates events within the antennal lobes that influence odorant sensitivity.

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

    PubMed

    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. B cell development in mice that lack one or both immunoglobulin kappa light chain genes.

    PubMed Central

    Chen, J; Trounstine, M; Kurahara, C; Young, F; Kuo, C C; Xu, Y; Loring, J F; Alt, F W; Huszar, D

    1993-01-01

    We have generated mice that lack the ability to produce immunoglobulin (Ig) kappa light chains by targeted deletion of J kappa and C kappa gene segments and the intervening sequences in mouse embryonic stem cells. In wild type mice, approximately 95% of B cells express kappa light chains and only approximately 5% express lambda light chains. Mice heterozygous for the J kappa C kappa deletion have approximately 2-fold more lambda+ B cells than wild-type littermates. Compared with normal mice, homozygous mutants for the J kappa C kappa deletion have about half the number of B cells in both the newly generated and the peripheral B cell compartments, and all of these B cells express lambda light chains in their Ig. Therefore, homozygous mutant mice appear to produce lambda-expressing cells at nearly 10 times the rate observed in normal mice. These findings demonstrate that kappa gene assembly and/or expression is not a prerequisite for lambda gene assembly and expression. Furthermore, there is no detectable rearrangement of 3' kappa RS sequences in lambda+ B cells of the homozygous mutant mice, thus rearrangements of these sequences, per se, is not required for lambda light chain gene assembly. We discuss these findings in the context of their implications for the control of Ig light chain gene rearrangement and potential applications of the mutant animals. Images PMID:8458340

  20. Individual effects of the DR11-variable beta-chain residues 67, 71, and 86 upon DR(alpha,beta 1*1101)-restricted, peptide-specific T cell proliferation.

    PubMed

    McKinney, J S; Fu, X T; Swearingen, C; Klohe, E; Karr, R W

    1994-12-15

    The four members of the HLA-DR11 family of class II molecules vary only by three or fewer amino acids via dimorphisms among DR beta-chain residues 67, 71, and 86. However, they differ markedly in their abilities to induce proliferation of DR(alpha,beta 1*1101)-restricted, peptide-specific T cell clones. To dissect which DR11-variable residues, individually and in combination, mediate these functional differences, we used as APC transfectants expressing DR molecules with one of all possible permutations of DR11-variable sequences, including the four DR11 family members, and four additional DR11 variant mutants. The abilities of the wild-type or mutant molecules to present two distinct influenza peptide Ags, HA307-19 and HA128-45, to T cells was assessed in in vitro T cell proliferation assays. Of the naturally dimorphic DR11 positions, residue beta 71 variation significantly influenced the ability of DR11 molecules to present both peptides to DR(alpha,beta 1*1101)-restricted T cells. Residue beta 86 variation had relatively less influence than reported in several other DR and peptide systems. Residue beta 67 variation usually appeared irrelevant to T cell proliferation, but in two mutants led to unexpected T cell proliferation independent of nominal peptide Ag. Peptide binding, assessed by flow cytometry, was not found to be altered by any mutations that disrupted DR(alpha,beta 1*1101)-like presentation. These data indicate that residue beta 71 exerts a central role in influencing the functional differences among DR11 molecules, whereas the widely studied dimorphism of residue beta 86 is not as generally influential in DR11 as in other alleles.

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

    PubMed

    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.

  2. Insertion/deletion-related polymorphisms in the human T cell receptor beta gene complex

    PubMed Central

    1989-01-01

    Insertion/deletion related polymorphisms (IDRP) involving stretches of 15-30 kb within the human TCR-beta gene complex were revealed by pulse- field gel electrophoresis. Two independent IDRP systems were detected by analysis of Sfi I- and Sal I-digested human DNA samples using probes for TCR C and V region gene segments. The allelic nature of these systems was verified in family studies, and mapping data allowed localization of one area of insertion/deletion among the V gene segments and the other near the C region genes. All but one of 50 individuals tested could be typed for the two allelic systems, and gene frequencies for the two allelic forms were 0.37/0.61 and 0.46/0.54, indicating that these polymorphisms are widespread. PMID:2571667

  3. Crop wild relative populations of Beta vulgaris allow direct mapping of agronomically important genes.

    PubMed

    Capistrano-Gossmann, Gina G; Ries, D; Holtgräwe, D; Minoche, A; Kraft, T; Frerichmann, S L M; Rosleff Soerensen, T; Dohm, J C; González, I; Schilhabel, M; Varrelmann, M; Tschoep, H; Uphoff, H; Schütze, K; Borchardt, D; Toerjek, O; Mechelke, W; Lein, J C; Schechert, A W; Frese, L; Himmelbauer, H; Weisshaar, B; Kopisch-Obuch, F J

    2017-06-06

    Rapid identification of agronomically important genes is of pivotal interest for crop breeding. One source of such genes are crop wild relative (CWR) populations. Here we used a CWR population of <200 wild beets (B. vulgaris ssp. maritima), sampled in their natural habitat, to identify the sugar beet (Beta vulgaris ssp. vulgaris) resistance gene Rz2 with a modified version of mapping-by-sequencing (MBS). For that, we generated a draft genome sequence of the wild beet. Our results show the importance of preserving CWR in situ and demonstrate the great potential of CWR for rapid discovery of causal genes relevant for crop improvement. The candidate gene for Rz2 was identified by MBS and subsequently corroborated via RNA interference (RNAi). Rz2 encodes a CC-NB-LRR protein. Access to the DNA sequence of Rz2 opens the path to improvement of resistance towards rhizomania not only by marker-assisted breeding but also by genome editing.

  4. Crop wild relative populations of Beta vulgaris allow direct mapping of agronomically important genes

    PubMed Central

    Capistrano-Gossmann, Gina G.; Ries, D.; Holtgräwe, D.; Minoche, A.; Kraft, T.; Frerichmann, S.L.M.; Rosleff Soerensen, T.; Dohm, J. C.; González, I.; Schilhabel, M.; Varrelmann, M.; Tschoep, H.; Uphoff, H.; Schütze, K.; Borchardt, D.; Toerjek, O.; Mechelke, W.; Lein, J. C.; Schechert, A. W.; Frese, L.; Himmelbauer, H.; Weisshaar, B.; Kopisch-Obuch, F. J.

    2017-01-01

    Rapid identification of agronomically important genes is of pivotal interest for crop breeding. One source of such genes are crop wild relative (CWR) populations. Here we used a CWR population of <200 wild beets (B. vulgaris ssp. maritima), sampled in their natural habitat, to identify the sugar beet (Beta vulgaris ssp. vulgaris) resistance gene Rz2 with a modified version of mapping-by-sequencing (MBS). For that, we generated a draft genome sequence of the wild beet. Our results show the importance of preserving CWR in situ and demonstrate the great potential of CWR for rapid discovery of causal genes relevant for crop improvement. The candidate gene for Rz2 was identified by MBS and subsequently corroborated via RNA interference (RNAi). Rz2 encodes a CC-NB-LRR protein. Access to the DNA sequence of Rz2 opens the path to improvement of resistance towards rhizomania not only by marker-assisted breeding but also by genome editing. PMID:28585529

  5. A novel fibrinogen B beta chain frameshift mutation causes congenital afibrinogenaemia.

    PubMed

    Zhang, Jian; Zhao, Xiaojuan; Wang, Zhaoyue; Yu, Ziqiang; Cao, Lijuan; Zhang, Wei; Bai, Xia; Ruan, Changgeng

    2013-07-01

    Congenital afibrinogenaemia is a rare autosomal recessive disorder caused by various mutations within the fibrinogen genes FGA, FGB and FGG. Ins/del mutations in FGB are extremely rare. We report a patient with afibrinogenaemia who suffered from umbilical cord bleeding and repeated bleeding episodes. His plasma fibrinogen levels could not be detected using the Clauss method and immunological methods. Molecular analyses revealed homozygosity in a novel four bases insertion in codon 40 of FGB exon 2 (g. 2833_2834 ins GTTT), which resulted in a truncated 50-residue polypeptide that contained 11 exceptional abnormal residues. In the transient expression experiments, mutant fibrinogen could be detected at higher level than wild-type fibrinogen in COS-7 cell lysates but not in culture media. These results suggest that the homozygous mutation in FGB could be responsible for congenital afibrinogenaemia in this patient. This frameshift mutation could impair fibrinogen assembly and secretion without influencing the protein synthesis.

  6. Screening for gap junction protein beta-2 gene mutations in Malays with autosomal recessive, non-syndromic hearing loss, using denaturing high performance liquid chromatography.

    PubMed

    Aishah, Z Siti; Khairi, M D Mohd; Normastura, A R; Zafarina, Z; Zilfalil, B A

    2008-12-01

    To determine the frequency and type of gap junction protein beta-2 gene mutations in Malay patients with autosomal recessive, non-syndromic hearing loss. A total of 33 Malay patients with autosomal recessive, non-syndromic hearing loss were screened for mutations in the Cx26 coding region. Deoxyribonucleic acid was extracted from buccal swab samples and subjected to polymerase chain reaction. Slow-reannealing was performed, followed by screening using denaturing high performance liquid chromatography. Eight of the samples (24.2 per cent) showed heterozygous peaks, and further sequencing of these samples revealed four patients (50.0 per cent) with the W24X mutation, two (25.0 per cent) with the V37I mutation and another two (25.0 per cent) with the G4D mutation. Analysis of buccal swab samples by denaturing high performance liquid chromatography is noninvasive and suitable for rapid and reliable screening of gap junction protein beta-2 gene mutations in patients with autosomal recessive, non-syndromic hearing loss. Malay patients with autosomal recessive, non-syndromic hearing loss have different kinds of gap junction protein beta-2 gene mutations which are rarely found in other populations.

  7. Allelic inclusion in a pre-B-cell line that generates immunoglobulin heavy chain genes in vitro.

    PubMed Central

    Beck-Engeser, G; Jäck, H M; Wabl, M

    1987-01-01

    In a pre-B-cell line that rearranges its heavy chain gene segments in vitro, we found that the rate of productive rearrangement on one allele was not influenced by the presence of heavy chain protein encoded by the other allele. This shows that allelic exclusion of heavy chain genes is not regulated at the genetic level. Images PMID:3103122

  8. A single-base change at a splice site in a beta 0-thalassemic gene causes abnormal RNA splicing.

    PubMed

    Treisman, R; Proudfoot, N J; Shander, M; Maniatis, T

    1982-07-01

    We have studied the structure and transcription of a cloned human beta-globin gene from a fetus diagnosed for beta 0 thalassemia. The sequence of the beta 0 gene differs from that of a normal beta-globin gene at positions 1 and 74 of the second intervening sequence (IVS2). The position 1 change alters the GT dinucleotide conserved at 5' splice sites, while the position 74 change is a common sequence polymorphism. When the cloned beta 0 gene is introduced into HeLa cells by use of an SV40-derived plasmid vector, two abnormally spliced cytoplasmic beta-globin RNAs are detected. The predominant RNA differs from normal beta-globin mRNA by the insertion of the first 47 nucleotides of IVS2 between exons 2 and 3. The less abundant RNA comprises the normal first exon spliced directly to the third. Analysis of nuclear RNA suggests that the beta 0 transcript is inefficiently spliced and that the removal of the two intervening sequences is coupled.

  9. A self-excising beta-recombinase/six cassette for repetitive gene deletion and homokaryon purification in Neurospora crassa

    USDA-ARS?s Scientific Manuscript database

    In a previous study we developed a cassette employing a bacterial beta-recombinase acting on six recognition sequences (beta-rec/six), which allowed repetitive site-specific gene deletion and marker recycling in Neurospora crassa. However, only one positive selection marker was used in the cassette...

  10. The crystal structure of progesterone 5beta-reductase from Digitalis lanata defines a novel class of short chain dehydrogenases/reductases.

