Chromosomal heterogeneity of various Mycoplasma hyopneumoniae field strains.

Restriction enzyme digestion and field inversion gel electrophoresis were used to analyze the chromosomes of strains of Mycoplasma hyopneumoniae and the related organism Mycoplasma flocculare. The chromosome size for the M. hyopneumoniae type strain was calculated from individual fragments to be 1,011.3 +/- 32.9 kbp. The chromosomes of M. hyopneumoniae field strains were approximately the same size. The restriction patterns obtained for the chromosomes of phenotypically similar M. hyopneumoniae strains were quite different. Therefore, the species M. hyopneumoniae seems to be very heterogeneous. A field inversion gel electrophoresis analysis of the entire chromosomes allowed us to distinguish M. hyopneumoniae strains easily and hence to characterize further the species M. hyopneumoniae. The chromosome size for M. flocculare was calculated to be 988.3 +/- 39.5 kbp. Restriction enzyme XhoI, which statistically should cut the M. hyopneumoniae chromosome frequently, did not cut the DNA of any of the M. hyopneumoniae strains but did digest M. flocculare DNA, indicating that there is a site-specific modification at CTCGAG which probably belongs to a restriction modification system in M. hyopneumoniae and is absent in M. flocculare.     

Comparative Analysis of the Genomes of Two Field Isolates of the Rice Blast Fungus Magnaporthe oryzae

Rice blast caused by Magnaporthe oryzae is one of the most destructive diseases of rice worldwide. The fungal pathogen is notorious for its ability to overcome host resistance. To better understand its genetic variation in nature, we sequenced the genomes of two field isolates, Y34 and P131. In comparison with the previously sequenced laboratory strain 70-15, both field isolates had a similar genome size but slightly more genes. Sequences from the field isolates were used to improve genome assembly and gene prediction of 70-15. Although the overall genome structure is similar, a number of gene families that are likely involved in plant-fungal interactions are expanded in the field isolates. Genome-wide analysis on asynonymous to synonymous nucleotide substitution rates revealed that many infection-related genes underwent diversifying selection. The field isolates also have hundreds of isolate-specific genes and a number of isolate-specific gene duplication events. Functional characterization of randomly selected isolate-specific genes revealed that they play diverse roles, some of which affect virulence. Furthermore, each genome contains thousands of loci of transposon-like elements, but less than 30% of them are conserved among different isolates, suggesting active transposition events in M. oryzae. A total of approximately 200 genes were disrupted in these three strains by transposable elements. Interestingly, transposon-like elements tend to be associated with isolate-specific or duplicated sequences. Overall, our results indicate that gain or loss of unique genes, DNA duplication, gene family expansion, and frequent translocation of transposon-like elements are important factors in genome variation of the rice blast fungus.     

Analysis of distribution indicates diverse functions of simple sequence repeats in Mycoplasma genomes

Simple sequence repeats (SSRs) composed of extensive tandem iterations of a single nucleotide or a short oligonucleotide are rare in most bacterial genomes, but they are common among Mycoplasma. Some of these repeats act as contingency loci in association with families of surface antigens. By contraction or expansion during replication, these SSRs increase genetic variance of the population and facilitate avoidance of the immune response of the host. Occurrence and distribution of SSRs are analyzed in complete genomes of 11 Mycoplasma and 3 related Mollicutes in order to gain insights into functional and evolutionary diversity of the SSRs in Mycoplasma. The results revealed an unexpected variety of SSRs with respect to their distribution and composition and suggest that it is unlikely that all SSRs function as contingency loci or recombination hot spots. Various types of SSRs are most abundant in Mycoplasma hyopneumoniae, whereas Mycoplasma penetrans, Mycoplasma mobile, and Mycoplasma synoviae do not contain unusually long SSRs. Mycoplasma hyopneumoniae and Mycoplasma pulmonis feature abundant short adenine and thymine runs periodically spaced at 11 and 12 bp, respectively, which likely affect the supercoiling propensities of the DNA molecule. Physiological roles of long adenine and thymine runs in M. hyopneumoniae appear independent of location upstream or downstream of genes, unlike contingency loci that are typically located in protein-coding regions or upstream regulatory regions. Comparisons among 3 M. hyopneumoniae strains suggest that the adenine and thymine runs are rarely involved in genome rearrangements. The results indicate that the SSRs in the Mycoplasma genomes play diverse roles, including modulating gene expression as contingency loci, facilitating genome rearrangements via recombination, affecting protein structure and possibly protein-protein interactions, and contributing to the organization of the DNA molecule in the cell.     

