981 resultados para Gene Locus Pum


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Intrathymic T-cell development requires temporally regulated rearrangement and expression of T-cell receptor (TCR) genes. To assess the role of the TCR beta gene transcriptional enhancer (Ebeta) in this process, mouse strains in which Ebeta is deleted were generated using homologous recombination techniques. We report that mice homozygous for the Ebeta deletion, whether a selectable marker gene is present or not, show a block in alphabeta T-cell development at the CD4-CD8- double-negative cell stage, whereas the number of gammadelta+ T cells is normal, few CD4+CD8+ double-positive thymocytes and no alphabeta+ T cells are produced. DNA-PCR and RNA-PCR analyses of thymic cells from homozygous mutants showed no evidence of TCR beta gene rearrangement although germ-line Vbeta transcripts were detected at a low level, in heterozygous T cells, the targeted allele is not rearranged. Thus, deletion of Ebeta totally prevents rearrangement, but not transcription, of the targeted beta locus. These data formally establish the critical role played by Ebeta in cis-activation of the TCR beta locus for V(D)J recombination during alphabeta T-cell development.

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The core oligosaccharide Glc3Man9GlcNAc2 is assembled at the membrane of the endoplasmic reticulum on the lipid carrier dolichyl pyrophosphate and transferred to selected asparagine residues of nascent polypeptide chains. This transfer is catalyzed by the oligosaccharyl transferase complex. Based on the synthetic phenotype of the oligosaccharyl transferase mutation wbp1 in combination with a deficiency in the assembly pathway of the oligosaccharide in Saccharomyces cerevisiae, we have identified the novel ALG9 gene. We conclude that this locus encodes a putative mannosyl transferase because deletion of the gene led to accumulation of lipid-linked Man6GlcNAc2 in vivo and to hypoglycosylation of secreted proteins. Using an approach combining genetic and biochemical techniques, we show that the assembly of the lipid-linked core oligosaccharide in the lumen of the endoplasmic reticulum occurs in a stepwise fashion.

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Analyses of the human PAX-5 locus and of the 5' region of the mouse Pax-5 gene revealed that transcription from two distinct promoters results in splicing of two alternative 5' exons to the common coding sequences of exons 2-10. Transcription from the upstream promoter initiates downstream of a TATA box and occurs predominantly in B-lymphocytes, whereas the TATA-less downstream promoter is active in all Pax-5-expressing tissues. The human PAX-5 gene is located on chromosome 9 in region p13, which is involved in t(9;14)(pl3;q32) translocations recurring in small lymphocytic lymphomas of the plasmacytoid subtype and in derived large-cell lymphomas. A previous molecular analysis of a t(9;14) breakpoint from a diffuse large-cell lymphoma (KIS-1) demonstrated that the immunoglobulin heavy-chain (IgH) locus on 14q32 was juxtaposed to chromosome 9p13 sequences of unknown function [Ohno, H., Furukawa, T., Fukuhara, S., Zong, S. Q., Kamesaki, H., Shows, T. B., Le Beau, M. M., McKeithan, T. W., Kawakami, T. & Honjo, T. (1990) Proc. Natl. Acad. Sci. USA 87,628-632]. Here we show that the KIS-1 translocation breakpoint is located 1807 base pairs upstream of exon 1A of PAX-5, thus bringing the potent Emu enhancer of the IgH gene into close proximity of the PAX-5 promoters. These data suggest that deregulation of PAX-5 gene transcription by the t(9;14)(pl3;q32) translocation contributes to the pathogenesis of small lymphocytic lymphomas with plasmacytoid differentiation.

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Genomic mapping has been used to identify a region of the host genome that determines resistance to fusiform rust disease in loblolly pine where no discrete, simply inherited resistance factors had been previously found by conventional genetic analysis over four decades. A resistance locus, behaving as a single dominant gene, was mapped by association with genetic markers, even though the disease phenotype deviated from the expected Mendelian ratio. The complexity of forest pathosystems and the limitations of genetic analysis, based solely on phenotype, had led to an assumption that effective long-term disease resistance in trees should be polygenic. However, our data show that effective long-term resistance can be obtained from a single qualitative resistance gene, despite the presence of virulence in the pathogen population. Therefore, disease resistance in this endemic coevolved forest pathosystem is not exclusively polygenic. Genomic mapping now provides a powerful tool for characterizing the genetic basis of host pathogen interactions in forest trees and other undomesticated, organisms, where conventional genetic analysis often is limited or not feasible.

