Large Heterologies Impose Their Gene Conversion Pattern onto Closely Linked Point Mutations

We have studied the meiotic non-Mendelian segregation (NMS) pattern of seven large heterologous combinations located in the b2 ascospore gene of Ascobolus. The NMS patterns of these aberration heterozygotes widely differ from each other and from those of point mutations located in the same genetic region. They give lower gene conversion frequencies than point mutations, no postmeiotic segregations (PMS), and either parity or disparity that favors the wild type allele. Two related deletions, G234 and G40, were studied for their effects on the conversion behavior of closely linked point mutations. We found that, when heterozygous, the deletions impose their own NMS pattern onto close mutations. These effects occur on both sides of the heterologies. The effects upon PMS and disparity of linked point mutations gradually disappear as point mutations become more distant. The effects on NMS frequencies and on aberrant 4:4 are polar. They persist for all mutations located downstream from the high conversion end of the gene. This last effect can reflect a blockage of symmetric hDNA formation by large heterologies, whereas the epistasis of the NMS pattern of large heterologies over that of closely linked point mutations suggests that large heterologies and point mutations undergo conversion by means of distinct pathways.     

Gene conversion spectrum of 15 mutants giving post-meiotic segregation in the b2 locus of Ascobolus immersus

Summary The conversion spectrum of fifteen mutants giving post-meiotic segregations and located in the b2 locus of Ascobolus immersus was studied in 77 mutant x wild-type crosses. These mutants all yield aberrant 4:4 asci, mutants located in the right portion of the locus yielding more aberrant 4:4 than left mutants. The basic frequency of conversion is higher in the left portion. The frequency of hybrid DNA, its symmetrical or asymmetrical distribution and the frequency of correction of the mismatch in hybrid DNA were estimated. The left region shows a higher frequency of hybrid DNA formation than the right region. The fraction of hybrid DNA with a symmetrical distribution tends to increase from left to right in the locus. The frequencies of mismatch correction show considerable variation from one mutant to another and have no relationship to their location. The implications of these observations on the molecular models of genetic recombination are discussed.     

The intergradation, genetic interchangeability and interpretation of gene conversion spectrum types

In the Pasadena strains of Ascobolus immersus, the gene conversion propperties of 29 induced (nine UV, nine NG, and 11 ICR-170) and nine spontaneous white-ascospore mutations have been studied. Each mutant was crossed to three types of derived wild-type strains; single mutants often gave very different conversion results in the three types of crosses, with any or all of the following changes in: percentage with post-meiotic segregation among aberrant-ratio asci; percentage with conversion to wild type among aberrant-ratio asci; and in total conversion frequency. - These results are compared with those of Leblon (1972 a, b) from Ascobolus immersus and Yu-Sun, Wickramaratne and Whitehouse (1977) from Sordaria brevicollis. It is shown that conversion spectrum types are not necessarily distinct, but can completely intergrade, on the criteria of both post-meiotic segregation frequency and direction of correction. Genetic differences between strains in the present work resulted in much interchangeability of spectrum types for the same mutation in different crosses; e.g., from type C in one cross to type B/D type in another cross, although the mutation is presumably of the same molecular type (addition or deletion frame shift, or base substitution) in each cross. These changes of conversion properties for a given mutation in different crosses mean that previous interpretations of spectrum types in terms of specific conversion properties for various molecular types of mutation are inapplicable, or inadequate on their own, to explain the present data. Other factors, such as heterozygous cryptic mutations or conversion control genes, are probably involved. Because of asymmetric hybrid DNA formation, correction properties may differ from observed conversion properties.     

Genetic recombination at the buff spore colour locus in Sordaria brevicollis

Summary Asci showing aberrant segregation at the buff spore colour locus in Sordaria brevicollis were selected from crosses between buff mutants and wild type in the presence of closely-linked flanking markers. The frequency of crossing-over associated with aberrant segregations was calculated and corrected to allow for crossovers between the flanking markers incidental to the aberrant segregation. The average frequency of crossing over was found to be related to the class of aberrant ascus studied. 5+:3m and 3+:5m asci showed 16% associated marker recombination while 6+:2m and 2+:6m asci showed 27% recombination. The frequency of tritype and tetratype postmeiotic segregation asci was calculated. Only 3% tetratypes were found and this is thought to indicate a low frequency of symmetric hybrid DNA formation.     