    PubMed

    Thorn, Andrea; Egerer-Sieber, Claudia; Jäger, Christof M; Herl, Vanessa; Müller-Uri, Frieder; Kreis, Wolfgang; Muller, Yves A

    2008-06-20

    Progesterone 5beta-reductase (5beta-POR) catalyzes the stereospecific reduction of progesterone to 5beta-pregnane-3,20-dione and is a key enzyme in the biosynthetic pathway of cardenolides in Digitalis (foxglove) plants. Sequence considerations suggested that 5beta-POR is a member of the short chain dehydrogenase/reductase (SDR) family of proteins but at the same time revealed that the sequence motifs that in standard SDRs contain the catalytically important residues are missing. Here we present crystal structures of 5beta-POR from Digitalis lanata in complex with NADP(+) at 2.3A and without cofactor bound at 2.4A resolution together with a model of a ternary complex consisting of 5beta-POR, NADP(+), and progesterone. Indeed, 5beta-POR displays the fold of an extended SDR. The architecture of the active site is, however, unprecedented because none of the standard catalytic residues are structurally conserved. A tyrosine (Tyr-179) and a lysine residue (Lys-147) are present in the active site, but they are displayed from novel positions and are part of novel sequence motifs. Mutating Tyr-179 to either alanine or phenylalanine completely abolishes the enzymatic activity. We propose that the distinct topology reflects the fact that 5beta-POR reduces a conjugated double bond in a steroid substrate via a 1-4 addition mechanism and that this requires a repositioning of the catalytically important residues. Our observation that the sequence motifs that line the active site are conserved in a number of bacterial and plant enzymes of yet unknown function leads us to the proposition that 5beta-POR defines a novel class of SDRs.

  11. Harnessing the theoretical foundations of the exponential and beta-Poisson dose-response models to quantify parameter uncertainty using Markov Chain Monte Carlo.

    PubMed

    Schmidt, Philip J; Pintar, Katarina D M; Fazil, Aamir M; Topp, Edward

    2013-09-01

    Dose-response models are the essential link between exposure assessment and computed risk values in quantitative microbial risk assessment, yet the uncertainty that is inherent to computed risks because the dose-response model parameters are estimated using limited epidemiological data is rarely quantified. Second-order risk characterization approaches incorporating uncertainty in dose-response model parameters can provide more complete information to decisionmakers by separating variability and uncertainty to quantify the uncertainty in computed risks. Therefore, the objective of this work is to develop procedures to sample from posterior distributions describing uncertainty in the parameters of exponential and beta-Poisson dose-response models using Bayes's theorem and Markov Chain Monte Carlo (in OpenBUGS). The theoretical origins of the beta-Poisson dose-response model are used to identify a decomposed version of the model that enables Bayesian analysis without the need to evaluate Kummer confluent hypergeometric functions. Herein, it is also established that the beta distribution in the beta-Poisson dose-response model cannot address variation among individual pathogens, criteria to validate use of the conventional approximation to the beta-Poisson model are proposed, and simple algorithms to evaluate actual beta-Poisson probabilities of infection are investigated. The developed MCMC procedures are applied to analysis of a case study data set, and it is demonstrated that an important region of the posterior distribution of the beta-Poisson dose-response model parameters is attributable to the absence of low-dose data. This region includes beta-Poisson models for which the conventional approximation is especially invalid and in which many beta distributions have an extreme shape with questionable plausibility.

  12. Interaction between two discontiguous chain segments from the beta-sheet of Escherichia coli thioredoxin suggests an initiation site for folding.

    PubMed

    Tasayco, M L; Fuchs, J; Yang, X M; Dyalram, D; Georgescu, R E

    2000-09-05

    The approach of comparing folding and folding/binding processes is exquisitely poised to narrow down the regions of the sequence that drive protein folding. We have dissected the small single alpha/beta domain of oxidized Escherichia coli thioredoxin (Trx) into three complementary fragments (N, residues 1-37; M, residues 38-73; and C, residues 74-108) to study them in isolation and upon recombination by far-UV CD and NMR spectroscopy. The isolated fragments show a minimum of ellipticity of ca. 197 nm in their far-UV CD spectra without concentration dependence, chemical shifts of H(alpha) that are close to the random coil values, and no medium- and long-range NOE connectivities in their three-dimensional NMR spectra. These fragments behave as disordered monomers. Only the far-UV CD spectra of binary or ternary mixtures that contain N- and C-fragments are different from the sum of their individual spectra, which is indicative of folding and/or binding of these fragments. Indeed, the cross-peaks corresponding to the rather hydrophobic beta(2) and beta(4) regions of the beta-sheet of Trx disappear from the (1)H-(15)N HSQC spectra of isolated labeled N- and C-fragments, respectively, upon addition of the unlabeled complementary fragments. The disappearing cross-peaks indicate interactions between the beta(2) and beta(4) regions, and their reappearance at lower temperatures indicates unfolding and/or dissociation of heteromers that are predominantly held by hydrophobic forces. Our results argue that the folding of Trx begins by zippering two discontiguous and rather hydrophobic chain segments (beta(2) and beta(4)) corresponding to neighboring strands of the native beta-sheet.

  13. Low autocrine interferon beta production as a gene therapy approach for AIDS: Infusion of interferon beta-engineered lymphocytes in macaques chronically infected with SIVmac251.

    PubMed

    Gay, Wilfried; Lauret, Evelyne; Boson, Bertrand; Larghero, Jérome; Matheux, Franck; Peyramaure, Sophie; Rousseau, Véronique; Dormont, Dominique; De Maeyer, Edward; Le Grand, Roger

    2004-09-25

    The aim of this study was to evaluate gene therapy for AIDS based on the transduction of circulating lymphocytes with a retroviral vector giving low levels of constitutive macaque interferon beta production in macaques chronically infected with a pathogenic isolate of SIVmac251. Two groups of three animals infected for more than one year with a pathogenic primary isolate of SIVmac251 were included in this study. The macaques received three infusions of their own lymphocytes transduced ex vivo with the construct encoding macaque IFN-beta (MaIFN-beta or with a vector carrying a version of the MaIFN-beta gene with a deletion preventing translation of the mRNA. Cellular or plasma viremia increased transiently following injection in most cases, regardless of the retroviral construct used. Transduced cells were detected only transiently after each infusion, among the peripheral blood mononuclear cells of all the animals, with copy numbers of 10 to 1000 per 106 peripheral mononuclear cells. Long-term follow-up indicated that the transitory presence of such a small number of cells producing such small amounts of MaIFN-beta did not prevent animals from the progressive decrease in CD4+ cell count typical of infection with simian immunodeficiency virus. These results reveal potential pitfalls for future developments of gene therapy strategies of HIV infection.

  14. Molecular analysis of the beta-glucuronidase gene: novel mutations in mucopolysaccharidosis type VII and heterogeneity of the polyadenylation region.

    PubMed

    Vervoort, R; Buist, N R; Kleijer, W J; Wevers, R; Fryns, J P; Liebaers, I; Lissens, W

    1997-04-01

    We used polymerase chain reaction (PCR)/single-strand conformation polymorphism analysis and direct sequencing of the coding region of the beta-glucuronidase cDNA and gene to detect mutations causing beta-glucuronidase enzyme deficiency in five MPS VII patients. Four patients presented with hydrops fetalis, one with an early infantile form of the disease. Genetic heterogeneity of MPS VII alleles was further confirmed in this study. Recurrent mutations were observed in patients of related origin. Previously unknown alleles detected were RII0X, F361delta9, 1270 + 1G-->A, S52F and 1480delta4. Reverse transcription/PCR analysis of the 1270 + 1G-->A messenger showed aberrant splicing: inclusion of intron 7 or skipping of exons 6-7 and 9. Messenger RNA transcribed from the R110X and 1480delta4 alleles was unstable. We detected a 2154A/G change in the 3' non-coding region of the gene, in the neighbourhood of the two consensus polyadenylation sites. 3'-Rapid amplification of cDNA ends/PCR of fibroblast cDNA revealed equal usage of two alternative polyadenylation sites. The 2154A/G substitution did not influence adenylation-site choice, nor the amount of stable messenger produced. The finding that 2 out of 30 normal controls carried the 2154G allele indicated that the 2154A/G substitution is a harmless polymorphism. The S52F and F361delta9 cDNAs were constructed in vitro and used to transfect COS cells transiently. Both mutations completely abolished enzyme activity.

  15. Beta-strength and anti-neutrino spectra from total absorption spectroscopy of a decay chain 142Cs→142Ba→142La

    NASA Astrophysics Data System (ADS)

    Wolińska-Cichocka, Marzena; Rasco, B. Charles; Rykaczewski, Krzysztof P.; Brewer, Nathan T.; Stracener, Daniel; Grzywacz, Robert; Gross, Carl J.; kowska, Aleksandra Fijał; Goetz, K. Callie; Karny, Marek; King, Toby; Go, Shintaro; McCutchan, Elizabeth A.; Nesaraja, Caroline; Sonzogni, Alejandro A.; Wang, Enhong; Winger, Jeff A.; Xiao, Yongchi; Zachary, Christopher J.; Zganjar, Edward F.

    2017-09-01

    Beta decays of mass A = 142 isobaric chain starting from 142Cs have been investigated by means of Modular Total Absorption Spectrometer (MTAS) and on-line mass separation at Oak Ridge National Laboratory. The beta strength distribution derived for 142Cs decay from MTAS spectra is showing significant differences in β-feeding pattern when compared to the values listed at nuclear databases. MTAS results are shifting the associated anti-neutrino energy spectrum towards lower energies. A decay pattern deduced for 142Ba is similar to earlier reported results.

  16. Increased gene dosage plays a predominant role in the initial stages of evolution of duplicate TEM-1 beta lactamase genes.

    PubMed

    Dhar, Riddhiman; Bergmiller, Tobias; Wagner, Andreas

    2014-06-01

    Gene duplication is important in evolution, because it provides new raw material for evolutionary adaptations. Several existing hypotheses about the causes of duplicate retention and diversification differ in their emphasis on gene dosage, subfunctionalization, and neofunctionalization. Little experimental data exist on the relative importance of gene expression changes and changes in coding regions for the evolution of duplicate genes. Furthermore, we do not know how strongly the environment could affect this importance. To address these questions, we performed evolution experiments with the TEM-1 beta lactamase gene in Escherichia coli to study the initial stages of duplicate gene evolution in the laboratory. We mimicked tandem duplication by inserting two copies of the TEM-1 gene on the same plasmid. We then subjected these copies to repeated cycles of mutagenesis and selection in various environments that contained antibiotics in different combinations and concentrations. Our experiments showed that gene dosage is the most important factor in the initial stages of duplicate gene evolution, and overshadows the importance of point mutations in the coding region. © 2014 The Author(s). Evolution © 2014 The Society for the Study of Evolution.

  17. Beta-myosin heavy-chain mutations R403QLW, V606M, K615N and R663H in patients with hypertrophic cardiomyopathy.

    PubMed

    Atay, Sevcan; Tetik, Aslı; Bozok Çetintaş, Vildan; Yakar Tülüce, Selcen; Tülüce, Kamil; Kayıkçıoğlu, Meral; Eroğlu, Zuhal

    2014-05-01

    Hypertrophic cardiomyopathy (HCM) is a disease of the myocardium with an autosomal-dominant pattern of inheritance mainly caused by single heterozygous mutations in sarcomere genes. In this study we aimed to detect the presence of R403QLW, V606M, K615N, and R663H mutations in beta-myosin heavy-chain gene (MYH7) and figure out the genotype-phenotype correlations in Turkish patients with HCM. This case-control study based on genotype-phenotype correlation included 69 patients (mean age, years: 50±13.16) diagnosed with HCM constituting the study group and 50 healthy individuals (mean age, years: 52±1.4) constituting the control group. DNA was extracted from peripheral blood and the genotyping of mutations was performed by real-time PCR technique and high resolution melting analysis. Associations between categoric variables were determined using chi-square tests. Differences between two groups were compared with unpaired Student's t-test for continuous variables. None of the patients in the HCM group were carrying the index mutations. One healthy individual was found to be heterozygous for the R663H mutation with mildly abnormal IVS and LVPW thickness. The allele frequency for R663H (G>A) mutation was found to be 0.01% in control group. We performed a mutational screening of 6 HCM-associated mutations in 69 Turkish HCM patients (not previously studied except R403Q). There was no significant difference in the prevalence of the mutations between the patients with HCM and the healthy controls (p>0.05).

