Inter- and intraspecific genomic variability of the 16S-23S intergenic spacer regions (ISR) in representatives of Acidithiobacillus thiooxidans and Acidithiobacillus ferrooxidans

The complete sequences of 32 intergenic spacer regions (ISR) from Acidithiobacillus strains, including 29 field strains isolated from coal, copper, molybdenum mine wastes or sediment of different geoclimatic regions in China, reference strain ATCC19859 and the type strains of the two species were determined. These data, together with other sequences available in the GenBank database, were used to carry out the first detailed assessment of the inter- and intraspecific genomic variability of the ISR sequences and to infer phylogenetic relationships within the genus. The total length of the 16S-23S rRNA intergenic spacer regions of the Acidithiobacillus thiooxidans and Acidithiobacillus ferrooxidans strains ranged from 451 to 490 bp, and from 434 to 456 bp, respectively. The degree of intrageneric ISR sequence similarity was higher than the degree of intergeneric similarity, and the overall similarity values of the ISRs varied from 60.49% to 84.71% between representatives of different species of the genus Acidithiobacillus. Sequences from the spacer of the A. thiooxidans and A. ferrooxidans strains ranged from 86.71% to 99.56% and 92.36% to 100% similarity, respectively. All Acidithiobacillus strains were separated into three phylogenetic major clusters and seven phylogenetic groups. ISR may be a potential target for the development of in situ hybridization probe aimed at accurately detecting acidithiobacilli in the various acidic environments.     

Mobilization of Thiobacillus ferrooxidans plasmids among Escherichia coli strains

Nonconjugative Thiobacillus ferrooxidans plasmids were mobilized at high frequencies among Escherichia coli strains by the IncP plasmid RP4 and at low frequencies by the IncN plasmid R46, but not by the IncW plasmid pSa. The mobilization region of a nonconjugative T. ferrooxidans plasmid was located on a 5.3-kilobase T. ferrooxidans DNA fragment.     

Mobilization of Thiobacillus ferrooxidans plasmids among Escherichia coli strains.

Nonconjugative Thiobacillus ferrooxidans plasmids were mobilized at high frequencies among Escherichia coli strains by the IncP plasmid RP4 and at low frequencies by the IncN plasmid R46, but not by the IncW plasmid pSa. The mobilization region of a nonconjugative T. ferrooxidans plasmid was located on a 5.3-kilobase T. ferrooxidans DNA fragment.     

Genomic island identification in Vibrio vulnificus reveals significant genome plasticity in this human pathogen

Genomic islands (GIs) are large chromosomal regions present in a subset of bacterial strains that increase the fitness of the organism under specific conditions. We compared the complete genome sequences of two Vibrio vulnificus strains YJ016 and CMCP6 and identified 14 regions (ranging in size from 14 to 117 kb), which had the characteristics of GIs. Bioinformatic analysis of these 14 GI regions identified the presence of phage-like integrase genes, aberrant GC content and genome signature (dinucleotide frequency) within each GI compared with the core genome indicating that these regions were acquired from an anomalous source. We examined the distribution of the nine GIs from strain YJ016 among 27 V. vulnificus isolates and found that most GIs were absent from the majority of these isolates. The chromosomal insertion sites of three GIs were adjacent to tRNA sites, which contained novel horizontally acquired DNA in all six available sequenced Vibrionaceae genomes. Supplementary information: Supplementary data are available at Bioinformatics online.     

Comprehensive Sieve Analysis of Breakthrough HIV-1 Sequences in the RV144 Vaccine Efficacy Trial

