Genes involved in the control of nuclear fusion during the sexual cycle of Saccharomyces cerevisiae

Summary Mutants of Saccharomyces cerevisiae defective for nuclear fusion have been isolated. Their mutations have been characterized by meiotic analysis, dominance-recessivity and complementation. Twelve of the mutations are allelic to the previously described kar 1–1; five affect a second gene designated KAR 2 and three affect a third gene designated KAR 3. There is evidence suggesting that other two mutants are affected in a gene different from the three mentioned.Mutations in KAR 1 and KAR 2 genes are recessive and do not cause obvious effects other than the failure of the karyogamy. Mutations in KAR 3 are semidominant and do cause pleiotropic effects affecting both mitosis and meiosis.     

Allelspezifische suppressormutationen vonSchizosaccharomyces pombe

By comparing the intragenic distribution of suppressor sensitive mutants in fine structure maps, 13 allele specific suppressor mutations (isolated from revertants in adenine dependent mutants of constitutionad ₇) have been analyzed for their allele specific patterns of action in three different groups of mutants blocked in adenine biosynthesis. The 13 suppressor mutations, which have resulted from mutations at seven different suppressor loci, are characterized by four different suppression patterns. Three of these patterns, which partially overlap, are not locus specific since they include sensitive mutants at each of the three lociad ₇, ad₆ andad ₁ studied. The relative frequency of mutants sensitive to one or the other of the suppressors of this type, the absence of osmotic-remedial strains among the suppressor sensitive mutants, and the polarized complementation behaviour of one suppressiblead ₆ mutant and two suppressiblead ₁ mutants capable of interallelic complementation, suggest that the suppression mechanism involves misreading of a mutant triplet of the nonsense type.     

Functional relationships between genes of the Shaker gene complex of Drosophila

Different mutations belonging to the HLI and HLII complementation groups of the haplolethal (HL) region of the Shaker complex (ShC) are described. The HLI complementation group includes viable (hdp), recessive lethals [l(1)1614], semidominant lethals [l(1)8384] and dominant lethals [l(1)5051,l(1)9916, l(1)13193], lack-of-function alleles that affect nervous system, cuticle and muscle development. The HLI complementation group encodes troponin I. HLII lack-of-function mutations [l(1)174 and l(l)4058] affect nervous system development. The semidominant lethal HLI mutation 1(1)8384 shows differential complementation with other mutations in the ME and HL regions of ShC. Thus, heterozygous combinations of l(1)8384 with ME mutations l(1)162 and l(1)387 are poorly viable. The same phenomenon is observed for heterozygotes of l(1)8384 with HL mutations l(1)1199, l(1)2288 and l(1)3014. These specific interactions indicate the existence of functional relationships among the genetic elements of ShC. The implications for the understanding of the functional organization of ShC are discussed.     

The Origin of Spontaneous Mutation in SACCHAROMYCES CEREVISIAE

Characterization of two antimutator loci in yeast shows that both are members of the same mutagenic repair system known to be responsible for almost all induced mutation (Lawrence and Christensen 1976, 1979a,b; Prakash 1976). One of the these newly isolated antimutator mutations is an allele of rev3 (Lemontt 1971b). Two other alleles of rev3 were tested and were also found to be antimutators. Double mutants carrying rev3 and mutator mutations of rad3, rad51 or rad18 are like rev3 single mutants with respect to spontaneous mutation rate, supporting the hypothesis (Hastings, Quah and von Borstel 1976) that many mutators in yeast act by channelling spontaneous lesions from accurate to mutagenic repair. However, the enhanced mutation rate seen in a radiation-resistant mutator mutant mut1 is not dependent on REV3, but is dependent on another gene designated ANT1. An additive effect on the reduction in spontaneous mutation, seen in the ant1 rev3 double-mutant strain, leads to the conclusion that at least 90% of spontaneous mutations seen in the wild type are caused by mutagenic repair of spontaneous lesions.     

