975 resultados para major histocompatibility complex gene


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Transcription of the Bacillus anthracis structural genes for the anthrax toxin proteins and biosynthetic operon for capsule are positively regulated by AtxA, a transcription regulator with unique properties. Consistent with the role of atxA in virulence factor expression, a B. anthracis atxA-null mutant is avirulent in a murine model for anthrax. In batch culture, multiple signals impact atxA transcript levels, and the timing and steady state level of atxA expression is critical for optimal toxin and capsule synthesis. Despite the apparent complex control of atxA transcription, only one trans-acting protein, the transition state regulator AbrB, has been demonstrated to directly interact with the atxA promoter. The AbrB-binding site has been described, but additional cis-acting control sequences have not been defined. Using transcriptional lacZ fusions, electrophoretic mobility shift assays, and Western blot analysis, the cis-acting elements and trans-acting factors involved in regulation of atxA in B. anthracis strains containing either both virulence plasmids, pXO1 and pXO2, or only one plasmid, pXO1, were studied. This work demonstrates that atxA transcription from the major start site P1 is dependent upon a consensus sequence for the housekeeping sigma factor SigA, and an A+T-rich upstream element (UP-element) for RNA polymerase (RNAP). In addition, the data show that a trans-acting protein(s) other than AbrB negatively impacts atxA transcription when it binds specifically to a 9-bp palindrome within atxA promoter sequences located downstream of P1. Mutation of the palindrome prevents binding of the trans-acting protein(s) and results in a corresponding increase in AtxA and anthrax toxin production in a strain- and culture-dependent manner. The identity of the trans-acting repressor protein(s) remains elusive; however, phenotypes associated with mutation of the repressor binding site have revealed that the trans-acting repressor protein(s) indirectly controls B. anthracis development. Mutation of the repressor binding site results in misregulation and overexpression of AtxA in conditions conducive for development, leading to a marked sporulation defect that is both atxA- and pXO2-61-dependent. pXO2-61 is homologous to the sensor domain of sporulation sensor histidine kinases and is proposed to titrate an activating signal away from the sporulation phosphorelay when overexpressed by AtxA. These results indicate that AtxA is not only a master virulence regulator, but also a modulator of proper B. anthracis development. Also demonstrated in this work is the impact of the developmental regulators AbrB, Spo0A, and SigH on atxA expression and anthrax toxin production in a genetically incomplete (pXO1+, pXO2-) and genetically complete (pXO1+, pXO2+) strain background. AtxA and anthrax toxin production resulting from deletion of the developmental regulators are strain-dependent suggesting that factors on pXO2 are involved in control of atxA. The only developmental deletion mutant that resulted in a prominent and consistent strain-independent increase in AtxA protein levels was an abrB-null mutant. As a result of increased AtxA levels, there is early and increased production of anthrax toxins in an abrB-null mutant. In addition, the abrB-null mutant exhibited an increase in virulence in a murine model for anthrax. In contrast, virulence of the atxA promoter mutant was unaffected in a murine model for anthrax despite the production of 5-fold more AtxA than the abrB-null mutant. These results imply that AtxA is not the only factor impacting pathogenesis in an abrB-null mutant. Overall, this work highlights the complex regulatory network that governs expression of atxA and provides an additional role for AtxA in B. anthracis development.

