Mating type regulates the radiation-associated stimulation of reciprocal translocation events in Saccharomyces cerevisiae

Both ultraviolet (UV) and ionizing radiation were observed to stimulate mitotic, ectopic recombination between his3 recombinational substrates, generating reciprocal translocations in Saccharomyces cervisiae (yeast). The stimulation was greatest in diploid strains competent for sporulation and depends upon both the ploidy of the strain and heterozygosity at the MAT locus. The difference in levels of stimulation between MATa/MAT diploid and MAT haploid strains increases when cells are exposed to higher levels of UV radiation (sevenfold at 150 J/m²), whereas when cells are exposed to higher levels of ionizing radiation (23.4 krad), only a twofold difference is observed. When the MAT gene was introduced by DNA transformation into a MATa/mat::LEU2 ⁺ diploid, the levels of radiation-induced ectopic recombination approach those obtained in a strain that is heterozygous at MAT. Conversely, when the MATA gene was introduced by DNA transformation into a MAT haploid, no enhanced stimulation of ectopic recombination was observed when cells were irradiated with ionizing radiation but a threefold enhancement was observed when cells were irradiated with UV The increase in radiation-stimulated ectopic recombination resulting from heterozygosity at MAT correlated with greater spontaneous ectopic recombination and higher levels of viability after irradiation. We suggest that MAT functions that have been previously shown to control the level of mitotic, allelic recombination (homolog recombination) also control the level of mitotic, radiation-stimulated ectopic recombination between short dispersed repetitive sequences on non-homologous chromosomes.     

Isolation and characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by the mating hormone a-factor

Summary Nine independent mutants which are supersensitive (ssl ^–) to G1 arrest by the mating hormone a-factor were isolated by screening mutagenized Saccharomyces cerevisiae MAT cells on solid medium for increased growth inhibition with a-factor. These mutants carried lesions in two complementation groups, ssl1 and ssl2. Mutations at the ssl1 locus were mating type specific: MAT ssl1 ^– cells were supersensitive to -factor but MAT ssl1 ^– were not supersensitive to -factor. In contrast, mutations at the ssl2. locus conferred supersensitivity to the mating hormone of the opposite mating type on both MAT, and MATa cells. The -cell specific capacity to inactivate externally added a-factor was shown to be lacking in MAT ssl1 ^– mutants whereas MAT ssl2. cells were able to inactivate a-factor. Complementation analysis showed that ssl2 and sst2, a mutation originally isolated as conferring supersensitivity to -factor to MATa cells, are lesions in the same gene. The ssl1 gene was mapped 30.5 centi-Morgans distal to ilv5 on chromosome XII.     

Molecular cloning of Saccharomyces cerevisiae MGC1/YDR473c gene which is essential for cell growth

Saccharomyces cerevisiae haploid cells undergo morphological changes in response to mating pheromones, a- and -factors, during sexual conjugation. As a first step to elucidate the mechanism, I had previously identified the mgc1 mutation which affected the morphogenesis induced by mating pheromones. The mutation had been designated mgc1 for morphogenesis control. In the present study I cloned the MGC1 gene. Sequencing analysis indicates that the MGC1 gene corresponds to the YDR473c gene. The MGC1 gene was shown to be essential for cell growth and required for the transition from the G1 to S phase of cell cycle. Protein-protein interaction of Mgc1 protein was shown by using yeast two-hybrid system. Mgc1 protein was also proposed to be localized in the nucleus in yeast cells.     

A long region upstream of the IME1 gene regulates meiosis in yeast

Summary Meiosis and sporulation in yeast are subject to two types of regulation. The first depends on environmental conditions. The second depends on a genetic pathway which involves the control of the positive regulatory gene IME1 by RME1, which is in turn controlled by the MAT locus. The presence of IME1 on a multicopy plasmid enables cells to undergo meiosis regardless of their genotype at MAT or RME1. We show here that a multicopy plasmid carrying IME1 also enables meiosis, regardless of the environment. Therefore, both kinds of regulation appear to act through IME1. Furthermore, the behavior of multicopy plasmids carrying various segments from the IME1 region suggests that the region upstream of IME1 contains both positive and negative regulatory sites. Control of IME1 by the environment and by the MAT pathway both act through negative regulatory sites.     

