猪链球菌2型次黄嘌呤核苷酸脱氢酶基因的克隆与鉴定

根据GenBank猪链球菌2型(SS2)报道序列,对江苏分离株SS2-H部分测序,发现位于已知毒力基因orf2与mrp之间存在两个新的开放阅读框序列。选取可能含有抗原决定簇肽段对应的核酸序列,该阅读框(2738~3694)编码319个氨基酸残基,分子量为33.5kDa,与已知任何基因无同源性。通过InterPro、PHD、DNAstar分析阅读框,并定向克隆至pET-32α( )载体中,转化至大肠杆菌BL21,表达出分子量为48kDa的融合蛋白,蛋白免疫转印可被SS2的抗血清识别,具有免疫原性;并且含有IMP dehydrogenase结构域,催化IMP生成XMP;流式细胞仪检测该蛋白可明显影响HEp-2细胞周期。     

Innate immunity functional gene polymorphisms and gout susceptibility

Gout is a common autoinflammatory disease characterized with elevated serum urate and recurrent attacks of intra-articular crystal deposition of monosodium urate. Accumulating evidence has demonstrated that MSU crystal-induced inflammation is a paradigm of innate immunity and the TLRs, NALP3 inflammasome and IL1R pathways are involved in gout development. Innate immunity components containing TLR2, TLR4, CD14, NALP3, ASC, Caspase-1 and CARD-8 are essential in the development of gouty inflammation. Recent studies suggest that innate immunity component gene functional mutations contribute to the development of autoinflammatory diseases including hereditary periodic fever syndrome, arthritis as well as inflammatory bowel disease. Taking into account these genetic findings, we would like to propose a novel hypothesis that the gene functional mutations might make innate immunity components as attractive susceptibility candidates and genetic markers for gout. Further clinical genetic studies need to be performed to confirm the role of innate immunity in the etiology of gout.     

CD1 gene polymorphisms and phenotypic variability in X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is characterized by marked phenotypic variation ranging from adrenomyeloneuropathy (AMN) to childhood cerebral ALD (CCALD). X-ALD is caused by mutations in the ABCD1 gene, but no genotype-phenotype correlation has been established so far and modifier gene variants are suspected to modulate phenotypes. Specific classes of lipids, enriched in very long-chain fatty acids that accumulate in plasma and tissues from X-ALD patients are suspected to be involved in the neuroinflammatory process of CCALD. CD1 proteins are lipid- antigen presenting molecules encoded by five CD1 genes in human (CD1A-E). Association studies with 23 tag SNPs covering the CD1 locus was performed in 52 patients with AMN and 87 patients with CCALD. The minor allele of rs973742 located 4-kb downstream from CD1D was significantly more frequent in AMN patients (χ² = 7.6; P = 0.006). However, this association was no longer significant after Bonferroni correction for multiple testing. The other polymorphisms of the CD1 locus did not reveal significant association. Further analysis of other CD1D polymorphisms did not detect stronger association with X-ALD phenotypes. Although the association with rs973742 warrants further investigations, these results indicate that the genetic variants of CD1 genes do not contribute markedly to the phenotypic variance of X-ALD.     

Bacterial IMPDH gene used for the selection of mammalian cell transfectants

Stable cell transfection is used for the expression of exogenous genes or cDNAs in eukaryotic cells. Selection of these transfectants requires a dominant selectable marker. A variety of such markers has been identified and is currently in use. However, many of these are not suitable for all cell types or require unique conditions. Here we describe a simple and versatile dominant selectable marker that involves bacterial IMP dehydrogenase (IMPDH), an enzyme essential for the replication of mammalian and bacterial cells. Although IMPDH is evolutionarily conserved, the bacterial enzyme is orders of magnitude more resistant to the toxic effect of the drug mycophenolic acid, which is an IMPDH inhibitor. We have demonstrated that transfection of human, monkey or Chinese hamster cell lines with an expression vector containing bacterial IMPDH and mycophenolic acid treatment resulted in the selection of colonies with a strikingly increased resistance to mycophenolic acid toxicity. Analysis of cells derived from these colonies indicated that the acquisition of this resistance was associated with bacterial IMPDH protein expression. As a proof of principle, we showed that mammalian cell transfection with a bicistronic IMPDH/GFP expression vector and mycophenolic acid treatment can be used to successfully select transfectants that express the fluorescent protein. These results indicate that bacterial IMPDH is a practical dominant selectable marker that can be used for the selection of transfectants that express exogenous genes or cDNAs in mammalian cells.     