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My dissertation focuses on developing methods for gene-gene/environment interactions and imprinting effect detections for human complex diseases and quantitative traits. It includes three sections: (1) generalizing the Natural and Orthogonal interaction (NOIA) model for the coding technique originally developed for gene-gene (GxG) interaction and also to reduced models; (2) developing a novel statistical approach that allows for modeling gene-environment (GxE) interactions influencing disease risk, and (3) developing a statistical approach for modeling genetic variants displaying parent-of-origin effects (POEs), such as imprinting. In the past decade, genetic researchers have identified a large number of causal variants for human genetic diseases and traits by single-locus analysis, and interaction has now become a hot topic in the effort to search for the complex network between multiple genes or environmental exposures contributing to the outcome. Epistasis, also known as gene-gene interaction is the departure from additive genetic effects from several genes to a trait, which means that the same alleles of one gene could display different genetic effects under different genetic backgrounds. In this study, we propose to implement the NOIA model for association studies along with interaction for human complex traits and diseases. We compare the performance of the new statistical models we developed and the usual functional model by both simulation study and real data analysis. Both simulation and real data analysis revealed higher power of the NOIA GxG interaction model for detecting both main genetic effects and interaction effects. Through application on a melanoma dataset, we confirmed the previously identified significant regions for melanoma risk at 15q13.1, 16q24.3 and 9p21.3. We also identified potential interactions with these significant regions that contribute to melanoma risk. Based on the NOIA model, we developed a novel statistical approach that allows us to model effects from a genetic factor and binary environmental exposure that are jointly influencing disease risk. Both simulation and real data analyses revealed higher power of the NOIA model for detecting both main genetic effects and interaction effects for both quantitative and binary traits. We also found that estimates of the parameters from logistic regression for binary traits are no longer statistically uncorrelated under the alternative model when there is an association. Applying our novel approach to a lung cancer dataset, we confirmed four SNPs in 5p15 and 15q25 region to be significantly associated with lung cancer risk in Caucasians population: rs2736100, rs402710, rs16969968 and rs8034191. We also validated that rs16969968 and rs8034191 in 15q25 region are significantly interacting with smoking in Caucasian population. Our approach identified the potential interactions of SNP rs2256543 in 6p21 with smoking on contributing to lung cancer risk. Genetic imprinting is the most well-known cause for parent-of-origin effect (POE) whereby a gene is differentially expressed depending on the parental origin of the same alleles. Genetic imprinting affects several human disorders, including diabetes, breast cancer, alcoholism, and obesity. This phenomenon has been shown to be important for normal embryonic development in mammals. Traditional association approaches ignore this important genetic phenomenon. In this study, we propose a NOIA framework for a single locus association study that estimates both main allelic effects and POEs. We develop statistical (Stat-POE) and functional (Func-POE) models, and demonstrate conditions for orthogonality of the Stat-POE model. We conducted simulations for both quantitative and qualitative traits to evaluate the performance of the statistical and functional models with different levels of POEs. Our results showed that the newly proposed Stat-POE model, which ensures orthogonality of variance components if Hardy-Weinberg Equilibrium (HWE) or equal minor and major allele frequencies is satisfied, had greater power for detecting the main allelic additive effect than a Func-POE model, which codes according to allelic substitutions, for both quantitative and qualitative traits. The power for detecting the POE was the same for the Stat-POE and Func-POE models under HWE for quantitative traits.

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Increasingly, studies of genes and genomes are indicating that considerable horizontal transfer has occurred between prokaryotes. Extensive horizontal transfer has occurred for operational genes (those involved in housekeeping), whereas informational genes (those involved in transcription, translation, and related processes) are seldomly horizontally transferred. Through phylogenetic analysis of six complete prokaryotic genomes and the identification of 312 sets of orthologous genes present in all six genomes, we tested two theories describing the temporal flow of horizontal transfer. We show that operational genes have been horizontally transferred continuously since the divergence of the prokaryotes, rather than having been exchanged in one, or a few, massive events that occurred early in the evolution of prokaryotes. In agreement with earlier studies, we found that differences in rates of evolution between operational and informational genes are minimal, suggesting that factors other than rate of evolution are responsible for the observed differences in horizontal transfer. We propose that a major factor in the more frequent horizontal transfer of operational genes is that informational genes are typically members of large, complex systems, whereas operational genes are not, thereby making horizontal transfer of informational gene products less probable (the complexity hypothesis).

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During αβ thymocyte development, clonotype-independent CD3 complexes are expressed at the cell surface before the pre-T cell receptor (TCR). Signaling through clonotype-independent CD3 complexes is required for expression of rearranged TCRβ genes. On expression of a TCRβ polypeptide chain, the pre-TCR is assembled, and TCRβ locus allelic exclusion is established. We investigated the putative contribution of clonotype-independent CD3 complex signaling to TCRβ locus allelic exclusion in mice single-deficient or double-deficient for CD3ζ/η and/or p56lck. These mice display defects in the expression of endogenous TCRβ genes in immature thymocytes, proportional to the severity of CD3 complex malfunction. Exclusion of endogenous TCRβ VDJ (variable, diversity, joining) rearrangements by a functional TCRβ transgene was severely compromised in the single-deficient and double-deficient mutant mice. In contrast to wild-type mice, most of the CD25+ double-negative (DN) thymocytes of the mutant mice failed to express the TCRβ transgene, suggesting defective expression of the TCRβ transgene similar to endogenous TCRβ genes. In the mutant mice, a proportion of CD25+ DN thymocytes that failed to express the transgene expressed endogenous TCRβ polypeptide chains. Many double-positive cells of the mutant mice coexpressed endogenous and transgenic TCRβ chains or more than one endogenous TCRβ chain. The data suggest that signaling through clonotype-independent CD3 complexes may contribute to allelic exclusion of the TCRβ locus by inducing the expression of rearranged TCRβ genes in CD25+ DN thymocytes.