GENETICS OF SORDARIA FIMICOLA. V. ABERRANT SEGREGATION AT THE G LOCUS

Kitani , Y., L. S. Olive , and Arif S. El -Ani . (Columbia U., New York City.) Genetics of Sordaria fimicola. V. Aberrant segregation at the g locus. Amer. Jour. Bot. 49(7): 697–706. Illus. 1962.—Aberrant segregation of the gray-spore color locus in Sordaria fimicola was studied with the aid of closely linked markers. It was found that 6:2 and 5:3 asci occur with about the same frequency, but asci with an excess of wild-type spores occur with a frequency 5.5 times that of asci with an excess of gray spores. Also, the frequency of related crossing over (occurring close to the miscopied loeus and involving the miscopying strand) was much higher than the expected value, and in 5:3 asci it appears to be at least twice that found in 6:2 asci. Nine aberrant 4:4 asci, each with 2 spore pairs heterogeneous for color, were found. These are believed to have resulted from reciprocal double transreplication. The rarest aberrant type was represented by a single 7:1 ascus, which is difficult to explain on the basis of a single meiotic process. Miscopying is discussed with relation to an 8-strand model of paired homologues and the occurrence of localized chromosome pairing during prezygotene DNA synthesis. Several possible explanations for the occurrence of aberrant tetrads are considered. Miscopying has also been found to involve several spore-color loci not previously studied; whereas, several other such mutant loci fail to show evidence of it. One locus (m) shows abnormal segregation of the 6:2 but not the 5:3 type.     

Meiotic Gene Conversion Mutants in SACCHAROMYCES CEREVISIAE . I. Isolation and Characterization of pms1-1 and pms1-2

The pms1 mutants, isolated on the basis of sharply elevated meiotic prototroph frequencies for two closely linked his4 alleles, display pleiotropic phenotypes in meiotic and mitotic cells. Two isolates carrying recessive mutations in PMS1 were characterized. They identify a function required to maintain low postmeiotic segregation (PMS) frequencies at many heterozygous sites. In addition, they are mitotic mutators. In mutant diploids, spore viability is reduced, and among survivors, gene conversion and postmeiotic segregation frequencies are increased, but reciprocal exchange frequencies are not affected. The conversion event pattern is also dramatically changed in multiply marked regions in pms1 homozygotes. The PMS1 locus maps near MET4 on chromosome XIV. The PMS1 gene may identify an excision-resynthesis long patch mismatch correction function or a function that facilitates correction tract elongation. The PMS1 gene product may also play an important role in spontaneous mitotic mutation avoidance and correction of mismatches in heteroduplex DNA formed during spontaneous and UV-induced mitotic recombination. Based on meiotic recombination models emphasizing mismatch correction in heteroduplex DNA intermediates, this interpretation is favored, but alternative interpretations involving longer recombination intermediates in the mutants are also considered.     

Hybrid DNA tracts may start at different sites during meiotic recombination in gene b2 of Ascobolus

The initiation of hybrid DNA in the b2 spore colour gene was investigated by the analysis of non-Mendelian segregation asci in the progeny of crosses involving several allelic b2 mutations. These analyses showed that, instead of the unique initiation region of hybrid DNA in b2, assumed previously by Paquette and Rossignol (1978), there are multiple sites for hybrid DNA starting. They produce major and minor fractions of hybrid DNA. The major fraction starts upstream of the left end of the gene, propagates rightwards and can cover its entire length. The minor fractions do not span the left b2 end. One of them is detected within the left half of the gene, the others within its right half.     

Mechanism of gene conversion in Ascobolus immersus

Summary Sixty two ascospore colour mutants have been induced in Ascobolus immersus: 25 by an acridine (ICR₁₇₀), 18 by N-methyl-N-nitro-N-nitrosoguanidine (NG) and 19 by ethyl methanesulfonate (EMS). All these mutants have been crossed to the wild type strain and their conversion spectrum has been determined. It appears that the conversion spectrum is closely related to the origin of the mutants studied with respect to the mutagen by which they were induced. All NG mutants gave numerous asci with postmeiotic segregation and an excess of conversion to wild type over conversion to the mutant type. ICR mutants gave no postmeiotic segregation and an excess of conversion to the mutant type. The majority of EMS mutants behave like NG mutants, but some showed only meiotic segregation with either an excess of conversion to the mutant type or an excess of conversion to wild type. These data are in good agreement with the hypothesis that the nature of the mutation has a strong influence on the conversion spectrum.     

