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.     

Unstable ascospore color mutants of Ascobolus immersus

Summary In Ascobolus immersus, 16 unstable mutants with white ascospores were isolated. These mutants mutate back to the wild-type phenotype with brown ascospores. Only two mutants B and 301 are the object of the present study. At least in these two mutants it was demonstrated that true back-mutations occur. The frequencies of back-mutations are quite high and can reach 0.30 in the case of mutant B.The reversions appear at well defined but different for each mutant stages of the developmental cycle. These stages are relatively short and involve not more than about ten nuclear divisions cycles. In the case of mutant B, the back-mutations occur only in very young mycelia and at elevated temperature. Thus, the frequencies of back-mutations observed depend on the time of the transfer to higher temperature condition.The back-mutations of 301 mutant occur only in spermatized female organ and depend on the genotype of the female parent.The data established for Ascobolus immersus are compared with genic instability due to transposable elements described in corn by B. Mc Clintock.     

Unstable ascospore color mutants of Ascobolus immersus

Summary New unstable mutants of Ascobolus immersus involving the color or size of ascospores were sought among spontaneous mutants. Among the 34 unstable mutants isolated, 31 had white spores, 2 had pink spores and 1 had a large sized spores. The unstable mutants involve 11 loci whose mutation leads to white spores and 2 loci whose mutations give pink spores, among the 19 loci known to be implicated in this character; 1 locus is defined by only one large spore mutant. All these genes are localized on at least 7 different chromosomes. Unstable mutants of the same locus may correspond to several different sites, but the number of these sites is very limited.The frequency of unstable mutations was estimated: among 36 mutants belonging to 8 different genes, 20 were stable and 16 were unstable. This high frequency of unstable mutants is undoubtedly underestimated. The moment of reversion of 23 of these new mutants was also sought: 15 of them revert as does mutant B, previously studied, in the very young mycelium, at high temperature and with a reversion frequency of 0.004 to 0.34, according to the mutant; 5 of them revert as mutant 301, also previously studied, during the development of the fruit-body and with a frequency of 0.009 to 0.035; two of these mutants revert very early in the ascospore as soon as the first mitoses or in the very young mycelium at 22° C, with a very high reversion frequency that may reach 1.0; finally, the last mutant studied reverts in the fruit-body with a frequency reaching 0.40, but with modalities different from mutant 301. The mutants of the same locus may revert with different modalities. The same modality may correspond to different sites of the same gene.In unstable double-mutant strains involving two different genes, the reversion of one is independent of the reversion of the other, whether or not the reversion modailities of each mutant are identical.Results indicate the existence of inducers common to several unstable mutants which present the same modalities of reversion.These data support the previously formulated hypothesis of transposable elements.     

Intragenic location of an unstable insertion element within gene b5 of the fungus Ascobolus immersus

Summary A fine structure map of gene b5 has been established in Ascobolus immersus and the unstable mutant site b5-301 (phenotype: ascospore coloration) has been found to map within this gene. This map was constructed using seven b5 mutants induced by ICR170 and is based on the additivity of recombinant frequencies and confirmed by three point tests. The unstable site 301 is located between the induced sites. In particular, mutant 249 is located to the left of site 301, whereas sites 601 and 754 are located to the right.Previous studies showed that the inducing gene of mutant b5-301 reversions are either closely linked to the b5 locus or within it in certain strains. The study of asci resulting from reciprocal recombination between unstable mutant site and several induced mutant sites showed that neither crossovers located on the left nor the right of site 301, separate the unstable site from the inducing gene. Thus, the inducing gene was found to map within gene b5 as did the inducible site.These results constitute a genetic argument showing the presence of an insertion element. In this case, the insertion structure contains at least the integration site (inducible site) and the inducing gene which allows the excision.     

The estimation of conversion parameters and the control of conversion in Ascobolus immersus

Summary Pasadena strains of Ascobolus immersus were used to study the controls of gene conversion. Formulae were derived for estimating conversion parameters according to different hybrid DNA models of recombination. Genetic factors influencing conversion were determined by using genetically different isolates and using alleles before and after they had undergone conversion. Conversion properties at particular sites were greatly affected by genetic factors very near to those sites: the nature of the mutations and factors elsewhere in the genome were much less important. These genetic factors and temperature both affected the frequency of hybrid DNA formation at particular sites, the efficiency of mispair correction and the relative frequencies of correction to wild-type or mutant. The effects of temperature on conversion parameters were usually complex.     

