The relative roles of three DNA repair pathways in preventing Caenorhabditis elegans mutation accumulation

Mutation is a central biological process whose rates and spectra are influenced by a variety of complex and interacting forces. Although DNA repair pathways are generally known to play key roles in maintaining genetic stability, much remains to be understood about the relative roles of different pathways in preventing the accumulation of mutations and the extent of heterogeneity in pathway-specific repair efficiencies across different genomic regions. In this study we examine mutation processes in base excision repair-deficient (nth-1) and nucleotide excision repair-deficient (xpa-1) Caenorhabditis elegans mutation-accumulation (MA) lines across 24 regions of the genome and compare our observations to previous data from mismatch repair-deficient (msh-2 and msh-6) and wild-type (N2) MA lines. Drastic variation in both average and locus-specific mutation rates, ranging two orders of magnitude for the latter, was detected among the four sets of repair-deficient MA lines. Our work provides critical insights into the relative roles of three DNA repair pathways in preventing C. elegans mutation accumulation and provides evidence for the presence of pathway-specific DNA repair territories in the C. elegans genome.     

Frequent germline mutations and somatic repeat instability in DNA mismatch-repair-deficient Caenorhabditis elegans

Mismatch-repair-deficient mutants were initially recognized as mutation-prone derivatives of bacteria, and later mismatch repair deficiency was found to predispose humans to colon cancers (HNPCC). We generated mismatch-repair-deficient Caenorhabditis elegans by deleting the msh-6 gene and analyzed the fidelity of transmission of genetic information to subsequent generations. msh-6-defective animals show an elevated level of spontaneous mutants in both the male and female germline; also repeated DNA tracts are unstable. To monitor DNA repeat instability in somatic tissue, we developed a sensitive system, making use of heat-shock promoter-driven lacZ transgenes, but with a repeat that puts this reporter gene out of frame. In genetic msh-6-deficient animals lacZ+ patches are observed as a result of somatic repeat instability. RNA interference by feeding wild-type animals dsRNA homologous to msh-2 or msh-6 also resulted in somatic DNA instability, as well as in germline mutagenesis, indicating that one can use C. elegans as a model system to discover genes involved in maintaining DNA stability by large-scale RNAi screens.     

An Inducible,Isogenic Cancer Cell Line System for Targeting the State of Mismatch Repair Deficiency

The DNA mismatch repair system (MMR) maintains genome stability through recognition and repair of single-base mismatches and small insertion-deletion loops. Inactivation of the MMR pathway causes microsatellite instability and the accumulation of genomic mutations that can cause or contribute to cancer. In fact, 10-20% of certain solid and hematologic cancers are MMR-deficient. MMR-deficient cancers do not respond to some standard of care chemotherapeutics because of presumed increased tolerance of DNA damage, highlighting the need for novel therapeutic drugs. Toward this goal, we generated isogenic cancer cell lines for direct comparison of MMR-proficient and MMR-deficient cells. We engineered NCI-H23 lung adenocarcinoma cells to contain a doxycycline-inducible shRNA designed to suppress the expression of the mismatch repair gene MLH1, and compared single cell subclones that were uninduced (MLH1-proficient) versus induced for the MLH1 shRNA (MLH1-deficient). Here we present the characterization of these MMR-inducible cell lines and validate a novel class of rhodium metalloinsertor compounds that differentially inhibit the proliferation of MMR-deficient cancer cells.     