A new high-throughput AFLP approach for identification of new genetic polymorphism in the genome of the clonal microorganism Mycobacterium tuberculosis

We have here applied high-throughput amplified fragment length polymorphism (htAFLP) analysis to strains belonging to the five classical species of the Mycobacterium tuberculosis complex. Using 20 strains, three enzyme combinations and eight selective amplification primer pairs, 24 AFLP reactions were performed per strain. Overall, this resulted in 480 DNA fingerprints and more than 1200 htAFLP-amplified PCR fragments were visualised per strain. The cumulative dendrogram correctly clustered strains from the various species, albeit within a distance of 6.5% for most of them. The single isolate of Mycobacterium canettii presented separately at 19% distance. All over, 169 fragments (14%) appeared to be polymorphic. Sixty-eight were specific for M. canetti and forty-five for Mycobacterium bovis. For the 10 different M. tuberculosis strains included in the present analysis, 56 polymorphic markers were identified. Upon sequencing 20 of these marker regions and comparisons with the H37Rv genome sequence, 25% appeared to share homology to members of the antigenically variable PE/PPE surface protein encoding gene family confirming previous findings on the genetic heterogeneity within these genes. In addition, homologues for phage genes and insertion element-encoded genes were detected. Forty-five percent of the sequences derived from ORFs with a currently unknown function, which was corroborated by genome sequence comparison for the clinical M. tuberculosis CD 1551 isolate. Sequence variation in M. tuberculosis was assessed in more detail for a subset of these loci by newly designed PCR restriction fragment length polymorphism (RFLP) tests and direct sequencing. Fourteen novel PCR RFLP tests were developed and twelve novel single nucleotide polymorphisms (SNPs) were identified, all suited for epidemiological analysis of M. tuberculosis. The tests allowed for identification of the major Mycobacterium species and M. tuberculosis variants and clones.     

Whole-genome comparison of Mycobacterium tuberculosis clinical and laboratory strains

Virulence and immunity are poorly understood in Mycobacterium tuberculosis. We sequenced the complete genome of the M. tuberculosis clinical strain CDC1551 and performed a whole-genome comparison with the laboratory strain H37Rv in order to identify polymorphic sequences with potential relevance to disease pathogenesis, immunity, and evolution. We found large-sequence and single-nucleotide polymorphisms in numerous genes. Polymorphic loci included a phospholipase C, a membrane lipoprotein, members of an adenylate cyclase gene family, and members of the PE/PPE gene family, some of which have been implicated in virulence or the host immune response. Several gene families, including the PE/PPE gene family, also had significantly higher synonymous and nonsynonymous substitution frequencies compared to the genome as a whole. We tested a large sample of M. tuberculosis clinical isolates for a subset of the large-sequence and single-nucleotide polymorphisms and found widespread genetic variability at many of these loci. We performed phylogenetic and epidemiological analysis to investigate the evolutionary relationships among isolates and the origins of specific polymorphic loci. A number of these polymorphisms appear to have occurred multiple times as independent events, suggesting that these changes may be under selective pressure. Together, these results demonstrate that polymorphisms among M. tuberculosis strains are more extensive than initially anticipated, and genetic variation may have an important role in disease pathogenesis and immunity.     

The genome sequence of Mycoplasma hyopneumoniae strain 232, the agent of swine mycoplasmosis

We present the complete genome sequence of Mycoplasma hyopneumoniae, an important member of the porcine respiratory disease complex. The genome is composed of 892,758 bp and has an average G+C content of 28.6 mol%. There are 692 predicted protein coding sequences, the average protein size is 388 amino acids, and the mean coding density is 91%. Functions have been assigned to 304 (44%) of the predicted protein coding sequences, while 261 (38%) of the proteins are conserved hypothetical proteins and 127 (18%) are unique hypothetical proteins. There is a single 16S-23S rRNA operon, and there are 30 tRNA coding sequences. The cilium adhesin gene has six paralogs in the genome, only one of which contains the cilium binding site. The companion gene, P102, also has six paralogs. Gene families constitute 26.3% of the total coding sequences, and the largest family is the 34-member ABC transporter family. Protein secretion occurs through a truncated pathway consisting of SecA, SecY, SecD, PrsA, DnaK, Tig, and LepA. Some highly conserved eubacterial proteins, such as GroEL and GroES, are notably absent. The DnaK-DnaJ-GrpR complex is intact, providing the only control over protein folding. There are several proteases that might serve as virulence factors, and there are 53 coding sequences with prokaryotic lipoprotein lipid attachment sites. Unlike other mycoplasmas, M. hyopneumoniae contains few genes with tandem repeat sequences that could be involved in phase switching or antigenic variation. Thus, it is not clear how M. hyopneumoniae evades the immune response and establishes a chronic infection.     

