Role of uracil-DNA glycosylase in mutation avoidance by Streptococcus pneumoniae.

Uracil-DNA glycosylase activity was found in Streptococcus pneumoniae, and the enzyme was partially purified. An ung mutant lacking the activity was obtained by positive selection of cells transformed with a plasmid containing uracil in its DNA. The effects of the ung mutation on mutagenic processes in S. pneumoniae were examined. The sequence of several malM mutations revertible by nitrous acid showed them to correspond to A.T----G.C transitions. This confirmed a prior deduction that nitrous acid action on transforming DNA gave only G.C----A.T mutations. Examination of malM mutant reversion frequencies in ung strains indicated that G.C----A.T mutation rates generally were 10-fold higher than in wild-type strains, presumably owing to lack of repair of deaminated cytosine residues in DNA. No effect of ung on mutation avoidance by the Hex mismatch repair system was observed, which means that uracil incorporation and removal from nascent DNA cannot be solely responsible for producing strand breaks that target nascent DNA for correction after replication. One malM mutation corresponding to an A.T----G.C transition showed a 10-fold-higher spontaneous reversion frequency than other such transitions in a wild-type background. This "hot spot" was located in a directly repeated DNA sequence; it is proposed that transient slippage to the wild-type repeat during replication accounts for the higher reversion frequency.     

Mutagenesis of uracil-DNA glycosylase deficient mutants of the extremely thermophilic eubacterium Thermus thermophilus

Thermus thermophilus is an extremely thermophilic, aerobic, and gram-negative eubacterium that grows optimally at 70-75 degrees C, pH 7.5. In extremely high temperature environment, DNA damages in cells occur at a much higher frequency in thermophiles than mesophiles such as E. coli. When temperature rises, the deamination of cytosine residues in double-strand DNA is expected to increase greatly. T. thermophilus HB27 has two putative uracil-DNA glycosylase genes (udgA and udgB). Expression level of udgA gene was 2-3 times higher than that of udgB at 70, 74, and 78 degrees C when it was monitored by beta-glucosidase reporter assay. We developed hisD(3110), hisD(3113), hisD(3115), and hisD(174) marker allele that can specifically detect G:C-->A:T, C:G-->A:T, T:A-->A:T, and A:T-->G:C base-substitutions, respectively, by His(+) reverse mutations. We then disrupted udgA and udgB by thermostable kanamycin-resistant gene (htk) or pyrE gene insertion in each hisD background, and their spontaneous His(+) reversion frequencies were compared. A udgA,B double mutant showed a pronounced increase in G:C-->A:T reversion frequency compared with each single udg mutant, udgA or udgB. Estimated mutation rates of the udgA,B mutant cultured at 60, 70, and 78 degrees C were about 2, 12, and 117 His(+)/10(8)/generation, respectively. At 70 degrees C culture, increased ratio of the mutation rate compared with the udg(+) strain was 12-fold in udgA, 3-fold in udgB, and 56-fold in udgA,B mutant. On the other hand, no difference was observed in other mutations of C:G-->A:T, T:A-->A:T, and A:T-->G:C between udgA,B double mutant and the parent udg(+) strain. The present results indicated that gene products of udgB as well as udgA functioned in vivo to remove uracil in DNA and prevent G:C-->A:T transition mutations.     

A new reversion assay for measuring all possible base pair substitutions in Saccharomyces cerevisiae

A TRP5-based reversion system that allows the rates of all possible base pair substitutions to be measured when the TRP5 locus is in both orientations relative to a defined origin of replication has been developed. This system should be useful for a wide variety of mutation and repair studies in yeast.     

