Frequency Of Gene Conversion Research Articles

Non-locus-specific polygenes giving responses to selection for gene conversion frequencies in Ascobolus immersus.

Selection for higher and lower meiotic conversion frequencies was investigated in the fungus Ascobolus immersus. Strains carrying the same known gene conversion control factors, which have major effects on conversion frequencies at their specific target locus, sometimes gave significant differences in conversion frequency. Selection for high or low conversion frequencies at the w1-78 site was practiced for five generations, giving significant responses in both directions. These responses were due to polygenes, or genes of minor effect, not to new conversion control factors of major effect. Crosses of selected strains to strains with other mutations showed that the genes' effects were not specific to w1-78, but could affect conversion frequencies of another mutation, w1-3C1, at that locus and of two other loci, w-BHj and w9, which are unlinked to w1 or to each other. The proportional changes in gene conversion frequency due to selection varied according to the locus and site involved and according to the conversion control factor alleles present. There were differences of > or = 277% in conversion frequency between "high" and "low" strains. Selection for conversion frequency had little effect on other features of conversion, such as the frequency of postmeiotic segregation or the relative frequencies of conversion to mutant or wild type.

Modulation of bleomycin-induced mitotic recombination in yeast by the aminothiols cysteamine and WR-1065.

The cancer chemotherapy drug bleomycin (BLM) is a potent inducer of genetic damage in a wide variety of assays. The radioprotectors cysteamine (CSM) and WR-1065 have been shown in previous studies to potentiate the induction of micronuclei and chromosome aberrations by BLM in Go human lymphocytes. By contrast, WR-1065 is reported to reduce the induction of hprt mutations by BLM in Chinese hamster cells. To elucidate the basis for these interactions, we examined the effects of CSM and WR-1065 on the induction of mitotic gene conversion by BLM in the yeast Saccharomyces cerevisiae. Treatment with BLM causes a dose-dependent increase in the frequency of mitotic gene conversion and gene mutations. Unlike its potentiation of BLM in Go lymphocytes, WR-1065 protected against the recombinagenicity of BLM in yeast. CSM was also strongly-antirecombinagenic under, some conditions, but the nature of the interaction depended strongly on the treatment conditions. Under hypoxic conditions, cysteamine protected against BLM, but under oxygen-rich conditions CSM potentiated the genetic activity of BLM. The protective effect of aminothiols against BLM may be ascribed to the depletion of oxygen required for the activation of BLM and the processing of BLM-induced damage. Aminothiols may potentiate the effect of BLM by acting as an electron source for the activation of BLM and/or by causing conformational alterations that make DNA more accessible to BLM. The results indicate that aminothiols have a strong modulating influence on the genotoxicity of BLM in yeast as they do in other genetic assays. Moreover, the modulation differs markedly depending on physiological conditions. Thus, yeast assays help to explain why aminothiols have been observed to potentiate BLM in some genetic systems and to protect against it in others.

A determination of the frequency of gene conversion in unmanipulated mouse sperm.

Gene conversion, sometimes also called micro gene conversion or gene conversion-like events, has been proposed to act on a number of genes in higher eukaryotes, such as gamma-globin, beta-tubulin, major urinary protein, and amyloid A genes. In the immune system, immunoglobulin genes and major histocompatibility complex class I and class II genes have been implicated. The notion that integral segments of DNA have been transferred from one gene to another in these cases has, however, met considerable resistance. We have devised a PCR assay detecting only the molecule that results if the E beta d-derived fragment analogous to that introduced in the bm12 mutation is transferred to the A beta k gene. We have proceeded to analyze sperm from the F1 cross C3H/HeJ (haplotype k) x BALB/c (haplotype d). In our assay, we find that the frequency for conversion of this particular DNA segment is 2 x 10(-6). This frequency is relevant only in the germ line; when liver cells were tested as an example of somatic cells, no events were observed, implying a frequency of < 2 x 10(-8) in liver. Fragments > 100 bp seem to be possible to transfer in this conversion.