DNA错配修复与癌症的发生及治疗

DNA错配修复是细胞复制后的一种修复机制,具有维持DNA复制保真度,控制基因变异的作用。DNA错配修复缺陷使整个基因组不稳定,最终会导致肿瘤和癌症的发生。DNA错配修复系统不仅通过矫正在DNA重组和复制过程中产生的碱基错配而保持基因组的稳定,而且通过诱导DNA损伤细胞的凋亡而消除由突变细胞生长形成的癌变。错配修复缺陷细胞的抗药性也引起了癌症化疗研究方面的关注。大多数情况下,错配修复健全型细胞对肿瘤化疗药物敏感,而错配修复缺陷细胞却有较高的抗性。DNA错配修复系统通过修复和诱导细胞凋亡维护基因组稳定的功能,显示了错配修复途径在癌症生物学和分子医学中的重要性。     

Mlh1-dependent suppression of specific mutations induced in vivo by the food-borne carcinogen 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP)

Disruption of the DNA mismatch repair (MMR) pathway results in elevated mutation rates, inappropriate survival of cells bearing DNA damage, and increased cancer risk. Relatively little is known about the potential impact of environmentally relevant carcinogens on cancer risk in individuals with MMR-deficiency. We determined the effect of MMR status (Mlh1+/+ versus Mlh1-/-) on mutagenesis induced by the cooked-meat mutagen, 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) within cII and supFG1 transgene reporters. Despite being a lymphomagen in mice, PhIP was not mutagenic in thymus. In colon, PhIP exposure induced 3-fold more mutations in Mlh1-deficient mice compared to their Mlh1+/+ littermates. Similar induction was seen in Mlh1-/- small intestine. Analysis of mutational spectra revealed that G/C to T/A transversions, the "signature PhIP mutation", were induced to similar levels regardless of Mlh1 status. In contrast, Mlh1-/- mice exhibited hypermutability to frameshifts, G/C to A/T transitions, and G/C to C/G transversions. Thus, both the level and types of mutation induced by PhIP are influenced by the activity of the MMR system. MMR may suppress PhIP-induced mutation through recognition and processing of specific mispairs (PhIP-G/T, PhIP-G/G, and PhIP-G/loop mispairs). In contrast, the PhIP-G/A mispair is unlikely to be a MMR substrate. In addition, the similar induction of both transversions and transitions in Mlh1-/- mice suggests that mutagenic bypass of PhIP-G is similarly efficient with dATP, dTTP, and dGTP, in contrast to previously published conclusions. Our data suggests that MMR-deficiency would increase the likelihood of PhIP-induced carcinogenic mutations. Further evaluation of the risk that consumption of heterocyclic amines may impart to MMR-deficient individuals therefore is warranted.     

Mutation of msh-2 in Neurospora crassa does not reduce the incidence of recombinants with multiple patches of donor chromosome sequence

The Neurospora homologue msh-2 of the Escherichia coli mismatch repair gene mutS was mutated by repeat-induced point mutation (RIP) of a 1.9-kb duplication covering 1661bp of the coding sequence and 302 bp 5' of the gene. msh-2(RIP-LK1) exhibited a mutator phenotype conferring a 17-fold increase in the frequency of spontaneous mitotic reversion of his-3 allele K458. In msh-2(RIP-LK1) homozygotes, recombination frequency at the his-3 locus increased up to 2.9-fold over that in msh-2(+) diploids. Progeny of crosses homozygous msh-2(RIP-LK1), like those from crosses homozygous msh-2(+) frequently had multiple patches of donor chromosome sequence, suggesting that patchiness in msh-2(+) crosses is not explained by incomplete repair of heteroduplex DNA by MSH-2. These findings are consistent with data from the analysis of events in a Neurospora translocation heterozygote that suggested multiple patches of donor chromosome sequence arising during recombination reflect multiple template switches during DNA repair synthesis.     