The RV144 clinical trial showed the partial efficacy of a vaccine regimen with an estimated vaccine efficacy (VE) of 31% for protecting low-risk Thai volunteers against acquisition of HIV-1. The impact of vaccine-induced immune responses can be investigated through sieve analysis of HIV-1 breakthrough infections (infected vaccine and placebo recipients). A V1/V2-targeted comparison of the genomes of HIV-1 breakthrough viruses identified two V2 amino acid sites that differed between the vaccine and placebo groups. Here we extended the V1/V2 analysis to the entire HIV-1 genome using an array of methods based on individual sites, k-mers and genes/proteins. We identified 56 amino acid sites or “signatures” and 119 k-mers that differed between the vaccine and placebo groups. Of those, 19 sites and 38 k-mers were located in the regions comprising the RV144 vaccine (Env-gp120, Gag, and Pro). The nine signature sites in Env-gp120 were significantly enriched for known antibody-associated sites (p = 0.0021). In particular, site 317 in the third variable loop (V3) overlapped with a hotspot of antibody recognition, and sites 369 and 424 were linked to CD4 binding site neutralization. The identified signature sites significantly covaried with other sites across the genome (mean = 32.1) more than did non-signature sites (mean = 0.9) (p < 0.0001), suggesting functional and/or structural relevance of the signature sites. Since signature sites were not preferentially restricted to the vaccine immunogens and because most of the associations were insignificant following correction for multiple testing, we predict that few of the genetic differences are strongly linked to the RV144 vaccine-induced immune pressure. In addition to presenting results of the first complete-genome analysis of the breakthrough infections in the RV144 trial, this work describes a set of statistical methods and tools applicable to analysis of breakthrough infection genomes in general vaccine efficacy trials for diverse pathogens.     

The use of RAPD genomic fingerprinting to study relatedness in strains of Acidithiobacillus ferrooxidans

Twelve strains of Acidithiobacillus ferrooxidans were recovered from acid mine drainage (AMD) sites from three different geographical locations: Copper Cliff, Ontario, Canada; Mineral City, OH, USA; and Cornwall, England. The spread-plate technique and various culture media were used to isolate and purify all strains. DNA was extracted from each purified culture and amplified using PCR and twenty, 10-mer primers. Amplification products were separated by gel electrophoresis and photographed under UV light. The RAPD (Randomly Amplified Polymorphic DNA) profiles were compared on the basis of the presence or absence of each DNA band and a data matrix was constructed. Strain diversity was calculated using the Jaccard's coefficient and UPGMA (Unweighted Pair-Group Arithmetic Average Clustering) cluster analysis. The variations in the banding patterns indicated genomic variability among the twelve A. ferrooxidans strains tested. The primers used in this study grouped the twelve strains into five major groups. Similarities between the strains ranged from 5.49% to 85.14%. These results show that the strains have a high degree of genomic diversity and that the RAPD procedure is a powerful technique to assess strain variability in this bacterium.     

Molecular evolution of cadherin-related neuronal receptor/protocadherin(alpha) (CNR/Pcdh(alpha)) gene cluster in Mus musculus subspecies

The mouse cadherin-related neuronal receptor/protocadherin (CNR/Pcdh) gene clusters are located on chromosome 18. We sequenced single-nucleotide polymorphisms (SNPs) of the CNR/Pcdh(alpha)-coding region among 12 wild-derived and four laboratory strains; these included the four major subspecies groups of Mus musculus: domesticus, musculus, castaneus, and bactrianus. We detected 883 coding SNPs (cSNPs) in the CNR/Pcdh(alpha) variable exons and three in the constant exons. Among all the cSNPs, 586 synonymous (silent) and 297 nonsynonymous (amino acid exchanged) substitutions were found; therefore, the K(a)/K(s) ratio (nonsynonymous substitutions per synonymous substitution) was 0.51. The synonymous cSNPs were relatively concentrated in the first and fifth extracellular cadherin domain-encoding regions (ECs) of CNR/Pcdh(alpha). These regions have high nucleotide homology among the CNR/Pcdh(alpha) paralogs, suggesting that gene conversion events in synonymous and homologous regions of the CNR/Pcdh(alpha) cluster are related to the generation of cSNPs. A phylogenetic analysis revealed gene conversion events in the EC1 and EC5 regions. Assuming that the common sequences between rat and mouse are ancestral, the GC content of the third codon position has increased in the EC1 and EC5 regions, although biased substitutions from GC to AT were detected in all the codon positions. In addition, nonsynonymous substitutions were extremely high (11 of 13, K(a)/K(s) ratio 5.5) in the laboratory mouse strains. The artificial environment of laboratory mice may allow positive selection for nonsynonymous amino acid variations in CNR/Pcdh(alpha) during inbreeding. In this study, we analyzed the direction of cSNP generation, and concluded that subspecies-specific nucleotide substitutions and region-restricted gene conversion events may have contributed to the generation of genetic variations in the CNR/Pcdh genes within and between species.     

