Two Chromosomal Genes Required for Killing Expression in Killer Strains of SACCHAROMYCES CEREVISIAE

The killer character of yeast is determined by a 1.4 x 10⁶ molecular weight double-stranded RNA plasmid and at least 12 chromosomal genes. Wild-type strains of yeast that carry this plasmid (killers) secrete a toxin which is lethal only to strains not carrying this plasmid (sensitives). ——— We have isolated 28 independent recessive chromosomal mutants of a killer strain that have lost the ability to secrete an active toxin but remain resistant to the effects of the toxin and continue to carry the complete cytoplasmic killer genome. These mutants define two complementation groups, kex1 and kex2. Kex1 is located on chromosome VII between ade5 and lys5. Kex2 is located on chromosome XIV, but it does not show meiotic linkage to any gene previously located on this chromosome. ——— When the killer plasmid of kex1 or kex2 strains is eliminated by curing with heat or cycloheximide, the strains become sensitive to killing. The mutant phenotype reappears among the meiotic segregants in a cross with a normal killer. Thus, the kex phenotype does not require an alteration of the killer plasmid. ——— Kex1 and kex2 strains each contain near-normal levels of the 1.4 x 10⁶ molecular weight double-stranded RNA, whose presence is correlated with the presence of the killer genome.     

Dominant chromosomal mutation bypassing chromosomal genes needed for killer RNA plasmid replication in yeast

Yeast strains carrying a double-stranded RNA plasmid of 1.4–1.7 x 10⁶ daltons encapsulated in virus-like particles secrete a toxin that kills strains lacking this plasmid. The plasmid requires at least 24 chromosomal genes (pets, and mak1 through mak23) for its replication or maintenance. We have detected dominant Mendelian mutations (called KRB1 for killer replication bypass) that bypass two chromosomal genes, mak7 and pets, normally needed for plasmid replication. Strains mutant in mak7 and carrying the bypass mutation (mak7–1 KRB1) are isolated as frequent K⁺R⁺ sectors of predominantly K^-R ^- segregants from crosses of mak7–1 with a wild-type killer. All KRB1 mutations isolated in this way are inherited as single dominant centromere-linked chromosomal changes. They define a new centromere. KRB1 is not a translational suppressor. KRB1 strains contain a genetically normal killer plasmid and ds RNA species approximately the same in size and amount as do wild-type killers. Bypass of both mak7 and pets by one mutation suggests that these two genes are functionally related.

Two properties of the inheritance of KRB1 indicate an unusually high reversion frequency: (1) Heat or cycloheximide (treatments known to cure strains of the wild-type killer plasmid) readily induce conversion of mak7–1 KRB1 strains from killers to nonkillers with concomitant disappearance of KRB1 as judged by further crosses, and (2) mating two strains of the type mak7–1 KRB1 with each other yields mostly 2 K⁺R⁺: 2 K^-R^- segregation, although the same KRB1 mutation and the same killer plasmid are present in both parents.

     

Genetic and Morphological Study of Aggregation in the Cellular Slime Mold POLYSPHONDYLIUM VIOLACEUM

A system for genetic analysis in the cellular slime mold P. violaceum has been developed. Two growth-temperature-sensitive mutants were isolated in a haploid strain and used to select rare diploid heterozygotes arising by spontaneous fusion of the haploid cells. A recessive mutations to cycloheximide resistance in one strain enables selection of segregants, which often appear to be aneuploid.—Aggregation-defective (ag^- ) mutants having a wide range of phenotypes were isolated in both temperature-sensitive strains after nitrosoguanidine treatment, and complementation tests were performed between pairs of these mutants. Of 380 diploids isolated, 32 showed defective aggregation and were considered to contain 2 noncomplementing ag^- mutations. Among noncomplementing mutants interallelic complementation is common. Noncomplementing mutants fall into 4 complementation groups, and those within each complementation group are phenotypically similar. Statistical analysis of the results suggests that the number of complementation units involved in aggregation is about 50.     