Genetic and molecular analysis of hybrids in the genus Saccharomyces involving S. cerevisiae, S. uvarum and a new species, S. douglasii

We have studied the phenomenon of infertility of yeast hybrids obtained with physiological conditions under the control of compatible mating systems. The yeasts investigated are three Saccharomyces species: S. cerevisiae, S. uvarum and a new species, S. douglasii. The diploid hybrids from crosses between these species sporulate well but are essentially infertile. The rare viable spores, one per 10(4) to 10(5) asci, that have been examined carry a complete genome comprised of chromosomes contributed by both parents but invariably have extra chromosomes, i.e. they are generally disomic for at least two or three chromosomes. This observation is consistent with a failure, in meiosis I, of the pairing and disjunction of homologous chromosomes which in most cases results in spores with an incomplete set of chromosomes. This apparent lack of pairing of 'homeologous' chromosomes in meiosis I was analysed in most detail with S. cerevisiae/S. douglasii hybrids. As a genetic tool we studied frequencies of recombination, taking advantage of an S. douglasii breeding stock of some 50 identified mutations in non-switching haploids. Recombination, although markedly reduced, could be observed at both the chromosomal and allelic levels, implying a sporadic pairing in meiosis to allow genetic exchange. Meiotic recombination frequencies were studied for 14 gene pairs and generally found to be reduced ten-fold. Heteroallelic recombination (gene conversion) frequencies were measured at 22 loci and were judged to be reduced at least two- to 100-fold. DNA hybridization experiments with S. cerevisiae gene probes gave results consistent with low DNA sequence homologies between S. cerevisiae and S. douglasii. Moreover, by change, our experiments disclosed another Saccharomyces strain (CBS2908, originally classified as S. cerevisiae) with hybridization patterns identical to S. douglasii except for the hybridization with the Ty transposon probes. Crosses between CBS2908 and S. douglasii yielded diploid hybrids with 80-90% spore viability, thus establishing a second member of the S. douglasii species.

Analysis of a recombination hotspot for gene conversion occurring at the HIS2 gene of Saccharomyces cerevisiae.

The properties of gene conversion as measured in fungi that generate asci containing all the products of meiosis imply that meiotic recombination initiates at specific sites. The HIS2 gene of Saccharomyces cerevisiae displays a high frequency of gene conversion, indicating that it is a recombination hotspot. The HIS2 gene was cloned and sequenced, and the cloned DNA was used to make several different types of alterations in the yeast chromosome by transformation; these alterations were used to determine the location of the sequences necessary for the high levels of meiotic conversion observed at HIS2. Previous work indicated that the gene conversion polarity gradient is high at the 3' end of the gene, and that the promoter of the gene is not necessary for the high frequency of conversion observed. Data presented here suggest that at least some of the sequences necessary for high levels of conversion at HIS2 are located over 700 bp downstream of the end of the coding region, extend over (at least) several hundred base pairs, and may be quite complex, perhaps involving chromatin structure. Additional data indicate that multiple single base heterologies within a 1-kb interval contribute little to the frequency of gene conversion. This contrasts with other reports about the role of heterologies at the MAT locus.

Interaction between mismatch repair and genetic recombination in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae encodes a set of genes that show strong amino acid sequence similarity to MutS and MutL, proteins required for mismatch repair in Escherichia coli. We examined the role of MSH2 and PMS1, yeast homologs of mutS and mutL, respectively, in the repair of base pair mismatches formed during meiotic recombination. By using specifically marked HIS4 and ARG4 alleles, we showed that msh2 mutants displayed a severe defect in the repair of all base pair mismatches as well as 1-, 2- and 4-bp insertion/deletion mispairs. The msh2 and pms1 phenotypes were indistinguishable, suggesting that the wild-type gene products act in the same repair pathway. A comparison of gene conversion events in wild-type and msh2 mutants indicated that mismatch repair plays an important role in genetic recombination. (1) Tetrad analysis at five different loci revealed that, in msh2 mutants, the majority of aberrant segregants displayed a sectored phenotype, consistent with a failure to repair mismatches created during heteroduplex formation. In wild type, base pair mismatches were almost exclusively repaired toward conversion rather than restoration. (2) In msh2 strains 10-19% of the aberrant tetrads were Ab4:4. (3) Polarity gradients at HIS4 and ARG4 were nearly abolished in msh2 mutants. The frequency of gene conversion at the 3' end of these genes was increased and was nearly the frequency observed at the 5' end. (4) Co-conversion studies were consistent with mismatch repair acting to regulate heteroduplex DNA tract length. We favor a model proposing that recombination events occur through the formation and resolution of heteroduplex intermediates and that mismatch repair proteins specifically interact with recombination enzymes to regulate the length of symmetric heteroduplex DNA.