A gene affecting liver activities of three glycosidases in the house mouse

A gene locus is described controlling liver activities in the house mouse of three glycosidases, i.e., -galactosidase, -glucuronidase, and N-acetyl--hexosaminidase. An allele conferring low activity is present in the inbred strain LIS/A, and an allele for high activity is present in A/Br Af mice. The three enzyme activities are correlated with each other. The possible linkage between this gene and the Bgs locus on chromosome 9 is discussed.     

Role of cyclooxygenase-2 functional gene polymorphisms in Helicobacter pylori induced gastritis and gastric atrophy

In India, the role of host genetic factors is poorly studied for Helicobacter pylori associated diseases. Therefore, we evaluated the association of functionally relevant COX-2 gene polymorphisms (-765 G>C and +8473 T>C) in gastritis and precancerous lesions susceptibility. After upper GI endoscopy, 130 rapid urease test positive patients with non-ulcer dyspepsia, also showed positivity for H. pylori using modified Geimsa staining and anti-CagA IgG serology were included. All patients and 260 asymptomatic controls were genotyped for COX-2 variations using PCR-RFLP. COX-2 -765 (GC+CC) genotypes, -765 C allele, +8473 CC genotype, +8473 (TC+CC) genotypes, +8473 C allele, and variant haplotypes imparted high risk for gastritis (P = 0.036, OR = 1.82; P = 0.007, 1.92; P = 0.025, OR = 2.13; P = 0.017, OR = 1.80; P = 0.017, OR = 1.45; P = 0.010, OR = 2.40; P = 0.023, OR = 1.50 and P = 0.012, OR = 2.20 folds, respectively). In contrast, COX-2 -765 C allele carriers had low risk for lymphocyte (P = 0.020, OR = 0.35), plasma cell infiltrations (P = 0.016, OR = 0.33), and gastric atrophy (GA) development (P = 0.019, OR = 0.35). In conclusion, COX-2 variant allele/genotype/haplotype carriers may be at high risk for gastritis. However, COX-2 -765 C allele carriers may be at low risk for GA development.     

Comparative modeling of marsupial MHC class I molecules identifies structural polymorphisms affecting functional motifs

Major histocompatibility complex (MHC) class I molecules are transmembrane glycoproteins that present antigenic peptides to CD8+ T cells and are subsequently important for the initiation of an immune response. In this study novel MHC class I sequences from the tammar wallaby (Macropus eugenii) have been characterized. Analysis and comparative modeling of these and existing marsupial molecules reveals potential functional polymorphisms within peptide-binding grooves, MHC assembly motifs and the T cell receptor recognition interface. In addition, we show that a previously identified marsupial-specific insertion is within a region, which is known as a putative NK cell receptor (Ly49A) binding site in the mouse, suggesting that this site may be functionally active in marsupials. Further, the analysis highlighted differences in structural and sequence based grouping of marsupial MHC class I molecules.     

Von Willebrand factor gene polymorphisms in three Brazilian ethnic groups

Von Willebrand factor gene polymorphisms were studied in three Brazilian ethnic groups: Euro-Brazilians, Afro-Brazilians, and Amerindians. Six polymorphic sites were analyzed: RsaI (exon 18), NlaIV (exon 20), HphI, KpnI, D1472H, and V1565L (all four in exon 28). The allele frequencies were significantly different between Euro- and Afro-Brazilians for RsaI, HphI, D1472H, and V1565L, while in Amerindians NlaIV and HphI showed significant differences among tribes. This is the first report of these allele frequencies in Amerindians. Eighteen haplotypes were observed, and they showed significant differences between Euro- and Afro-Brazilians, among Amerindian tribes, and among the three ethnic groups. These results furnish important background data for evolutionary and anthropological investigations; in addition, they will be useful for establishing the origin and molecular characterization of the different forms of von Willebrand disease, as well as for detecting carriers and offering genetic counseling to those with this condition.     