Cloning and characterization of DST2, the gene for DNA strand transfer protein beta from Saccharomyces cerevisiae.

The gene encoding the 180-kDa DNA strand transfer protein beta from the yeast Saccharomyces cerevisiae was identified and sequenced. This gene, DST2 (DNA strand transferase 2), was located on chromosome VII. dst2 gene disruption mutants exhibited temperature-sensitive sporulation and a 50% longer generation time during vegetative growth than did the wild type. Spontaneous mitotic recombination in the mutants was reduced severalfold for both intrachromosomal recombination and intragenic gene conversion. The mutants also had reduced levels of the intragenic recombination that is induced during meiosis. Meiotic recombinants were, however, somewhat unstable in the mutants, with a decrease in recombinants and survival upon prolonged incubation in sporulation media. spo13 or spo13 rad50 mutations did not relieve the sporulation defect of dst2 mutations. A dst1 dst2 double mutant has the same phenotype as a dst2 single mutant. All phenotypes associated with the dst2 mutations could be complemented by a plasmid containing DST2.     

Single and Coincident Intragenic Mutations Attributable to Gene Conversion in a Human Cell Line

Two polymorphic sites are located within the heterozygous TK1 locus in the human lymphoblastoid cell line TK6: an inactivating frameshift in exon 4 of the nonfunctional allele and a phenotypically silent frameshift in exon 7 of the functional allele. Through the use of these intragenic polymorphisms and microsatellite markers that flank TK1, we demonstrate that partial gene conversion accounts for 3/75 (0.04) spontaneous and 9/163 (0.06) X-ray-induced TK1(-) mutants, thus comprising a significant component of forward mutations at this locus. In all cases, the conversion tract is <1 cM, rendering double exchange a remote alternate explanation for these results. Sequence analysis of full length TK1 cDNA provides rigorous exclusion of deletion events as a mechanism for generation of these allelotypes. Detailed examination of allelotypes in TK1(-) mutants identified two mechanisms for the generation of coincident sequence alterations that sometimes accompanied gene conversions. Mutations within the conversion tract were attributed to either error-prone gap filling synthesis during recombinational repair or mismatch repair within a heteroduplex region following branch migration. These findings suggest that a proportion of point mutations may not be targeted to sites of DNA base damage, but rather may arise as secondary consequences from the repair of DNA strand breaks.     

Cytoplasmic inheritance of antimycin A resistance in Saccharomyces cerevisiae

Summary Three antimycin resistant mutants of Saccharomyces cerevisiae are characterized genetically. The mutations have been shown to be cytoplasmically inherited by four criteria. The phenotype persists in diploids formed by a cross with a ⁰ strain of yeast of the opposite mating type. Diploids heterozygous for the antimycin marker, however, show segregation of the resistance and sensitivity during mitosis. Tetrad analysis indicated a non-Mendelian segregation (4:0 and 0:4) of the mutations. The antimycin marker can be eliminated by ethidium bromide treatment under conditions that should have deleted all of the mitochondrial DNA.     

Hybrid DNA Extension and Reciprocal Exchanges: Alternative Issues of an Early Intermediate during Meiotic Recombination?

Large heterologies in gene b2 strongly increase the frequencies of reciprocal exchanges on their left border, towards the high conversion end. In a previous study, we observed that heterozygous point mutations located in the high conversion end (region F) stimulate the reciprocal exchanges instigated by the large heterology 138. We have defined some properties of this stimulation. The effect does not depend on the nature of the large heterology used. It is effective only with point mutations located on the left side of the large heterology. It does not depend on the number of heterozygosities accumulated in region F. It is not specific on the location of point mutations in region F: it decreases from region F (left end) to region E (middle part of b2). It is correlated with the mismatch correction efficiencies of the point mutations used. It is not observed in the absence of a large heterology. Point mutation heterozygosities which stimulate reciprocal exchanges also decrease the frequency of HDNA formation in gene b2. We propose a model in which reciprocal exchanges on the one hand and hybrid DNA formation on the other hand correspond to alternative processings of a common recombination intermediate.     