The isolation of Vibrio fluvialis on the territory of the USSR]

For the first time V. fluvialis strains were detected on the territory of the USSR. The taxonomic position of these vibrios was determined by their nucleotide DNA composition (the content of guanine + cytosine was 49.3-51.0 mole%) and the characteristic features of their phenotype. The individual features of the strains consisted in their capacity for agglutination with cholera antisera, groups 01 and Inaba, in diagnostic dilutions in the presence of differences in genomes and phenotypes with cholera vibrios. Molecular hybridization DNA-DNA also gave no confirmation of their relationship to cholera vibrios (23-26% homology). The comparative study of V. fluvialis strains from the USSR and other countries by a broader set of their phenotypical signs confirmed their identity.     

Reevaluation of amino acid variability of the human immunodeficiency virus type 1 gp120 envelope glycoprotein and prediction of new discontinuous epitopes

To elucidate the evolutionary mechanisms of the human immunodeficiency virus type 1 gp120 envelope glycoprotein at the single-site level, the degree of amino acid variation and the numbers of synonymous and nonsynonymous substitutions were examined in 186 nucleotide sequences for gp120 (subtype B). Analyses of amino acid variabilities showed that the level of variability was very different from site to site in both conserved (C1 to C5) and variable (V1 to V5) regions previously assigned. To examine the relative importance of positive and negative selection for each amino acid position, the numbers of synonymous and nonsynonymous substitutions that occurred at each codon position were estimated by taking phylogenetic relationships into account. Among the 414 codon positions examined, we identified 33 positions where nonsynonymous substitutions were significantly predominant. These positions where positive selection may be operating, which we call putative positive selection (PS) sites, were found not only in the variable loops but also in the conserved regions (C1 to C4). In particular, we found seven PS sites at the surface positions of the alpha-helix (positions 335 to 347 in the C3 region) in the opposite face for CD4 binding. Furthermore, two PS sites in the C2 region and four PS sites in the C4 region were detected in the same face of the protein. The PS sites found in the C2, C3, and C4 regions were separated in the amino acid sequence but close together in the three-dimensional structure. This observation suggests the existence of discontinuous epitopes in the protein's surface including this alpha-helix, although the antigenicity of this area has not been reported yet.     

Active site organization of bacterial type I fatty acid synthetase

Four kinds of active sites of bacterial fatty acid synthetase were mapped on distinct regions within a subunit. Active sites were specifically labeled with radioactive substrates and active-site-directed inhibitors. Labeled enzymes were cleaved with proteases, and the fragments thus produced were identified with respect to specific labels by SDS-polyacrylamide gel electrophoresis and a fluorographic technique. The linear alignment of such fragments in the original subunit was established and when the results were combined with those of our previous work, five active sites were located in three regions as follows. Starting from the N-terminal of the subunit, we located acetyl, malonyl and palmitoyl transferases in the first region, the acyl carrier site in the second region (Morishima & Ikai (1985) Biochim. Biophys. Acta 832, 297-307), and beta-ketoacyl synthetase in the third region. The observed order of active sites of bacterial fatty acid synthetase can be correlated with that of the yeast enzyme, which has two kinds of subunits.     

蕨类植物叶绿体rps12基因的分子进化研究

以68种蕨类植物和2种石松类植物的rps12基因为对象,在系统发育背景下,结合最大似然法,使用HyPhy和PAML软件对该基因进行进化速率和适应性进化研究。结果显示:位于IR区的外显子2~3,其替换率明显降低,rps12基因编码序列的替换率也随之降低,且rps12基因密码子第3位的GC含量明显升高;在蕨类植物的进化过程中,3′-rps12更倾向定位于IR区,以保持较低的替换率;rps12基因编码的123个氨基酸位点中,共检测到4个正选择位点和116个负选择位点。研究结果表明基因序列进入到IR区后,显示出降低的替换率;强烈的负选择压力表明RPS12蛋白的高度保守性以及rps12基因的功能和结构已经趋于稳定。     

Crosslinking of transcription factor TFIIIA to ribosomal 5S RNA from X. laevis by trans-diamminedichloroplatinum (II)

Trans-diamminnedichloroplatinum (II) was used to induce reversible crosslinks between 5S rRNA and TFIIIA within the 7S RNP particle from X. laevis immature oocyte. The crosslinked fragments have been unambiguously identified. These fragments exclusively arise from three RNA regions centered around the hinge region at the junction of the three helical domains. Major crosslinking sites are located in region 9-21 (comprising loops A and helix II) and region 54-71 (comprising loop B, helices II and V). A minor site is also found in the 3' part of helix I and helix V (region 100-120). Our results point to the crucial role of the junction region and of the three-dimensional folding of the RNA in the recognition of the 5S rRNA by TFIIIA.     