Polarity of meiotic gene conversion in fungi: contrasting views.

The frequency of meiotic gene conversion often varies linearly from one end of the gene to the other. This phenomenon has been called 'polarity'. In this review, we will primarily studies of polarity that have been done in the yeast Saccharomyces cerevisiae (ARG4 and HIS4 loci) and in Ascobolus (b2 locus) with an emphasis on possible mechanisms. The genetic and physical data obtained at these 'hotspots' of recombination strongly suggests that the formation of a polarity gradient reflects both the frequency of heteroduplex formation and the processing of this recombination intermediate by mismatch-repair-dependent processes.

Analysis of the role of the NUC1 endo/exonuclease in yeast mitochondrial DNA recombination.

Mitochondrial DNA recombination was reduced in an yeast mutant lacking the NUC1 endo/exonuclease. Between linked markers in either the omega or cob region the frequency of recombination decreased nearly 50% compared to wild-type. Gene conversion frequencies in the var1 gene and in the omega region were also lower in the mutant strain. In particular, the gradient of gene conversion at omega was most affected by the absence of the NUC1 nuclease. In crosses between nuclease-deficient and wild-type strains, gene conversion frequencies at omega were reduced only when the omega+ allele was contributed to the zygote by the nuclease-deficient parent. We propose that the 5' exonuclease activity of the NUC1 nuclease functions during recombination to enlarge heteroduplex tracts following a double-strand break in DNA. In crosses between nuclease-deficient and wild-type strains, the anisotropy in gene conversion frequencies at omega is hypothesized to be due to the slow mixing of parental mitochondrial membranes as they fuse in the zygote.

Radiation-induced mitotic gene conversion frequency in yeast in modulated by the conditions allowing DNA double-strand break repair

Repair of DNA double-strand breaks (DSB)_involves recombinational process which may lead to gene conversion (intragenic recombination). Using the diploid yeast mutant rad54-3 heteroallelic for his1 (his1-7/his1-1) and temperature conditional for DSB rejoining, radiation induced gene conversion was investigated as dependent on DSB rapair under different postirradiation conditions. Gene conversion is negligible under conditions preventing DSB repair (36°C). In contrast, gene conversion is observed when cells are incubated at the permissive temperature (23°C) both under growth and nongrowth condtions. However, there is a much higher yield of convertants for cells incubated under growth as opposed to nongrowth conditions. These results can most plausibly be explained by the cell cycle regulated enhancement of the expression of genes such as PMS and POL3 known to be involved in gene conversion processes and/or the enhanced recombination in transcriptionally active genes. ‘Nutrient stress’ inducible responses and/or cell specific recombination pathways leading to gene conversion events preferentially in S-phase cells seem to be less likely.

The control in cis of the position and the amount of the ARG4 meiotic double-strand break of Saccharomyces cerevisiae.

During meiosis, a transient DNA double-strand break (DSB) occurs in the promoter region (positions -200/-185) of the Saccharomyces cerevisiae ARG4 gene and is a likely intermediate in the initiation of meiotic gene conversion events in this region. We report here a functional analysis of the ARG4 DSB based on the study of various deletions in this chromosomal region. We have identified several cis-acting elements located within the -465/+3 region of the ARG4 promoter that control the formation of this DSB. The -465/-317 region includes a transcription terminator and is necessary for a normal amount of ARG4 DSB, but not for its positioning. The -316/-140 region can be replaced by an unrelated DNA sequence where a meiotic DSB then occurs, suggesting that the site of DSB is not sequence-specific, but is positioned at a fixed distance from the adjacent -139/+3 region. Also, in all strains constructed, the amount of meiotic DSB is correlated with the frequency of gene conversion in ARG4, which provides a strong argument for the initiation of gene conversion by a DSB in this region of the yeast genome.