Genetic analysis of endogenous xenotropic murine leukemia viruses: association with two common mouse mutations and the viral restriction locus Fv-1.

We have defined 40 endogenous xenotropic virus (Xmv) loci from several common inbred strains of mice by examining provirus-cell DNA junction fragments in recombinant inbred mice. Some inbred strains carried unique proviruses, but most Xmv loci were present in several strains, indicating that many Xmv integration events preexisted modern inbreeding. It was also clear that most Xmv junction fragment variation between inbred strains resulted from independent integration events and not modification or restriction site polymorphism following integration. Chromosomal assignments were determined for 32 Xmv loci by comparing their recombinant inbred strain distribution patterns to those of known genetic markers. The Xmv loci were generally dispersed throughout the genome, but several chromosomal regions contained more than one provirus. Furthermore, several close genetic associations with cellular genes were discovered. Four Xmv loci were closely linked to Fv-1b, a dominant viral resistance gene present in C57BL/6J, BALB/cJ, A/J, and several other strains. Xmv-28 was closely linked to rd (retinal degeneration), and Xmv-10 was closely linked to a (non-agouti), both of which are old mutations as inferred from their broad distribution in mice. We suggest that Xmv integration contributed to genetic diversity in the past and that much of this diversity exists today in common laboratory strains.     

Mosaic genealogy of the Mus musculus genome revealed by 21 nuclear genes from its three subspecies

Patterns of genetic variation provide insight into the evolutionary history of a species. Mouse (Mus musculus) is a good model for this purpose. Here we present the analysis of genealogies of the 21 nuclear loci and one mitochondrial DNA region in M. musculus based on our nucleotide sequences of nine inbred strains from three M. musculus subspecies (musculus, domesticus, and castaneus) and one M. spicilegus strain as an outgroup. The mitochondrial DNA gene genealogy of those strains confirmed the introgression pattern of one musculus strain. When all the nuclear DNA data were concatenated to produce a phylogenetic tree of nine strains, musculus and domesticus strains formed monophyletic clusters with each other, while the two castaneus strains were paraphyletic. When each DNA region was treated independently, the phylogenetic networks revealed an unnegligibly high level of subspecies admixture and the mosaic nature of their genome. Estimation of ancestral and derived population sizes and migration rates suggests the effects of ancestral polymorphism and gene flow on the pattern of genetic variation of the current subspecies. Gene genealogies of Fut4 and Dfy loci also suggested existence of the gene flow between M. musculus and M. spicilegus or other distant species.     

A physical and genetic map of the Mycoplasma hyopneumoniae strain J genome

A macrorestriction map of the genome of Mycoplasma hyopneumoniae strain J, the type strain of the causative agent of enzootic pneumonia in pigs, was constructed using pulsed-field gel electrophoresis (PFGE) and DNA hybridization. The size of the genome as determined by PFGE was approximately 1070 kb. Assembly of the M. hyopneumoniae genomic map was facilitated and complimented by the simultaneous construction of an ordered cosmid library. Five contigs of overlapping cosmids were assembled, which together represent coverage of approximately 728 kb. Forty-two genetic markers (including three types of repeated elements) were placed on the M. hyopneumoniae map. Closer examination of an ApaI restriction fragment contained entirely within a single cosmid insert suggests that the genome size may be overestimated by PFGE.     

Mycoplasma genitalium: an efficient strategy to generate genetic variation from a minimal genome

Mycoplasma genitalium, a human pathogen associated with sexually transmitted diseases, is unique in that it has smallest genome of any known free-living organism. The goal of this study was to investigate if and how M. genitalium uses a minimal genome to generate genetic variations. We analysed the sequence variability of the third gene (MG192 or mgpC) of the M. genitalium MgPa adhesion operon, demonstrated that the MG192 gene is highly variable among and within M. genitalium strains in vitro and in vivo, and identified MG192 sequence shifts in the course of in vitro passage of the G37 type strain and in sequential specimens from an M. genitalium-infected patient. In order to establish the origin of the MG192 variants, we examined nine genomic loci containing partial copies of the MgPa operon, known as MgPar sequences. Our analysis suggests that the MG192 sequence variation is achieved by recombination between the MG192 expression site and MgPar sequences via gene cross-over and, possibly, also by gene conversion. It appears plausible that M. genitalium has the ability to generate unlimited variants from its minimized genome, which presumably allows the organism to adapt to diverse environments and/or to evade host defences by antigenic variation.     