Mammalian DNA replication: mutation biases and the mutation rate

Experimental studies have shown that the fidelity of DNA replication can be affected by the concentrations of free deoxyribonucleotides present in the cell. Replication of mammalian chromosomes is achieved using pools of newly-synthesized deoxyribonucleotides which fluctuate during the cell cycle. Since regions of mammalian chromosomes are replicated sequentially, there is the potential for differences among mammalian loci in both the relative and absolute frequencies of the various transitional and transversional mutations which may occur. Where these mutations are effectively neutral, at silent sites in genes and in non-coding sequences, this may result in different rates of evolution and in different base compositions, as have been observed in data from mammalian genes. A simple model of the DNA replication process is developed to describe how the mutation rate could be affected by the G + C contents of the deoxyribonucleotide pools and of the replicating DNA. Mutation rates are predicted to vary from locus to locus; only in the particular case of identical G + C contents in the DNA locus and the deoxyribonucleotide pools, and no proofreading, will the mutation rate be uniform over all loci.     

An assessment of the importance of error-prone repair and point mutations to forward mutation to L-azetidine-2-carboxylic acid resistance in Escherichia coli

By comparison of E. coli WP2 with CM891 (uvrA- pKM101) we found that pKM101 plasmid and uvrA- mutation considerably enhanced both spontaneous and chemically-induced reversion at the trp locus. However, little or no increase was observed for forward mutation at the A2C locus. Furthermore, mutation frequency decline was considerably greater for trp reversion than for mutation to A2Cr. Thus neither error-prone repair nor point mutation seemed likely to be the major mechanism for forward mutation at the A2C locus. Results for spontaneous mutation of recA-, polA- and gyrA- strains showed that polA- and gyrA- gave good increases in forward mutation but not in reversion. It was inferred that deletion, transposition and/or larger chromosomal effects rather than point mutation were mainly responsible for most forward mutation.     

Variation in mutation dynamics across the maize genome as a function of regional and flanking base composition

We examine variation in mutation dynamics across a single genome (Zea mays ssp. mays) in relation to regional and flanking base composition using a data set of 10,472 SNPs generated by resequencing 1776 transcribed regions. We report several relationships between flanking base composition and mutation pattern. The A + T content of the two sites immediately flanking the mutation site is correlated with rate, transition bias, and GC --> AT pressure. We also observe a significant CpG effect, or increase in transition rate at CpG sites. At the regional level we find that the strength of the CpG effect is correlated with regional A + T content, ranging from a 1.7-fold increase in transition rate in relatively G + C-rich regions to a 2.6-fold increase in A + T-rich regions. We also observe a relationship between locus A + T content and GC --> AT pressure. This regional effect is in opposition to the influence of the two immediate neighbors in that GC --> AT pressure increases with increasing locus A + T content but decreases with increasing flanking base A + T content and may represent a relationship between genome location and mutation bias. The data indicate multiple context effects on mutations, resulting in significant variation in mutation dynamics across the genome.     

中华鳖GHRL基因SNPs的筛选及生长性状的关联分析

为探究GHRL基因多态性对中华鳖(Pelodiscus sinensis)生长性状的影响, 采用直接测序法在GHRL基因上检测到14个单核苷酸多态性位点C289T、G501T、T738C、G776T、A841G、T885C、T2960C、A2987T、G3390A、A3857C、G4718A、T4820C、A4850C、T4979C。随机选取同批繁殖的120只中华鳖用飞行时间质谱法进行SNPs位点的分型, 并分析与生长性状的相关性。检测结果显示, 所有SNP位点均符合Hardy-Weinberg平衡状态(P>0.05)。方差分析结果显示, C289T位点CT、CC基因型的5项生长数据均显著高于TT基因型(P<0.05)。S2位点AB基因型的体重、背甲长、背甲宽和裙边宽4项数据均显著高于AA基因型(P<0.05)。G3390A位点AG基因型的背甲长、背甲宽2项数据显著高于AA基因型(P<0.05)。G4718A位点AG基因型的背甲长、背甲宽、裙边宽3项数据显著高于AA基因型(P<0.05)。在GHRL基因上获得的SNP位点可能影响着中华鳖的生长性状或与之紧密连锁, 可为中华鳖分子辅助育种提供助力与参考。     

Analysis of Y-chromosomal diallelic loci polymorphism in populations of the Volga-Ural region