The primary structure of the yeast hexokinase PII gene (HXK2) which is responsible for glucose repression

The nucleotide sequence of the Saccharomyces cerevisiae gene encoding the glycolytic isoenzyme hexokinase PII (HXK2), which is responsible for triggering glucose repression, has been determined. The reading frame was identified by comparison with the N-terminal undecameric amino acid (aa) sequence, determined previously [Schmidt and Colowick, Arch. Biochem. Biophys. 158 (1973) 458-470]. The codon sequence was not random, with 82.1% of the aa specified by only 25 codons. The structural gene sequence corresponded to 1455 bp, coding for 485 aa residues, corresponding to the Mr of 53 800 for the HXK2 monomer. Five initiation regions spanning 162 bp and three termination sites spanning 29 bp were detected. Sequences with similarities to a 5'-TATAAA-3' sequence were located 24-39 bp upstream of each initiation region. The most pronounced initiation region corresponded to the 5'-TATAAA-3' sequence at position -152. Two of the minor initiation sites were inside the coding sequence in front of two ATG codons.     

Variability of the env gene in cynomolgus macaques persistently infected with human immunodeficiency virus type 2 strain ben.

The sequence variability of distinct regions of the proviral env gene of human immunodeficiency virus type 2 strain ben (HIV-2ben) isolated sequentially over 3 to 4 years from six experimentally infected macaques was studied. The regions investigated were homologous to the V1, V2, V3, V4, V5, and V7 hypervariable regions identified in the env genes of HIV-1 and simian immunodeficiency virus SIVmac, respectively. In contrast to findings with HIV-1 and SIVmac, the V1- and V2-homologous regions were found to be highly conserved during the course of the HIV-2ben infection in macaques. The V3-homologous region showed a degree of variation comparable to that of HIV-1 but not of SIV. In the V4-, V5-, and V7-homologous regions, mutation hot spots were detected in most reisolates of the infected monkeys. Most of these mutations occurred during the first 10 weeks after infection. After 50 weeks, new mutations were rarely detected. At most mutation sites, a dynamic equilibrium between the mutated viral isotype and the infecting predominant wild type was present. This equilibrium might prevent an accumulation of mutations in isolates later in the course of infection.     

The Ig kappa L chain allelic groups among the Ig kappa haplotypes and Ig kappa crossover populations suggest a gene order

The Ig kappa complex locus of inbred mice found on chromosome 6 contains one constant (C kappa), five joining (J kappa), and 100 to 300 variable (V kappa) exons and spans an estimated 500 to 2000 kbp of DNA. The V kappa exons are organized into groups of highly homologous coding regions (approximately 300 bp) separated by approximately 10 kbp of intervening sequence. A group contains from 1 to 30 or more exons (exon refers to uninterrupted coding region DNA which is capable of encoding all or part of V kappa gene) that can be detected with specific DNA probes in conjunction with restriction endonuclease fragments (REF) from genomic DNA. Thirteen DNA probes specific for different V kappa exon groups and one DNA probe specific for J kappa and C kappa exons were used in conjunction with 55 inbred strains in an attempt to detect RFLP that could be used to establish Ig kappa allelic groups and Ig kappa haplotypes. Each probe detected two to four different REF patterns (allelic groups) among the panel of inbred mice examined. Size estimates of the REF were made, and each probe detected 4.2 to 107.7 kbp of DNA, including faint REF, 675.6 to 723.6 kbp of DNA could be detected within a single haplotype. Based on these allelic groups, seven haplotypes were identified among the 55 inbred strains of mice. No subline differences were detected, and the distribution of allelic groups implied common ancestry among many of the inbred strains examined. The DNA probes were also used in conjunction with recombinant inbred, congenic strains and backcross populations of mice. By using the analysis of known Ig kappa r populations, and assuming a common ancestry among the inbred strains, a gene order was predicted: Centromere-Hd-(Ig kappa-V11, Ig kappa-V24, Ig kappa-V9-26)-(Ig kappa-V1, Ig kappa-V9)-(Ig kappa-V4, Ig kappa-V8, Ig kappa-V10, Ig kappa-V12, 13, Ig kappa-V19)-(Ig kappa-V28, Rn7s-6)-Ig kappa-V23-(Ig kappa-V21, Ig kappa-J, Ig kappa-C)-(Ly2, Ly3)-wa-1.