Isolation and Characterization of Schizosaccharomyces Pombe Mutants Affected in Mitotic Recombination

A haploid Schizosaccharomyces pombe strain carrying a heteroallelic duplication of the ade6 gene was used to isolate mitotic recombination-deficient mutants. Recombination between the different copies of the ade6 gene can lead to Ade+ segregants. These are observed as growing papillae when colonies of a suitable size are replicated onto selective medium. We isolated mutants which show an altered papillation phenotype. With two exceptions, they exhibit a decrease in the frequency of mitotic recombination between the heteroalleles of the duplication. The two other mutants display a hyper-recombination phenotype. The 12 mutations were allocated to at least nine distinct loci by recombination tests. Of the eight rec mutants analyzed further, six were also affected in mitotic intergenic recombination in the intervals cen2-mat or cen3-arg 1. No effect on mitotic intragenic recombination was observed. These data suggest that mitotic gene conversion and crossing over can be separated mutationally. Meiotic recombination occurs at the wild-type frequency in all mutants investigated.     

Meiotic Recombination-Deficient Mutants of Schizosaccharomyces Pombe

A mutant screen employing the ade6-M26 recombination hotspot was developed and used to isolate Schizosaccharomyces pombe mutants deficient in meiotic recombination. Nine rec mutations were recessive, defining six complementation groups, and reduced ade6 meiotic recombination 3-fold to greater than or equal to 300-fold when homozygous. Three recessive rec mutations analyzed further also reduced meiotic intragenic recombination at ura4 on chromosome III and intergenic recombination between pro2 and arg3 on chromosome I. The observed non-co-ordinate reductions of the recombinant frequencies in the three test intervals suggest a degree of locus (or intragenic vs. intergenic) specificity of the corresponding rec+ gene products. None of the mutations specifically inactivated the ade6-M26 hotspot. Additional rec genes may be identified with these methods.     

Isolation of Temperature-Sensitive Mutants of Schizosaccharomyces pombe

Seventy-one mutants of the yeast Schizosaccharomyces pombe that were able to grow on complete medium at 25 C but not at 37 C were isolated. Strains selected for further study showed: (i) single gene mutation and (ii) cell lengthening at the restrictive temperature. Preliminary characterization of 13 mutants is reported. Seven of them have a less pronounced synthesis of deoxyribonucleic acid at the restrictive temperature, and four of them seem to be affected in cell division.     

The Rec8 Gene of Schizosaccharomyces Pombe Is Involved in Linear Element Formation, Chromosome Pairing and Sister-Chromatid Cohesion during Meiosis

The fission yeast Schizosaccharomyces pombe does not form tripartite synaptonemal complexes during meiotic prophase, but axial core-like structures (linear elements). To probe the relationship between meiotic recombination and the structure, pairing, and segregation of meiotic chromosomes, we genetically and cytologically characterized the rec8-110 mutant, which is partially deficient in meiotic recombination. The pattern of spore viability indicates that chromosome segregation is affected in the mutant. A detailed segregational analysis in the rec8-110 mutant revealed more spores disomic for chromosome III than in a wild-type strain. Aberrant segregations are caused by precocious segregation of sister chromatids at meiosis I, rather than by nondisjunction as a consequence of lack of crossovers. In situ hybridization further showed that the sister chromatids are separated prematurely during meiotic prophase. Moreover, the mutant forms aberrant linear elements and shows a shortened meiotic prophase. Meiotic chromosome pairing in interstitial and centromeric regions is strongly impaired in rec8-110, whereas the chromosome ends are less deficient in pairing. We propose that the rec8 gene encodes a protein required for linear element formation and that the different phenotypes of rec8-110 reflect direct and indirect consequences of the absence of regular linear elements.     

Isolation and Partial Characterization of Regulatory Mutants of the Pyrimidine Pathway in Salmonella typhimurium

Mutants of Salmonella typhimurium affected in the regulation of pyrimidine biosynthesis were isolated by two methods. The first involved screening for bacteria able to feed a pyrimidine-requiring indicator strain, and the second involved selection for bacteria simultaneously resistant to two pyrimidine analogues, 5-fluorouracil and 5-fluorouridine, in a S. typhimurium strain unable to degrade 5-fluorouridine. Among the mutants isolated by these methods are constitutive mutants, producing high levels of pyrimidine biosynthetic enzymes in the presence or absence of pyrimidines, and feedback modified mutants, in which aspartate transcarbamylase is partially desensitized to its inhibitor, cytidine triphosphate. No fully desensitized mutant has been found. The partially desensitized character cotransduces with the pyrB locus, that of aspartate transcarbamylase. The constitutive character has been determined in a few cases to be localized in the region of leu and pro on the Salmonella map.     

Respiration deficient mutants of Schizosaccharomyces Pombe I

Summary By use of N-methyl-N-nitro-N-nitrosoguanidin (NG) respiration deficient (RD) mutants were induced. They could be selected by replica-plating on glycerol medium. RD mutants were also induced by UV irradiation, enriched by use of 2,3,5-triphenyltetrazolium chloride (TTC) and tested for their inability to grow on glycerol medium. The RD mutants were characterized enzymatically for their decrease or loss in cytochrome c oxidase activity and in succinate- cytochrome c reductase activity. These assays allowed the localization of the mutational blocks in complexes II, III and IV of the respiratory chain. Tetrad analysis and random spore analysis demonstrated that all mutants contained chromosomal defects.     