  18. Activation of the beta myosin heavy chain promoter by MEF-2D, MyoD, p300, and the calcineurin/NFATc1 pathway.

    PubMed

    Meissner, Joachim D; Umeda, Patrick K; Chang, Kin-Chow; Gros, Gerolf; Scheibe, Renate J

    2007-04-01

    Calcium is a key element in intracellular signaling in skeletal muscle. Changes in intracellular calcium levels are thought to mediate the fast-to-slow transformation of muscle fiber type. One factor implicated in gene regulation in adult muscle is the nuclear factor of activated T-cells (NFAT) isoform c1, whose dephosphorylation by the calcium/calmodulin-dependent phosphatase calcineurin facilitates its nuclear translocation. Here, we report that differentiated C2C12 myotubes predominantly expressing fast-type MyHCII protein undergo fast-to-slow transformation following calcium-ionophore treatment, with several transcription factors and a transcriptional coactivator acting in concert to upregulate the slow myosin heavy chain (MyHC) beta promoter. Transient transfection assays demonstrated that the calcineurin/NFATc1 signaling pathway is essential for MyHCbeta promoter activation during transformation of C2C12 myotubes but is not sufficient for complete fast MyHCIId/x promoter inhibition. Along with NFATc1, myocyte enhancer factor-2D (MEF-2D) and the myogenic transcription factor MyoD transactivated the MyHCbeta promoter in calcium-ionophore-treated myotubes in a calcineurin-dependent manner. To elucidate the mechanism involved in regulating MyHCbeta gene expression, we analyzed the -2.4-kb MyHCbeta promoter construct for cis-regulatory elements. Using electrophoretic mobility shift assays (EMSAs), chromatin immunoprecipitation assays (ChIP), and nuclear complex coimmunoprecipitation (NCcoIP) assays, we demonstrated calcium-ionophore-induced binding of NFATc1 to a NFAT consensus site adjacent to a MyoD-binding E-box. At their respective binding sites, both NFATc1 and MyoD recruited the transcriptional coactivator p300, and in turn, MEF-2D bound to the MyoD complex. The calcium-ionophore-induced effects on the MyHCbeta promoter were shown to be calcineurin-dependent. Together, our findings demonstrate calcium-ionophore-induced activation of the beta MyHC promoter by

  19. The human and mouse sex-determining SRY genes repress the Rspol/beta-catenin signaling.

    PubMed

    Lau, Yun-Fai Chris; Li, Yunmin

    2009-04-01

    The sex-determining region Y (SRY) is the gene on the Y chromosome responsible for switching on male sex determination during mammalian embryogenesis. In its absence, ovaries develop in the embryo. Hence, ovarian determination and differentiation is considered to be a default, or passive, developmental pathway. Recently this classical paradigm of sex determination has been challenged with the discovery of the R-spondin 1 (RSPO1) as an active ovarian determinant. Mutations of RSPO1 cause a female-to-male sex reversal. RSPO1 synergizes with WNT4 in activating an ovarian development in the bipotential gonad via the canonical Wnt signaling. Early studies showed that SRY represses such Wnt signaling, but also generated discrepancies on whether only mouse Sry is capable of inhibiting such Wnt signaling and whether both human and mouse SRY proteins are able to interact with beta-catenin, the intracellular messenger responsible for executing the Wnt signals. Our studies show that both human SRY and mouse Sry are capable of repressing the Rspo1/Wnt/beta-catenin signaling. However, the repression activities vary among different SRY/Sry proteins and paradoxically related to the presence and/or size of an acidic/glutamine-rich domain. The HMG box of human SRY could bind directly to beta-catenin while the mouse Sry binds to beta-catenin via its HMG box and glutamine-rich domain. The results clarify some of the initial discrepancies, and raise the possibility that SRY interacts with beta-catenin in the nucleus and represses the transcriptional activation of the Rspo1/Wnt target genes involved in ovarian determination, thereby switching on testis determination.

  20. beta. amyloid gene duplication in Alzheimer's disease and karyotypically normal Down syndrome

    SciTech Connect

    Delabar, J.; Goldgaber, D.; Lamour, Y.; Nicole, A.; Huret, J.; De Groucy, J.; Brown, P.; Gajdusek, D.C.; Sinet, P.

    1987-03-13

    With the recently cloned complementary DNA probe, lambdaAm4 for the chromosome 21 gene encoding brain amyloid polypeptide (..beta.. amyloid protein) of Alzheimer's disease, leukocyte DNA from three patients with sporadic Alzheimer's disease and two patients with karyotypically normal Down syndrome was found to contain three copies of this bene. Because a small region of chromosome 21 containing the ets-2 gene is duplicated in patients with Alzheimer's disease, as well as in karyotypically normal Down syndrome, duplication of a subsection of the critical segment of chromosome 21 that is duplicated in Down syndrome may be the genetic defect in Alzeimer's disease.

  1. Targets for dioxin: Genes for plasminogen activator inhibitor-2 and interleukin-1. beta

    SciTech Connect

    Sutter, T.R.; Guzman, K.; Dold, K.M. ); Greenlee, W.F. )

    1991-10-18

    Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD), a widespread environmental contaminant, may elicit its effects by altering gene expression in susceptible cells. Five TCDD-responsive complementary DNA clones were isolated from a human keratinocyte cell line. One of these clones encodes plasminogen activator inhibitor-2, a factor that influences growth and differentiation by regulating proteolysis of the extracellular matrix. Another encodes the cytokine interleukin-1{beta}. Thus, TCDD alters the expression of growth regulator genes and has effects similar to those of other tumor-promoting agents that affect both inflammation and differentiation.

  2. TGF-Beta Gene Polymorphisms in Food Allergic versus Non-Food Allergic Eosinophilic Esophagitis

    DTIC Science & Technology

    2014-12-01

    AD_________________ Award Number: W81XWH-11-1-0741 TITLE: Gene Food Allerg PRINCIPAL INVESTIGATOR: David Broide MB ChB CONTRACTING...TYPE Final 3. DATES COVERED (From - To) 15 Sep 2011 to 2014 4. TITLE AND SUBTITLE TGF-Beta Gene Polymorphisms in Food Allergic 5a. CONTRACT NUMBER...versus Non- Food Allergic Eosinophilic Esophagitis 5b. GRANT NUMBER W81XWH-11-1-0741 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) David Broide MB ChB

  3. Globin synthesis in fractionated Normoblasts of beta-thalassemia heterozygotes.

    PubMed Central

    Wood, W G; Stamatoyannopoulos, G

    1975-01-01

    Globin chain synthesis was examined in erythroid cells of increasing maturity, fractionated from the whole bone marrow of beta-thalassemia heterozygotes by a density gradient centrifugation procedure. In experiments using total cell "globin," a gradient of alpha/beta chain ratios was observed, increasing with erythroid cell maturation from unity in the basophilic cells up to 2.0 in reticulocytes. Gel filtration of the lysates from these marrow fractions revealed the presence of free alpha chains even in the most immature cells, the amount of which increased with erythroid cell age; the total alpha/beta ratio derived from gel filtration experiments showed a gradient similar to that observed in the total globin experiments. However, the alpha/beta ratio of the hemoglobin fraction obtained by gel filtration remained constant throughout maturation at an average of 0.65. This latter finding is incompatible with balanced synthesis at any stage of red cell development and excludes the possibility that total beta chain production is higher in the early cells than in the later cells or that alpha chain production in the early cells is reduced to the level of beta chain synthesis. Furthermore, in a Hb S/beta-thalassemia marrow examined, the beta A/beta S ratio remained constant throughout maturation while the alpha/non-alpha ratio showed an increase like that observed in the simple beta-thalassemia heterozygotes. This argues strongly against increased synthesis from either the thalassemic or nonthalassemic beta chain gene being responsible for the balanced synthesis in the immature cells. These findings lead us to suggest that, in beta-thalassemia heterozygotes, a large alpha chain pool is present throughout erythroid cell maturation and that the observed increase in alpha/beta ratios is a function of the ability of those cells to degrade the excess alpha chains. Images PMID:1167870

  4. Gene-gene interaction between PPAR gamma 2 and ADR beta 3 increases obesity risk in children and adolescents.

    PubMed

    Ochoa, M C; Marti, A; Azcona, C; Chueca, M; Oyarzábal, M; Pelach, R; Patiño, A; Moreno-Aliaga, M J; Martínez-González, M A; Martínez, J A

    2004-11-01

    Multiple genes are likely to be involved in obesity and these genes may interact with environmental factors to influence obesity risk. Our aim was to explore the synergistic contribution of the two polymorphisms: Pro12Ala of the PPAR gamma 2 gene and Trp64Arg of the ADR beta 3 gene to obesity risk in a Spanish children and adolescent population. We designed a sex- and age-matched case-control study. Participants were 185 obese and 185 control children (aged 5-18 y) from the Navarra region, recruited through Departments of Pediatrics (Hospital Virgen del Camino, Navarra University Clinic and several Primary Health Centers). The obesity criterion (case definition) was BMI above the 97th percentile according to Spanish BMI reference data for age and gender. Anthropometric parameters were measured by standard protocols. The genotype was assessed by PCR-RFLP after digestion with BstUI for PPAR gamma 2 mutation and BstNI for ADR beta 3 variants. Face-to-face interviews were conducted to assess the physical activity. Using a validated physical activity questionnaire, we computed an activity metabolic equivalent index (METs h/week), which represents the physical exercise during the week for each participant. Statistical analysis was performed by conditional logistic regression, taking into account the matching between cases and controls. Carriers of the polymorphism Pro12Ala of the PPAR gamma 2 gene had a significantly higher obesity risk than noncarriers (odds ratio (OR)=2.18, 95% CI=1.09-4.36) when we adjusted for sex, age and physical activity. Moreover, the risk of obesity was higher (OR=2.59, 95% CI=1.17-5.34) when family history of obesity was also taken into account in the model. The OR for obesity linked to both polymorphisms (PPAR gamma 2 and ADR beta 3) was 5.30 (95% CI=1.08-25.97) when we adjusted for sex, age and physical activity. After adjustment for family history of obesity, the OR for carriers of both polymorphisms was 19.5 (95% CI=2.43-146.8). A

  5. Use of microarray analysis to unveil transcription factor and gene networks contributing to Beta cell dysfunction and apoptosis.

    PubMed

    Eizirik, Decio L; Kutlu, Burak; Rasschaert, Joanne; Darville, Martine; Cardozo, Alessandra K

    2003-11-01

    The beta cell fate following immune-mediated damage depends on an intricate pattern of dozens of genes up- or downregulated in parallel and/or sequentially. We are utilizing microarray analysis to clarify the pattern of gene expression in primary rat beta cells exposed to the proapoptotic cytokines, IL-1beta and/or IFN-gamma. The picture emerging from these experiments is that beta cells are not passive bystanders of their own destruction. On the contrary, beta cells respond to damage by activating diverse networks of transcription factors and genes that may either lead to apoptosis or preserve viability. Of note, cytokine-exposed beta cells produce and release chemokines that may contribute to the homing and activation of T cells and macrophages during insulitis. Several of the effects of cytokines depend on the activation of the transcription factor, NF-kappaB. NF-kappaB blocking prevents cytokine-induced beta cell death, and characterization of NF-kappaB-dependent genes by microarray analysis indicated that this transcription factor controls diverse networks of transcription factors and effector genes that are relevant for maintenance of beta cell differentiated status, cytosolic and ER calcium homeostasis, attraction of mononuclear cells, and apoptosis. Identification of this and additional "transcription factor networks" is being pursued by cluster analysis of gene expression in insulin-producing cells exposed to cytokines for different time periods. Identification of complex gene patterns poses a formidable challenge, but is now technically feasible. These accumulating evidences may finally unveil the molecular mechanisms regulating the beta cell "decision" to undergo or not apoptosis in early T1DM.