Sequence variability of three alleles of the self-incompatibility gene of Nicotiana alata.

Three alleles of the self-incompatibility gene of Nicotiana alata have been cloned and sequenced. A comparison of the sequences shows a surprisingly low level of homology (56%) and the presence of defined regions of homology and variability. The homologous regions include the N-terminal sequence, most of the cysteine residues and glycosylation sites, as well as other blocks throughout the sequence. We interpret these conserved regions as "framework" and nonconserved regions as "hypervariable," following the terminology used to describe analogous regions in the IgG supergene family. The low level of overall homology forms the basis of a general method for isolating S-allele-specific cDNAs. Allele-specific antibodies can be generated using synthetic peptides corresponding to one of the variable regions. When applied to sections of the pistil, these antibodies label the intercellular matrix in the stigma and transmitting tissue of the style and the cell walls in the epidermis of the placenta. HindIII digestion of genomic DNA generates a characteristic pattern of S-gene fragments for each genotype. These restriction fragment length polymorphisms can be used to assign S-genotype to progeny arising from breeding experiments.     

Haplotype variability and identification of new functional alleles at the Rdg2a leaf stripe resistance gene locus

The barley Rdg2a locus confers resistance to the leaf stripe pathogen Pyrenophora graminea and, in the barley genotype Thibaut, it is composed of a gene family with three highly similar paralogs. Only one member of the gene family (called as Rdg2a) encoding for a CC-NB-LRR protein is able to confer resistance to the leaf stripe isolate Dg2. To study the genome evolution and diversity at the Rdg2a locus, sequences spanning the Rdg2a gene were compared in two barley cultivars, Thibaut and Morex, respectively, resistant and susceptible to leaf stripe. An overall high level of sequence conservation interrupted by several rearrangements that included three main deletions was observed in the Morex contig. The main deletion of 13,692 bp was most likely derived from unequal crossing over between Rdg2a paralogs leading to the generation of a chimeric Morex rdg2a gene which was not associated to detectable level of resistance toward leaf stripe. PCR-based analyses of genic and intergenic regions at the Rdg2a locus in 29 H. vulgare lines and one H. vulgare ssp. spontaneum accession indicated large haplotype variability in the cultivated barley gene pool suggesting rapid and recent divergence at this locus. Barley genotypes showing the same haplotype as Thibaut at the Rdg2a locus were selected for a Rdg2a allele mining through allele re-sequencing and two lines with polymorphic nucleotides leading to amino acid changes in the CC-NB and LRR encoding domains, respectively, were identified. Analysis of nucleotide diversity of the Rdg2a alleles revealed that the polymorphic sites were subjected to positive selection. Moreover, strong positively selected sites were located in the LRR encoding domain suggesting that both positive selection and divergence at homologous loci are possibly representing the molecular mechanism for the generation of high diversity at the Rdg2a locus in the barley gene pool.     

Computational refinement of functional single nucleotide polymorphisms associated with ATM gene

Background

Understanding and predicting molecular basis of disease is one of the major challenges in modern biology and medicine. SNPs associated with complex disorders can create, destroy, or modify protein coding sites. Single amino acid substitutions in the ATM gene are the most common forms of genetic variations that account for various forms of cancer. However, the extent to which SNPs interferes with the gene regulation and affects cancer susceptibility remains largely unknown.

Principal findings

We analyzed the deleterious nsSNPs associated with ATM gene based on different computational methods. An integrative scoring system and sequence conservation of amino acid residues was adapted for a priori nsSNP analysis of variants associated with cancer. We further extended our approach on SNPs that could potentially influence protein Post Translational Modifications in ATM gene.

Significance

In the lack of adequate prior reports on the possible deleterious effects of nsSNPs, we have systematically analyzed and characterized the functional variants in both coding and non coding region that can alter the expression and function of ATM gene. In silico characterization of nsSNPs affecting ATM gene function can aid in better understanding of genetic differences in disease susceptibility.     