表达猪肺炎支原体免疫原性基因的重组猪霍乱沙门氏菌对小鼠的免疫原性

【目的】构建表达猪肺炎支原体免疫原性基因的重组猪霍乱沙门氏菌,对重组菌株的生物学特性以及对小鼠的免疫原性进行研究。【方法】分别将猪肺炎支原体的免疫原性基因p36、p46、p65和p97R1-Nrdf克隆到pYA3493,得到重组质粒pYA-36、pYA-46、pYA-65和pYA-97R1-Nrdf。重组质粒和空质粒pYA3493分别电转asd基因缺失株C500ˉ,获得重组菌株C36(pYA-36)、C46(pYA-46)、C65(pYA-65)、C97R1-Nrdf(pYA-97R1-Nrdf)和空质粒菌株CpYA(pYA3493)。研究重组菌株的生物学特性,并以小鼠为动物模型评价重组菌株在口服、肌注两种不同免疫途径下的免疫原性。【结果】成功构建表达猪肺炎支原体免疫原性基因的重组猪霍乱沙门氏菌,重组菌株能表达外源蛋白,生化和生长特性未发生改变,插入的外源基因亦稳定存在。小鼠的免疫原性结果显示:口服C36+C46+C65+C97R1-Nrdf组的猪肺炎支原体抗体极显著高于口服C36+C46+C65组和肌注商品疫苗组(P<0.01),但与肌注C36+C46+C65组无显著性差异(P>0.05);IFN-γ为肌注C36+C46+C65组显著高于肌注商品疫苗组(P<0.05),而与口服C36+C46+C65或C36+C46+C65+C97R1-Nrdf组差异均不显著(P>0.05);IL-4水平为口服C36+C46+C65组>口服C36+C46+C65+C97R1-Nrdf组>肌注商品疫苗组>肌注C36+C46+C65组,但各组之间差异均不显著(P>0.05)。对照组的猪肺炎支原体抗体、IFN-γ以及IL-4均与试验组差异极显著(P<0.01)。【结论】构建的表达猪肺炎支原体免疫原性基因的重组猪霍乱沙门氏菌,采用肌注免疫时具有较好的免疫原性,有望发展为猪肺炎支原体的基因工程疫苗。     

Genetic profile of alloxan-induced diabetes-susceptible mice (ALS) and-resistant mice (ALR)

Two inbred strains from a foundation stock derived from Crj: CD-1 (ICR) mice were established after more than 20 generations of full-sib mating, and by simultaneous selective breeding for developing and not developing diabetes after alloxan administration (45 mg/kg in males, 47 mg/kg in females). To elucidate the genetic background of the two inbred strains, i. e., alloxan-induced diabetes-susceptible (ALS) strain and alloxan-induced diabetes-resistant (ALR) strain, their biochemical genetic markers and immunogenetic markers were examined. 1) For both strains, investigation of biochemical genetic markers at 19 loci and immunogenetic markers at 11 loci revealed no variation in any gene within the same strain, showing to be homogeneous, thus indicating establishment of the inbred strains. 2) The two strains differed from each other at 2 loci of biochemical genetic markers and at 5 loci of immunogenetic markers. 3) The ALS and ALR strains can be regarded as new inbred strains derived from ICR mice. 4) The results show that the marker genes of the two strains are different at 7 loci, but it remains unclear whether or not these genes are involved in the difference between the two strains in susceptibility to alloxan.     

Complete genome sequence of Mycoplasma hyopneumoniae strain 168

Mycoplasma hyopneumoniae strain 168, a pathogenic strain prevalent in China, was isolated in 1974. Although this strain has been widespread for a long time, the genome sequence had not been determined. Here, we announce the complete genome sequence of M. hyopneumoniae strain 168.     