  6. Cloning and characterization of a novel human clathrin heavy chain gene (CLTCL)

    SciTech Connect

    Long, K.R.; Trofatter, J.A.; McCormick, M.K.

    1996-08-01

    An exon representing a novel clathrin heavy chain gene (CLTCL) was isolated during gene identification studies and transcription mapping of human chromosome 22. Isolation and sequencing of cDNA clones corresponding to this exon revealed extensive similarity of the predicted amino acid sequence of this gene product to those of clathrin heavy chain genes of other species. Northern blot analysis has revealed an apparent developmental expression pattern of an approximately 6-kb mRNA. The gene appears to be expressed ubiquitously in the limited number of fetal tissues that were tested, but is selectively expressed in certain adult tissues, particularly in skeletal muscle. In addition, alternative splicing of an exon was observed near the carboxyl terminus of the predicted gene product. Its location overlaps the domain putatively involved in clathrin light chain binding and is adjacent to the heavy chain self-assembly (or trimerization) region, suggesting that alternative splicing may be involved in regulating one or both of these interactions. The expression pattern of this gene, in addition to its potential role in receptor-mediated endocytosis and signal transduction, suggests that is may be important in some developmental processes. The location of CLTCL on human chromosome 22 near the region commonly deleted in DiGeorge and other apparent haploinsufficiency syndromes warrants further investigation into its relationship with these developmental disorders. 34 refs., 3 figs., 1 tab.

  7. Increase in expression level of alpha- and beta-tubulin genes in Arabidopsis seedlings under hypergravity conditions

    NASA Astrophysics Data System (ADS)

    Saito, Y.; Soga, K.; Wakabayashi, K.; Hoson, T.

    Hypergravity, a gravitational force of more than 1 g, suppresses elongation growth of shoots of various plants. The analysis of the changes in gene expression by hypergravity treatment in Arabidopsis hypocotyls by the differential display method showed that a gene encoding alpha-tubulin, which is a component of microtubules, was up-regulated by hypergravity [Yoshioka et al. (2003) Adv. Space Res. 31: 2187-2193]. However, the detailed relation between hypergravity treatment and the changes in alpha-tubulin gene levels has not been determined yet. Microtubules are formed by the spontaneous polymerization of dimers consisting of one alpha- and one beta-tubulin protein molecule. Thus, the expression levels of beta-tubulin genes may also be affected by hypergravity. In the present study, we examined the dose-response and the time course relations of change in the expression of both alpha- and beta-tubulin genes in Arabidopsis hypocotyls grown under hypergravity conditions. Elongation growth of Arabidopsis hypocotyls was suppressed by increasing gravity. The expression levels of both alpha- and beta-tubulin genes were increased depending on the magnitude of gravity. At 300 g, expression levels of alpha- and beta-tubulin genes were about 400% and 350% of the 1 g control, respectively. The increases in expression of both tubulin genes were detected within a few hours, when the seedlings grown at 1 g were transferred to hypergravity conditions. The increase in alpha- and beta-tubulin genes preceded or occurred at the same time as growth suppression. These results suggest that Arabidopsis hypocotyls regulate the expression level of alpha- and beta-tubulin genes promptly in response to gravity stimuli. The increase in the amount of microtubules due to the activation of tubulin gene expression may be involved in the regulation by gravity signal of shoot growth.

  8. Influence of coding region polymorphism on the peripheral expression of a human TCR V[beta] gene

    SciTech Connect

    Vissinga, C.S.; Charmley, P.; Concannon, P. )

    1994-02-01

    A number of human TCR V[beta] gene segments are reported to be polymorphic, with alleles differing by one or a small number of amino acid substitutions. In the absence of detailed structural information regarding the interaction of specific positions in the TCR with Ag or MHC, the significance of such variation is difficult to assess. In this report the relative use of the two common alleles of the human V[beta]6.7 gene, 6.7a and 6.7b, which differ by two nonconservative amino acid substitutions, and the use of two common alleles of the V[beta]12.2 gene, which differ by only silent substitutions, were measured in PBL derived from individuals heterozygous for these alleles. Equal use of V[beta]12.2 alleles was observed, consistent with the inability of selection mechanisms to discriminate between the products of these alleles that are indistinguishable at the amino acid level. However, statistically significant skewing in the use of V[beta]6.7 alleles was observed in 15 of 16 individuals studied. Expression levels for each allele ranged from 16 to 84% of the total V[beta]6.7 signal in heterozygous individuals, with either the 6.7a or the 6.7b allele predominant in different individuals. Based on segregation studies in families, it seems unlikely that other unidentified polymorphism in the TCR[beta] locus, such as in the V[beta]6.7 promoter, was responsible for the differential allele expression. Family studies provided no evidence for an association between specific HLA haplotypes and V[beta]6.7 allele use. These results indicate that even modest allelic variation in human TCR V[beta] coding regions can have a significant impact on the expression of human V[beta] genes in the peripheral repertoire. 29 refs., 4 figs.

  9. Functional analysis of the human neurofilament light chain gene promoter.

    PubMed Central

    Yazdanbakhsh, K; Fraser, P; Kioussis, D; Vidal, M; Grosveld, F; Lindenbaum, M

    1993-01-01

    We have carried out a structural and functional analysis on the human NF-L (H-NF-L) gene. It contains a methylation-free island, spanning the 5' flanking sequences and the first exon and a number of neuronal-specific DNase I hypersensitive sites have been identified in the upstream region as well as within the body of the gene. Analysis in cell lines and transgenic mice using a combination of these sites has revealed the presence of a conserved element(s) between -300bp and -190bp which is required for neuronal-specific expression. Images PMID:8441658

  10. Beta-Arrestin2 regulates RANKL and ephrins gene expression in response to bone remodeling in mice.

    PubMed

    Pierroz, Dominique D; Rufo, Anna; Bianchi, Estelle N; Glatt, Vaida; Capulli, Mattia; Rucci, Nadia; Cavat, Fanny; Rizzoli, René; Teti, Anna; Bouxsein, Mary L; Ferrari, Serge L

    2009-05-01

    PTH-stimulated intracellular signaling is regulated by the cytoplasmic adaptor molecule beta-arrestin. We reported that the response of cancellous bone to intermittent PTH is reduced in beta-arrestin2(-/-) mice and suggested that beta-arrestins could influence the bone mineral balance by controlling RANKL and osteoprotegerin (OPG) gene expression. Here, we study the role of beta-arrestin2 on the in vitro development and activity of bone marrow (BM) osteoclasts (OCs) and Ephrins ligand (Efn), and receptor (Eph) mRNA levels in bone in response to PTH and the changes of bone microarchitecture in wildtype (WT) and beta-arrestin2(-/-) mice in models of bone remodeling: a low calcium diet (LoCa) and ovariectomy (OVX). The number of PTH-stimulated OCs was higher in BM cultures from beta-arrestin2(-/-) compared with WT, because of a higher RANKL/OPG mRNA and protein ratio, without directly influencing osteoclast activity. In vivo, high PTH levels induced by LoCa led to greater changes in TRACP5b levels in beta-arrestin2(-/-) compared with WT. LoCa caused a loss of BMD and bone microarchitecture, which was most prominent in beta-arrestin2(-/-). PTH downregulated Efn and Eph genes in beta-arrestin2(-/-), but not WT. After OVX, vertebral trabecular bone volume fraction and trabecular number were lower in beta-arrestin2(-/-) compared with WT. Histomorphometry showed that OC number was higher in OVX-beta-arrestin2(-/-) compared with WT. These results indicate that beta-arrestin2 inhibits osteoclastogenesis in vitro, which resulted in decreased bone resorption in vivo by regulating RANKL/OPG production and ephrins mRNAs. As such, beta-arrestins should be considered an important mechanism for the control of bone remodeling in response to PTH and estrogen deprivation.

  11. Interaction of SOCS3 with NonO attenuates IL-1beta-dependent MUC8 gene expression.

    PubMed

    Song, Kyoung Seob; Kim, Kyubo; Chung, Kwang Chul; Seol, Jae Hong; Yoon, Joo-Heon

    2008-12-19

    The intracellular negatively regulatory mechanism which affects IL-1beta-induced MUC8 gene expression remains unclear. We found that SOCS3 overexpression suppressed IL-1beta-induced MUC8 gene expression in NCI-H292 cells, whereas silencing of SOCS3 restored IL-1beta-induced MUC8 gene expression. Sequentially activated ERK1/2, RSK1, and CREB by IL-1beta were not affected by SOCS3, indicating that SOCS3 has an independent mechanism of action. Using immunoprecipitaion and nano LC mass analysis, we found that SOCS3 bound NonO (non-POU-domain containing, octamer-binding domain protein) in the absence of IL-1beta, whereas IL-1beta treatment dissociated the direct binding of SOCS3 and NonO. A dominant-negative SOCS3 mutant (Y204F/Y221F) did not bind to NonO. Interestingly, SOCS3 overexpression dramatically suppressed MUC8 gene expression in cells transfected with wild-type or siRNA of NonO. Moreover, silencing of SOCS3 dramatically increased NonO-mediated MUC8 gene expression caused by IL-1beta compared to NonO overexpression alone, suggesting that SOCS3 acts as a suppressor by regulating the action of NonO.

  12. Species-dependent expression of the hyoscyamine 6 beta-hydroxylase gene in the pericycle.

    PubMed

    Kanegae, T; Kajiya, H; Amano, Y; Hashimoto, T; Yamada, Y

    1994-06-01

    The tropane alkaloid scopolamine is synthesized in the pericycle of branch roots in certain species of the Solanaceae. The enzyme responsible for the synthesis of scopolamine from hyoscyamine is hyoscyamine 6 beta-hydroxylase (H6H). The gene for H6H was isolated from Hyoscyamus niger. It has an exon/intron organization very similar to those for ethylene-forming enzymes, suggesting a common evolutionary origin. The 827-bp 5' flanking region of the H6H gene was fused to the beta-glucuronidase (GUS) reporter gene and transferred to three solanaceous species by Agrobacterium-mediated transformation systems: H. niger and belladonna (Atropa belladonna), which have high and low levels, respectively, of H6H mRNA in the root, and tobacco (Nicotiana tabacum), which has no endogenous H6H gene. Histochemical analysis showed that GUS expression occurred in the pericycle and at the root meristem of transgenic H. niger hairy roots, but only at the root meristem of transgenic H. niger hairy roots, but only at the root meristem of hairy roots and plants of transgenic tobacco. In transgenic hairy roots and regenerated plants of belladonna, the root meristem was stained with GUS activity, except for a few transformants in which the vascular cylinder was also stained. These studies indicate that the cell-specific expression of the H6H gene is controlled by some genetic regulation specific to scopolamine-producing plants.

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

    SciTech Connect

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

    2015-12-04

    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. - Highlights: • TLX overexpression in MIN6 cell causes significant expression changes of 225 genes. • TLX overexpression promotes MIN6 cell proliferation and decreases cell apoptosis. • TLX overexpression does not cause impairment of insulin secretion.

  14. Expression of the Acremonium chrysogenum cefT gene in Penicillum chrysogenum indicates that it encodes an hydrophilic beta-lactam transporter.