Dependence of inessential late gene expression on early meiotic events in Saccharomyces cerevisiae

Summary SPR3 is one of at least nine genes which are expressed in sporulating Saccharomyces cerevisiae cells at the time of meiosis I. We show below that strains homozygous for null alleles of SPR3 are capable of normal meiosis and the production of viable ascospores. We have also monitored SPR3 expression in a series of strains that are defective in meiotic development, using an SPR3: lacZ fusion carried on a single copy plasmid. -Galactosidase activity occurred at wild-type levels in diploid strains homozygous for mutations in spo13, rad50, rad57 and cdc9, but was greatly reduced in strains carrying cdc8 or spo7 defects. We conclude that SPR3 expression is a valid monitor of early meiotic development, even though the gene is inessential for the sporulation process.     

Interchromosomal and intrachromosomal recombination in rad 18 mutants of Saccharomyces cerevisiae

Summary The frequency of intra- and interchromosomal recombination was determined in RAD18 and rad18 deletion and rad18-3 mutant strains. It was found that spontaneous interchromosomal recombination at trp5, his1, ade2, and MAT was elevated 10- to 70-fold in the rad18-3 and rad18 mutants as compared to the RAD ⁺ strains. On the other hand the frequencies of spontaneous intrachromosomal recombination for the his33, his35 and the his4C ^–, his4A ^– duplications and for heterothallic mating type switching were only marginally elevated in the rad18 deletion mutant, and recombination between ribosomal DNA repeats was only 2-fold elevated in the rad18-3 mutant. These differences may be due to a haploid versus diploid specific difference. However interchromosomal recombination was elevated 40-fold and intrachromosomal recombination was only marginally (1.5-fold) elevated in a diploid homozygous for rad18, arguing against a haploid versus diploid specific difference. Possible explanations for the difference in the elevated levels of intra- versus interchromosomal spontaneous recombination are discussed.     

Electron microscopic examination of sporulation-deficient mutants of the fission yeast Schizosaccharomyces pombe

A homothallic haploid strain of the fission yeast Schizosaccharomyces pombe initiates sexual reproduction (mating, meiosis and sporulation) in nitrogen-free sporulation medium. Cellular fine structures of eleven sporulation-deficient mutants (spo2, spo3, spo4, spo5, spo6, spo13, spo14, spo15, spo18, spo19 and spo20) of S. pombe in sporulation medium were examined by serial section-electron microscopy. The striking features of these spo mutants were: 1) the disappearance of the spindle pole bodies (SPBs) after the second meiotic division, and 2) the accumulation of unorganized structures. Based on histochemical staining, these structures were presumably unorganized spore wall precursors. In some mutants (spo3, spo5, spo6, spo19 and spo20), diploid zygotes contained four spore-like bodies which had walls similar to complete spore walls but failed to enclose any nuclei. After completion of the second meiotic division the nuclei were abnormally distributed in zygotic diploid cells. In the spo5, spo13, spo14, spo15 and spo19 mutants, the nuclei remained attached to each other. In spo5 and spo19, the inner membrane of the nuclear envelope was separated, but its outer membrane was shared by two sister nuclei. These observations suggest that the spo⁺ gene products play important roles in spatial and temporal organization of cellular structures during ascospore development.Abbreviations SPB spindle pole body - PTA-Cr phosphotungstic acid and chromic acid - PATAg periodic acid, thiocarbohydrazide and silver proteinate     

Assessment ofGibberella fujikuroi mating type by PCR

Mating type (MAT)-specific fragments of the two idiomorphs ofGibberella fujikuroi (anamorph,Fusarium moniliforme) were obtained by PCR amplification using primers to conserved regions ofMAT homologs from other fungal species and used to assign mating type by molecular criteria rather than the arbitrary historical designation. Mating type—strains of mating populations A-E and a mating type+strain of mating population F carry an α-box motif and should therefore be designatedMAT-1. Mating type+strains of mating populations A-E and a mating type—strain of mating population F carry an HMG-box motif and should be designatedMAT-2. Thus, assessment of mating type ofG. fujikurol strains can be easily achieved usingMAT-specific primers.     