Cytotoxic and genotoxic properties of tert.-butyl- p-quinone (TBQ) in an in vitro assay system with Chinese hamster V79 cells and in strain D7 of Saccharomyces cerevisiae

tert.-Butyl- p-quinone (TBQ), a major metabolite of the phenolic antioxidant tert.-butyl-4-hydroxyanisole (BHA), was examined for cytotoxic and genotoxic properties in an in vitro assay system with Chinese hamster V79 cells and in diploid strain D7 of Saccharomyces cerevisiae. TBQ was prepared from BHA by oxidative demethylation with sodium nitrite at low pH. Spectroscopic analyses identified the crystalline reaction product as TBQ with a purity close to 100%. Cytotoxicity of TBQ was determined by cloning efficiency in the absence of hepatocyte activation. TBQ reduced colony size of V79 cells at 0.4 μg/ml, prevented growth of 50% of the cells at 0.6 μg/ml, and was lethal to 100% of the cells at concentrations above 1.0 μg/ml. TBQ was 6–7 times more cytotoxic to V79 cells than TBHQ, a related BHA metabolite, and 100 times more cytotoxic than BHA. At dose levels of 0.2, 0.4 and 0.6 μg/ml of medium, TBQ did not increase significantly the frequency of sister-chromatid exchanges (SCE) in V79 cells and did not consistently increase the frequency of mutation to thioguanine resistance (TG R) at the hgprt gene locus either alone or with activation by rat hepatocytes. Incubation with TBQ for 4 h at pH 3.6 without activation resulted in only small increases in the frequency of gene conversion and reverse mutation in strain D7 of Saccharomyces cerevisiae. However, exposure to TBQ alone in growth medium for 24 h, produced inconsistent results. From these studies it was concluded that TBQ was cytotoxic but not genotoxic to V79 cells; and may be weakly genotoxic to strain D7 of S. cerevisiae.

A sequence-specific endonuclease, Endo.SceI, can efficiently induce gene conversion in yeast mitochondria lacking a major exonuclease.

Endo.SceI most likely initiates homologous recombination of yeast mitochondrial genome through sequence-specific double-strand scission of DNA. According to the double-strand break-repair model for the mechanism of homologous recombination, DNA ends created by sequence-specific endonucleases have to be processed by exonucleases. The major mitochondrial exonuclease (encoded by NUC1) has been shown to greatly affect the length of conversion tracts at the 21S rRNA locus when site-specific gene conversion is induced by omega endonuclease. In order to examine the role of the NUC1 nuclease in the Endo.SceI-induced recombination, recombination frequencies were measured after crossing of parental strains either in the presence or absence of NUC1 nuclease activity. The frequency of gene conversion in the oli2 locus induced by Endo.SceI was not reduced by disruption of the NUC1 gene. This result strongly implicates the presence of multiple exonucleases for the processing of the DNA ends created by sequence-specific endonucleases.

Urban air pollution: Use of different mutagenicity assays to evaluate environmental genetic hazard

The genotoxic activities associated with airborne particulate matter collected in Parma (northern Italy) have been determined. The airborne particle extracts were tested for mutagenicity using Salmonella frameshift (TA98) and base-substitution (TA100) tester strains with and without S9 microsomal activation and Saccharomyces cerevisiae strain D7 in order to determine the frequency of mitotic gene conversion and i[v]-92 mutant reversion in cells harvested at stationary and logarithmic growth phase. The relationship between mitochondrial DNA mutations and ageing, degenerative diseases and cancer prompted us to take into account the mitochondrial informational target, i.e., the respiratory-deficient (RD) mutants. The results obtained show a variability in the response for the different test systems during different months. The Salmonella mutagenicity trend was directly correlated with carbon monoxide, nitrogen oxides (NO x ) and Pb concentration in airborne particulates and inversely correlated with temperature, whereas the mitochondrial genotoxic effect was higher during spring and late summer. These data suggest that the genotoxic risk assessment is a time-dependent value strictly correlated with the evaluation system being tested.

High-frequency germ line gene conversion in transgenic mice.

Gene conversion is the nonreciprocal transfer of genetic information between two related genes or DNA sequences. It can influence the evolution of gene families, having the capacity to generate both diversity and homogeneity. The potential evolutionary significance of this process is directly related to its frequency in the germ line. While measurement of meiotic inter- and intrachromosomal gene conversion frequency is routine in fungal systems, it has hitherto been impractical in mammals. We have designed a system for identifying and quantitating germ line gene conversion in mice by analyzing transgenic male gametes for a contrived recombination event. Spermatids which undergo the designed intrachromosomal gene conversion produce functional beta-galactosidase (encoded by the lacZ gene), which is visualized by histochemical staining. We observed a high incidence of lacZ-positive spermatids (approximately 2%), which were produced by a combination of meiotic and mitotic conversion events. These results demonstrate that gene conversion in mice is an active recombinational process leading to nonparental gametic haplotypes. This high frequency of intrachromosomal gene conversion seems incompatible with the evolutionary divergence of newly duplicated genes. Hence, a process may exist to uncouple gene pairs from frequent conversion-mediated homogenization.