Analysis of yeast pms1, msh2, and mlh1 mutators points to differences in mismatch correction efficiencies between prokaryotic and eukaryotic cells

Genetic stability relies in part on the efficiency with which post-replicative mismatch repair (MMR) detects and corrects DNA replication errors. In Escherichia coli, endogenous transition mispairs and insertion/deletion (ID) heterologies are corrected with similar efficiencies – but much more efficiently than transversion mispairs – as revealed by mutation rate increases in MMR mutants. To assess the relative efficiencies with which these mismatches are corrected in the yeast Saccharomyces cerevisiae, we examined repair of defined mismatches on heteroduplex plasmids and compared the spectra for >1000 spontaneous SUP4-o mutations arising in isogenic wild-type or MMR-deficient (pms1, mlh1, msh2) strains. Heteroduplexes containing G/T mispairs or ID heterologies were corrected more efficiently than those containing transversion mismatches. However, the rates of single base-pair insertion/deletion were increased much more (82-fold or 34-fold, respectively) on average than the rate of base pair substitutions (4.4-fold), with the rates for total transitions and transversions increasing to similar extents. Thus, the relative efficiencies with which mismatches formed during DNA replication are repaired appear to differ in prokaryotic and eukaryotic cells. In addition, our results indicate that in yeast, and probably other eukaryotes, these efficiencies may not mirror those obtained from an analysis of heteroduplex correction.     

Analysis of yeast pms1, msh2, and mlh1 mutators points to differences in mismatch correction efficiencies between prokaryotic and eukaryotic cells.

Genetic stability relies in part on the efficiency with which post-replicative mismatch repair (MMR) detects and corrects DNA replication errors. In Escherichia coli, endogenous transition mispairs and insertion/deletion (ID) heterologies are corrected with similar efficiencies – but much more efficiently than transversion mispairs – as revealed by mutation rate increases in MMR mutants. To assess the relative efficiencies with which these mismatches are corrected in the yeast Saccharomyces cerevisiae, we examined repair of defined mismatches on heteroduplex plasmids and compared the spectra for >1000 spontaneous SUP4-o mutations arising in isogenic wild-type or MMR-deficient (pms1, mlh1, msh2) strains. Heteroduplexes containing G/T mispairs or ID heterologies were corrected more efficiently than those containing transversion mismatches. However, the rates of single base-pair insertion/deletion were increased much more (82-fold or 34-fold, respectively) on average than the rate of base pair substitutions (4.4-fold), with the rates for total transitions and transversions increasing to similar extents. Thus, the relative efficiencies with which mismatches formed during DNA replication are repaired appear to differ in prokaryotic and eukaryotic cells. In addition, our results indicate that in yeast, and probably other eukaryotes, these efficiencies may not mirror those obtained from an analysis of heteroduplex correction. Received: 15 November 1998 / Accepted: 4 February 1999     

Evolutionary implications of the frequent horizontal transfer of mismatch repair genes

Mutation and subsequent recombination events create genetic diversity, which is subjected to natural selection. Bacterial mismatch repair (MMR) deficient mutants, exhibiting high mutation and homologous recombination rates, are frequently found in natural populations. Therefore, we have explored the possibility that MMR deficiency emerging in nature has left some "imprint" in the sequence of bacterial genomes. Comparative molecular phylogeny of MMR genes from natural Escherichia coli isolates shows that, compared to housekeeping genes, individual functional MMR genes exhibit high sequence mosaicism derived from diverse phylogenetic lineages. This apparent horizontal gene transfer correlates with hyperrecombination phenotype of MMR-deficient mutators. The sequence mosaicism of MMR genes may be a hallmark of a mechanism of adaptive evolution that involves modulation of mutation and recombination rates by recurrent losses and reacquisitions of MMR gene functions.     

Comparison of geno- and cytotoxicity of methylnitrosourea on MMR-proficient and MMR-deficient human tumor cell lines