    PubMed

    Ullán, Ricardo V; Teijeira, Fernando; Martín, Juan F

    2008-09-01

    The Acremonium chryrsogenum cefT gene encoding a membrane protein of the major facilitator superfamily implicated in the cephalosporin biosynthesis in A. chrysogenum was introduced into Penicillium chrysogenum Wisconsin 54-1255 (a benzylpenicillin producer), P. chrysogenum npe6 pyrG(-) (a derivative of Wisconsin 54-1255 lacking a functional penDE gene) and P. chrysogenum TA98 (a deacetylcephalosporin producer containing the cefD1, cefD2, cefEF and cefG genes from A. chrysogenum). RT-PCR analysis revealed that the cefT gene was expressed in P. chrysogenum strains. HPLC analysis of the culture broths of the TA98 transformants showed an increase in the secretion of deacetylcephalosporin C and hydrophilic penicillins (isopenicillin N and penicillin N). P. chrysogenum Wisconsin 54-1255 strain transformed with cefT showed increased secretion of the isopenicillin N intermediate and a drastic decrease in the benzylpenicillin production. Southern and northern blot analysis indicated that the untransformed P. chrysogenum strains contain an endogenous gene similar to cefT that may be involved in the well-known secretion of the isopenicillin N intermediate. In summary, the cefT transporter is a hydrophilic beta-lactam transporter that is involved in the secretion of hydrophilic beta-lactams containing alpha-aminoadipic acid side chain (isopenicillin N, penicillin N and deacetylcephalosporin C).

  15. Prognostic value of CpG island hypermethylation at PTGS2, RAR-beta, EDNRB, and other gene loci in patients undergoing radical prostatectomy.

    PubMed

    Bastian, Patrick J; Ellinger, Jörg; Heukamp, Lukas C; Kahl, Philip; Müller, Stefan C; von Rücker, Alexander

    2007-03-01

    To evaluate CpG island hypermethylation in a set of candidate genes in prostate cancer (pCA) and its relationship to clinicopathologic parameters and a nomogram predicting prostate-specific antigen (PSA) recurrence after radical prostatectomy. Tissues of 78 prostate carcinomas, 32 benign prostate hyperplasias (BPHs), and prostate cell lines (LNCaP, DU145, PC3, BPH-1) were examined with MethyLight polymerase chain reaction at 13 gene loci (APC, CDC6, CTNNB1, E-Cadherin, EDNRB, FGFR2, GSTP1, NAB2, PKCmu, PTGS2, RAR-beta, RASL11A, WWOX). APC, RAR-beta, PTGS2, GSTP1, EDNRB, and CTNNB1 (83%, 71%, 65%, 33%, 14%, 9%, respectively) were methylated in pCA but rarely or not methylated in BPH. NAB2 and CDC6 were hypermethylated frequently in pCA (92%, 67%, respectively) and in BPH (91%, 59%, respectively). FGFR2, WWOX, E-Cadherin, PKCmu, and RASLL1A did not display noteworthy methylation in pCA (0-1%) or in BPH. CpG island hypermethylation at APC, retinoic acid receptor beta (RAR-beta), and PTGS2 discriminated with a sensitivity of 65-83% and a specificity of 97-100% between BPH and pCA. The combination of various genes increased the diagnostic expressiveness. PTGS2 hypermethylation correlated with seminal vesicle infiltration (p=0.047), capsular penetration (p=0.004), and pT stage (p=0.014). RAR-beta methylation was accompanied by a higher cumulative Gleason score (p=0.042). The probability of PSA-free-survival calculated with a Kattan nomogram correlated inversely with CpG island hypermethylation at EDNRB, RAR-beta, and PTGS2. All prostate cancer cell lines displayed a varying degree of demethylation after 5-aza-2'deoxycytidine treatment. CpG island hypermethylation at various gene loci is frequent in prostate cancer and can distinguish between neoplastic and noncancerous tissue. Furthermore, hypermethylation at PTGS2, RAR-beta, and EDNRB inversely correlated with PSA-free-survival according to a Kattan nomogram and has potential prognostic value.

  16. Role of Na+/H+ exchange by interferon-gamma in enhanced expression of JE and I-A beta genes

    SciTech Connect

    Prpic, V.; Yu, S.F.; Figueiredo, F.; Hollenbach, P.W.; Gawdi, G.; Herman, B.; Uhing, R.J.; Adams, D.O.

    1989-04-28

    The rapid transductional sequences initiated by interferon-gamma (IFN-gamma) on binding to its receptor regulate functional and genomic responses in many cells but are not well defined. Induction of macrophage activation is an example of such functional and genomic changes in response to IFN-gamma. Addition of IFN-gamma to murine macrophages, at activating concentrations, produced rapid (within 60 seconds) alkalinization of the cytosol and a concomitant, rapid influx of /sup 22/Na+. Amiloride inhibited the ion fluxes and the accumulation of specific messenger RNA for two genes induced by IFN-gamma (the early gene JE and the beta chain of the class II major histocompatibility complex gene I-A). The data indicate that IFN-gamma initiates rapid exchange of Na+ and H+ by means of the Na+/H+ antiporter and that these amiloride-sensitive ion fluxes are important to some of the genomic effects of IFN-gamma.

  17. Beta-rolls, beta-helices, and other beta-solenoid proteins.

    PubMed

    Kajava, Andrey V; Steven, Alasdair C

    2006-01-01

    Beta-rolls and beta-helices belong to a larger group of topologically similar proteins with solenoid folds: because their regular secondary structure elements are exclusively beta-strands, they are referred to as beta-solenoids. The number of beta-solenoids whose structures are known is now large enough to support a systematic analysis. Here we survey the distinguishing structural features of beta-solenoids, also documenting their notable diversity. Appraisal of these structures suggests a classification based on handedness, twist, oligomerization state, and coil shape. In addition, beta-solenoids are distinguished by the number of chains that wind around a common axis: the majority are single-stranded but there is a recently discovered subset of triple-stranded beta-solenoids. This survey has revealed some relationships of the amino acid sequences of beta-solenoids with their structures and functions-in particular, the repetitive character of the coil sequences and conformations that recur in tracts of tandem repeats. We have proposed the term beta-arc for the distinctive turns found in beta-solenoids and beta-arch for the corresponding strand-turn-strand motifs. The evolutionary mechanisms underlying these proteins are also discussed. This analysis has direct implications for sequence-based detection, structural prediction, and de novo design of other beta-solenoid proteins. The abundance of virulence factors, toxins and allergens among beta-solenoids, as well as commonalities of beta-solenoids with amyloid fibrils, imply that this class of folds may have a broader role in human diseases than was previously recognized. Thus, identification of genes with putative beta-solenoid domains promises to be a fertile direction in the search for viable targets in the development of new antibiotics and vaccines.

  18. Restricted alpha/beta receptor gene usage of idiotype-specific major histocompatibility complex-restricted T cells: selection for CDR3-related sequences.

    PubMed

    Snodgrass, H R; Fisher, A M; Bruyns, E; Bogen, B

    1992-08-01

    We have sequenced the T cell receptor (TcR) V alpha and V beta genes of seven independent BALB/c CD4+ T cell clones specific for the immunoglobulin lambda 2 light chain produced by the MOPC 315 myeloma (lambda 2(315)). All the clones recognize a peptide of residues 91-101 of lambda 2(315) and are restricted by the major histocompatibility complex (MHC) molecule I-E(d). The results indicate that in BALB/c mice, this anti-idiotypic response uses a very limited number of TcR. The four clones which cross-react between Phe94 and Tyr94 peptide analogues use very similar receptors (V alpha 3, J alpha 1, V beta 6, J beta 1.1). The V alpha 3 gene used by all of these clones is identical and has not been previously described. Although the four clones differ in nucleotide sequence in the V/J borders, two had identical receptors at the amino acid level. One of the cross-reactive clones exhibits a heteroclitic response to the Tyr94 peptide variant resulting from a single amino acid exchange in the V/J junction of the alpha chain. The three remaining clones which recognize only the Phe94 and not the Tyr94 peptide have somewhat more diverse TcR, however, two of these three clones use V beta 6. One of these non-crossreacting clones is alloreactive, the specificity of which can be attributed to differences in the N-D-J sequences. Taken together these data indicate that this T cell response to an immunoglobulin idiotope is very restricted in terms of the TcR used. These data in conjunction with recently published results indicate that, although there can be strong preference for individual V alpha or V beta gene segments, certain V alpha/V beta combinations are preferentially selected for interacting with a given peptide/MHC combination, and that the CDR3-related regions are crucial for antigen fine specificity and alloreactivity.

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

    PubMed

    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.

  20. Poly(beta-amino esters): procedures for synthesis and gene delivery.

    PubMed

    Green, Jordan J; Zugates, Gregory T; Langer, Robert; Anderson, Daniel G

    2009-01-01

    Non-viral gene delivery systems are promising as they avoid many problems of viral gene therapy by having increased design flexibility, high safety, large DNA cargo capacity, and ease of manufacture. Here, we describe the use of polymeric vectors, in particular biodegradable poly(beta-amino esters) (PBAEs), for non-viral gene delivery. These polymers are able to self-assemble with DNA and form positively charged gene delivery nanoparticles. Methods for synthesis of these polymers, particle self-assembly, and transfection using these particles are delineated. A standard protocol is presented as well as a high-throughput screening technique that can be used to more quickly optimize transfection parameters for efficient delivery.

  1. Lactogenic hormonal induction of long distance interactions between beta-casein gene regulatory elements.

    PubMed

    Kabotyanski, Elena B; Rijnkels, Monique; Freeman-Zadrowski, Courtneay; Buser, Adam C; Edwards, Dean P; Rosen, Jeffrey M

    2009-08-21

    Lactogenic hormone regulation of beta-casein gene expression in mammary epithelial cells provides an excellent model in which to study the mechanisms by which steroid and peptide hormone signaling control gene expression. Prolactin- and glucocorticoid-mediated induction of beta-casein gene expression involves two principal regulatory regions, a proximal promoter and a distal enhancer located in the mouse approximately -6 kb upstream of the transcription start site. Using a chromosome conformation capture assay and quantitative real time PCR, we demonstrate that a chromatin loop is created in conjunction with the recruitment of specific transcription factors and p300 in HC11 mammary epithelial cells. Stimulation with both prolactin and hydrocortisone is required for the induction of these long range interactions between the promoter and enhancer, and no DNA looping was observed in nontreated cells or cells treated with each of the hormones separately. The lactogenic hormone-induced interaction between the proximal promoter and distal enhancer was confirmed in hormone-treated primary three-dimensional mammary acini cultures. In addition, the developmental regulation of DNA looping between the beta-casein regulatory regions was observed in lactating but not in virgin mouse mammary glands. Furthermore, beta-casein mRNA induction and long range interactions between these regulatory regions were inhibited in a progestin-dependent manner following stimulation with prolactin and hydrocortisone in HC11 cells expressing human PR-B. Collectively, these data suggest that the communication between these regulatory regions with intervening DNA looping is a crucial step required to both create and maintain active chromatin domains and regulate transcription.

  2. Mutation in a structural gene for a beta-tubulin specific to testis in Drosophila melanogaster.

    PubMed Central

    Kemphues, K J; Raff, R A; Kaufman, T C; Raff, E C

    1979-01-01

    By two-dimensional gel electrophoresis of tubulins prepared from tissues of Drosophila melanogaster we have identified a beta-tubulin subunit that is present only in the testis. Furthermore, we have isolated, as a male sterile, a third chromosome dominant mutation [ms(3)KKD] in the structural gene for this beta-tubulin. Males heterozygous for this mutation produce no motile spermatozoa. Beginning with meiosis, all processes in spermatogenesis are abnormal to some extent. Many microtubules (including both cytoplasmic microtubules and doublet tubules of the axoneme) show aberrant structure in cross section, and the overall morphology of the developing spermatids is disorganized. Testes from these males were shown, by two-dimensional gel electrophoresis, to contain both the normal testis-specific beta-tubulin and an electrophoretic variant of this tubulin in equal amounts. Both wild-type and mutant testis-specific beta-tubulins were characterized by vinblastine sulfate precipitation, coassembly with purified Drosophila embryo tubulin, and peptide mapping. Images PMID:115008

  3. Presence of the bla(Z) beta-lactamase gene in isolates of Staphylococcus aureus that appear penicillin susceptible by conventional phenotypic methods.