Rapid genotyping of three polymorphisms in the LBP gene using a triplex pyrosequencing approach

We described a triplex polymerase chain reaction (PCR) and triplex pyrosequencing assay which allowed a simultaneous determination of three tag single nuleotide polymorphisms (tag SNPs) in the lipopolysaccharide-binding protein (LBP) gene: rs1780623, rs11536972 and rs2232618. This method enables a fast and cost-effective genotyping and a simultaneous determination of the three tag SNPs.     

Single-nucleotide polymorphisms of the Trypanosoma cruzi MSH2 gene support the existence of three phylogenetic lineages presenting differences in mismatch-repair efficiency

We have identified single-nucleotide polymorphisms (SNPs) in the mismatch-repair gene TcMSH2 from Trypanosoma cruzi. Phylogenetic inferences based on the SNPs, confirmed by RFLP analysis of 32 strains, showed three distinct haplogroups, denominated A, B, and C. Haplogroups A and C presented strong identity with the previously described T. cruzi lineages I and II, respectively. A third haplogroup (B) was composed of strains presenting hybrid characteristics. All strains from a haplogroup encoded the same specific protein isoform, called, respectively, TcMHS2a, TcMHS2b, and TcMHS2c. The classification into haplogroups A, B, and C correlated with variation in the efficiency of mismatch repair in these cells. When microsatellite loci of strains representative of each haplogroup were analyzed after being cultured in the presence of hydrogen peroxide, new microsatellite alleles were definitely seen in haplogroups B and C, while no evidence of microsatellite instability was found in haplogroup A. Also, cells from haplogroups B and C were considerably more resistant to cisplatin treatment, a characteristic known to be conferred by deficiency of mismatch repair in eukaryotic cells. Altogether, our data suggest that strains belonging to haplogroups B and C may have decreased mismatch-repair ability when compared with strains assigned to the haplogroup A lineage.     

Inter-ethnic differences in genetic polymorphisms of xenobiotic-metabolizing enzymes (CYP1A1, CYP2D6, NAT1 and NAT2) in healthy populations: correlation with the functional in silico prediction

Several studies have shown that many polymorphisms of the xenobiotic-metabolizing enzymes (XME) affect either enzymatic functions or are associated with various aspects of human health. Owing to the presence of these single nucleotide variants (SNVs), differences in detoxification capacity have been observed between many ethnicities. The aim of this investigation was to study the prevalence of four polymorphisms in XME among various ethnic groups. Attention was focused on polymorphisms of CYP2D6 (rs1058172, G>A, p.Arg365His), CYP1A1 (rs4646421, c.-26-728C>T), NAT1 (rs4921880, c.-85-1014T>A) and NAT2 (rs1208, A>G, p.Arg268Lys). These polymorphisms were analyzed in 261 healthy Tunisians individuals in comparison with different ethnic backgrounds from hapmap database. In addition, in silico functional prediction was also performed to determine the loss of function variants. Our results demonstrated that population’s origins widely affect the genetic variability of XME enzymes and Tunisians show a characteristic pattern. In silico predictions showed a deleterious effect for p.Arg268Lys substitution on CYP2D6 function, findings confirmed its key role played in cancer susceptibility. These data show that detoxification genes structures depend on the studied population. This suggests that ethnic differences impact on disease risk or response to drugs and therefore should be taken into consideration in genetic association studies focusing on XME enzymes. Our results provide the first report on these SNV in Tunisian population and could be useful for further epidemiological investigations including targeted therapy.     