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The A subunit of DNA gyrase in Mycobacterium leprae, unlike its counterpart in Mycobacterium tuberculosis, is produced by protein splicing as its gene, gyrA, harbors a 1260-bp in-frame insertion encoding an intein, a putative homing endonuclease. Analysis of the gyrA locus from different mycobacterial species revealed the presence of inteins in Mycobacterium flavescens, Mycobacterium gordonae and Mycobacterium kansasii but not in 10 other pathogenic or saprophytic mycobacteria. In all four cases where intein coding sequences were found, they were localized in the same position in gyrA, immediately downstream of the codon for the key active-site residue Tyr-130. The intein products were similar, but not identical, in sequence and the splice junctions displayed all the features found in other polypeptides known to be produced by protein splicing from a precursor protein. Paired motifs, found in homing endonucleases encoded by some group I RNA introns, and inteins showing endonuclease activity, were present in the gyrA inteins as were other intein-specific signatures. Some strains of M. flavescens, M. gordonae, and M. kansasii were shown by PCR analysis to have inteinless gyrA genes, in contrast to the situation in M. leprae where all the isolates possessed insertions in gyrA. Sequencing of the corresponding regions revealed that, although the GyrA protein sequence was conserved, the nucleotide sequences differed in gyrA genes with and without inteins, suggesting that the homing endonuclease displays sequence specificity.

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A family of proteins involved in cell cycle progression, DNA recombination, and the detection of DNA damage has been recently identified. One of the members of this family, human ATM, is defective in the cells of patients with ataxia telangiectasia and is involved in detection and response of cells to damaged DNA. Other members include Mei-41 (Drosophila melanogaster), Mec1p (Saccharomyces cerevisiae), and Rad3 (Schizosaccharomyces pombe), which are required for the S and G2/M checkpoints, as well as FRAP (Homo sapiens) and Torl/2p (S. cerevisiae), which are involved in a rapamycin-sensitive pathway leading to G1 cell cycle progression. We report here the cloning of a human cDNA encoding a protein with significant homology to members of this family. Three overlapping clones isolated from a Jurkat T-cell cDNA library revealed a 7.9-kb open reading frame encoding a protein that we have named FRP1 (FRAP-related protein) with 2644 amino acids and a predicted molecular mass of 301 kDa. Using fluorescence in situ hybridization and a full-length cDNA FRP1 clone, the FRP1 gene has been mapped to the chromosomal locus 3q22-q24. FRP1 is most closely related to three of the PIK-related kinase family members involved in checkpoint function--Mei-41, Mec1p, and Rad3--and as such may be the functional human counterpart of these proteins.

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Parental origin-specific alterations of chromosome 11p15 in human cancer suggest the involvement of one or more maternally expressed imprinted genes involved in embryonal tumor suppression and the cancer-predisposing Beckwith-Wiedemann syndrome (BWS). The gene encoding cyclin-dependent kinase inhibitor p57KIP2, whose overexpression causes G1 phase arrest, was recently cloned and mapped to this band. We find that the p57KIP2 gene is imprinted, with preferential expression of the maternal allele. However, the imprint is not absolute, as the paternal allele is also expressed at low levels in most tissues, and at levels comparable to the maternal allele in fetal brain and some embryonal tumors. The biochemical function, chromosomal location, and imprinting of the p57KIP2 gene match the properties predicted for a tumor suppressor gene at 11p15.5. However, as the p57KIP2 gene is 500 kb centromeric to the gene encoding insulin-like growth factor 2, it is likely to be part of a large domain containing other imprinted genes. Thus, loss of heterozygosity or loss of imprinting might simultaneously affect several genes at this locus that together contribute to tumor and/or growth- suppressing functions that are disrupted in BWS and embryonal tumors.