Recombination of the bph (Biphenyl) Catabolic Genes from Plasmid pWW100 and Their Deletion during Growth on Benzoate

Pseudomonas sp. strain CB406 was isolated from polychlorinated biphenyl-contaminated soil and harbors a nontransmissible plasmid, pWW100, of approximately 200 kb which carries the genes required for biphenyl and 4-chlorobiphenyl catabolism. The catabolic phenotype was mobilized following the construction in vivo of a cointegrate plasmid containing functional upper and lower biphenyl operons inserted into the broad-host-range R plasmid RP4. The Bph⁺ phenotype carried by pWW100 was stable in nonselective media but was unstable during growth on benzoate, where the sequential selection of two species of bph deletion derivatives occurs at high frequency. This mirrors observations made with TOL plasmids (encoding toluene and xylene catabolism) grown under similar conditions. Subcloning of dioxygenase genes involved in biphenyl catabolism confirmed the localization of the bph genes on the wild-type plasmid and the RP4 cointegrate plasmid.     

Sexual differentiation in Mucor: Trisporic acid response mutants and mutants blocked in zygospore development

Spores of a minus strain of Mucor mucedo (Bref.) were treated with 1-methyl-[3-nitro]-1-nitro-soguanidine and mutants were isolated either by testing for zygophore induction with externally supplied trisporic acids (TA) or by mating with wild type plus colonies. Mutants were found defective (Tar^?) or temperature-sensitive (Tar-Ts) in their reaction towards trisporic acids, blocked or temperature-sensitive in their mating with plus strain (Mat^? or Mat-Ts) or temperature-sensitive in zygospore development (Zyg-Ts). The inability to react against externally supplied trisporic acids was not necessarily coupled with an inability to mate with plus strain (phenotype Tar^? Mat⁺). This indicated that the diffusion and uptake of trisporic acids is not a necessary prerequisite to the sexual interaction of Mucor mating types.     

Mitotic Crossing over and Nondisjunction in Translocation Heterozygotes of Aspergillus

To analyze mitotic recombination in translocation heterozygotes of A. nidulans two sets of well-marked diploids were constructed, homo- or heterozygous for the reciprocal translocations T1(IL;VIIR) or T2(IL;VIIIR) and heterozygous for selective markers on IL. It was found that from all translocation heterozygotes some of the expected mitotic crossover types could be selected. Such crossovers are monosomic for one translocated segment and trisomic for the other and recovery depends on the relative viabilities of these unbalanced types. The obtained segregants show characteristically reduced growth rates and conidiation dependent on sizes and types of mono- and trisomic segments, and all spontaneously produce normal diploid sectors. Such secondary diploid types either arose in one step of compensating crossing over in the other involved arm, or—more conspicuously—in two steps of nondisjunction via a trisomic intermediate.—In both of the analyzed translocations the segments translocated to IL were extremely long, while those translocated from IL were relatively short. The break in I for T1(I;VII) was located distal to the main selective marker in IL, while that of T2(I;VIII) had been mapped proximal but closely linked to it. Therefore, as expected, the selected primary crossover from the two diploids with T2( I;VIII) in coupling or in repulsion to the selective marker, showed the same chromosomal imbalance and poor growth. These could however be distinguished visually because they spontaneously produced different trisomic intermediates in the next step, in accordance with the different arrangement of the aneuploid segments. On the other hand, from diploids heterozygous for T1( I;VII) mitotic crossovers could only be selected when the selective markers were in coupling with the translocation; these crossovers were relatively well-growing and produced frequent secondary segregants of the expected trisomic, 2n+VII, type. For both translocations it was impossible to recover the reciprocal crossover types (which would be trisomic for the distal segments of I and monosomic for most of groups VII or VIII) presumably because these were too inviable to form conidia.—In addition to the selected segregants of expected types a variety of unexpected ones were isolated. The conditions of selection used favour visual detection of aneuploid types, even if these produce only a few conidial heads and are not at a selective advantage. For T2(I;VIII) these "non-selected" unbalanced segregants were mainly "reciprocal" crossovers of the same phenotype and imbalance as the selected ones. For T1(I;VII) two quite different types were obtained, both possibly originating with loss of the small VII–I translocation chromosome. One was isolated when the selective marker in repulsion to T1(I;VII) was used and, without being homo- or hemizygous for the selective marker, it produced stable sectors homozygous for this marker. The other was obtained from both coupling and repulsion diploids and showed a near-diploid genotype; it produced practically only haploid stable sectors of the type expected from monosomics, 2n–1 for the short translocation chromosome.     