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Polymers of N-substituted glycines (“peptoids”) containing chiral centers at the α position of their side chains can form stable structures in solution. We studied a prototypical peptoid, consisting of five para-substituted (S)-N-(1-phenylethyl)glycine residues, by NMR spectroscopy. Multiple configurational isomers were observed, but because of extensive signal overlap, only the major isomer containing all cis-amide bonds was examined in detail. The NMR data for this molecule, in conjunction with previous CD spectroscopic results, indicate that the major species in methanol is a right-handed helix with cis-amide bonds. The periodicity of the helix is three residues per turn, with a pitch of ≈6 Å. This conformation is similar to that anticipated by computational studies of a chiral peptoid octamer. The helical repeat orients the amide bond chromophores in a manner consistent with the intensity of the CD signal exhibited by this molecule. Many other chiral polypeptoids have similar CD spectra, suggesting that a whole family of peptoids containing chiral side chains is capable of adopting this secondary structure motif. Taken together, our experimental and theoretical studies of the structural properties of chiral peptoids lay the groundwork for the rational design of more complex polypeptoid molecules, with a variety of applications, ranging from nanostructures to nonviral gene delivery systems.

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Mice, homozygous for disrupted ganglioside GM2/GD2 synthase (EC 2.4.1.94) gene and lacking all complex gangliosides, do not display any major neurologic abnormalities. Further examination of these mutant mice, however, revealed that the males were sterile and aspermatogenic. In the seminiferous tubules of the mutant mice, a number of multinuclear giant cells and vacuolated Sertoli cells were observed. The levels of testosterone in the serum of these mice were very low, although testosterone production equaled that produced in wild-type mice. Testosterone was found to be accumulated in interstitial Leydig cells, and intratesticularly injected testosterone was poorly drained in seminiferous fluid in the mutant mice. These results suggested that complex gangliosides are essential in the transport of testosterone to the seminiferous tubules and bloodstream from Leydig cells. Our results provide insights into roles of gangliosides in vivo.

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Mutations of von Hippel–Lindau disease (VHL) tumor-suppressor gene product (pVHL) are found in patients with dominant inherited VHL syndrome and in the vast majority of sporadic clear cell renal carcinomas. The function of the pVHL protein has not been clarified. pVHL has been shown to form a complex with elongin B and elongin C (VBC) and with cullin (CUL)-2. In light of the structural analogy of VBC-CUL-2 to SKP1-CUL-1-F-box ubiquitin ligases, the ubiquitin ligase activity of VBC-CUL-2 was examined in this study. We show that VBC-CUL-2 exhibits ubiquitin ligase activity, and we identified UbcH5a, b, and c, but not CDC34, as the ubiquitin-conjugating enzymes of the VBC-CUL-2 ubiquitin ligase. The protein Rbx1/ROC1 enhances ligase activity of VBC-CUL-2 as it does in the SKP1-CUL-1-F-box protein ligase complex. We also found that pVHL associates with two proteins, p100 and p220, which migrate at a similar molecular weight as two major bands in the ubiquitination assay. Furthermore, naturally occurring pVHL missense mutations, including mutants capable of forming a complex with elongin B–elongin C-CUL-2, fail to associate with p100 and p220 and cannot exhibit the E3 ligase activity. These results suggest that pVHL might be the substrate recognition subunit of the VBC-CUL-2 E3 ligase. This is also, to our knowledge, the first example of a human tumor-suppressor protein being directly involved in the ubiquitin conjugation system which leads to the targeted degradation of substrate proteins.