三个近交系C57BL/6J小鼠群体微卫星遗传变异分析(英文）

应用微卫星遗传标记对近交系C57BL/6J(B6)小鼠遗传稳定性进行分析。用FAM标记的引物PCR扩增了来自北京和上海三个实验动物生产单位提供的三个B6小鼠群体共15个微卫星位点并进行分型。结果显示,所有位点均处于纯合状态,其中7个位点为多态位点。研究表明各B6群体虽然为高度近交群体,但不同生产单位维持的B6群体之间存在遗传分化。     

Spectrum of heart diseases in a new cardiac service in Nigeria: An echocardiographic study of 1441 subjects in Abeokuta

Background

The recent determination of the complete nucleotide sequence of several Mycobacterium tuberculosis (MTB) genomes allows the use of comparative genomics as a tool for dissecting the nature and consequence of genetic variability within this species. The multiple alignment of the genomes of clinical strains (CDC1551, F11, Haarlem and C), along with the genomes of laboratory strains (H37Rv and H37Ra), provides new insights on the mechanisms of adaptation of this bacterium to the human host.

Findings

The genetic variation found in six M. tuberculosis strains does not involve significant genomic rearrangements. Most of the variation results from deletion and transposition events preferentially associated with insertion sequences and genes of the PE/PPE family but not with genes implicated in virulence. Using a Perl-based software islandsanalyser, which creates a representation of the genetic variation in the genome, we identified differences in the patterns of distribution and frequency of the polymorphisms across the genome. The identification of genes displaying strain-specific polymorphisms and the extrapolation of the number of strain-specific polymorphisms to an unlimited number of genomes indicates that the different strains contain a limited number of unique polymorphisms.

Conclusion

The comparison of multiple genomes demonstrates that the M. tuberculosis genome is currently undergoing an active process of gene decay, analogous to the adaptation process of obligate bacterial symbionts. This observation opens new perspectives into the evolution and the understanding of the pathogenesis of this bacterium.     

Genetic analysis of Helicobacter pylori strain populations colonizing the stomach at different times postinfection

Genetic diversity of the human gastric pathogen Helicobacter pylori in an individual host has been observed; whether this diversity represents diversification of a founding strain or a mixed infection with distinct strain populations is not clear. To examine this issue, we analyzed multiple single-colony isolates from two to four separate stomach biopsies of eight adult and four pediatric patients from a high-incidence Mexican population. Eleven of the 12 patients contained isolates with identical random amplified polymorphic DNA, amplified fragment length polymorphism, and vacA allele molecular footprints, whereas a single adult patient had two distinct profiles. Comparative genomic hybridization using whole-genome microarrays (array CGH) revealed variation in 24 to 67 genes in isolates from patients with similar molecular footprints. The one patient with distinct profiles contained two strain populations differing at 113 gene loci, including the cag pathogenicity island virulence genes. The two strain populations in this single host had different spatial distributions in the stomach and exhibited very limited genetic exchange. The total genetic divergence and pairwise genetic divergence between isolates from adults and isolates from children were not statistically different. We also analyzed isolates obtained 15 and 90 days after experimental infection of humans and found no evidence of genetic divergence, indicating that transmission to a new host does not induce rapid genetic changes in the bacterial population in the human stomach. Our data suggest that humans are infected with a population of closely related strains that vary at a small number of gene loci, that this population of strains may already be present when an infection is acquired, and that even during superinfection genetic exchange among distinct strains is rare.     

Evidence for genetic hitchhiking effect associated with insecticide resistance in Aedes aegypti.

Information on genetic variation within and between populations is critical for understanding the evolutionary history of mosquito populations and disease epidemiology. Previous studies with Drosophila suggest that genetic variation of selectively neutral loci in a large fraction of genome may be constrained by fixation of advantageous mutations associated with hitchhiking effect. This study examined restriction fragment length polymorphisms of four natural Aedes aegypti mosquito populations from Trinidad and Tobago, at 16 loci. These populations have been subjected to organophosphate (OP) insecticide treatments for more than two decades, while dichlor-diphenyltrichlor (DDT) was the insecticide of choice prior to this period. We predicted that genes closely linked to the OP target loci would exhibit reduced genetic variation as a result of the hitchhiking effect associated with intensive OP insecticide selection. We also predicted that genetic variability of the genes conferring resistance to DDT and loci near the target site would be similar to other unlinked loci. As predicted, reduced genetic variation was found for loci in the general chromosomal region of a putative OP target site, and these loci generally exhibited larger F(ST) values than other random loci. In contrast, the gene conferring resistance to DDT and its linked loci show polymorphisms and genetic differentiation similar to other random loci. The reduced genetic variability and apparent gene deletion in some regions of chromosome 1 likely reflect the hitchhiking effect associated with OP insecticide selection.