粪便样品中大肠杆菌多态性分子研究

以粪便样品中分离到的大肠杆菌为研究对象,比较了3种不同方法在分离鉴定大肠杆菌过程中的应用。首先,通过传统方法从粪便样品中分离,筛选和确定了一批大肠杆菌疑似菌株,再用现代分子生物学方法对待鉴定的大肠杆菌疑似菌株,已知大肠杆菌MG1655以及几种其它细菌进行ARDRA(AmplifiedRibosomalDNARestrictionAnalysis)分析,最后利用ERIC-PCR技术在个体水平上分析菌株的多样性。结果表明,所有由传统方法确定的大肠杆菌疑似菌株和MG1655都属于同一ARDRA型,并与其它细菌的ARDRA条码型不同。这说明ARDRA分析得到的结果与传统分析方法的结果吻合,利用ARDRA分析可以区分大肠杆菌和其它肠道细菌。但是在本实验中ARDRA分析不能反映大肠杆菌中不同菌株之间的多样性,ERIC-PCR则可以区分它们。     

Polymorphism and selection of rpoS in pathogenic Escherichia coli

Background  

Though RpoS is important for survival of pathogenic Escherichia coli in natural environments, polymorphism in the rpoS gene is common. However, the causes of this polymorphism and consequential physiological effects on gene expression in pathogenic strains are not fully understood.     

Genotyping Cryptosporidium parvum with an hsp70 Single-Nucleotide Polymorphism Microarray

We investigated the application of an oligonucleotide microarray to (i) specifically detect Cryptosporidium spp., (ii) differentiate between closely related C. parvum isolates and Cryptosporidium species, and (iii) differentiate between principle genotypes known to infect humans. A microarray of 68 capture probes targeting seven single-nucleotide polymorphisms (SNPs) within a 190-bp region of the hsp70 gene of Cryptosporidium parvum was constructed. Labeled hsp70 targets were generated by PCR with biotin- or Cy3-labeled primers. Hybridization conditions were optimized for hybridization time, temperature, and salt concentration. Two genotype I C. parvum isolates (TU502 and UG502), two C. parvum genotype II isolates (Iowa and GCH1), and DNAs from 22 non-Cryptosporidium sp. organisms were used to test method specificity. Only DNAs from C. parvum isolates produced labeled amplicons that could be hybridized to and detected on the array. Hybridization patterns between genotypes were visually distinct, but identification of SNPs required statistical analysis of the signal intensity data. The results indicated that correct mismatch discrimination could be achieved for all seven SNPs for the UG502 isolate, five of seven SNPs for the TU502 isolate, and six of seven SNPs for both the Iowa and GCH1 isolates. Even without perfect mismatch discrimination, the microarray method unambiguously distinguished between genotype I and genotype II isolates and demonstrated the potential to differentiate between other isolates and species on a single microarray. This method may provide a powerful new tool for water utilities and public health officials for assessing point and nonpoint source contamination of water supplies.     

Polymorphism of genes encoding SOS polymerases in natural populations of Escherichia coli

High fidelity replicative DNA polymerases can be blocked during DNA replication by various base damages, which represents a potentially lethal event. Escherichia coli possesses three DNA polymerases, PolII, PolIV and PolV, that can continue replication over such lesions in template DNA, thus allowing for cell survival. Genes coding for these enzymes, polB, dinB, and umuCD respectively, belong to the stress-inducible SOS regulon. We have analyzed the patterns of nucleotide sequence variability of genes encoding for three SOS polymerases from E. coli natural isolates in order to identify the nature of selective forces that determine their evolution. The frequency of inferred inter-strain recombination events, and the frequency of synonymous and non-synonymous base substitutions within these genes do not deviate significantly from those observed for the control group composed of 2 genes coding for DNA polymerases PolI and PolIII and 10 metabolic genes. This suggests that the loci coding for SOS polymerases are subject to selective pressure for the maintenance of their function and specificity. The fact that genes coding for translesion-synthesis (TLS) polymerases, particularly dinB and umuC homologs, have been conserved during evolution and the present analysis suggest that their activity is essential for the cellular survival and fitness.     

Polymorphism in the dgt-dapD-tsf region of Escherichia coli K-12 strains.