Vegetative Incompatibility and the Mating-Type Locus in the Cellular Slime Mold DICTYOSTELIUM DISCOIDEUM

The genetic basis of vegetative incompatibility in the cellular slime mold, Dictyostelium discoideum, is elucidated. Vegetatively compatible haploid strains from parasexual diploids at a frequency of between 10^-6 and 10^-5, whereas "escaped" diploids are formed between vegetatively incompatible strains at a frequency of ~10^-8. There is probably only a single vegetative incompatibility site, which appears to be located at, or closely linked to, the mating-type locus. The nature of the vegetative incompatibility is deduced from parasexual diploid formation between wild isolates and tester strains of each mating type, examination of the frequency of formation of "escaped" diploids formed between vegetatively incompatible strains, and examination of the mating type and vegetative incompatibility of haploid segregants obtained from "escaped" diploids.     

Studies of the inheritance of virulence in the entomopathogenic fungus Metarhizium anisopliae

A forced heterocaryon was established between two auxotrophic conidial color mutants of Metarhizium anisopliae. From the heterocaryon, a prototrophic somatic diploid was selected which, in turn, yielded somatic segregants. The virulence of the original mutants, the somatic diploid, and the somatic segregants was evaluated on three species of mosquitoes as well as on Ostrinia nubilalis larvae. The virulence of the somatic diploid was comparable to that of the wild-type parental strain while the auxotrophic somatic segregants exhibited virulence approximately equal to that of the auxotrophic components of the heterocaryon. Putative somatic diploids were obtained between morphological mutants of the two species varieties (M. anisopliae var. minor and var. major). The presumptive diploids were avirulent for the insect species to which the parental strains exhibited virulence.     

The role of recombinational repair proteins in mating type switching in fission yeast cells

DNA double-strand breaks (DSBs) occur after exposing cells to ionizing radiation or under the action of various antitumor antibiotics. They can be also generated in the course cell processes, such as meiosis and mating type switching in yeast. The most preferential mechanism for the correction of DNA DSB in yeasts is recombinational repair controlled by RAD52 group genes. The role of recombinational repair in mating type switching of fission yeast cells was examined on the example of genes of this group, rhp51 ⁺ and rhp55 ⁺. We constructed homothallic strains of genotypes h ⁹⁰ rhp51 and h ⁹⁰ rhp55, and found that mutant cells yielded colonies with the mottled phenotype. In addition, h ⁹⁰ cells with deletions in these genes were shown to segregate heterothallic iodine-negative colonies h ^? and h ⁺. The genome region, responsible for the switching process in these segregants, was analyzed by DNA hybridization. As shown in this analysis, h ⁺ segregants had the h ^+N or h ⁹⁰ configuration of the mat region, whereas h ^?, the h ⁹⁰ configuration. Segregants h ^+N contained DNA duplication in the mat region. DNA rearrangements were not detected at the mating type locus, but the level of DNA DSB formation was drastically decreased in these segregants. Thus, our results show that genes rhp51 ⁺ and rhp55 ⁺ are involved not only in the repair of induced DNA DSB, but also in the mechanism of mating type switching in fission yeast.     

Induced somatic segregation inArmillaria mellea diploids

Armillaria mellea is a bifactorially heterothallic fungus with a fertile, diploid, vegetative phase. While diploids of this fungus are readily recovered by nutritional selection from compatible and incompatible pairings of haploids, genetic analyses have been limited both because this organism does not produce fruiting bodies reliably in the laboratory and because somatic segregation occurs infrequently in diploid cultures. In this study, diploids ofA. mellea heterozygous at nutritional and mating-type marker loci were treated with formaldehyde, para-fluorophenylalanine, benomyl, and ultraviolet light in order to recover somatic segregants. Of these agents, only benomyl dramatically increased the frequency of somatic segregation under the conditions used. Auxotrophic segregants were recovered from macerates of prototrophic, diploid mycelia that had been grown in the presence of benomyl. Many of these segregants differed from their diploid progenitors in mating behavior as well as in nutritional phenotype. The development of a reliable method for the recovery of somatic segregants from diploids ofA. mellea permits parasexual analysis and reduces the need forin vitro production of fruiting bodies in future genetic studies.     