High-frequency germ line gene conversion in transgenic mice.

Gene conversion is the nonreciprocal transfer of genetic information between two related genes or DNA sequences. It can influence the evolution of gene families, having the capacity to generate both diversity and homogeneity. The potential evolutionary significance of this process is directly related to its frequency in the germ line. While measurement of meiotic inter- and intrachromosomal gene conversion frequency is routine in fungal systems, it has hitherto been impractical in mammals. We have designed a system for identifying and quantitating germ line gene conversion in mice by analyzing transgenic male gametes for a contrived recombination event. Spermatids which undergo the designed intrachromosomal gene conversion produce functional beta-galactosidase (encoded by the lacZ gene), which is visualized by histochemical staining. We observed a high incidence of lacZ-positive spermatids (approximately 2%), which were produced by a combination of meiotic and mitotic conversion events. These results demonstrate that gene conversion in mice is an active recombinational process leading to nonparental gametic haplotypes. This high frequency of intrachromosomal gene conversion seems incompatible with the evolutionary divergence of newly duplicated genes. Hence, a process may exist to uncouple gene pairs from frequent conversion-mediated homogenization.

Genetic analysis of a new mutation conferring cysteine auxotrophy in Saccharomyces cerevisiae: updating of the sulfur metabolism pathway.

We have identified a mutation in a gene of Saccharomyces cerevisiae, STR1, that leads to a strict nutritional requirement for cysteine. The str1-1 mutation decreases to an undetectable level the cystathionine gamma-lyase activity. This enzyme catalyzes one of the two reactions involved in the transsulfuration pathway that yields cysteine from homocysteine with the intermediary formation of cystathionine. The phenotype induced by this mutation implies that, in S. cerevisiae, the sulfur atom of sulfide resulting from the reductive assimilation of sulfate is incorporated into a four carbon backbone yielding homocysteine, which, in turn, is the precursor of the biosynthesis of both cysteine and methionine. This also reveals that the direct synthesis of cysteine by incorporation of the sulfur atom into a three carbon backbone as found in Escherichia coli does not occur in S. cerevisiae. The study of the meiotic progeny of diploid strains heterozygous at the STR1 locus has shown that the str1-1 mutation undergoes a particularly high frequency of meiotic gene conversion.

Strategies for advancement of short-term mutagenicity tests: on the optimal ionic strength for the liver microsomal assay.

The aim of this work was to optimize the ionic strength (tau) in the liver microsomal assay (LMA) in performing short-term genotoxicity tests. tau optimization would increase the sensitivity (i.e. decrease false negatives) and at the same time increase the specificity (decrease false positives). Such optimization depends upon the relative activities and stabilities of the liver polysubstrate cytochrome P450- and FAD-containing monooxygenase-dependent metabolizing enzymes present in the incubation mixtures. With regard to phase-I pathway, the expression of various P450-like activities (IA1, IA2, IIB1, IIE1, IIIA P450 classes) and thiobenzamide s-oxidase (as FAD-MFO marker), were examined in terms of their exact incubation conditions for the LMA during a period of preincubation (1 h) over the tau range 0.06-1.40. As a comparison with the phase-II pathway, the behaviour of glutathione S-transferases (total and pi class), glutathione S-epoxide transferase, epoxide hydrolase and UDP-glucuronosyl transferase were studied. Lipid peroxidation (LP) was also determined. Experiments were performed on S9 fractions derived from sodium phenobarbital, beta-naphthoflavone, isosafrol, ethanol and pregnenolone 16-alpha carbonitrile super-induced mouse liver. The maximal value of the mean specific activity (Asp), up to a 46% increase, was found at tau = 0.864 for oxidative reactions considered. On the contrary, a slight modulation of Asp for post-oxidative reactions was seen. LP was not changed appreciably by varying tau. In vitro DNA binding of the well-known premutagenic agent [14C]dimethylnitrosamine ([14C]DMNA), mediated by mouse hepatic microsomal enzymes, showed a significant increase of specific activity at tau = 0.864 (2.25-fold) compared to the usual tau (0.06) used. Additional confirmation of these results stems from mutagenesis experiments using DMNA on the diploid D7 strain of Saccharomyces cerevisiae as a biological test system. Indeed, a significant enhancement of mitotic gene conversion (up to 1.8-fold), mitotic crossing-over (2.6-fold) and reverse point mutation (2.6-fold) frequencies was achieved at tau = 0.86 compared to tau = 0.06 (traditional). These data show that tau = 0.86 can provide more convenient conditions for in vitro bioactivation (as exemplified by an increased Asp phase-I/Asp phase-II ratio), as well as DNA binding and genotoxic response.