Restriction analysis of the dapD region cloned from several strains of Escherichia coli, revealed a restriction-fragment length polymorphism (RFLP). This RFLP, which includes the BamHI, EcoRI and SalI sites, may be useful in classification of various E. coli strains.     

A High-Density Single-Nucleotide Polymorphism Map of Xq25–q28

A high-density single-nucleotide polymorphism (SNP) map was developed for Xq25–q28 using a targeted approach to SNP discovery. This high-density map includes 217 new SNP markers, and 117 are informative in the CEPH parent population with >20% minor allele frequency. The average distance between SNP markers is 100 kb in the targeted regions. This is the densest genetic map of Xq25–q28 to date. The SNP markers are presented in order by their distance in megabases along the X chromosome, and the markers from the current genetic map are placed using the same scale to produce an integrated map of the region.     

qpure: A Tool to Estimate Tumor Cellularity from Genome-Wide Single-Nucleotide Polymorphism Profiles

Tumour cellularity, the relative proportion of tumour and normal cells in a sample, affects the sensitivity of mutation detection, copy number analysis, cancer gene expression and methylation profiling. Tumour cellularity is traditionally estimated by pathological review of sectioned specimens; however this method is both subjective and prone to error due to heterogeneity within lesions and cellularity differences between the sample viewed during pathological review and tissue used for research purposes. In this paper we describe a statistical model to estimate tumour cellularity from SNP array profiles of paired tumour and normal samples using shifts in SNP allele frequency at regions of loss of heterozygosity (LOH) in the tumour. We also provide qpure, a software implementation of the method. Our experiments showed that there is a medium correlation 0.42 (-value = 0.0001) between tumor cellularity estimated by qpure and pathology review. Interestingly there is a high correlation 0.87 (-value 2.2e-16) between cellularity estimates by qpure and deep Ion Torrent sequencing of known somatic KRAS mutations; and a weaker correlation 0.32 (-value = 0.004) between IonTorrent sequencing and pathology review. This suggests that qpure may be a more accurate predictor of tumour cellularity than pathology review. qpure can be downloaded from https://sourceforge.net/projects/qpure/.     

Single-Nucleotide Polymorphism Alleles in the Insulin Receptor Gene Are Associated with Typical Migraine

We have identified a migraine locus on chromosome 19p13.3/2 using linkage and association analysis. We isolated 48 single-nucleotide polymorphisms within the locus, of which we genotyped 24 in a Caucasian population comprising 827 unrelated cases and 765 controls. Five single-nucleotide polymorphisms within the insulin receptor gene showed significant association with migraine. This association was independently replicated in a case-control population collected separately. We used experiments with insulin receptor RNA and protein to investigate functionality for the migraine-associated single-nucleotide polymorphisms. We suggest possible functions for the insulin receptor in migraine pathogenesis.     

Methods for Human Demographic Inference Using Haplotype Patterns From Genomewide Single-Nucleotide Polymorphism Data

We propose a novel approximate-likelihood method to fit demographic models to human genomewide single-nucleotide polymorphism (SNP) data. We divide the genome into windows of constant genetic map width and then tabulate the number of distinct haplotypes and the frequency of the most common haplotype for each window. We summarize the data by the genomewide joint distribution of these two statistics—termed the HCN statistic. Coalescent simulations are used to generate the expected HCN statistic for different demographic parameters. The HCN statistic provides additional information for disentangling complex demography beyond statistics based on single-SNP frequencies. Application of our method to simulated data shows it can reliably infer parameters from growth and bottleneck models, even in the presence of recombination hotspots when properly modeled. We also examined how practical problems with genomewide data sets, such as errors in the genetic map, haplotype phase uncertainty, and SNP ascertainment bias, affect our method. Several modifications of our method served to make it robust to these problems. We have applied our method to data collected by Perlegen Sciences and find evidence for a severe population size reduction in northwestern Europe starting 32,500–47,500 years ago.     