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One-third of humans are infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. Sequence analysis of two megabases in 26 structural genes or loci in strains recovered globally discovered a striking reduction of silent nucleotide substitutions compared with other human bacterial pathogens. The lack of neutral mutations in structural genes indicates that M. tuberculosis is evolutionarily young and has recently spread globally. Species diversity is largely caused by rapidly evolving insertion sequences, which means that mobile element movement is a fundamental process generating genomic variation in this pathogen. Three genetic groups of M. tuberculosis were identified based on two polymorphisms that occur at high frequency in the genes encoding catalase-peroxidase and the A subunit of gyrase. Group 1 organisms are evolutionarily old and allied with M. bovis, the cause of bovine tuberculosis. A subset of several distinct insertion sequence IS6110 subtypes of this genetic group have IS6110 integrated at the identical chromosomal insertion site, located between dnaA and dnaN in the region containing the origin of replication. Remarkably, study of ≈6,000 isolates from patients in Houston and the New York City area discovered that 47 of 48 relatively large case clusters were caused by genotypic group 1 and 2 but not group 3 organisms. The observation that the newly emergent group 3 organisms are associated with sporadic rather than clustered cases suggests that the pathogen is evolving toward a state of reduced transmissability or virulence.

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The plastid genomes of some nonphotosynthetic parasitic plants have experienced an extreme reduction in gene content and an increase in evolutionary rate of remaining genes. Nothing is known of the dynamics of these events or whether either is a direct outcome of the loss of photosynthesis. The parasitic Scrophulariaceae and Orobanchaceae, representing a continuum of heterotrophic ability ranging from photosynthetic hemiparasites to nonphotosynthetic holoparasites, are used to investigate these issues. We present a phylogenetic hypothesis for parasitic Scrophulariaceae and Orobanchaceae based on sequences of the plastid gene rps2, encoding the S2 subunit of the plastid ribosome. Parasitic Scrophulariaceae and Orobanchaceae form a monophyletic group in which parasitism can be inferred to have evolved once. Holoparasitism has evolved independently at least five times, with certain holoparasitic lineages representing single species, genera, and collections of nonphotosynthetic genera. Evolutionary loss of the photosynthetic gene rbcL is limited to a subset of holoparasitic lineages, with several holoparasites retaining a full length rbcL sequence. In contrast, the translational gene rps2 is retained in all plants investigated but has experienced rate accelerations in several hemi- as well as holoparasitic lineages, suggesting that there may be substantial molecular evolutionary changes to the plastid genome of parasites before the loss of photosynthesis. Independent patterns of synonymous and nonsynonymous rate acceleration in rps2 point to distinct mechanisms underlying rate variation in different lineages. Parasitic Scrophulariaceae (including the traditional Orobanchaceae) provide a rich platform for the investigation of molecular evolutionary process, gene function, and the evolution of parasitism.

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The conserved organization of the Hox genes throughout the animal kingdom has become one of the major paradigms of evolutionary developmental biology. We have examined the organization of the Hox genes of the grasshopper, Schistocerca gregaria. We find that the grasshopper Hox cluster is over 700 kb long, and is not split into equivalents of the Antennapedia complex and the bithorax complex of Drosophila melanogaster. SgDax and probably also Sgzen, the grasshopper homologues of fushi-tarazu (ftz) and Zerknüllt (zen), respectively, are also in the cluster, showing that the non-homeotic Antp-class genes, “accessory genes,” are an ancient feature of insect Hox clusters.

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Asthma is a complex heritable inflammatory disorder of the airways associated with clinical signs of atopy and bronchial hyperresponsiveness. Recent studies localized a major gene for asthma to chromosome 5q31-q33 in humans. Thus, this segment of the genome represents a candidate region for genes that determine susceptibility to bronchial hyperresponsiveness and atopy in animal models. Homologs of candidate genes on human chromosome 5q31-q33 are found in four regions in the mouse genome, two on chromosome 18, and one each on chromosomes 11 and 13. We assessed bronchial responsiveness as a quantitative trait in mice and found it linked to chromosome 13. Interleukin 9 (IL-9) is located in the linked region and was analyzed as a gene candidate. The expression of IL-9 was markedly reduced in bronchial hyporesponsive mice, and the level of expression was determined by sequences within the qualitative trait locus (QTL). These data suggest a role for IL-9 in the complex pathogenesis of bronchial hyperresponsiveness as a risk factor for asthma.