Development of an integrative DNA transformation system for the yeast Hansenula anomala

A host-vector system for the yeast Hansenula anomala was developed. The system was based on an auxotrophic mutant host of H. anomala which was defective in orotidine-5′-phosphate decarboxylase (ODCase) activity. The H. anomala ODCase-negative mutant strains (ura3 strains) were isolated based on 5-fluoroorotic acid (5-FOA) resistance. A plasmid vector containing the H. anomala URA3 gene was used for transformation. Using this plasmid, all of the H. anomala ura3 strains tested could be transformed to Ura⁺ phenotypes. In all of Ura⁺ transformants, the introduced plasmid was integrated into the chromosomal URA3 locus by homologous recombination. The Ura⁺ phenotype of the transformants was stably maintained after nonselective growth.     

Defective Interference in the Killer System of Saccharomyces cerevisiae

The K₁ killer virus (or plasmid) of Saccharomyces cerevisiae is a noninfectious double-stranded RNA genome found intracellularly packaged in an icosahedral capsid. This genome codes for a protein toxin and for resistance to that toxin. Defective interfering virus mutants are deletion derivatives of the killer virus double-stranded RNA genome; such mutants are called suppressive. Unlike strains carrying the wild-type genome, strains with these deletion derivatives are neither toxin producers nor toxin resistant. If both the suppressive and the wildtype virus are introduced into the same cell, most progeny become toxin-sensitive nonkillers (J. M. Somers, Genetics 74:571-579, 1973). Diploids formed by the mating of a killer with a suppressive strain were grown in liquid culture, and RNA was extracted from samples taken up to 41 generations after the mating. The ratio of killer RNA to suppressive RNA decreased with increasing generations; by 41 generations the killer RNA was barely detectable. The copy numbers of the suppressive genome and its parental killer were virtually the same in isogenic strains, as were the growth rates of diploid strains containing either virus alone. Therefore, suppressiveness, not being due to segregation or overgrowth by faster growing segregants, is likely due to preferential replication or maintenance of the suppressive genome. Three suppressive viruses, all derivatives of the same killer virus (T. K. Sweeney et al., Genetics 84:27-42, 1976), did not coexist stably. The evidence strongly indicates that the largest genome of the three slowly suppressed both of the smaller genomes, showing that larger genomes can suppress smaller ones and that suppression can occur between two suppressives. Of 48 isolates of strains carrying the suppressive viruses, 5 had newly detectable RNA species, all larger than the original suppressive genomes. At least seven genes necessary for maintenance of the wild-type killer virus (MAK genes) were needed by a suppressive mutant. No effect of ski mutations (affecting regulation of killer virus double-stranded RNA replication) on suppressiveness was observed.     

Deletion of mitochondrial DNA bypassing a chromosomal gene needed for maintenance of the killer plasmid of yeast

Strains of Saccharomyces cerevisiae carrying a 1.4 x 10⁶ dalton double-stranded (ds) RNA in virus-like particles (the killer plasmid or virus) secrete a toxin that is lethal to strains not carrying this plasmid (virus). The mak10 gene is one of 24 chromosomal genes (called pets, mak1, mak2,...) that are needed to maintain and replicate the killer plasmid. We report here isolation of spontaneous and induced mutants in which the killer plasmid is maintained and replicated in spite of a defect in the mak10 gene. The bypass (or suppressor) mutations in these strains are in the mitochondrial genome. Respiratory deficiency produced by various chromosomal pet mutations, by chloramphenicol, or by antimycin A, does not bypass the mak10–1 mutation. Several spontaneous mak10–1 killer strains have about 12-fold more of the killer plasmid ds RNA than do wild-type killers. Although the absence of mitochondrial DNA bypasses mak10–1, it does not bypass pets–1, mak1–1, mak3–1, mak4–1, mak5–1, mak6–1, mak7–1, or mak8–1.     