Three diallelic polymorphisms of human Y chromosome, DYS287 (Y Alu polymorphism, YAP), T/C transition at the RBF5 locus (Tat), and G/A transition at the LLY22 locus, were studied in eight ethnic populations of the Volga-Ural region, representing Turkic (Bashkirs, Tatars, and Chuvashes) and Finno-Ugric (Maris, Mordovians, Udmurts, Komi-Zyryans, and Komi-Permyaks) branches of the Uralic linguistic family, and in the group of Slavic migrants, belonging to the Indo-European linguistic family (Russians). Ethnic populations of the Volga-Ural region were characterized by a low frequency of the Y chromosome Alu insertion. Examination of an association between the Alu polymorphism and Tat mutation revealed absolute C/YAP linkage. Analysis of the haplotype frequency distribution patterns constructed from the data on the DYS287 and RBF5 polymorphisms revealed substantial differences between Udmurts and the other ethnic populations. The differences were also observed between Komi-Zyryans and the populations of Bashkirs, Mordovians, Komi-Permyaks, and Russians. Analysis of the degree of genetic differentiation pointed to high level of genetic differentiation of the male lineages of the Finno-Ugric ethnic groups. The data on the linkage between mutations of the RBF5 and the LLY22 loci indicated the common origin of the Tat mutation in Bashkirs, Mordovians, Udmurts, and Komi-Zyryans, and of a number of ancestral C allele-bearing chromosomes in Tatars, Maris, and Chuvashes.     

Asymmetric directional mutation pressures in bacteria

Background

When there are no strand-specific biases in mutation and selection rates (that is, in the substitution rates) between the two strands of DNA, the average nucleotide composition is theoretically expected to be A = T and G = C within each strand. Deviations from these equalities are therefore evidence for an asymmetry in selection and/or mutation between the two strands. By focusing on weakly selected regions that could be oriented with respect to replication in 43 out of 51 completely sequenced bacterial chromosomes, we have been able to detect asymmetric directional mutation pressures.

Results

Most of the 43 chromosomes were found to be relatively enriched in G over C and T over A, and slightly depleted in G+C, in their weakly selected positions (intergenic regions and third codon positions) in the leading strand compared with the lagging strand. Deviations from A = T and G = C were highly correlated between third codon positions and intergenic regions, with a lower degree of deviation in intergenic regions, and were not correlated with overall genomic G+C content.

Conclusions

During the course of bacterial chromosome evolution, the effects of asymmetric directional mutation pressures are commonly observed in weakly selected positions. The degree of deviation from equality is highly variable among species, and within species is higher in third codon positions than in intergenic regions. The orientation of these effects is almost universal and is compatible in most cases with the hypothesis of an excess of cytosine deamination in the single-stranded state during DNA replication. However, the variation in G+C content between species is influenced by factors other than asymmetric mutation pressure.

     

Comparison among patterns of macromolecular synthesis in Escherichia coli B/r at growth rates of less and more than one doubling per hour at 37 degrees C.

In Escherichia coli B/r, the relationship between the patterns of chromosome replication and of synthesis of envelope components differs at various growth rates. At growth rates greater than 1.0 doubling per h at 37 degrees C, the average mass and age at initiation of rounds of chromosome replication are similar to those at increase in incorporation of precursors into a major outer membrane protein and phosphatidylethanolamine. At growth rates less than 1.0 doubling per h at 37 degrees C the average mass and age at increase in the synthesis of these envelope components differ from those at initiation of chromosome replication. The average cell mass per chromosomal origin at initiation of rounds of chromosome replication is not a constant and varies between growth rates greater and less than 1.0 doubling per h.     

Ribosomal RNA Multigene Loci: Nomads of the Triticeae Genomes

The nucleolus organizing regions (NORs) on the short arms of chromosomes 1A(m) and 5A(m) of diploid wheat, Triticum monococcum L., are at the most distal loci in the linkage maps of these two chromosome arms. This distal location differs from the interstitial location of the Nor loci on chromosome arms 1BS of tetraploid Triticum turgidum L. and hexaploid T. aestivum L., 5DS of T. aestivum and diploid Ae. tauschii Coss., and 5HS of barley. Moreover, the barley 5HS locus is at a different location than the 5DS locus. However, other markers, including the centromeres, are colinear. These findings showed that the major Nor loci have repeatedly changed position in the chromosome arms during the radiation of species in the tribe Triticeae without rearrangements of the linkage groups. It is suggested that Nor loci may change position via dispersion of minor loci, that are shown here to exist in the T. monococcum genome, magnification of gene copy numbers in these minor loci, and subsequent deletion of the original major loci. Implications of these findings for the use of rRNA nucleotide sequences in phylogenetic reconstructions are pointed out.     