Deficient mismatch repair (MMR) is identified as a mutation of one of four major MMR genes and(or) microsatellite instability. These genomic changes are used as markers of MMR status of the heredity nonpolyposis colorectal cancer (HNPCC) spectrum tumors--familial and sporadic tumors of colon and extracolonic cancers fulfilling Amsterdam clinical criteria II. MMR-deficiency results in mutator phenotype and resistance to geno- and cytotoxicity of alkylating agents. The main cytotoxic damage to DNA in response to chemical methylation is O6-methylguanine (O6-mG). The secondary DNA strand breaks, which are formed during the MMR functioning, are proposed to be required for methylation induced cytotoxicity. We have assumed that the secondary double stand breaks (DSB) upon DNA methylation are able to represent functional efficiency of MMR in cells. The purpose of the paper was to test this assumption on human tumor cells differing in MMR-status and pulse-treated with methylnitrosourea (MNU). We used 3 cell lines: HeLa (MMR-competent endometrial tumor cells), HCT116 (MMR-deficient colorectal carcinoma cells), and Colo320 (sigmoid intestine tumor cells with uncharacterized MMR status). DSBs were evaluated with neutral comet assay. Cytotoxicity/viability was evaluated with MTT-asay and apoptotic index (frequency of morphologically determined apoptotic cells). We show that 1) cytotoxic effect of MNU (250 microM) on HeLa cells was exhibited 3 days after pulse-treatment of cells with MNU; 2) DSBs occurred 48 h after the drug treatment but prior to the onset of apoptosis of HeLa cells; 3) MMR-deficient HCT116 cells were resistant to the drug: no decreased viability, DSBs and apoptosis were observed during 3 days after cell treatment. Both cell lines exhibited high sensitivity to etoposide, classical inductor of unrepairable DSBs and p53. Etoposide has been found to induce DSBs in 6-12 h, which was followed by apoptosis (in 24 h). Colo320 cells exhibited intermediate position between HeLa and HCT116 cell lines in regard to sensitivity to MNU according to MTT-assay and the number of secondary DSBs formed in MNU-treated cells. Nevertheless, in contrast to HeLa cells, these breaks did not induce apoptosis in Colo320 cells. Our data confirm the assumption about case/effect relationship between secondary DNA double strand breaks, induced by monofunctional methylating agent MNU, and functioning of MMR in human tumor cells.     

The role of DNA mismatch repair in cisplatin mutagenicity

Cisplatin (DDP) is used with varying success for the treatment of a wide spectrum of human cancers. The most abundant lesions produced in DNA are intrastrand crosslinks, which are believed to account for not only the cytotoxic action but also the mutagenicity of the drug. The molecular basis for the mutagenicity of DDP adducts is believed to be related to bypass replication across the adducts by DNA polymerase. This results in misincorporation of non-complimentary bases by polymerase beta which, if left unpaired, will generate point or frameshift mutations. An important replication-associated correction function is provided by the post-replicative DNA mismatch repair (MMR) system. Loss of MMR activity is well documented to result in increased mutation rates and instability of genomic DNA. Inactivation of the MMR system also augments the intrinsic mutagenicity of DDP and enhances the risk of developing cells resistant to other drugs commonly used in combination with DDP. A future challenge will be to assess the clinical significance of the presence of MMR-deficient cells in tumors, and investigate new approaches to circumvent such multidrug resistance.     

Neurofibromatosis type 1 gene as a mutational target in a mismatch repair-deficient cell type

DNA mismatch repair (MMR) is the process by which incorrectly paired DNA nucleotides are recognized and repaired. A germline mutation in one of the genes involved in the process may be responsible for a dominantly inherited cancer syndrome, hereditary nonpolyposis colon cancer. Cancer progression in predisposed individuals results from the somatic inactivation of the normal copy of the MMR gene, leading to a mutator phenotype affecting preferentially repeat sequences (microsatellite instability, MSI). Recently, we identified children with a constitutional deficiency of MMR activity attributable to a mutation in the h MLH1 gene. These children exhibited a constitutional genetic instability associated with clinical features of de novo neurofibromatosis type 1 (NF1) and early onset of extracolonic cancer. Based on these observations, we hypothesized that somatic NF1 gene mutation was a frequent and possibly early event in MMR-deficient cells. To test this hypothesis, we screened for NF1 mutations in cancer cells. Genetic alterations were identified in five out of ten tumor cell lines with MSI, whereas five MMR-proficient tumor cell lines expressed a wild-type NF1 gene. Somatic NF1 mutations were also detected in two primary tumors exhibiting an MSI phenotype. Finally, a 35-bp deletion in the murine Nf1 coding region was identified in mlh1-/- mouse embryonic fibroblasts. These observations demonstrate that the NF1 gene is a mutational target of MMR deficiency and suggest that its inactivation is an important step of the malignant progression of MMR-deficient cells.     