Time-dependent changes in gene conversion inAscobolus immersus

SummaryThe stability of conversion frequencies with time was investigated inAscobolus immersus. There were usually marked reductions in gene conversion frequencies at locuswlas crosses matured, to about one third of the initial values. This applied to all sixwlalleles tested, irrespective of their molecular nature, and at all temperatures used. Asci dehiscing early from an apothecium had much higher conversion frequencies than those dehiscing late, but there were no differences between apothecia maturing at different times within a cross. Alleles at four loci unlinked towlwere also tested. All four showed significant changes, though not in all crosses; three loci showed decreases in conversion frequency with time, while one showed an increase. The relative frequencies of different conversion classes often changed with time. These changes appear to result from alterations in locus-specific recombination initiation and in repair of base mispairs in hybrid DNA, not from differential maturation rates of different ascal segregation classes. These effects could cause misinterpretation of quantitative tests of recombination models from gene conversion data.

Extensive 3′-overhanging, single-stranded DNA associated with the meiosis-specific double-strand breaks at the ARG4 recombination initiation site

Meiosis-specific double-strand breaks occur at the initiation site for melotic gene conversion in the yeast ARG4 gene. Here we show that the break fragments end in extensive 3′-overhanging, single-stranded talls. The single-stranded talls vary in length, generating a gradlent of single-strandedness that parallels the gradient of gene conversion frequencies in ARG4. In strains carrying a rad50S mutation, which blocks meiotic recombination, the extensive single-stranded tails do not form, suggesting that their generation is an obligatory step in melotic recombination. Using the rad50S mutant, we have mapped the site of the ARG4 break to a small region within the genetically defined recombination initiation site. These results strongly support the double-strand break model of melotic recombination.

A defect in mismatch repair in Saccharomyces cerevisiae stimulates ectopic recombination between homeologous genes by an excision repair dependent process.

Null mutations in three recombination and DNA repair genes were studied to determine their effects on mitotic recombination between the duplicate AdoMet (S-adenosylmethionine) synthetase genes (SAM1 and SAM2) in Saccharomyces cerevisiae. SAM1 and SAM2, located on chromosomes XII and IV, respectively, encode functionally equivalent although differentially regulated AdoMet synthetases. These similar but not identical (homeologous) genes are 83% homologous at the nucleotide level and this identity is limited solely to the coding regions of the genes. Single frameshift mutations were introduced into the 5' end of SAM1 and the 3' end of SAM2 by restriction site ablation. The sequences surrounding these mutations differ significantly in their degree of homology to the corresponding area of the other gene. Mitotic ectopic recombination between the mutant sam genes occurs at a rate of 8.4 x 10(-9) in a wild-type genetic background. Gene conversion of the marker within the region of greater sequence homology occurs 20-fold more frequently than conversion of the marker within the region of relative sequence diversity. The relative orientation of the two genes prevents the recovery of translocations. Mitotic recombination between the sam genes is completely dependent on the DNA repair and recombination gene RAD52. A mutation in PMS1, a mismatch repair gene, causes a 4.5-fold increase in the rate of ectopic recombination. RAD1, an excision repair gene, is required to observe this increased rate of ectopic conversion. In addition, RAD1 is involved in modulating the pattern of coconversion during recombination between the homeologous sam genes. These results suggest that interactions between mismatch repair, excision repair and recombinational repair functions are involved in determining the ectopic gene conversion frequency between the sam genes.