In organello replication and viral affinity of linear, extrachromosomal DNA of the ascomycete Ascobolus immersus

Summary Linear, extrachromosomal DNA's of the filamentous fungus Ascobolus immersus are localized within the mitochondria. These linear plasmids have no homology to the high molecular weight mtDNA (hmw mtDNA). For analysis of plasmid replication an in organello DNA synthesis system was developed, in which radionucleotides were incorporated into intact mitochondria. Plasmid DNA is labelled preferentially in this system. From replication analysis of a specific plasmid there is evidence of a virus-like protein-primed replication. Sequence analysis of this plasmid reveals that a viral DNA polymerase is encoded. Thus, these genetic elements presumably are viral remnants rather than true plasmids.     

The detection of post-meiotic segregation without tetrad analysis in Ustilago maydis

Summary Post-meiotic segregation (PMS) results in the formation of mixed genotypes from single meiotic products. A method is described in which single members of tetrads are selected, and these are then tested for their genetic homogeneity. The method is applied to Ustilago maydis using crosses which are heteroallelic for nar 1, the structural gene for nitrate reductase. In the absence of PMS, meiotic products containing a nar ⁺ recombinant are genetically pure (the equivalent of a 6 mutant: 2 wild-type octad). With PMS, a nar ⁺ recombinant clone arises in association with a nar ^- clone and these are otherwise genetically identical (the equivalent of a 7 mutant: 1 wild type octad). The procedure will make it possible to search for mutant strains which are defective in the correction of mismatched bases in hybrid DNA formed during recombination. Among 26 nar ⁺ recombinants from a control cross, PMS was detected on 3 occasions. In an equivalent cross, both parents were uvs 3, a mutant defective in the excision of pyrimidine dimers from DNA. Among 43 nar ⁺ recombinants, 7 arose from PMS. Thus the frequency of PMS for the nar alleles is about 15% and the excision of pyrimidine dimers is probably unrelated to the repair of mismatched bases in hybrid DNA.     

Gene centromere mapping and detection of post-meiotic segregation in angiosperms by genotyping the polar nucleus

A technique using biochemical markers for determining the genotype of the polar nucleus is suggested. This technique involves the use of allozyme markers in the endosperm which are sensitive to allele dosage. A method is suggested to detect crossovers in tetrasporic angiosperms having adoxa and drusa type of embryo sac development, and to detect post-meiotic segregations in monosporic angiosperms having polygonum and oenothera type of embryo sac development, by genotyping the polar nucleus.     

Integration defective mutants of bacteriophage P2

Summary Mutants of phage P2 unable by themselves to be integrated as prophages have been isolated. These mutants (int) are complemented by the wild type allele and may then yield stable lysogenic strains carrying an int prophage at location I in Escherichia coli C. These lysogens produce either no phage or little phage, depending on the int mutant used. All int mutants isolated appear to belong to a single complementation group.Exceptional lysogens carrying two or more int prophages may be obtained: they may produce spontaneously even more phage than normal lysogens, and they segregate out defective, singly lysogenic clones at low frequency. These exceptional lysogens carry both prophages in location I, presumably in tandem.Strains carrying two or more int prophages but defective in phage production were also isolated. One of these carries its prophages at two different, not closely linked, chromosomal locations.     

Intragenic recombination in the CSR1 locus of Arabidopsis

Four classes of herbicides are known to inhibit plant acetolactate synthase (ALS). In Arabidopsis, ALS is encoded by a single gene, CSR1. The dominant csr1-1 allele encodes an ALS resistant to chlorsulfuron and triazolopyrimidine sulfonamide while the dominant csr1-2 allele encodes an ALS resistant to imazapyr and pyrimidyl-oxy-benzoate. The molecular distance between the point mutations in csr1-1 and csr1-2 is 1369 bp. Here we used multiherbicide resistance as a stringent selection to measure the intragenic recombination frequency between these two point mutations. We found this frequency to be 0.008 ± 0.0028. The recombinant multiherbicide-resistant allele, csr1-4, provides an ideal marker for plant genetic transformation.     

The base-alteration spectrum of spontaneous and ultraviolet radiation-induced forward mutations in the URA3 locus of Saccharomyces cerevisiae

Summary A forward mutation system has been developed to obtain rapidly clonable mutants at the URA3 locus in yeast by means of selection for 5-fluoroorotic acid resistance. We have used this system to determine base changes in 35 spontaneous and 34 ultraviolet radiation-induced ura3 base substitution mutants. Other mutants (frameshift, deletion, duplication, replacement) were detected as well. Evidence is reported which suggests cyclobutane dimers are the principal mutagenic lesions induced by UV radiation in stationary phase cells of the yeast Saccharomyces cerevisiae. Since most of the induced lesions are at 5-TT-3 sites, the results suggest that the A-rule, preferential insertion of adenine residues opposite poorly pairing sites in DNA, does not apply for yeast cells irradiated in stationary phase, whereas the spontaneous mutation data indicate that the A-rule applies for cells in logarithmic phase. Most of the spontaneous mutations are transversions. UV-induced transitions and transversions occur at approximately equal frequencies.     