Control of sporulation in yeast: SAD1-a mating-type specific, unstable alteration that uncouples sporulation from mating-type control

Summary Normally, in order to sporulate, a diploid yeast cell must be heterozygous (MAT a/MAT) at the mating-type locus. In a new mutant, this requirement is circumvented by SAD1. This alteration is mating-type specific; it allows sporulation of MAT/ MAT. MAT/mat a-1, and MAT/mat-2 diploid cells, but not MAT a/MAT a or MAT a/0 (monosomic) strains. Other than acquiring the ability to sporulate, SAD1 cells behave as wild-type MAT/MAT strains; they exhibit medial budding, normal mating factor production and response and mate with normal mating efficiencies and kinetics. The segregation of SAD1 is often bizarre; for example, MAT/MAT strains which were constructed to be heterozygous SAD1/+ often segregate 4 SAD1:0+progeny and strains which were constructed to be homozygous SAD1/ SAD1 sometimes segregated 1 SAD1:3+progeny. The genetic analyses of SAD1 suggest that it is dominant and is located 30 cM from MAT on chromosome III.     

The switching gene swi6 affects recombination and gene expression in the mating-type region of Schizosaccharomyces pombe

Summary The products of 11 switching (swi) genes are required for efficient mating-type (MT) switching in homothallic (h ⁹⁰) strains of Schizosaccharomyces pombe. The MT region of h ⁹⁰ comprises three cassette genes: the expression site mat1: 1 and two silent loci, mat2: 2 and mat3: 3. Besides reducing MT switching, the swi6 mutation leads to deletions in the MT region caused by intrachromosomal cross-overs between two paired cassettes. These deletions only arise if DNA double-strand breaks are present at mat1: 1, which initiate MT switching. Furthermore, swi6 allows meiotic recombination in the K region, a region of 16 kb between mat2: 2 and mat3: 3; in wild-type strains no recombination occurs in K. swi6 also allows the simultaneous expression of two different cassettes in the same haploid cell. Thus swi6 may have an influence on the general chromatin structure in the MT region.     

Selection of Optimal Host Strain for Molecular Pathogenesis Studies on <Emphasis Type="Italic">Cryptococcus gattii</Emphasis>

Encapsulated yeast, Cryptococcus gattii (Cg) is a primary and emerging fungal pathogen in North America. It has a predilection for invading the central nervous system of both healthy and immunocompromised humans and animals. Recently, we initiated molecular pathogenesis studies in Cg strain NIH444 (ATCC 32609). In this report, we compared the biology and pathogenic potential of NIH444 to those of WM276, an Australian environmental isolate that is being used for the whole genome-sequencing project. Our data indicated that NIH444 is comparatively more virulent in a mouse model of cryptococcosis than is WM 276. We found robust mating of NIH444, and no mating of WM276, when tested against Cg MATa strain, NIH198. WM276 but not NIH444 was defective in filamentation and sporulation (haploid fruiting). Interestingly, NIH444 has a VGII/AFLP6 genotype similar to that of the genotype of the recent outbreak strains from Vancouver Island, British Columbia, Canada. Additionally, comparisons of nucleotide sequences of various genes also showed differences between NIH444 and WM276. Based on these observations, we conclude that NIH444 should remain the strain of choice for understanding Cg pathogenesis, especially on the North American continent.     