    PubMed

    El Feghaly, Rana E; Stamm, Jennifer E; Fritz, Stephanie A; Burnham, Carey-Ann D

    2012-12-01

    Beta-lactamase production may not be reliably detected by commonly used susceptibility testing methods such as Kirby-Bauer penicillin disk diffusion and nitrocefin beta-lactamase detection. We assayed 105 apparently penicillin-susceptible Staphylococcus aureus isolates using multiple methods to detect beta-lactamase production. The bla(Z) beta-lactamase gene was detected by polymerase chain reaction in 10 (9.5%) of the 105 isolates. The average disk diffusion zone diameter was 34 and 38 mm for the bla(Z)-positive and -negative isolates, respectively (P < 0.001). Qualitative description of the zone edge was observer-dependent. The "cloverleaf assay" was positive in 6 of the 10 phenotypically susceptible isolates possessing bla(Z). The results of this study suggest that conventional methods for S. aureus penicillin susceptibility testing may not reliably detect penicillin resistance in all isolates; however, increasing the disk diffusion zone size interpretive criteria to 35 mm for this antimicrobial/organism combination from the current 29-mm breakpoint may improve the sensitivity of phenotypic penicillin susceptibility testing.

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

    PubMed

    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.

  5. Effects of different activity and inactivity paradigms on myosin heavy chain gene expression in striated muscle

    NASA Technical Reports Server (NTRS)

    Baldwin, K. M.; Haddad, F.

    2001-01-01

    The goal of this mini-review is to summarize findings concerning the role that different models of muscular activity and inactivity play in altering gene expression of the myosin heavy chain (MHC) family of motor proteins in mammalian cardiac and skeletal muscle. This was done in the context of examining parallel findings concerning the role that thyroid hormone (T(3), 3,5,3'-triiodothyronine) plays in MHC expression. Findings show that both cardiac and skeletal muscles of experimental animals are initially undifferentiated at birth and then undergo a marked level of growth and differentiation in attaining the adult MHC phenotype in a T(3)/activity level-dependent fashion. Cardiac MHC expression in small mammals is highly sensitive to thyroid deficiency, diabetes, energy deprivation, and hypertension; each of these interventions induces upregulation of the beta-MHC isoform, which functions to economize circulatory function in the face of altered energy demand. In skeletal muscle, hyperthyroidism, as well as interventions that unload or reduce the weight-bearing activity of the muscle, causes slow to fast MHC conversions. Fast to slow conversions, however, are seen under hypothyroidism or when the muscles either become chronically overloaded or subjected to intermittent loading as occurs during resistance training and endurance exercise. The regulation of MHC gene expression by T(3) or mechanical stimuli appears to be strongly regulated by transcriptional events, based on recent findings on transgenic models and animals transfected with promoter-reporter constructs. However, the mechanisms by which T(3) and mechanical stimuli exert their control on transcriptional processes appear to be different. Additional findings show that individual skeletal muscle fibers have the genetic machinery to express simultaneously all of the adult MHCs, e.g., slow type I and fast IIa, IIx, and IIb, in unique combinations under certain experimental conditions. This degree of

  6. Effects of different activity and inactivity paradigms on myosin heavy chain gene expression in striated muscle

    NASA Technical Reports Server (NTRS)

    Baldwin, K. M.; Haddad, F.

    2001-01-01

    The goal of this mini-review is to summarize findings concerning the role that different models of muscular activity and inactivity play in altering gene expression of the myosin heavy chain (MHC) family of motor proteins in mammalian cardiac and skeletal muscle. This was done in the context of examining parallel findings concerning the role that thyroid hormone (T(3), 3,5,3'-triiodothyronine) plays in MHC expression. Findings show that both cardiac and skeletal muscles of experimental animals are initially undifferentiated at birth and then undergo a marked level of growth and differentiation in attaining the adult MHC phenotype in a T(3)/activity level-dependent fashion. Cardiac MHC expression in small mammals is highly sensitive to thyroid deficiency, diabetes, energy deprivation, and hypertension; each of these interventions induces upregulation of the beta-MHC isoform, which functions to economize circulatory function in the face of altered energy demand. In skeletal muscle, hyperthyroidism, as well as interventions that unload or reduce the weight-bearing activity of the muscle, causes slow to fast MHC conversions. Fast to slow conversions, however, are seen under hypothyroidism or when the muscles either become chronically overloaded or subjected to intermittent loading as occurs during resistance training and endurance exercise. The regulation of MHC gene expression by T(3) or mechanical stimuli appears to be strongly regulated by transcriptional events, based on recent findings on transgenic models and animals transfected with promoter-reporter constructs. However, the mechanisms by which T(3) and mechanical stimuli exert their control on transcriptional processes appear to be different. Additional findings show that individual skeletal muscle fibers have the genetic machinery to express simultaneously all of the adult MHCs, e.g., slow type I and fast IIa, IIx, and IIb, in unique combinations under certain experimental conditions. This degree of

  7. Expression of the human BETA-globin gene after retroviral transfer into murine erythroleukemia cells and human BFU-E cells

    SciTech Connect

    Bender, M.A.; Miller, A.D.; Gelinas, R.E.

    1988-04-01

    Replication-defective amphotropic retrovirus vectors containing either the human ..beta..-globin gene with introns or an intronless ..beta..-globin minigene were constructed and used to study ..beta..-globin expression following gene transfer into hematopoietic cells. The ..beta..-globin genes were marked by introducing a 6-base-pair insertion into the region corresponding to the 5' untranslated region of the ..beta..-globin mRNA to allow detection of RNA encoded by the new gene in human cells expressing normal human ..beta..-globin RNA. Introduction of a virus containing the ..beta..-globin gene with introns into murine erythroleukemia cells resulted in inducible expression of human ..beta..-globin RNA and protein, while the viruses containing the minigene were inactive. The introduced human ..beta..-globin gene was 6 to 110% as active as the endogenous mouse ..beta../sup maj/-globin genes in six randomly chosen cell clones. Introduction of the viruses into human BFU-E cells, followed by analysis of marked and unmarked globin RNAs in differentiated erythroid colonies, revealed that the introduced ..beta..-globin gene was about 5% as active as the endogenous genes in these normal human erythroid cells and that again the minigene was inactive. These data are discussed in terms of the potential treatment of genetic disease by gene therapy.

  8. Expression of the human beta-globin gene after retroviral transfer into murine erythroleukemia cells and human BFU-E cells.

    PubMed Central

    Bender, M A; Miller, A D; Gelinas, R E

    1988-01-01

    Replication-defective amphotropic retrovirus vectors containing either the human beta-globin gene with introns or an intronless beta-globin minigene were constructed and used to study beta-globin expression following gene transfer into hematopoietic cells. The beta-globin genes were marked by introducing a 6-base-pair insertion into the region corresponding to the 5' untranslated region of the beta-globin mRNA to allow detection of RNA encoded by the new gene in human cells expressing normal human beta-globin RNA. Introduction of a virus containing the beta-globin gene with introns into murine erythroleukemia cells resulted in inducible expression of human beta-globin RNA and protein, while the viruses containing the minigene were inactive. The introduced human beta-globin gene was 6 to 110% as active as the endogenous mouse beta maj-globin genes in six randomly chosen cell clones. Introduction of the viruses into human BFU-E cells, followed by analysis of marked and unmarked globin RNAs in differentiated erythroid colonies, revealed that the introduced beta-globin gene was about 5% as active as the endogenous genes in these normal human erythroid cells and that again the minigene was inactive. These data are discussed in terms of the potential treatment of genetic disease by gene therapy. Images PMID:3288863

  9. Cationic amphiphiles with fatty acyl chain asymmetry of coconut oil deliver genes selectively to mouse lung.

    PubMed

    Chandrashekhar, Voshavar; Srujan, Marepally; Prabhakar, Rairala; Reddy, Rakesh C; Sreedhar, Bojja; Rentam, Kiran K R; Kanjilal, Sanjit; Chaudhuri, Arabinda

    2011-03-16

    Recent structure-activity studies have revealed a dramatic influence of hydrophobic chain asymmetry in enhancing gene delivery efficacies of synthetic cationic amphiphiles (Nantz, M. H. et al. Mol. Pharmaceutics2010, 7, 786-794; Koynova, R. et al. Mol. Pharmaceutics2009, 6, 951-958). The present findings demonstrate for the first time that such a transfection enhancing influence of asymmetric hydrocarbon chains observed in pure synthetic cationic amphiphiles also works for cationic amphiphiles designed with natural, asymmetric fatty acyl chains of a food-grade oil. Herein, we demonstrate that cationic amphiphiles designed with the natural fatty acyl chain asymmetry of food-grade coconut oil are less cytotoxic and deliver genes selectively to mouse lung. Despite lauroyl chains being the major fatty acyl chains of coconut oil, both the in vitro and In vivo gene transfer efficiencies of such cationic amphiphiles were found to be remarkably superior (>4-fold) to those of their pure dilauroyl analogue. Mechanistic studies involving the technique of fluorescence resonance energy transfer (FRET) revealed higher biomembrane fusibility of the cationic liposomes of the coconut amphiphiles than that of the symmetric dilauroyl analogue. AFM study revealed pronounced fusogenic nonlamellar structures of the liposomes of coconut amphiphiles. Findings in the FRET and cellular uptake study, taken together, support the notion that the higher cellular uptake resulting from the more fusogenic nature of the liposomes of coconut amphiphiles 1 are likely to play a dominant role in making the coconut amphiphiles transfection competent.

  10. Editing of the heavy chain gene of Bombyx mori using transcription activator like effector nucleases.

    PubMed

    Wang, Yujun; Nakagaki, Masao

    2014-07-18

    The silk gland of Bombyx mori represents an established in vivo system for producing recombinant proteins. However, low yields of recombinant proteins have limited the system's further development because endogenous silk proteins were present. Transcription activator-like effector nucleases (TALENs) tool which work in pairs to bind and cleave DNA at specific sites, have recently been shown to be effective for genome editing in various organisms, including silkworms. To improve the yield of recombinant proteins synthesized in the silkworm by eliminated competition with endogenous fibroin synthesis, the heavy chain (H-chain) gene was knocked out using transcription activator-like effector nucleases (TALENs). A pair of TALENs that targets the 1st exon in the H-chain gene was synthesized and microinjected into silkworm embryos; the injected silkworms were screened for H-chain gene knock out (H-KO) based on their sericin cocoon-making characteristics. Sequence analysis revealed that the H-chain of the mutation was successfully edited. The TALENs was very efficient in editing the genome DNA of silkworm. By being eliminated competition with the H-chain, the production of recombinant proteins would be expected to increase markedly if this H-KO system is used.

  11. Systemic hyperosmolality improves beta-glucuronidase distribution and pathology in murine MPS VII brain following intraventricular gene transfer.