Methionine biosynthesis in Saccharomyces cerevisiae: Mutations at the regulatory locus ETH2

Summary Homoallelic and heteroallelic diploids involving the eth2-1, eth2-2 and eth2-7 alleles have been studied on the basis of several criteria used for the study of haploid strains: resistance towards ethionine, overproduction of either methionine or/and S-adenosylmethionine, repressibility of methionine biosynthetic enzymes. Complete recessivity of the three alleles over the wild type allele has been observed, when resistance and methionine synthesis are considered. However, with the eth2-2 allele, repressibility corresponds more to a dose effect of the ETH2 allele than to recessivity. The implications of these findings have been discussed. Results obtained for heteroallelic combinations show significant deviations from the expected values. These results have been interpreted as indicating possible interactions between two differently impaired products of gene ETH2. They render likely that the product of this gene is at least an homopolymer.     

Chromatin structure of the yeast SUC2 promoter in regulatory mutants

Summary We have previously suggested that two positioned nucleosomes are removed from the promoter of the Saccharomyces cerevisiae SUC2 gene upon derepression by glucose starvation. To gain further insight into the changes accompanying derepression at the chromatin level we have studied the chromatin structure of the SUC2 promoter in several mutants affecting SUC2 expression. The non-derepressible mutants snf1, snf2 and snf5 present a chromatin structure characteristic of the repressed state, irrespective of the presence or absence of glucose. The non-repressible mutants, mig1 and ssn6, as well as the double mutant snfs sn6 exhibit an opened chromatin structure even in the presence of glucose. These results suggest that the DNA-binding protein encoded by MIG1 is necessary to produce the characteristic pattern of repressed chromatin and that the SNF1 protein kinase is sufficient to produce the derepressed chromatin pattern. A model is presented for the transitions that result in opening up of the chromatin structure.     

Response of ad-2 mutants of Saccharomyces cerevisiae to carbon dioxide

Summary Confirmation that the ad-2 locus of yeast controls the carboxylation of aminoimidazole ribonucleotide (AIR) to 5-amino-4-imidazole carboxylate ribonucleotide (CAIR) is provided by the observation that 21 out of a sample of 113 ad-2 mutants were affected by CO₂. 19 of the mutants were stimulated by CO₂ and 2 were inhibited. The majority of the CO₂-stimulated mutants were confined to one section of the complementation map of the ad-2 locus.     

Gene sequence of recA+ and construction of recA mutants of Vibrio cholerae

The recA ⁺ gene of Vibrio cholerae O1 has been cloned, its nucleotide sequence determined and the product characterized. A deletion mutation was constructed in the recA gene and mutants showed the typical sensitivity to UV and to DNA-damaging agents, as well as an inability to mediate homologous DNA recombination. The chromosomal recA deletion mutants in V. cholerae do not show altered virulence in the infant mouse cholera model and are thus ideal strains for use in complementation studies.     

Genetic characterization of the mei-41 locus in Drosophila melanogaster

Summary The mutagen-sensitive mutant mus(1)104 ^D1 of Drosophila melanogaster maps to a position on the X chromosome very close to the meiotic mutant mei-41 ^D5 . Both mutants have been characterized as mutagen-sensitive and defective in post-replication repair. In the present report we show by complementation studies that mus(1)104 and mus(1)103 are allelic with mei-41. In addition, two reported alleles of mus(1)104 lie between the mei-41 alleles A10 and D5. The size of the mei-41 locus is estimated to be about 0.1 centimorgans (cM). Because several alleles of mei-41 have been shown to reduce recombination and increase meiotic chromosome loss and nondisjunction, mus(1)104 ^D1 females were examined for defects in meiosis. Although there was no evidence for reduced recombination on the second chromosome in homozygous mus(1)104 ^D1 females, heterozygous mus(1)104 ^D1 /mei-41 ^>D5 and mus(1)104 ^D1 /deficiency females showed reduced levels of recombination. However, there was no evidence of an increase in nondijunction in these females.We dedicate this article to the memory of Larry Sandler, who passed away suddenly on February 7, 1987