Isolation of a dnaA mutant of Bacillus subtilis defective in initiation of replication: amount of DnaA protein determines cells'' initiation potential.

The dnaA gene is essential for initiation of chromosomal replication in Escherichia coli. A gene homologous with the E. coli dnaA was found in the replication origin region of the Bacillus subtilis chromosome. We have now isolated a temperature sensitive mutant of the B. subtilis dnaA by in vitro mutagenesis of the cloned gene. At a nonpermissive temperature, 49 degrees C, DNA replication stops completely after 60% increase in a rich medium, while cell mass continues to increase exponentially at 2.5 times the rate at 30 degrees C. A ratio of gene frequency between purA (origin marker) and metB (terminus marker) changes gradually from 2.7 at 30 degrees C to 1.0 in 45 min at 49 degrees C, indicating completion of the ongoing replication cycle. Upon the temperature shift down to 30 degrees C after the incubation at 49 degrees C for 60 min, DNA replication resumes without delay, and the purA/metB ratio increases rapidly to 6, i.e. consecutive initiation of more than two rounds of replication. Addition of chloramphenicol at the time of the temperature shift down did not inhibit the increase in the purA/metB ratio, while rifampicin inhibited the re-initiation completely. The mutation is a single base change from C to T in the dnaA gene resulting in an amino acid substitution from Ser to Phe in the DnaA protein. The mutation was responsible for both temperature sensitive growth and the defect in initiation of chromosomal replication. We observed a remarkable correlation between the amount of DnaA protein and the amount of initiation potential accumulated during incubation at the non-permissive temperature.     

Comparison of Somatic Reversions between the Ivory Allele and Transposon-Caused Mutant Alleles at the White Locus of DROSOPHILA MELANOGASTER after Larval Treatment with X Rays and Ethyl Methanesulfonate

Somatic reversion of strains with the ivory (wi) allele, a mutation associated with a tandem duplication of a DNA sequence at the white locus, increased with the age of larvae at the time of X-irradiation as expected from the increase in the number of target cells. In contrast, two independently isolated strains with unstable w+ loci associated with insertion of transposable elements showed higher reversion frequencies after treatment with X rays or ethyl methanesulfonate (EMS) at early larval stages than at late stages. Nevertheless, both the wi strain and the two unstable w+ strains reverted at nearly equal rates after treatment with X rays or EMS at early larval stages. Possible similarity in "hot spot" structure for the high reversibility of the two types of mutations is discussed in relation to production of presumed "mutator-type" cofactors specific to the transposon-caused mutations at early larval stages.     

The Genetics of the Dras3-Roughened-Ecdysoneless Chromosomal Region (62b3-4 to 62d3-4) in Drosophila Melanogaster: Analysis of Recessive Lethal Mutations