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To facilitate large-scale genotype analysis, an efficient PCR-based multiplex approach has been developed. For simultaneously amplifying the target sequences at a large number of genetic loci, locus-specific primers containing 5' universal tails are used. Attaching the universal tails to the target sequences in the initial PCR steps allows replacement of all specific primers with a pair of primers identical to the universal tails and converts the multiplex amplification into "uniplex." Simultaneous amplification of 26 genetic loci with this approach is described. The multiplex amplification can be coupled with genotype determination. By incorporating a single-base mismatch between a primer and the template into the target sequences, a polymorphic site can be converted into a desirable restriction fragment length polymorphism when it is necessary. In this way, the allelic PCR products for the polymorphic loci can be discriminated by gel electrophoresis after restriction enzyme digestion. In this study, 32 loci were typed in such a multiplex way.

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Mapping the insertion points of 16 signature-tagged transposon mutants on the Salmonella typhimurium chromosome led to the identification of a 40-kb virulence gene cluster at minute 30.7. This locus is conserved among all other Salmonella species examined but is not present in a variety of other pathogenic bacteria or in Escherichia coli K-12. Nucleotide sequencing of a portion of this locus revealed 11 open reading frames whose predicted proteins encode components of a type III secretion system. To distinguish between this and the type III secretion system encoded by the inv/spa invasion locus known to reside on a pathogenicity island, we refer to the inv/spa locus as Salmonella pathogenicity island (SPI) 1 and the new locus as SPI2. SPI2 has a lower G+C content than that of the remainder of the Salmonella genome and is flanked by genes whose products share greater than 90% identity with those of the E. coli ydhE and pykF genes. Thus SPI2 was probably acquired horizontally by insertion into a region corresponding to that between the ydhE and pykF genes of E. coli. Virulence studies of SPI2 mutants have shown them to be attenuated by at least five orders of magnitude compared with the wild-type strain after oral or intraperitoneal inoculation of mice.

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An experimental strategy to facilitate correction of single-base mutations of episomal targets in mammalian cells has been developed. The method utilizes a chimeric oligonucleotide composed of a contiguous stretch of RNA and DNA residues in a duplex conformation with double hairpin caps on the ends. The RNA/DNA sequence is designed to align with the sequence of the mutant locus and to contain the desired nucleotide change. Activity of the chimeric molecule in targeted correction was tested in a model system in which the aim was to correct a point mutation in the gene encoding the human liver/bone/kidney alkaline phosphatase. When the chimeric molecule was introduced into cells containing the mutant gene on an extrachromosomal plasmid, correction of the point mutation was accomplished with a frequency approaching 30%. These results extend the usefulness of the oligonucleotide-based gene targeting approaches by increasing specific targeting frequency. This strategy should enable the design of antiviral agents.

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The pre-T-cell receptor, composed of the T-cell receptor (TCR) beta chain (TCRbeta), pre-Talpha (pTalpha) chain, and CD3 molecules, has been postulated to be a transducer of signals during the early stages of T-cell development. To examine the function of the transmembrane pTalpha chain during tbymocyte development, we generated pTalpha-/- embryonic stem cells and assayed their ability to differentiate into lymphoid cells in vivo after injection into recombination-activating gene (RAG)-2-deficient blastocysts. Thymocytes representing all stages of T-cell differentiation were detected in the thymus of pTalpha-/- chimeric mice, indicating that thymocyte development can occur without pTalpha. However, greatly reduced thymocyte numbers and substantially increased percentages of both CD4-CD8- thymocytes and TCRgammadelta+ thymocytes suggest that pTalpha plays a critical role in thymocyte expansion. To investigate the role of the pTalpha chain in allelic exclusion at the TCRbeta locus, a functionally rearranged TCRbeta minigene was introduced into pTalpha-/- and pTalpha+/- embryonic stem cells, which were subsequently assayed by RAG-2-deficient blastocyst complementation. In the absence of pTalpha, expression of the transgenic TCRbeta inhibited rearrangement of the endogenous TCRbeta locus to an extent similar to that seen in normal TCRbeta transgenic mice, suggesting that pTalpha may not be required for signaling allelic exclusion at the TCRbeta locus.

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The genome of the pufferfish (Fugu rubripes) (400 Mb) is approximately 7.5 times smaller than the human genome, but it has a similar gene repertoire to that of man. If regions of the two genomes exhibited conservation of gene order (i.e., were syntenic), it should be possible to reduce dramatically the effort required for identification of candidate genes in human disease loci by sequencing syntenic regions of the compact Fugu genome. We have demonstrated that three genes (dihydrolipoamide succinyltransferase, S31iii125, and S20i15), which are linked to FOS in the familial Alzheimer disease focus (AD3) on human chromosome 14, have homologues in the Fugu genome adjacent to Fugu cFOS. The relative gene order of cFOS, S31iii125, and S20i15 was the same in both genomes, but in Fugu these three genes lay within a 12.4-kb region, compared to >600 kb in the human AD3 locus. These results demonstrate the conservation of synteny between the genomes of Fugu and man and highlight the utility of this approach for sequence-based identification of genes in human disease loci.