Differences in genome-wide repeat sequence instability conferred by proofreading and mismatch repair defects

Mutation rates are used to calibrate molecular clocks and to link genetic variants with human disease. However, mutation rates are not uniform across each eukaryotic genome. Rates for insertion/deletion (indel) mutations have been found to vary widely when examined in vitro and at specific loci in vivo. Here, we report the genome-wide rates of formation and repair of indels made during replication of yeast nuclear DNA. Using over 6000 indels accumulated in four mismatch repair (MMR) defective strains, and statistical corrections for false negatives, we find that indel rates increase by 100 000-fold with increasing homonucleotide run length, representing the greatest effect on replication fidelity of any known genomic parameter. Nonetheless, long genomic homopolymer runs are overrepresented relative to random chance, implying positive selection. Proofreading defects in the replicative polymerases selectively increase indel rates in short repetitive tracts, likely reflecting the distance over which Pols δ and ϵ interact with duplex DNA upstream of the polymerase active site. In contrast, MMR defects hugely increase indel mutagenesis in long repetitive sequences. Because repetitive sequences are not uniformly distributed among genomic functional elements, the quantitatively different consequences on genome-wide repeat sequence instability conferred by defects in proofreading and MMR have important biological implications.     

Role of the mismatch repair system and p53 in the clastogenicity and cytotoxicity induced by bleomycin

The mismatch repair (MMR) system and p53 protein play a pivotal role in maintaining genomic stability and modulate cell chemosensitivity. Aim of this study was to examine the effects of either MMR-deficiency or p53 inactivation, or both, on cellular responses to bleomycin. The MMR-deficient colon carcinoma cell line HCT116 and its MMR-proficient subline HCT116/3-6, both expressing wild-type p53, were transfected with an expression vector encoding a dominant-negative p53 mutant, or with the empty vector. Four transfected clones, having the following phenotypes, MMR-proficient/p53 wild-type, MMR-proficient/p53 mutant, MMR-deficient/p53 wild-type, MMR-deficient/p53 mutant, were subjected to treatment with bleomycin. Loss of MMR function alone was associated with increased resistance to apoptosis, chromosomal damage and inhibition of colony formation caused by bleomycin. Loss of p53 alone resulted in abrogation of G1 arrest and increased sensitivity to apoptosis and chromosomal damage induced by the drug, but did not affect clonogenic survival after bleomycin treatment. Disabling both p53 and MMR function led to abrogation of G1 arrest and to a moderate impairment of drug-induced apoptosis. Chromosomal damage was reduced in the MMR-deficient/p53 mutant clone with respect to the MMR-proficient/p53 wild-type one, when evaluated 48 h after bleomycin treatment, but was comparable in both clones 96 h after drug exposure. Clonogenic survival of the MMR-deficient/p53 mutant clone was similar to that of the MMR-deficient/p53 wild-type one. The effects of MMR-deficiency on cellular responses to bleomycin were confirmed using the MMR-proficient lymphoblastoid cell line TK6 and its MMR-deficient subline MT1, both expressing wild-type p53. In conclusion, our data show that loss of MMR and p53 function exerts opposite and independent effects on apoptosis and chromosomal damage induced by bleomycin. Moreover, inactivation of MMR confers resistance to the cytotoxic activity of the anticancer agent in cells expressing either wild-type or mutant p53.     