Female gametophytic mutants of Arabidopsis thaliana identified in a gene trap insertional mutagenesis screen

In plants, the male and female gametophytes represent the haploid generation that alternates with the diploid sporophytic generation. Male and female gametophytes develop from haploid micro- and megaspores, respectively. In flowering plants (angiosperms), the spores themselves arise from the sporophyte through meiotic divisions of sporogenous cells in the reproductive organs of the flower. Male and female gametophytes contain two pairs of gametes that participate in double fertilization, a distinctive feature of angiosperms. In this paper, we describe the employment of a transposon-based gene trap system to identify mutations affecting the gametophytic phase of the plant life cycle. Mutants affecting female gametogenesis were identified in a two-step screen for (i) reduced fertility (seed abortion or undeveloped ovules) and (ii) segregation ratio distortion. Non-functional female gametophytes do not initiate seed development, leading to semi-sterility such that causal or linked alleles are transmitted at reduced frequency to the progeny (non-Mendelian segregation). From a population of 2,511 transposants, we identified 54 lines with reduced seed set (2%). Examination of their distorted segregation ratios and seed phenotypes led to the isolation of 12 gametophytic mutants, six of which are described herein. Chromosomal sequences flanking the transposon insertions were identified and physically mapped onto the genome sequence of Arabidopsis thaliana. Surprisingly, the insertion sites were often associated with chromosomal rearrangements, making it difficult to assign the mutant phenotypes to a specific gene. The mutants were classified according to the process affected at the time of arrest, i.e. showing mitotic, karyogamic, maternal or degenerative phenotypes.     

Rare occurrence of the tetratype tetrads in Saccharomycodes ludwigii.

Genetic data suggesting the absence of crossover in Saccharomycodes ludwigii have been described. Tetrad data obtained from 888 asci from 60 pairs of genes with 22 genetic markers showed the absence of tetratype asci, except for 5 asci in which a single pair of alleles showed tetratype segregation to the other genetic markers in each ascus. Spore arrays in the linear asci showed that the + - + - and + - - + (or - + + -) asci occurred at almost equal frequencies. The two coherent spores at each end of an ascus were always marked with different alleles of a gene.     

Spontaneous Mitotic Recombination in Yeast: The Hyper-Recombinational Rem1 Mutations Are Alleles of the Rad3 Gene

The RAD3 gene of Saccharomyces cerevisiae is required for UV excision-repair and is essential for cell viability. We have identified the rem1 mutations (enhanced spontaneous mitotic recombination and mutation) of Saccharomyces cerevisiae as alleles of RAD3 by genetic mapping, complementation with the cloned wild-type gene, and DNA hybridization. The high levels of spontaneous mitotic gene conversion, crossing over, and mutation conferred upon cells by the rem1 mutations are distinct from the effects of all other alleles of RAD3. We present preliminary data on the localization of the rem1 mutations within the RAD3 gene. The interaction of the rem1 mutant alleles with a number of radiation-sensitive mutations is also different than the interactions reported for previously described (UV-sensitive) alleles of RAD3. Double mutants of rem1 and a defect in the recombination-repair pathway are inviable, while double mutants containing UV-sensitive alleles of RAD3 are viable. The data presented here demonstrate that: (1) rem1 strains containing additional mutations in other excision-repair genes do not exhibit elevated gene conversion; (2) triple mutants containing rem1 and mutations in both excision-repair and recombination-repair are viable; (3) such triple mutants containing rad52 have reduced levels of gene conversion but wild-type frequencies of crossing over. We have interpreted these observations in a model to explain the effects of rem1. Consistent with the predictions of the model, we find that the size of DNA from rem1 strains, as measured by neutral sucrose gradients, is smaller than wild type.     

Frequency and Directionality of Gene Conversion Events Involving the CYC7-H3 Mutation in SACCHAROMYCES CEREVISIAE

The CYC7-H3 mutation is a 5-kb deletion that causes overproduction of iso-2 cytochrome c. Unlike most mutations in yeast, the CYC7-H3 mutation is preferentially lost when it is involved in a gene conversion event. We have shown that cloned copies of CYC7-H3 DNA that are inserted into the yeast genome are associated with a high frequency of recombination and aberrant segregation events. Since parity in conversion frequency was observed when the extensive insertion/deletion heterozygosity at this locus was eliminated, we conclude that the CYC7-H3 sequences are inherently capable of acting as donors or recipients in gene conversion events, although they are unlikely to act as donors when they are located opposite a large heterology. DNA sequence comparisons revealed similarities between the CYC7-H3 junction region and the 2-micron circle DNA region that is involved in site-specific recombination.     