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To probe genetic variation in the regulation of sexual dimorphism, we have characterized the mouse protein Slp, coded by the gene sex-limited protein (Slp). Slp expression in many strains is limited to males and is androgen-dependent. However, female expression is also observed in rare strains, due to nonlinked gene(s) termed regulator of sex-limitation (rsl). In this report we demonstrate that female expression of Slp results from homozygous recessive allele(s) at a single autosomal locus that maps to a 2.2-centimorgan interval on chromosome 13. This conclusion was supported by extensive genetic analyses including the use of polymorphic microsatellites to type numerous backcross progeny and a recombinant inbred series and to identify the congenic interval in three independently derived congenic strains. Four attractive candidate genes were identified by the localization of rsl. Interestingly, rsl was found not only to enable expression in females but to also increase expression in males. The findings suggest that the expression of Slp and perhaps other sexually dimorphic proteins is regulated by two pathways, one that is dependent upon rsl but not androgens and another that is rsl-independent but requires androgens.

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High-density mutational spectra have been established for exon 3 of the gene encoding adenine phosphoribosyltransferase (APRT) of the Chinese hamster ovary (CHO) cell line derivative D422 and closely related and/or modified lines by using the mutagen ethyl methanesulfonate (EMS). The total number of selectable sites (GC-->AT transitions yielding a selectable APRT- phenotype) was estimated at 31 based on our own accumulated data base of 136 sequenced exon 3 mutations and on literature reports. D422 and two other APRT hemizygous lines each yielded very similar spectra and showed two populations of mutable sites: (i) 24 "baseline" sites that followed the Poisson distribution and therefore were equally susceptible to mutation and (ii) two hotspots, one comprising a cluster at nucleotides 1293-1309 and the other at nucleotide 1365. Collectively, the latter sites were about 10-fold more frequently mutated than the others. CHO cells are mer- as they lack the repair enzyme O6-methylguanidine methyltransferase (EC 2.1.1.63). In modified repair-proficient CHO cells, the distribution of mutations among all of the 31 sites was random, with only 3 of the 19 GC-->AT transitions in the above hotspots. To determine whether the distribution was locus-dependent, two independent lines carrying single copies of transfected APRT genes were generated from a derivative of D422 carrying a deletion in the endogenous APRT gene. Nucleotides 1293-1309 were again no longer preferentially mutated, but the site at nucleotide 1365 was still a hotspot. We conclude that mutational spectra in mer- cells are at least in part locus dependent and that some sequences are particularly susceptible to EMS mutagenesis and perhaps also to methyltransferase repair.

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UDP-N-acetylgalactosamine (GalNAc): polypeptide N-acetylgalactosaminyltransferase (polypeptide GalNAc-T) catalyzes transfer of the monosaccharide GalNAc to serine and threonine residues, thereby initiating O-linked oligosaccharide biosynthesis. Previous studies have suggested the possibility of multiple polypeptide GalNAc-Ts, although attachment of saccharide units to polypeptide or lipid in generating oligosaccharide structures in vertebrates has been dependent upon the activity of single gene products. To address this issue and to determine the relevance of Oglycosylation variation in T-cell ontogeny, we have directed Cre/loxP mutagenic recombination to the polypeptide GalNAc-T locus in gene-targeted mice. Resulting deletion in the catalytic region of polypeptide GalNAc-T occurred to completion on both alleles in thymocytes and was found in peripheral T cells, but not among other cell types. Thymocyte O-linked oligosaccharide formation persisted in the absence of a functional targeted polypeptide GalNAc-T allele as determined by O-glycan-specific lectin binding. T-cell development and colonization of secondary lymphoid organs were also normal. These results indicate a complexity in vertebrate O-glycan biosynthesis that involves multiple polypeptide GalNAc-Ts. We infer the potential for protein-specific O-glycan formation governed by distinct polypeptide GalNAc-Ts.