Multigeneration maximum-likelihood analysis applied to mutation-accumulation experiments in Caenorhabditis elegans

We develop a maximum-likelihood (ML) approach to estimate genomic mutation rates (U) and average homozygous mutation effects (s) from mutation-accumulation (MA) experiments in which phenotypic assays are carried out in several generations. We use simulations to compare the procedure's performance with the method of moments traditionally used to analyze MA data. Similar precision is obtained if mutation effects are small relative to the environmental standard deviation, but ML can give estimates of mutation parameters that have lower sampling variances than those obtained by the method of moments if mutations with large effects have accumulated. The inclusion of data from intermediate generations may improve the precision. We analyze life-history trait data from two Caenorhabditis elegans MA experiments. Under a model with equal mutation effects, the two experiments provide similar estimates for U of approximately 0.005 per haploid, averaged over traits. Estimates of s are more divergent and average at -0.51 and -0.13 in the two studies. Detailed analysis shows that changes of mean and variance of genetic values of MA lines in both C. elegans experiments are dominated by mutations with large effects, but the analysis does not rule out the presence of a large class of deleterious mutations with very small effects.     

Measurement of DNA mismatch repair activity in live cells

Loss of DNA mismatch repair (MMR) function leads to the development and progression of certain cancers. Currently, assays for DNA MMR activity involve the use of cell extracts and are technically challenging and costly. Here, we report a rapid, less labor-intensive method that can quantitatively measure MMR activity in live cells. A G–G or T–G mismatch was introduced into the ATG start codon of the enhanced green fluorescent protein (EGFP) gene. Repair of the G–G or T–G mismatch to G–C or T–A, respectively, in the heteroduplex plasmid generates a functional EGFP gene expression. The heteroduplex plasmid and a similarly constructed homoduplex plasmid were transfected in parallel into the same cell line and the number of green cells counted by flow cytometry. Relative EGFP expression was calculated as the total fluorescence intensity of cells transfected with the heteroduplex construct divided by that of cells transfected with the homoduplex construct. We have tested several cell lines from both MMR-deficient and MMR-proficient groups using this method, including a colon carcinoma cell line HCT116 with defective hMLH1 gene and a derivative complemented by transient transfection with hMLH1 cDNA. Results show that MMR-proficient cells have significantly higher EGFP expression than MMR-deficient cells, and that transient expression of hMLH1 alone can elevate MMR activity in HCT116 cells. This method is potentially useful in comparing and monitoring MMR activity in live cells under various growth conditions.     

Mismatch recognition function of Arabidopsis thaliana MutSγ

Genetic stability depends in part on an efficient DNA lesion recognition and correction by the DNA mismatch repair (MMR) system. In eukaryotes, MMR is initiated by the binding of heterodimeric MutS homologue (MSH) complexes, MSH2–MSH6 and MSH2–MSH3, which recognize and bind mismatches and unpaired nucleotides. Plants encode another mismatch recognition protein, named MSH7. MSH7 forms a heterodimer with MSH2 and the protein complex is designated MutSγ. We here report the effect the expression of Arabidopsis MSH2 and MSH7 alone or in combination exert on the genomic stability of Saccharomyces cerevisiae. AtMSH2 and AtMutSγ proteins failed to complement the hypermutator phenotype of an msh2 deficient strain. However, overexpressing AtMutSγ in MMR proficient strains generated a 4-fold increase in CAN1 forward mutation rate, when compared to wild-type strains. Can^r mutation spectrum analysis of AtMutSγ overproducing strains revealed a substantial increase in the frequency of base substitution mutations, including an increased accumulation of base pair changes from G:C to A:T and T:A to C:G, G:C or A:T. Taken together, these results suggest that AtMutSγ affects yeast genomic stability by recognizing specific mismatches and preventing correction by yeast MutSα and MutSβ, with subsequent inability to interact with yeast downstream proteins needed to complete MMR.     