The genetic organization of interval 62B3-4 to 62D3-4 on the Drosophila third chromosome was investigated. The region (designated DRE) includes four known loci: Roughened (R; 3-1.4), defined by a dominant mutation disrupting eye morphology; the nonvital locus Aprt, structural gene for adenine phosphoribosyltransferase; Dras3, a homolog of the vertebrate ras oncogene; and 1(3)ecdysoneless (1(3)ecd), a gene that has been implicated in the regulation of larval molting hormone (ecdysteroid) synthesis. Overlapping chromosomal deletions of the region were generated by gamma-ray-induced reversion of the R mutation. Recessive lethal mutations were isolated based upon failure to complement the recessive lethality of Df(3L)RR2, a deletion of the DRE region that removes 16-18 polytene chromosome bands. A total of 117 mutations were isolated following ethyl methanesulfonate and gamma-ray mutagenesis. These and two additional define 13 lethal complementation groups. Mutations at two loci were recovered at disproportionately high rates. One of these loci is preferentially sensitive to radiation-induced mutational alterations. Additionally, an unusually low recovery rate for cytologically detectable rearrangement breakpoints within the gamma-ray-sensitive locus suggests that an interval of the DRE region closely linked to the R locus may be dominantly sensitive to position effects. Lethal phase analysis of mutant hemizygotes indicates that a high proportion of DRE-region loci (11 of 13) are necessary for larval development. Mutations in five loci cause predominantly first-instar larval lethality, while mutations in four other loci cause predominantly second-instar lethality. Mutations in two loci cause late-larval lethality associated with abnormal imaginal disc development. A temperature-sensitive allele of one newly identified complementation group blocks ecdysteroid-induced pupariation. This developmental block is overcome by dietary 20-hydroxyecdysone, suggesting that a second locus in the region in addition to l(3)ecd may play a role in the regulation of late larval ecdysteroid levels.     

Evidence for Mitotic Recombination in W(ei)/+ Heterozygous Mice

A number of alleles at coat color loci of the house mouse give rise to areas of wild-type pigmentation on the coats of otherwise mutant animals. Such unstable alleles include both recessive and dominant mutations. Among the latter are several alleles at the W locus. In this report, phenotypic reversions of the Wei allele at the W locus were studied Mice heterozygous in repulsion for both Wei and buff (bf) [i.e. Wei+/+bf] were examined for the occurrence of phenotypic reversion events. Buff (bf) is a recessive mutation, which lies 21 cM from W on the telomeric side of chromosome 5 and is responsible for the khaki colored coat of nonagouti buff homozygotes (a/a; bf/bf). Two kinds of fully pigmented reversion spots were recovered on the coats of a/a; Wei+/+bf mice: either solid black or khaki colored. Furthermore phenotypic reversions of Wei/+ were enhanced significantly following X-irradiation of 9.25-day-old Wei/+ embryos (P less than 0.04). These observations are consistent with the suggestion of a role for mitotic recombination in the origin of these phenotypic reversions. In addition these results rise the intriguing possibility that some W mutations may enhance mitotic recombination in the house mouse.     

Association of the widespread A149P hereditary fructose intolerance mutation with newly identified sequence polymorphisms in the aldolase B gene.

Hereditary fructose intolerance (HFI) is a potentially fatal autosomal recessive disease resulting from the catalytic deficiency of fructose 1-phosphate aldolase (aldolase B) in fructose-metabolizing tissues. The A149P mutation in exon 5 of the aldolase B gene, located on chromosome 9q21.3-q22.2, is widespread and the most common HFI mutation, accounting for 57% of HFI chromosomes. The possible origin of this mutation was studied by linkage to polymorphisms within the aldolase B gene. DNA fragments of the aldolase B gene containing the polymorphic marker loci from HFI patients homozygous for the A149P allele were amplified by PCR. Absolute linkage to a common PvuII RFLP allele was observed in 10 A149P homozygotes. In a more informative study, highly heterozygous polymorphisms were detected by direct sequence determination of a PCR-amplified aldolase B gene fragment. Two two-allele, single-base-pair polymorphisms, themselves in absolute linkage disequilibrium, in intron 8 (C at nucleotide 84 and A at nucleotide 105, or T at 84 and G at 105) of the aldolase B gene were identified. Mendelian segregation of these polymorphisms was confirmed in three families. Allele-specific oligonucleotide (ASO) hybridizations with probes for both sequence polymorphisms showed that 47% of 32 unrelated individuals were heterozygous at these loci; the calculated PIC value was .37. Finally, ASO hybridizations of PCR-amplified DNA from 15 HFI patients homozygous for the A149P allele with probes for these sequence polymorphisms revealed absolute linkage disequilibrium between the A149P mutation and the 84T/105G allele. These results are consistent with a single origin of the A149P allele and subsequent spread by genetic drift.