Long-Term Experimental Evolution in Escherichia coli. VIII. Dynamics of a Balanced Polymorphism

We describe the short- and long-term dynamics of a phenotypic polymorphism that arose in a population of Escherichia coli while it was serially propagated for almost 20,000 generations in a glucose-limited minimal medium. The two types, designated L and S, differ conspicuously in the size of the colonies they form on agar plates as well as the size of their individual cells, and these differences are heritable. The S type reached a detectable frequency (>1%) at generation 6,000, and it remained above that frequency throughout the subsequent generations. In addition to morphological differences, L and S diverged in important ecological properties. With clones isolated at 18,000 generations, L has a maximal growth rate in fresh medium that is approximately 20% higher than that of S. However, experiments with conditioned media demonstrate that L and S secrete one or more metabolites that promote the growth of S but not of L. The death rate of L during stationary phase also increases when S is abundant, which suggests that S may either secrete a metabolite that is toxic to L or remove some factor that enables the survival of L. One-day competition experiments with the clones isolated at generation 18,000 show that their relative fitness is frequency dependent, with each type having an advantage when rare. When these two types are grown together for a period of several weeks, they converge on an equilibrium frequency that is consistent with the 1-d competition experiments. Over the entire 14,000-generation period of coexistence, however, the frequency of the S type fluctuated between approximately 10% and 85%. We offer several hypotheses that may explain the fluctuations in this balanced polymorphism, including the possibility of coevolution between the two types.     

A Single-Nucleotide Polymorphism of Human Neuropeptide S Gene Originated from Europe Shows Decreased Bioactivity

Using accumulating SNP (Single-Nucleotide Polymorphism) data, we performed a genome-wide search for polypeptide hormone ligands showing changes in the mature regions to elucidate genotype/phenotype diversity among various human populations. Neuropeptide S (NPS), a brain peptide hormone highly conserved in vertebrates, has diverse physiological effects on anxiety, fear, hyperactivity, food intake, and sleeping time through its cognate receptor-NPSR. Here, we report a SNP rs4751440 (L⁶-NPS) causing non-synonymous substitution on the 6^th position (V to L) of the NPS mature peptide region. L⁶-NPS has a higher allele frequency in Europeans than other populations and probably originated from European ancestors ∼25,000 yrs ago based on haplotype analysis and Approximate Bayesian Computation. Functional analyses indicate that L⁶-NPS exhibits a significant lower bioactivity than the wild type NPS, with ∼20-fold higher EC₅₀ values in the stimulation of NPSR. Additional evolutionary and mutagenesis studies further demonstrate the importance of the valine residue in the 6^th position for NPS functions. Given the known physiological roles of NPS receptor in inflammatory bowel diseases, asthma pathogenesis, macrophage immune responses, and brain functions, our study provides the basis to elucidate NPS evolution and signaling diversity among human populations.     

High-Throughput Multiplex Single-Nucleotide Polymorphism (SNP) Analysis in Genes Involved in Methionine Metabolism

Hyperhomocysteinemia is a well-known independent marker factor for atherothrombotic diseases and may result from acquired and genetic influences. Several polymorphisms are suspected to be associated with hyperhomocysteinemia, but data are limited and inconsistent. High-throughput genotyping technologies, such as GenomeLab SNPStream, are now available. Moreover, an appropriate selection of SNPs to be analyzed could represent a strong resource to define the role of genetic risk factors. We developed a multiplex PCR-oligonucleotide extension approach by GenomeLab platform. We selected 72 SNPs based on their putative function and frequency in the candidate genes AHCY, BHMT, BHMT2, CBS, ENOSF1, FOLH1, MTHFD1, MTHFR, MTR, MTRR, NNMT, PON1, PON2, SLC19A1, SHMT1, TCN2, and TYMS. We were able to analyze 57 of the SNPs (79%). For MTHFR C677T and A1298C and MTR A2756G SNPs, we compared data obtained with an electronic microchip technology and found 99.2% concordance. We also performed a haplotype analysis. This approach could represent a useful tool to investigate the genotype-phenotype correlation and the association of these genes with hyperhomocysteinemia and correlated diseases.     