Effects of camptothecin on double-strand break repair by non-homologous end-joining in DNA mismatch repair-deficient human colorectal cancer cell lines

Loss of a functional mismatch repair (MMR) system in colorectal cancer (CRC) cells is associated with microsatellite instability and increased sensitivity to topoisomerase inhibitors. In this study, we have investigated whether a defect in double-strand break (DSB) repair by non-homologous end-joining (NHEJ) could explain why MMR-deficient CRC cells are hypersensitive to camptothecin (CPT), a topoisomerase I inhibitor. To evaluate the efficiency and the fidelity of DSB repair, we have transiently transfected plasmids containing cohesive or non-complementary ends in cells with various MMR defects. We have observed that the repair efficiency of DSB with cohesive and non-complementary ends is comparable in all cell lines. In contrast to the MMR-proficient cell line HT29, the MMR-deficient cell lines were highly accurate in repairing DSB with cohesive ends, but this characteristic could not be directly assigned to the primary MMR deficiency. Furthermore, CPT treatment had no detectable effect on the repair of cohesive ends but significantly decreased the repair efficiency of non-complementary DSB. In conclusion, although our observations show that DSB repair efficiency by NHEJ decreases upon treatment with CPT, which possibly contributes to its cytotoxicity, it is quite unlikely that it accounts for the hypersensitivity of MMR-deficient cells to topoisomerase inhibitors.     

Evidence for the lack of mismatch-repair directed antirecombination during mouse meiosis

Meiotic recombination was studied in DNA mismatch repair (MMR)-deficient mice using a strain carrying a Pms2 knockout mutation. Using single-sperm typing, recombination was analyzed over five intervals on four chromosomes in four Pms2 -/- animals. A total of 1936 meioses were studied and compared to 1848 meioses from three Pms2 +/+ controls. A smaller study was carried out on a single interval in each of two chromosomes in an MMR-deficient mouse homozygous for the Msh2 knockout mutation. A total of 792 meioses were examined in the Msh2 -/- and 880 meioses in the Msh2 +/+ animal. Recombination fractions were not significantly different in either of the MMR-deficient mouse strains when compared to MMR-proficient controls. Our results appear to conflict with mouse embryonic stem (ES) cell gene-targeting experiments where MMR plays a major role in determining the efficiency of homologous recombination between nonidentical sequences. A number of possibilities could explain the apparent lack of a significant effect on meiosis.     

Genotype-environment interactions of spontaneous mutations for vegetative fitness in the human pathogenic fungus Cryptococcus neoformans

Spontaneous mutation is the ultimate source of all genetic variation. By interacting with environmental factors, genetic variation determines the phenotype and fitness of individuals in natural populations. However, except in a few model organisms, relatively little is known about the patterns of genotype-environment interactions of spontaneous mutations. Here I examine the rates of spontaneous mutation and the patterns of genotype-environment interaction of mutations affecting vegetative growth in the human fungal pathogen Cryptococcus neoformans. Eight mutation accumulation (MA) lines were established from a single clone on the nutrient-rich medium YEPD for each of two temperatures, 25 degrees and 37 degrees. Cells from generations 100, 200, 400, and 600 for each of the 16 MA lines were stored and assayed for vegetative growth rates under each of four conditions: (i) 25 degrees on SD (a synthetic dextrose minimal medium); (ii) 25 degrees on YEPD; (iii) 37 degrees on SD; and (iv) 37 degrees on YEPD. Both MA conditions and assay environments for vegetative growth showed significant influence on the estimates of genomic mutation rates, average effect per mutation, and mutational heritability. Significant genotype-environment interactions were detected among the newly accumulated spontaneous mutations. Overall, clones from MA lines maintained at 37 degrees showed less decline in vegetative fitness than those maintained at 25 degrees. The result suggests that a high-temperature environment might be very important for the maintenance of the ability to grow at a high temperature. Results from comparisons between clinical and environmental samples of C. neoformans were consistent with laboratory experimental population analyses. This study calls into question our long-standing view that warm-blooded mammals were only occasional and accidental hosts of this human fungal pathogen.