Randomness tests on the sequence of ascal segregation classes in Neurospora crassa.

Various statistical tests for randomness were made on the order of ascal classes in groups of asci from wild-type X asco crosses. There was no significant nonrandom clustering of asci of the same segregation class, nor a regular twinning of similar asci. Any apparent observed clustering of similar ascal classes is probably an artefact or due to chance. 2 x n X2 tests showed that frequencies of individual ascus classes from different perithecia were generally homogeneous, as were second division segregation frequencies. The tests described here for randomness in sequences of occurrences could be of general use in other areas of genetics.     

Genetic Evidence That the Meiotic Recombination Hotspot at the His4 Locus of Saccharomyces Cerevisiae Does Not Represent a Site for a Symmetrically Processed Double-Strand Break

In the yeast Saccharomyces cerevisiae, the binding of the Rap1 protein to a site located between the 5' end of the HIS4 gene and the 3' end of BIK1 stimulates meiotic recombination at both flanking loci. By using strains that contain mutations located in HIS4 and BIK1, we found that most recombination events stimulated by the binding of Rap1 involve HIS4 or BIK1, rather than bidirectional events including both loci. The patterns of aberrant segregation indicate that most of the Rap1-stimulated recombination events do not represent the symmetric processing of a double-strand DNA break located at the Rap1-binding site.     

Genetic Recombination at the Buff Spore Color Locus in SORDARIA BREVICOLLIS. II. Analysis of Flanking Marker Behavior in Crosses between Buff Mutants

Aberrant asci containing one or more wild-type spores were selected from crosses between pairs of alleles of the buff locus in the presence of closely linked flanking markers. Data were obtained relating to the site of aberrant segregation and the position of any associated crossover giving recombination of flanking markers. Aberrant segregation at a proximal site within the buff gene may be associated with a crossover proximal to the site of aberrant segregation or, with equal frequency, with a crossover distal to the site of the second mutant present in the cross. Similarly, segregation at a distal site may be associated with a crossover distal to the site or, with lower frequency, with a crossover proximal to the site of the proximal mutant present in the cross. Crossovers between the alleles were rare. This evidence for the relationship between hybrid DNA and crossing over is discussed in terms of current models for the mechanism of recombination.     

X-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa. IV. Irreparable mutants of genotype ad-3A and ad-3B result from multilocus deletion and an unexpectedly high frequency of multiple-locus mutations

The induction of specific-locus mutations in the ad-3 region of Neurospora crassa after X-irradiation was studied in a two-component heterokaryon to determine: (1) the ratio of reparable ad-3 mutants (presumed gene/point mutations, designated ad-3R) to irreparable ad-3 mutants (presumed multilocus deletions, designated ad-3IR), and (2) the induction kinetics of each class (Webber and de Serres, 1965). More extensive genetic tests made subsequently (de Serres, 1989a) on the 832 X-ray-induced specific-locus mutations recovered in those experiments showed that unexpected high frequencies of reparable and irreparable ad-3 mutants are actually multiple-locus mutants that have additional, but separate, sites of recessive lethal (RLCL) damage in the immediately adjacent genetic regions (designated ad-3R + RLCL or ad-3IR + RLCL). The frequencies of these X-ray-induced multiple-locus mutants in the ad-3 region are orders of magnitude higher than expected on the basis of target theory (where the frequency of the double mutant is expected to be the product of the frequencies of each single mutant) and classical models of chromosome structure during interphase (de Serres, 1989a). In the present paper, a random sample of 832 X-ray-induced ad-3 mutants of genotype ad-3A or ad-3B that are irreparable have been subjected to more extensive genetic fine-structure analysis. These experiments were designed to determine the extent of the functional inactivation in individual mutants in the ad-3 and immediately adjacent genetic regions in mutants classified as presumptive multilocus deletions or multiple-locus mutations. These experiments have shown that in Neurospora crassa most X-ray-induced irreparable mutants of genotype ad-3A or ad-3B map as a series of overlapping multilocus deletions. Among the 29 irreparable mutants of genotype ad-3A, there are 16 different subgroups of complementation patterns; and among the 63 irreparable mutants of genotype ad-3B, there are also 16 different subgroups. In addition, mutants classified as presumptive multiple-locus mutants result from a variety of separate, but closely linked, sites of genetic damage.(ABSTRACT TRUNCATED AT 400 WORDS)