α-mating-type-specific suppression and a-mating-type-specific enhancement by tunicamycin of sexual agglutinability in the yeast Saccharomyces cervisiae

Effects of tunicamycin (TM) on the sexual agglutinability and zygote formation of Saccharomyces cerevisiae were studied using the two kinds of haploid strains, inducible and constitutive for sexual agglutinability. Induction of sexual agglutinability by opposite mating type sex pheromone of inducible strains was inhibited by TM in mating type but not in a mating type. The recovery by temperature-shift-down from the temperature-suppressed sexual agglutinability of constitutive strains was enhanced by TM in a mating type but rather inhibited in mating type. Pretreatment with TM of constitutive strains enhanced sexual agglutinability in a mating type but not in mating type. The above-mentioned a-mating-type-specific agglutinability-enhancing actions of TM were discussed in relation to the action mechanism of pheromone which induces or enhances the sexual agglutinability of a cells.Zygote formation was inhibited by TM in both constitutive and inducible strains at concentrations which showed only partially inhibitory effect on sexual agglutinability.Abbreviations AI agglutination index - TM tunicamycin     

The effect of spo0 mutations on the expression of spo0A- and spo0F-lacZ fusions

Summary We have constructedspo0A-lacZ andspo0F-lacZ fusions with a temperate phage vector and have investigated howspo0 gene products are involved in the expression of each of these genes. The expression ofspo0A-lacZ andspo0F-lacZ was stimulated at about the time of cessation of vegetative growth in Spo⁺ cells. This stimulation ofspo0A-lacZ was impaired by mutations in thespo0B, D, E, F orH genes but was not affected by mutations in thespo0J orK genes. Similar results were obtained with thespo0F-lacZ fusion. The effect of thespo0A mutation onspo0A-lacZ expression was characteristic: thespo0A-directed β-galactosidase activity found during vegetative growth was significantly enhanced in thespo0A mutant. This result suggests thatspo0A gene expression is autoregulated being repressed by its own gene product. Another remarkable observation was the effect of thesof-1 mutation, which is known to be aspo0A allele; it suppressed the sporulation deficiency ofspo0B, spo0D andspo0F mutants. Thespo0A-lacZ stimulation, which is impaired by any one of thesespo0 mutations, was restored by the additionalsof-1 mutation.     

Cloning of the mating-type gene MATA of the yeast Yarrowia lipolytica

Summary The mating type gene MA TA of the dimorphic yeast Yarrowia lipolytica was cloned. The strategy used was based on the presumed function of this gene in the induction of sporulation. A diploid strain homozygous for the mating type B was transformed with an integrative gene bank from an A wild-type strain. A sporulating transformant was isolated, which contained a plasmid with an 11.6 kb insert. This sequence was rescued from the chromosomal DNA of the transformant and deletion mapping was performed to localize the MAT insert. The MAT gene conferred both sporulating and non-mating phenotypes on a B/B diploid. A LEU2 sequence targeted to this locus segregated like a mating type-linked gene. The A strain did not contain silent copies of the MAT gene.     

Fumonisin B1 production and vegetative compatibility of strains from Gibberella fujikuroi mating population “A” (Fusarium moniliforme)

We examined 25 strains of Fusarium moniliforme from eight states known to be associated with equine leukoencephalomalacia, a disease caused by the mycotoxin fumonisin B₁. We determined the mating population, mating type, and vegetative compatibility group to which each of these strains belonged. All 25 strains were in the A mating population; 12 were A⁺ and 13 were A^–. Seventeen of the 25 strains were female fertile; these strains also averaged higher levels of fumonisin B₁ production than did the strains that were female sterile. Nitrate non-utilizing (nit) mutants were generated in all 25 strains and each strain was assigned to a unique vegetative compatibility group based on the inability of the derived nit mutants to form a prototrophic heterokaryon with complementary nit mutants derived from any of the other strains examined. From these data, we concluded that the production of fumonisin B₁ is a general characteristic of strains from the A mating population of Gibberella fujikuroi associated with equine leukoencephalomalacia, since all 25 of the isolates that we examined were genetically distinct individuals.     