Isolation and Preliminary Characterization of Developmental Mutants from Microsporum gypseum

Developmental mutants affected in either sporulation or spore germination have been isolated from Microsporum gypseum with the aid of nitrosoguanidine or as spontaneously occurring mutants. The time course levels of several proteins temporally associated with conidial development have been assayed in the wild-type and mutant strains. The spore germination characteristics of two of the mutants are described. The relationship of alkaline protease accumulation to tyrosinase accumulation and spore germination is discussed.     

The Mutator Gene Swi8 Effects Specific Mutations in the Mating-Type Region of Schizosaccharomyces Pombe

The swi8(+) gene of Schizosaccharomyces pombe appears to be involved in the termination step of copy synthesis during mating-type (MT) switching. Mutations in swi8 confer a general mutator phenotype and, in particular, generate specific mutations in the MT region. Sequencing of the MT cassettes of the h(90) swi8-137 mutant revealed three altered sites. One is situated at the switching (smt) signal adjacent to the H1 homology box of the expression locus mat1:1. It reduces the rate of MT switching. The alteration at the smt signal arose frequently in other h(90) swi8 strains and is probably caused by gene conversion in which the sequence adjacent to the H1 box of mat2:2 is used as template. This change might be generated during the process of MT switching when hybrid DNA formation is anomalously extended into the more heterologous region flanking the H1 homology box. In addition to the gene conversion at mat1:1, two mutations were found in the H3 homology boxes of the silent cassettes mat2:2 and mat3:3.     

M26 Recombinational Hotspot and Physical Conversion Tract Analysis in the Ade6 Gene of Schizosaccharomyces Pombe

At the ade6 locus of Schizosaccharomyces pombe flanking markers have been introduced as well as five silent restriction site polymorphisms: four in the 5' upstream region and one in the middle of the gene. The mutations ade6-706, ade6-M26 (both at the 5' end) and ade6-51 (middle of the gene) were used as partners for crosses with the 3' mutation ade6-469. From these three types of crosses, wild-type recombinants were selected and analyzed genetically to assess association with crossing-over and physically to determine conversion tract lengths. The introduced restriction site polymorphisms (five vs. only one) neither influenced the pattern of recombinant types nor the distribution of conversion tracts. The hotspot mutation M26 enhances crossing-over and conversion to the same proportion. M26 not only stimulates conversion at the 5' end, but does this also (to a lower extent) at the 3' end of ade6 at a distance of more than 1 kb. The majority of meiotic conversion tracts are continuous and postmeiotic segregation of polymorphic sites is rare. Conversion tracts are slightly shorter with M26 in comparison with its control 706. The mean minimal length of tracts varies from 670 bp (M26) to 890 bp (706) to 1290 bp (51). It is concluded that M26 acts as an initiation site of recombination or enhances initiation of recombination. M26 does not act by termination of conversion. A region of recombination initiation exists at the 5' end of the ade6 gene also in the absence of the ade6-M26 hotspot mutation.     

Isolation and Characterization of Thymineless Mutants from Ultraviolet-Sensitive Bacillus subtilis Strains

Thymineless mutants of Bacillus subtilis 168 ind, hcr-9 were isolated by using trimethoprim. These and other Thy⁻ strains differed drastically from Thy⁺ ones in their patterns of [³H]thymidine uptake and growth in trimethoprim-containing medium. Transformation was negligible between most mutants derived from the ultraviolet-sensitive strain 168 ind, hcr-9 but significant between 168 ind, thy and these mutants. The latter and these new mutants all grow in the presence of trimethoprim plus thymidine or thymine and fail to grow if thymine or thymidine is omitted.     

Isolation and Characterization of Aminopeptidase-Deficient Lactobacillus bulgaricus Mutants

Lactobacillus bulgaricus CNRZ 397 is able to hydrolyze many amino-acyl- and dipeptidyl-β-naphthylamides. Analysis of heat inactivation kinetics, protease inhibitor effects, and the subcellular location of aminopeptidase (AP) activities from the parental strain and mutant derivatives dificient in alanyl- or leucyl-β-naphthylamide hydrolysis pointed out the existence of four APs. All mutants isolated were totally deficient in AP II, a cell wall metallo-enzyme with a broad substrate specificity but that is specifically responsible for lysyl-AP activity and is characterized by a molecular mass of 95,000 daltons. AP I and AP III are cytoplasmic enzymes that exhibit arginyl-AP activity; both enzymes are inducible during growth in rich peptide MRS medium (Difco Laboratories, Detroit, Mich.). The existence of a fourth AP (AP IV) that is involved in leucyl-AP activity was suggested. Moreover, we showed that X-prolyl-dipeptidyl-AP activity, which was not catalyzed by an AP, involved an enzyme(s) that is controlled by a regulatory mechanism that is common to that of AP II.