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The region of human chromosome 22q11 is prone to rearrangements. The resulting chromosomal abnormalities are involved in Velo-cardio-facial and DiGeorge syndromes (VCFS and DGS) (deletions), “cat eye” syndrome (duplications), and certain types of tumors (translocations). As a prelude to the development of mouse models for VCFS/DGS by generating targeted deletions in the mouse genome, we examined the organization of genes from human chromosome 22q11 in the mouse. Using genetic linkage analysis and detailed physical mapping, we show that genes from a relatively small region of human 22q11 are distributed on three mouse chromosomes (MMU6, MMU10, and MMU16). Furthermore, although the region corresponding to about 2.5 megabases of the VCFS/DGS critical region is located on mouse chromosome 16, the relative organization of the region is quite different from that in humans. Our results show that the instability of the 22q11 region is not restricted to humans but may have been present throughout evolution. The results also underscore the importance of detailed comparative mapping of genes in mice and humans as a prerequisite for the development of mouse models of human diseases involving chromosomal rearrangements.

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The unc-52 gene encodes the nematode homologue of mammalian perlecan, the major heparan sulfate proteoglycan of the extracellular matrix. This is a large complex protein with regions similar to low-density lipoprotein receptors, laminin, and neural cell adhesion molecules (NCAMs). In this study, we extend our earlier work and demonstrate that a number of complex isoforms of this protein are expressed through alternative splicing. We identified three major classes of perlecan isoforms: a short form lacking the NCAM region and the C-terminal agrin-like region; a medium form containing the NCAM region, but still lacking the agrin-like region; and a newly identified long form that contains all five domains present in mammalian perlecan.  Using region-specific antibodies and unc-52 mutants, we reveal a complex spatial and temporal expression pattern for these UNC-52 isoforms. As well, using a series of mutations affecting different regions and thus different isoforms of UNC-52, we demonstrate that the medium NCAM-containing isoforms are sufficient for myofilament lattice assembly in developing nematode body-wall muscle. Neither short isoforms nor isoforms containing the C-terminal agrin-like region are essential for sarcomere assembly or muscle cell attachment, and their role in development remains unclear.

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B-type cyclins are rapidly degraded at the transition between metaphase and anaphase and their ubiquitin-mediated proteolysis is required for cells to exit mitosis. We used a novel enrichment to isolate new budding mutants that arrest the cell cycle in mitosis. Most of these mutants lie in the CDC16, CDC23, and CDC27 genes, which have already been shown to play a role in cyclin proteolysis and encode components of a 20S complex (called the cyclosome or anaphase promoting complex) that ubiquitinates mitotic cyclins. We show that mutations in CDC26 and a novel gene, DOC1, also prevent mitotic cyclin proteolysis. Mutants in either gene arrest as large budded cells with high levels of the major mitotic cyclin (Clb2) protein at 37°C and cannot degrade Clb2 in G1-arrested cells. Cdc26 associates in vivo with Doc1, Cdc16, Cdc23, and Cdc27. In addition, the majority of Doc1 cosediments at 20S with Cdc27 in a sucrose gradient, indicating that Cdc26 and Doc1 are components of the anaphase promoting complex.

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Previously, we established that natural killer (NK) cells from C57BL/6 (B6), but not BALB/c, mice lysed Chinese hamster ovary (CHO) cells, and we mapped the locus that determines this differential CHO-killing capacity to the NK gene complex on chromosome 6. The localization of Chok in the NK gene complex suggested that it may encode either an activating or an inhibitory receptor. Here, results from a lectin-facilitated lysis assay predicted that Chok is an activating B6 NK receptor. Therefore, we immunized BALB/c mice with NK cells from BALB.B6–Cmv1r congenic mice and generated a mAb, designated 4E4, that blocked B6-mediated CHO lysis. mAb 4E4 also redirected lysis of Daudi targets, indicating its reactivity with an activating NK cell receptor. Furthermore, only the 4E4+ B6 NK cell subset mediated CHO killing, and this lysis was abrogated by preincubation with mAb 4E4. Flow cytometric analysis indicated that mAb 4E4 specifically reacts with Ly-49D but not Ly-49A, B, C, E, G, H, or I transfectants. Finally, gene transfer of Ly-49DB6 into BALB/c NK cells conferred cytotoxic capacity against CHO cells, thus establishing that the Ly-49D receptor is sufficient to activate NK cells to lyse this target. Hence, Ly-49D is the Chok gene product and is a mouse NK cell receptor capable of directly triggering natural killing.