A dominant mutation (SAD) bypassing the requirement for the a mating type locus in yeast sporulation

Summary SAD (suppressor of a deficiencies) is a mutation that allows -mater diploids such as / or a1^-/ strains to sporulate. This mutation is unstable and reverts to wildtype (sad ⁺) even in strains homozygous for SAD. SAD is dominant to sad ⁺: / and a1^-/ sad ¹/SAD diploids are sporulation-proficient. SAD is located on chromosome III, 40 cM distal to the mating type locus, between THR4 and HMR a. The ability of SAD to support sporulation requires the presence of an mating type locus with an active 2 function. Possible models for the action of SAD are (1) SAD bypasses the need for a1 function in sporulation, and (2) SAD provides a1 function to MAT a1^- mutants by supplying a1 function itself, for example, by allowing expression of a silent copy of MAT a.     

Lysine Dendrimers and Their Starburst Polymer Derivatives: Possible Application for DNA Compaction and in vitro Delivery of Genetic Constructs

We attempted to find some compounds for the effective delivery of gene constructs into cells and obtained two trispherical dendrimers on the basis of lysine, (Lys)₈-(,-Lys)₄-(,-Lys)₂-(,-Lys)-Ala-NH₂ (D1) and ((Lys)₈-(,-Lys)₄-(,-Lys)₂-,-Lys)-Ala-[Lys(Plm)]₂-Ala-NH₂ (D2), as well as the starburst polymeric derivatives of D1, (pVIm) ₈ -D1 and (pLys) _n -D1, containing poly(N-vinylimidazole) and polylysine chains single-point bound to the dendrimer amino groups. The conditions of dendrimer–plasmid DNA complex formation were studied. The intracellular localization of these complexes and the expression of gene constructs delivered with their help were analyzed in transfection experiments on the HeLa cell cultures of human epithelial carcinoma and on mouse C2C12 myoblasts. It was found that the chemical structure of dendrimer D1 and its derivatives significantly affected the structure and properties of complex.     

The pso4-1 mutation reduces spontaneous mitotic gene conversion and reciprocal recombination in Saccharomyces cerevisiae.

Summary Spontaneous mitotic recombination was examined in the haploid pso4-1 mutant of Saccharomyces cerevisiae and in the corresponding wild-type strain. Using a genetic system involving a duplication of the his4 gene it was shown that the pso4-1 mutation decreases at least fourfold the spontaneous rate of mitotic recombination. The frequency of spontaneous recombination was reduced tenfold in pso4-1 strains, as previously observed in the rad52-1 mutant. However, whereas the rad52-1 mutation specifically reduces gene conversion, the pso4-1 mutation reduces both gene conversion and reciprocal recombination. Induced mitotic recombination was also studied in pso4-1 mutant and wild-type strains after treatment with 8-methoxypsoralen plus UVA and 254 nm UV irradiation. Consistent with previous results, the pso4-1 mutation was found strongly to affect recombination induction.     

Genetic structure and domains of DNA polymerase III of Bacillus subtilis

Summary We have determined the nucleotide sequence of the polC gene of Bacillus subtilis which codes for DNA polymerase III. Our recent analysis has revealed that the gene comprises 4311 nucleotides, from the start to the stop codon, 306 nucleotides more than we reported earlier. The plasmid reported by us and by N.C. Brown's laboratory contained a sequence at the end of the gene which is not related to the polC region of B. subtilis. We have isolated the rest of the gene, the sequence of which is presented in this paper. The new stop codon is followed by a hyphenated palindromic sequence of 13 nucleotides. The C-terminus' of the coding region contains the novel mutation, dnaF, which results in a defect in the initiation of replication due to a change in the codon TCC to TTC (serine to phenylalanine). The hypermutator mutation mut-1 is due to two point mutations in the 3 to 5 exonuclease domain, the proof reading function. The codon changes are GGA to GAA (glycine to glutamic acid) and AGC to AAC (serine to asparagine). The elongation defective mutation, polC26, affecting the catalytic site that adds nucleotides to the growing chain, is due to a change in the codon GTC to GAC (valine to aspartic acid). It is separated from the mutation reported earlier, azp-12, by 306 nucleotides. Knowing the locations of the mutational sites allowed us to deduce the domains of the gene and the enzyme it encodes, and permitted us to present a precise map of the gene at the molecular level.Abbreviations HPUra p-hydroxyphenyl azouracil - nt nucleotide - PCR polymerase chain reaction