Genetic structure and regulation of a replicon of plasmid lambdadv.

Strains of Saccharomyces cerevisiae carrying a small double-stranded RNA species (the killer plasmid) secrete a toxin which is lethal only to strains not carrying this plasmid.We have isolated mutants in eight chromosomal genes essential for replication or maintenance of the killer plasmid, called mak1 through mak8. Seven of these genes have been mapped. mak4 and mak5 are on chromosome II; mak1 and mak8 are on chromosome XV; mak3 and mak6 are on chromosome XVI; and mak7 is on chromosome VIII. We have not yet located mak2. Two other chromosomal genes, m and pets, have been shown to be required for replication or maintenance of the killer plasmid.One allele of mak1 results in temperature sensitivity for host growth. Two independent pets isolates also result in the petite phenotype, as well as temperature sensitivity for growth.Wild-type killer strains have been reported to carry two species of doublestranded RNA of 2.5 × 10⁶ and 1.4 × 10⁶ molecular weight (designated L and M, respectively); wild-type non-killers carried only L. We estimate the size of the L and M species at 3.0 × 10⁶ and 1.7 × 10⁶ daltons, respectively. We have also detected a third species of double-stranded RNA of molecular weight 3.8 × 10⁶ (XL) present in all killer and non-killer strains examined.Mutation of any of mak1 through mak8 results in loss of the killer-associated species of double-stranded RNA (M; 1.7 × 10⁶). These mutants retain both the L species (3.0 × 10⁶) and the XL species (3.8 × 10⁶) of double-stranded RNA, and have acquired two new minor RNA species.     

The transfer RNA processing defect in Escherichia coli strains BN and CAN is not due to a mutation in RNAase D or RNAase II

Escherichia coli strains BN and CAN are unable to support the growth of bacteriophage T4 psu₁⁺-amber double mutants. For strain BN, this phenotype has been attributed to a defect in 3′ processing of the precursor to psu₁⁺ tRNA^Ser. Since RNAase D and RNAase II are the only well-characterized 3′ exoribonucleases to be implicated in tRNA processing, the status of these activities and their genes in the mutant strains was investigated. Although extracts of strains BN and CAN were defective for hydrolysis of the artificial tRNA precursor, tRNA-C-U, these strains contained normal levels of RNAase D and RNAase II, and purified RNAase D or RNAase II could only partially complement the mutant extracts. Introduction of the wild-type RNAase D gene into strains BN and CAN did not correct the mutant phenotype. Likewise, strains defective in RNAase D and/or RNAase II plated T4psu₁⁺-amber phage normally. These results indicate that the tRNA processing defect in strains BN and CAN is not due to a mutation in either RNAase U or RNAase II. The possibility that the mutation in these strains affects another exoribonuclease or a factor influencing the activity and specificity of RNAase D or RNAase II is discussed.     

The Origin of Mutants Under Selection: How Natural Selection Mimics Mutagenesis (Adaptive Mutation)

Selection detects mutants but does not cause mutations. Contrary to this dictum, Cairns and Foster plated a leaky lac mutant of Escherichia coli on lactose medium and saw revertant (Lac⁺) colonies accumulate with time above a nongrowing lawn. This result suggested that bacteria might mutagenize their own genome when growth is blocked. However, this conclusion is suspect in the light of recent evidence that revertant colonies are initiated by preexisting cells with multiple copies the conjugative F′lac plasmid, which carries the lac mutation. Some plated cells have multiple copies of the simple F′lac plasmid. This provides sufficient LacZ activity to support plasmid replication but not cell division. In nongrowing cells, repeated plasmid replication increases the likelihood of a reversion event. Reversion to lac⁺ triggers exponential cell growth leading to a stable Lac⁺ revertant colony. In 10% of these plated cells, the high-copy plasmid includes an internal tandem lac duplication, which provides even more LacZ activity—sufficient to support slow growth and formation of an unstable Lac⁺ colony. Cells with multiple copies of the F′lac plasmid have an increased mutation rate, because the plasmid encodes the error-prone (mutagenic) DNA polymerase, DinB. Without DinB, unstable and stable Lac⁺ revertant types form in equal numbers and both types arise with no mutagenesis. Amplification and selection are central to behavior of the Cairns–Foster system, whereas mutagenesis is a system-specific side effect or artifact caused by coamplification of dinB with lac. Study of this system has revealed several broadly applicable principles. In all populations, gene duplications are frequent stable genetic polymorphisms, common near-neutral mutant alleles can gain a positive phenotype when amplified under selection, and natural selection can operate without cell division when variability is generated by overreplication of local genome subregions.     