Association of functional polymorphisms in the MxA gene with susceptibility to enterovirus 71 infection

Myxovirus resistance A (MxA) is an antiviral protein induced by type I interferons α and β (IFN-α and IFN-β) that can inhibit virus replication. We examined whether the MxA polymorphisms were related to the risk and severity of enterovirus 71 (EV71) infection in Chinese populations. The MxA C-123A and G-88T polymorphisms were genotyped in two independent case–control populations in China by polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) analysis. Multivariate logistic regression analysis was used to calculate adjusted odds ratios (ORs) and 95 % confidence intervals (95 % CIs). MxA messenger RNA was quantified by real-time quantitative PCR in peripheral blood mononuclear cells (PBMCs) from 45 healthy children and 19 patients with EV71 infection. Significantly decreased susceptibility to EV71 infection was observed for the -123A allele and -88T allele carriers, with ORs (95 % CIs) estimated as 0.56 (0.39–0.81) and 0.64 (0.47–0.88), respectively, in the northern population. This association was confirmed in the southern population, with ORs (95 % CIs) estimated as 0.58 (0.38–0.89) and 0.67(0.47–0.95), respectively. The A_{- 123}T_{- 88} haplotype was also significantly associated with lower risk of EV71 infection in both the northern (OR = 0.62; 95 % CI = 0.44–0.85) and the southern population (OR = 0.63; 95 % CI = 0.43–0.92). Furthermore, we observed higher MxA messenger RNA levels in IFNβ1a-stimulated PBMCs from the -123A or -88T allele carriers compared with that from nocarriers. Our findings suggest that polymorphisms in the MxA promoter may play a role in mediating the susceptibility to EV71 infection in Chinese population.     

Use of three DNA polymorphisms of the LDL receptor gene in the diagnosis of familial hypercholesterolemia

Summary Familial hypercholesterolemia (FH) is an autosomal dominant metabolic disorder caused by several different mutations in the low density lipoprotein (LDL) receptor gene. This large number of different mutations, often undetectable in Southern blotting, makes it impossible directly to diagnose the disease. However, restriction fragment length polymorphisms (RFLPs) can be used to follow the inheritance of the defective gene in FH families. In the present study, we report the use of three RFLPs, detected by PvuII, ApaLI and AvaII restriction enzymes, to determine the haplotypes of normal and defective LDL receptor genes in 61 families with FH and in 128 normal individuals. Two of the nine haplotypes were significantly more often associated with the affected genes, whereas one was significantly less frequent. Although none of the associations was strong enough to allow diagnosis in individuals, it was possible, using the three RFLPs, to identify the haplotype of the affected gene in 57 families and to carry out unequivocal diagnosis in 67% of the cases. In four families, PvuII and AvaII detected an abnormal fragment co-segregating with the disease, thus increasing the percentage of diagnosis to 73.4% of the cases.     

Impact of single-nucleotide polymorphisms at the TP53-binding and responsive promoter region of BCL2 gene in modulating the phenotypic variability of LGMD2C patients

Apoptosis of skeletal muscle fibers is a well-known event occurring in patients suffering from muscular dystrophies. In this study, we hypothesized that functional polymorphisms in genes involved in the mitochondrial apoptotic pathway might modulate the apoptotic capacity underlying the muscle loss and contributing to intrafamilial and interfamilial variable phenotypes in LGMD2C (Limb Girdle Muscular Dystrophy type 2C) patients sharing the same c.521delT mutation in SGCG gene. Detection of apoptosis was confirmed on muscle biopsies taken from LGMD2C patients using the TUNEL method. We genotyped then ten potentially functional SNPs in TP53, BCL-2 and BAX genes involved in the mitochondrial apoptotic pathway. Potential genotype-dependent Bcl-2 and p53 protein expressed in skeletal muscle was investigated using western blot and ELISA assays. The result showed that muscle cells carrying the TP53-R72R and TP53-16?bp del/del genotypes displayed an increased p53 level which could be more effective in inducing apoptosis by activation of the pro-apoptotic gene expression. In addition, the BCL2-938 AA genotype was associated with increased Bcl-2 protein expression in muscle from LGMD2C patients compared to -938CC genotype, while there was no evidence of significant difference in the BAX haplotype. Our findings suggest that increased Bcl-2 protein expression may counteract pro-apoptotic pathways and thus reduce the muscle loss. To the best of our knowledge, this is a pioneer study evaluating the role of apoptotic BCL-2 and TP53 genes in contributing to the phenotypic manifestation of c.521delT mutation in LGMD2C patients. Larger studies are needed to validate these findings.