    PubMed

    Ghodsi, A; Stein, C; Derksen, T; Martins, I; Anderson, R D; Davidson, B L

    1999-11-01

    Mucopolysaccharidosis VII, a classical lysosomal storage disease, is caused by deficiency of the enzyme beta-glucuronidase. Central nervous system (CNS) manifestations are severe with accumulations of storage vacuoles in all cell types. Intraventricular gene transfer can lead to transduction of the ependyma, with production and secretion of beta-glucuronidase into the cerebral spinal fluid and underlying cortex resulting in reversal of disease pathology restricted to the periventricular areas. We tested if systemic hyperosmolality would increase the distribution of beta-glucuronidase in brain parenchyma after intraventricular virus injection. Mice were administered mannitol, intraperitoneally, 20 days after gene transfer and 1 day prior to sacrifice. Mannitol-induced systemic hyperosmolality caused a marked penetration of beta-glucuronidase into the brain parenchyma. If mannitol was administered at the time of the intraventricular injection of virus, there was penetration of vector across the ependymal cell layer, with infection of cells in the subependymal region. This also resulted in increased beta-glucuronidase activity throughout the brain. Sections of brains from beta-glucuronidase-deficient mice showed correction of cellular pathology in the subependymal region plus cortical structures away from the ventricular wall. These data indicate that virus-mediated gene transfer to the brain via the ventricles, coupled with systemic mannitol administration, can lead to extensive CNS distribution of beta-glucuronidase with concomitant correction of the storage defect. Our findings have positive therapeutic implications for the treatment of CNS disorders with gene transfer vectors and recombinant proteins.

  12. Evidence that Mls-2 antigens which delete V beta 3+ T cells are controlled by multiple genes.

    PubMed

    Pullen, A M; Marrack, P; Kappler, J W

    1989-05-01

    V beta 3+ T cells are eliminated in Mls-2a mice carrying some, but not all, H-2 types. Analysis of AKXD and BXD recombinant inbred strains showed that Mls-2a (formerly Mlsc) was not the product of a single gene and suggested that at least two non-H-2 genes control V beta 3 levels. Studies of the progeny of a B10.BR x (C3H/HeJ x B10.BR)F1 backcross confirmed the existence of two V beta 3+ T cell deleting genes: one unlinked and one linked to Ly-7, which we propose be called Mls-2 and Mls-3, respectively. Mls-2a induces partial deletion of V beta 3+ T cells with a bias toward deleting CD4+ cells. It stimulates V beta 3+ hybrids and may be linked to Mtv-13 on chromosome 4. A third non-H-2 gene is implicated in enhancing the presentation of Mls-2a. Mls-3a causes elimination of all V beta 3+ T cells in H-2k and H-2d mice but poorly stimulates V beta 3+ hybrids.

  13. Differential gene expression in well-regulated and dysregulated pancreatic beta-cell (MIN6) sublines.

    PubMed

    Lilla, Valérie; Webb, Gene; Rickenbach, Katharina; Maturana, Andres; Steiner, Donald F; Halban, Philippe A; Irminger, Jean-Claude

    2003-04-01

    To identify genes involved in regulated insulin secretion, we have established and characterized two sublines derived from the mouse pancreatic beta-cell line MIN6, designated B1 and C3. They have a similar insulin content, but differ in their secretory properties. B1 responded to glucose in a concentration- and cell confluence-dependent manner, whereas C3 did not. B1 cells were stimulated by phorbol 12-myristate 13-acetate, leucine, arginine, glibenclamide, isobutylmethylxanthine, and KCl, whereas C3 did not respond (leucine, arginine, and glibenclamide) or responded to a lesser extent (isobutylmethylxanthine, phorbol 12-myristate 13-acetate, and KCl). Although intracellular Ca(2+) rose in response to glucose in B1 but not C3 cells, KCl increased intracellular Ca(2+) in a similar manner in both sublines. GLUT-1, GLUT-2, Kir6.2, and SUR1 expression was not significantly different between B1 and C3 cells, whereas E-cadherin was more abundantly expressed in B1 cells. A more complete list of differentially expressed genes was established by suppression subtractive hybridization and high density (Affymetrix) oligonucleotide microarrays. Genes were clustered according to known or putative function. Those involved in metabolism, intracellular signaling, cytoarchitecture, and cell adhesion are of potential interest. These two sublines should be useful for identification of the genes and mechanisms involved in regulated insulin secretion of the pancreatic beta-cell.

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

    PubMed

    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.

  15. A Putatively Functional Haplotype in the Gene Encoding Transforming Growth Factor Beta-1 as a Potential Biomarker for Radiosensitivity

    SciTech Connect

    Schirmer, Markus A.; Brockmoeller, Juergen; Rave-Fraenk, Margret; Virsik, Patricia; Wilken, Barbara; Kuehnle, Elna; Campean, Radu; Hoffmann, Arne O.; Mueller, Katarina; Goetze, Robert G.; Neumann, Michael; Janke, Joerg H.; Nasser, Fatima; Wolff, Hendrik A.; Ghadimi, B. Michael; Schmidberger, Heinz; Hess, Clemens F.; Christiansen, Hans; Hille, Andrea

    2011-03-01

    Purpose: To determine whether genetic variability in TGFB1 is related to circulating transforming growth factor-{beta}1 (TGF-{beta}1) plasma concentrations after radiotherapy and to radiosensitivity of lymphoid cells. Patients and Methods: Transforming growth factor-{beta}1 plasma concentrations (n = 79) were measured in patients 1 year after radiotherapy and chromosomal aberrations (n = 71) ex vivo before therapy start. Furthermore, TGF-{beta}1 secretion and apoptosis were measured in isolated peripheral blood mononuclear cells of 55 healthy volunteers. These phenotypes were analyzed in relation to five germline polymorphisms in the 5' region of the TGFB1 gene. Because of high linkage disequilibrium, these five polymorphisms reflect frequent genetic variation in this region. A presumed impact of TGF-{beta}1 on DNA damage or repair was measured as micronucleus formation in 30 lymphoblastoid cell lines. Results: We identified a hypofunctional genetic haplotype termed H3 tagging the 5' region of the TGFB1 gene encoding TGF-{beta}1. H3 was associated with lower TGF-{beta}1 plasma concentrations in patients (p = 0.01) and reduced TGF-{beta}1 secretion in irradiated peripheral blood mononuclear cells (p = 0.003). Furthermore, cells with H3 were less prone to induction of chromosomal aberrations (p = 0.001) and apoptosis (p = 0.003) by irradiation. The hypothesis that high TGF-{beta}1 could sensitize cells to DNA damage was further supported by increased micronuclei formation in 30 lymphoblastoid cell lines when preincubated with TGF-{beta}1 before irradiation (p = 0.04). Conclusions: On the basis of TGF-{beta}1 plasma levels and radiation sensitivity of lymphoid cells, this study revealed a putatively hypofunctional TGFB1 haplotype. The significance of this haplotype and the suggested link between TGF-{beta}1 function and DNA integrity should be further explored in other cell types, as well as other experimental and clinical conditions.

  16. Mouse Cmu heavy chain immunoglobulin gene segment contains three intervening sequences separating domains.

    PubMed

    Calame, K; Rogers, J; Early, P; Davis, M; Livant, D; Wall, R; Hood, L

    1980-04-03

    The IgM molecule is composed of subunits made up of two light chain and two heavy chain (mu) polypeptides. The mu chain is encoded by several gene segments--variable (V), joining (J) and constant (Cmu). The Cmu gene segment is of particular interest for several reasons. First, the mu chain must exist in two very different environments--as an integral membrane protein in receptor IgM molecules (micrometer) and as soluble serum protein in IgM molecules into the blood (mus). Second, the Cmu region in mus is composed of four homology units or domains (Cmu1, Cmu2, Cmu3 and Cmu4) of approximately 110 amino acid residues plus a C-terminal tail of 19 residues. We asked two questions concerning the organisation of the Cmu gene segment. (1) Are the homology units separated by intervening DNA sequences as has been reported for alpha (ref. 5), gamma 1 (ref. 6) and gamma 2b (ref. 7) heavy chain genes? (2) Is the C-terminal tail separated from the Cmu4 domain by an intervening DNA sequence? If so, DNA rearrangements or RNA splicing could generate hydrophilic and hydrophobic C-terminal tails for the mus and micrometer polypeptides, respectively. We demonstrate here that intervening DNA sequences separate each of the four coding regions for Cmu domains, and that the coding regions for the Cmu4 domains and the C-terminal tail are directly contiguous.

  17. Characterization of methionine adenosyltransferase 2beta gene expression in skeletal muscle and subcutaneous adipose tissue from obese and lean pigs.

    PubMed

    Fang, Qian; Yin, Jingdong; Li, Fengna; Zhang, Jinxiao; Watford, Malcolm

    2010-06-01

    Methionine adenosyltransferase (MAT) catalyzes the biosynthesis of S-adenosylmethionine. Two genes (MAT1A and MAT2A) encode for the catalytic subunit of MAT, while a third gene (MAT2beta) encodes for a regulatory subunit (MAT II beta) that regulates the activity of the MAT2A-encoded isoenzyme and intracellular S-adenosylmethionine levels. Our previous work identified MAT2beta as a candidate gene for intramuscular fat (IMF) deposition in porcine skeletal muscle by microarray technology. Here, we cloned porcine MAT2beta cDNA and compared its expression pattern in subcutaneous adipose tissue and skeletal muscle from obese (Rongchang Breed) and lean (Pig Improvement Company, PIC) pigs (n = 6). The porcine MAT2beta cDNA was 1,800 bp long and encodes for 334 amino acids sharing high similarity with other species. MAT2beta is expressed at a higher level in liver and duodenum, followed by the stomach, fat and longissinus dorsi muscle. As expected, both subcutaneous fat content and IMF content were higher in obese than in lean pigs (both P < 0.01). MAT2beta mRNA abundance was lower in both subcutaneous adipose tissue and skeletal muscle in obese pigs compared with lean pigs (both P < 0.01). MAT II beta protein content was lower in skeletal muscle in obese than in lean pigs (P < 0.05), whereas the opposite was observed in subcutaneous adipose tissue (P < 0.01). These data demonstrated an obesity-related expression variation of the MAT II beta subunit in skeletal muscle and adipose tissue in pigs, and suggest a novel role for the MAT2beta gene in regulation of IMF deposition in skeletal muscle.

  18. Carbachol ameliorates lipopolysaccharide-induced intestinal epithelial tight junction damage by down-regulating NF-{kappa}{beta} and myosin light-chain kinase pathways

    SciTech Connect

    Zhang, Ying; Li, Jianguo

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer Carbachol reduced the lipopolysaccharide-induced intestinal barrier breakdown. Black-Right-Pointing-Pointer Carbachol ameliorated the lipopolysaccharide-induced ileal tight junction damage. Black-Right-Pointing-Pointer Carbachol prevented the LPS-induced NF-{kappa}{beta} and myosin light-chain kinase activation. Black-Right-Pointing-Pointer Carbachol exerted its beneficial effects in an {alpha}7 nicotinic receptor-dependent manner. -- Abstract: Carbachol is a cholinergic agonist that protects the intestines after trauma or burn injury. The present study determines the beneficial effects of carbachol and the mechanisms by which it ameliorates the lipopolysaccharide (LPS)-induced intestinal barrier breakdown. Rats were injected intraperitoneally with 10 mg/kg LPS. Results showed that the gut barrier permeability was reduced, the ultrastructural disruption of tight junctions (TJs) was prevented, the redistribution of zonula occludens-1 and claudin-2 proteins was partially reversed, and the nuclear factor-kappa beta (NF-{kappa}{beta}) and myosin light-chain kinase (MLCK) activation in the intestinal epithelium were suppressed after carbachol administration in LPS-exposed rats. Pretreatment with the {alpha}7 nicotinic acetylcholine receptor ({alpha}7nAchR) antagonist {alpha}-bungarotoxin blocked the protective action of carbachol. These results suggested that carbachol treatment can protect LPS-induced intestinal barrier dysfunction. Carbachol exerts its beneficial effect on the amelioration of the TJ damage by inhibiting the NF-{kappa}{beta} and MLCK pathways in an {alpha}7nAchR-dependent manner.

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

    PubMed

    Chewapreecha, Claire; Marttinen, Pekka; Croucher, Nicholas J; Salter, Susannah J; Harris, Simon R; Mather, Alison E; Hanage, William P; Goldblatt, David; Nosten, Francois H; Turner, Claudia; Turner, Paul; Bentley, Stephen D; Parkhill, Julian

    2014-08-01

    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.