Bisexual Mating Behavior in a Diploid of SACCHAROMYCES CEREVISIAE: Evidence for Genetically Controlled Non-Random Chromosome Loss during Vegetative Growth

A diploid strain of Saccharomyces cerevisiae has been isolated which exhabits bisexual mating behavior. The strain mates with either a or alpha strains with a relative mating efficiency of 1 to 2%. The efficiency of mating is correlated with the frequency with which subclones of this strain revert to a single mating type. Crosses of the bisexual diploid with a/a or alpha/alpha diploids yield bisexual segregants with a frequency of approximately 3%. Analysis of the segregation of the mating type alleles and other markers on chromosome III indicates that the primary event which leads to the bisexual phenotype is the loss of one homolog of chromosome III during vegetative growth to produce a monosomic (2n-1) diploid. Evidence is presented that the loss of chromosome III and possibly of other chromosomes during vegetative growth is affected by a recessive nuclear gene-her (hermaphrodite)-which is not closely linked to the mating type locus.     

Selection of dinB Alleles Suppressing Survival Loss upon dinB Overexpression in Escherichia coli

Escherichia coli strains overproducing DinB undergo survival loss; however, the mechanisms regulating this phenotype are poorly understood. Here we report a genetic selection revealing DinB residues essential to effect this loss-of-survival phenotype. The selection uses strains carrying both an antimutator allele of DNA polymerase III (Pol III) α-subunit (dnaE915) and either chromosomal or plasmid-borne dinB alleles. We hypothesized that dnaE915 cells would respond to DinB overproduction differently from dnaE⁺ cells because the dnaE915 allele is known to have an altered genetic interaction with dinB⁺ compared to its interaction with dnaE⁺. Notably, we observe a loss-of-survival phenotype in dnaE915 strains with either a chromosomal catalytically inactive dinB(D103N) allele or a low-copy-number plasmid-borne dinB⁺ upon DNA damage treatment. Furthermore, we find that the loss-of-survival phenotype occurs independently of DNA damage treatment in a dnaE915 strain expressing the catalytically inactive dinB(D103N) allele from a low-copy-number plasmid. The selective pressure imposed resulted in suppressor mutations that eliminated growth defects. The dinB intragenic mutations examined were either base pair substitutions or those that we inferred to be loss of function (i.e., deletions and insertions). Further analyses of selected novel dinB alleles, generated by single-base-pair substitutions in the dnaE915 strain, indicated that these no longer effect loss of survival upon overproduction in dnaE⁺ strains. These mutations are mapped to specific areas of DinB; this permits us to gain insights into the mechanisms underlying the DinB-mediated overproduction loss-of-survival phenotype.     

Transformation of the basidiomycete, Schizophyllum commune

Summary Protoplasts of aSchizophyllum commune tryptophan auxotroph (trp1), deficient in indole-3-glycerol phosphate synthetase (IGPS), were transformed to trp⁺ with plasmid DNA containing the SchizophyllumTRP1 sequence. Efficiencies up to 30 transformants per microgram of plasmid DNA were obtained. Southern blots reveal that the transforming DNA is integrated in chromosomal DNA. The trp⁺ phenotype of transformants is stable in meiosis and mitosis. Transformants possess IGPS activity comparable to wild-type cells.