Long-Term Experimental Evolution in Escherichia coli. XIII. Phylogenetic History of a Balanced Polymorphism

We investigated the phylogenetic history of a balanced polymorphism that evolved in an experimental population of Escherichia coli. Previous work showed that two ecologically and morphologically distinct types, designated L (large) and S (small), arose by generation 6000 and coexisted for more than 12,000 generations thereafter. Here, we performed RFLP analyses using Insertion Sequence elements to resolve the phylogenetic history of L and S. Specifically, we sought to determine whether the derived S morph was monophyletic, indicating a long history of coexistence with L or, alternatively, S was repeatedly regenerated from L, indicating a series of periods with only transiently stable coexistence. Phylogenetic analysis of some 200 clones collected throughout the history of this population demonstrates that S is monophyletic. We then performed competition assays using clones of both morphs from different generations to determine whether either or both lineages continued to undergo genetic adaptation. Indeed, both lineages continued to adapt, and their continued evolution contributed to fluctuations in their relative abundance over evolutionary time. Based on their phylogenetic history and independent evolutionary trajectories, S and L fulfill Cohan’s criteria for being different asexual species.[Reviewing Editor: Niles Lehman]     

Genetic Typing of Shiga Toxin 2 Variants of Escherichia coli by PCR-Restriction Fragment Length Polymorphism Analysis

Shiga toxins Stx1 and Stx2 play a prominent role in the pathogenesis of Shiga toxin-producing Escherichia coli (STEC) infections. Several variants of the stx₂ gene, encoding Stx2, have been described. In this study, we developed a PCR-restriction fragment length polymorphism system for typing stx₂ genes of STEC strains. The typing system discriminates eight described variants and allows the identification of new stx₂ variants and STEC isolates carrying multiple stx₂ genes. A phylogenetic tree, based on the nucleotide sequences of the toxin-encoding genes, demonstrates that stx₂ sequences with the same PvuII HaeIII HincII AccI type generally cluster together.     

Association Between Single-Nucleotide Polymorphism in CISH Gene and Susceptibility to Tuberculosis in Chinese Han Population

The cytokine-inducible SRC homology 2 domain (CISH) gene is up-regulated by IL-2 in response to infection, and inhibits microbial infection. The objective of the present study was to examine whether genetic variants of CISH (SNPs) are associated with increased susceptibility to tuberculosis (TB) in individuals of Chinese Han ethnicity. We sequenced five previously identified SNPs of CISH in patients with TB or healthy controls. Three of the SNPs, rs148685070 [position ?639; C/C], rs414171 [position ?292; A/T], and rs6768300 [position ?163; C/G]) are located in the promoter region, while the fourth (rs2239751 [position +1320; A/C]) near the translation start site, and the fifth (rs622502 [position +3415; C/G]) in the third intron. The AA genotypes of the SNPs rs2239751 and rs414171 were significantly associated with TB. Multivariate logistic regression analysis demonstrated that subjects with the rs414171 AA genotype were more likely to have TB than those with the AT genotype. By contrast, we did not observe genetic variants of the rs148685070 SNP. In conclusion, two genetic variants in CISH gene appear to increase susceptibility to TB in Chinese Han population.     

A Transmission/Disequilibrium Test That Allows for Genotyping Errors in the Analysis of Single-Nucleotide Polymorphism Data

The present study assesses the effects of genotyping errors on the type I error rate of a particular transmission/disequilibrium test (TDT(std)), which assumes that data are errorless, and introduces a new transmission/disequilibrium test (TDT(ae)) that allows for random genotyping errors. We evaluate the type I error rate and power of the TDT(ae) under a variety of simulations and perform a power comparison between the TDT(std) and the TDT(ae), for errorless data. Both the TDT(std) and the TDT(ae) statistics are computed as two times a log-likelihood difference, and both are asymptotically distributed as chi(2) with 1 df. Genotype data for trios are simulated under a null hypothesis and under an alternative (power) hypothesis. For each simulation, errors are introduced randomly via a computer algorithm with different probabilities (called "allelic error rates"). The TDT(std) statistic is computed on all trios that show Mendelian consistency, whereas the TDT(ae) statistic is computed on all trios. The results indicate that TDT(std) shows a significant increase in type I error when applied to data in which inconsistent trios are removed. This type I error increases both with an increase in sample size and with an increase in the allelic error rates. TDT(ae) always maintains correct type I error rates for the simulations considered. Factors affecting the power of the TDT(ae) are discussed. Finally, the power of TDT(std) is at least that of TDT(ae) for simulations with errorless data. Because data are rarely error free, we recommend that researchers use methods, such as the TDT(ae), that allow for errors in genotype data.