  20. A pseudodeficiency allele (D152N) of the human {beta}-glucuronidase gene

    SciTech Connect

    Vervoort, R.; Liebaers, I.; Lissens, W.

    1995-10-01

    We present evidence that a 480G{r_arrow}A transition in the coding region of the {Beta}glucuronidase gene, which results in an aspartic-acid-to-asparagine substitution at amino acid position 152 (D152N), produces a pseudodeficiency allele (GUSBp) that leads to greatly reduced levels of {Beta}-glucuronidase activity without apparent deleterious consequences. The 48OG{r_arrow}A mutation was found initially in the pseudodeficient mother of a child with mucopolysaccharidosis VII (MPSVII), but it was not on her disease-causing allele, which carried the L176F mutation. The 480G{r_arrow}A change was also present in an unrelated individual with another MPSVII allele who had unusually low {Beta}-glucuronidase activity, but whose clinical symptoms were probably unrelated to {Beta}-glucuronidase deficiency. This individual also had an R357X mutation, probably on his second allele. We screened 100 unrelated normal individuals for the 480G{r_arrow}A mutation with a PCR method and detected one carrier. Reduced {Beta}-glucuronidase activity following transfection of COS cells with the D152N cDNA supported the causal relationship between the D152N allele and pseudodeficiency. The mutation reduced the fraction of expressed enzyme that was secreted. Pulse-chase experiments indicated that the reduced activity in COS cells was due to accelerated intracellular turnover of the D152N enzyme. They also suggested that a potential glycosylation site created by the mutation is utilized in {approximately}50% of the enzyme expressed. 25 refs., 3 figs., 3 tabs.

  1. Assignment of human {alpha}-synuclein (SNCA) and {beta}-synuclein (SNCB) genes to chromosomes 4q21 and 5q35

    SciTech Connect

    Spillantini, M.G.; Goedert, M.; Divane, A.

    1995-05-20

    The authors previously identified two human brain proteins of 140 and 134 amino acids by virtue of their reactivity with a monoclonal antibody raised against a tangle preparation from Alzheimer disease brain. The 140-amino-acid protein is homologous to synuclein from Torpedo electroplaques and rat brain and identical to the precursor of the non-A{beta} component of Alzheimer disease amyloid plaques. The 134-amino-acid protein is homologous to bovine phosphoneuroprotein 14. In view fo the fact that both proteins are 61% identical in sequence, they have called them {alpha}-synuclein and {beta}-synuclein, respectively. Both proteins are present in nerve terminals, where they may be involved in the events mediating exocytosis of synaptic vesicles following nerve stimulation. They have now determined the chromosomal localizations of the {alpha}-synuclein and {beta}-synuclein genes. To map the two genes analyzed a panel of monochromosomal human-rodent somatic cell hybride by polymerase chain reaction and performed fluorescence in situ hybridization on metaphase spreads f human chromosomes.

  2. Glycosyl chains and 25-hydroxycholesterol contribute to the intracellular transport of amyloid beta (Aβ-42) in Jurkat T cells.

    PubMed

    Sharma, Neha; Baek, KeangOK; Phan, Huong T T; Shimokawa, Naofumi; Takagi, Masahiro

    2017-06-01

    Amyloid beta (Aβ) is a peptide responsible for the development of Alzheimer's disease (AD). Misfolding and accumulation of endogenous Aβ can lead to neural cell apoptosis through endoplasmic reticulum (ER) stress. Added exogenous Aβ can also result in ER stress, leading to neurotoxicity and apoptosis, which is identical to that caused by the endogenous peptide. We have speculated that the endocytic transport of Aβ causes ER stress and have previously shown that the oxysterol, in particular, 7-ketocholesterol (7-keto) induces more surface interaction between Aβ-42 and Jurkat cells than cholesterol. However, the interaction was not enough to induce intracellular transfer of the peptide. In this study, we investigated the effect of another oxysterol, 25-hydroxycholesterol (25-OH) on the membrane raft-dependent transport of Aβ-42 in Jurkat cells. Interestingly, intracellular transfer of Aβ-42 was observed in the presence of 25-OH only after the inclusion of cholera toxin B subunit (CT-B), a marker used to detect the raft domain. We speculated that 25-OH can induce intracellular movement of Aβ peptides. Furthermore, CT-B together with GM1 provided negative curvature, which resulted in the intracellular transport of Aβ-42. Notably, we used a protofibrillar species of Aβ-42 in this study. We have shown that the transport was microtubule-dependent since it could not be observed in depolymerized microtubules. These results demonstrate that oxysterols and glycosyl chains are important factors affecting intracellular transport. These compounds are also associated with aging and advanced glycation are risk factors for AD. Thus, this study should further understanding of the pathology of AD.

  3. Heavy chain transfer by tumor necrosis factor-stimulated gene 6 to the bikunin proteoglycan.

    PubMed

    Lamkin, Elliott; Cheng, Georgiana; Calabro, Anthony; Hascall, Vincent C; Joo, Eun Ji; Li, Lingyun; Linhardt, Robert J; Lauer, Mark E

    2015-02-20

    We present data that hyaluronan (HA) polysaccharides, about 14-86 monosaccharides in length, are capable of accepting only a single heavy chain (HC) from inter-α-inhibitor via transfer by tumor necrosis factor-stimulated gene 6 (TSG-6) and that this transfer is irreversible. We propose that either the sulfate groups (or the sulfation pattern) at the reducing end of the chondroitin sulfate (CS) chain of bikunin, or the core protein itself, enables the bikunin proteoglycan (PG) to accept more than a single HC and permits TSG-6 to transfer these HCs from its relatively small CS chain to HA. To test these hypotheses, we investigated HC transfer to the intact CS chain of the bikunin PG, and to the free chain of bikunin. We observed that both the free CS chain and the intact bikunin PG were only able to accept a single HC from inter-α-inhibitor via transfer by TSG-6 and that HCs could be swapped from the bikunin PG and its free CS chain to HA. Furthermore, a significant portion of the bikunin PG was unable to accept a single heavy chain. We discuss explanations for these observations, including the intracellular assembly of inter-α-inhibitor. In summary, these data demonstrate that the sulfation of the CS chain of bikunin and/or its core protein promote HC transfer by TSG-6 to its relatively short CS chain, although they are insufficient to enable the CS chain of bikunin to accept more than one HC in the absence of other cofactors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Heavy Chain Transfer by Tumor Necrosis Factor-stimulated Gene 6 to the Bikunin Proteoglycan*

    PubMed Central

    Lamkin, Elliott; Cheng, Georgiana; Calabro, Anthony; Hascall, Vincent C.; Joo, Eun Ji; Li, Lingyun; Linhardt, Robert J.; Lauer, Mark E.

    2015-01-01

    We present data that hyaluronan (HA) polysaccharides, about 14–86 monosaccharides in length, are capable of accepting only a single heavy chain (HC) from inter-α-inhibitor via transfer by tumor necrosis factor-stimulated gene 6 (TSG-6) and that this transfer is irreversible. We propose that either the sulfate groups (or the sulfation pattern) at the reducing end of the chondroitin sulfate (CS) chain of bikunin, or the core protein itself, enables the bikunin proteoglycan (PG) to accept more than a single HC and permits TSG-6 to transfer these HCs from its relatively small CS chain to HA. To test these hypotheses, we investigated HC transfer to the intact CS chain of the bikunin PG, and to the free chain of bikunin. We observed that both the free CS chain and the intact bikunin PG were only able to accept a single HC from inter-α-inhibitor via transfer by TSG-6 and that HCs could be swapped from the bikunin PG and its free CS chain to HA. Furthermore, a significant portion of the bikunin PG was unable to accept a single heavy chain. We discuss explanations for these observations, including the intracellular assembly of inter-α-inhibitor. In summary, these data demonstrate that the sulfation of the CS chain of bikunin and/or its core protein promote HC transfer by TSG-6 to its relatively short CS chain, although they are insufficient to enable the CS chain of bikunin to accept more than one HC in the absence of other cofactors. PMID:25561734

  5. Human alcohol dehydrogenase: structural differences between the beta and gamma subunits suggest parallel duplications in isoenzyme evolution and predominant expression of separate gene descendants in livers of different mammals.

    PubMed Central

    Bühler, R; Hempel, J; Kaiser, R; von Wartburg, J P; Vallee, B L; Jörnvall, H

    1984-01-01

    Human alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) occurs in multiple forms, which exhibit distinct electrophoretic mobilities and enzymatic properties. The homogeneous isoenzymes beta 1 beta 1 and gamma 1 gamma 1 were isolated from livers of Caucasians with "typical" ADH phenotype by double ternary complex affinity chromatography and ion exchange chromatography. The differences between the beta 1 and gamma 1 subunits were determined by structural analysis of all tryptic peptides from the carboxymethylated proteins. The human beta 1 and gamma 1 chains differ at 21 of the 373 positions (5.6%). Ten tryptic peptides account for the differences. All residue substitutions are compatible with one-base mutations and result in largely unaltered properties, but five lead to charge differences. Sixteen substitutions are at positions corresponding to the catalytic domain of the well-known horse enzyme; five correspond to the coenzyme-binding domain. Substitutions adjacent to important regions may correlate with differences in coenzyme binding, substrate specificities, and active-site relationships. The residue replacements between the beta 1 and gamma 1 subunits of human ADH are not identical to the known substitutions between ethanol-active (E) and steroid-active (S) subunits of horse ADH. Thus, the duplication leading to human beta 1 and gamma 1 subunits is separate and different from that leading to equine E and S subunits. Both duplications are likely to have occurred after the ancestral separation of human and equine ADH. Of the 21 residues that are different between beta 1/gamma 1, 13 in gamma 1 but only 6 in beta 1 are identical to those of the horse E chain. This suggests a closer relationship between gamma 1 and E, although beta 1 in man and E in the horse are the subunits recovered in highest yield from liver ADH preparations. Consequently, in these two mammalian species, relative activities of genes for an isoenzyme family appear to be

  6. Independent regulatory elements in the upstream region of the Drosophila beta 3 tubulin gene (beta Tub60D) guide expression in the dorsal vessel and the somatic muscles.

    PubMed

    Damm, C; Wolk, A; Buttgereit, D; Löher, K; Wagner, E; Lilly, B; Olson, E N; Hasenpusch-Theil, K; Renkawitz-Pohl, R

    1998-07-01

    The beta 3 tubulin gene (beta Tub60D) is a structural gene expressed during mesoderm development from the extended germ band stage onward. Expression within the individual mesodermal derivatives is guided by different control elements. The upstream regions allow expression in the dorsal vessel and the somatic mesoderm while enhancers localized in the first intron guide expression in the visceral mesoderm. Deletion analysis carried out in transgenic flies revealed independent regulatory elements for the dorsal vessel and the somatic mesoderm. For expression in the somatic mesoderm, a 279-bp region is absolutely essential. This region contains a binding site for the Drosophila myocyte-specific enhancer binding factor 2 (D-MEF2), a MADS-box transcription factor known to be essential for mesoderm development. Deletion or mutation of this D-MEF2 binding site strongly reduces transcription. This pattern is consistent with the strongly reduced expression of beta 3 tubulin in D-mef2 mutant embryos. This analysis furthermore reveals that the D-MEF2 binding site acts in concert with nearby cis regulatory elements. These data show that the upstream control region of the beta 3 tubulin gene is an early target of the D-MEF2 transcriptional activator.

  7. Immunoglobulin heavy chain gene organization and complexity in the skate, Raja erinacea.

    PubMed Central

    Harding, F A; Cohen, N; Litman, G W

    1990-01-01

    Immunoglobulin heavy chain genes from Raja erinacea have been isolated by cross hybridization with probes derived from the immunoglobulin genes