Physiological and genetic characterisation of osmosensitive mutants of Saccharomyes cerevisiae

The screening of 20,000 Saccharomyces cerevisiae random mutants to identify genes involved in the osmotic stress response yielded 14 mutants whose growth was poor in the presence of elevated concentrations of NaCl and glucose. Most of the mutant strains were more sensitive to NaCl than to glucose at the equivalent water activity (a_w) and were classified as salt-sensitive rather than osmosensitive. These mutants fell into 11 genetic complementation groups and were designated osr1–osr11 (osmotic stress response). All mutations were recessive and showed a clear 2⁺ : 2^– segregation of the salt-stress phenotype upon tetrad analysis when crossed to a wild-type strain. The complementation groups osr1, osr5 and osr11 were allelic to the genes PBS2, GPD1 and KAR3, respectively. Whereas intracellular and extracellular levels of glycerol increased in the wild-type strains when exposed to NaCl, all mutants demonstrated some increase in extracellular glycerol production upon salt stress, but a number of the mutants showed little or no increase in intracellular glycerol concentrations. The mutants had levels of glycerol-3-phosphate dehydrogenase, an enzyme induced by osmotic stress, either lower than or similar to those of the parent wild-type strain in the absence of osmotic stress. In the presence of NaCl, the increase in glycerol-3-phosphate dehydrogenase activity in the mutants did not match that of the parent wild-type strain. None of the mutants had defective ATPases or were sensitive to heat stress. It is evident from this study and from others that a wide spectrum of genes is involved in the osmotic stress response in S. cerevisiae. Received: 5 January 1998 / Accepted: 24 March 1998     

Genetic mapping of the G101 bacteriophage (Pseudomonas aeuruginosa by temperature-sensitive mutations

Temperature-sensitive (ts) mutations of the G101 phage were isolated after mutagenesis with hydroxylamine. A complementation analysis of 61ts mutants showed that these mutants may be divided into at least 12 complementation groups. Twots mutants probably originated in genes which control lytic functions of the G101 phage. It was shown by three factor crosses that all of the 12ts mutations tested are localized on that side of the “c” region where the probablecI repressor gene is positioned. Sevents mutations is closely linked to thecI ₂₆ clear marker, three exhibit a closer linkage and two do not exhibit any linkage withcI. All mutations isolated until now can be arrange linearly. According to the present knowledge the preliminary genetic map of the G101 phage is linear.     

Evidence for joint genic control of spontaneous mutation and genetic recombination during mitosis in Saccharomyces.

Summary A procedure for detection of mutants exhibiting either enhanced or reduced spontaneous mutation during mitosis and/or meiosis has been developed to probe the joint genic control of spontaneous mutation and recombination in yeast. A semidominant mutator,rem1-1, recovered by this technique, exhibits enhanced spontaneous mutation, intragenic recombination, and intergenic recombination during mitosis. Diploids homozygous forrem1-1 exhibit normal levels of meiotic intragenic and intergenic recombination and diminished ascospore viability.     

A new gene (madI) involved in the phototropic response of Phycomyces

Summary Only eight genes are known to be involved in the phototropic response of Phycomyces (madA-H). Mutants affected in these genes have played a major role in the analysis of photosensory transduction processes in this system. A set of new mutants isolated by Alvarez et al. (1989) that are unable to bend towards dim unilateral blue light were studied by complementation and recombination. Two of these mutants have mutations in madE, one has a mutation in madF and one is a double madE madF mutant. The three remaining mutants tested did not complement each other and showed positive complementation with strains carrying mutations in the genes madA, madB, and madC, indicating that they carried mutations in a new gene designated madI. Recombination analysis showed that madI is unlinked to madA, madB and madC.     

Genetic analysis of sterile mutants in the dpy-5 unc-13 (I) genomic region of Caenorhabditis elegans

Essential genes were identified in the 1.5-map unit dpy-5 unc-13 region of chromosome I in the Caenorhabditis elegans genome by rescuing lethal mutations using the duplication sDp2. In this paper, we report the mapping and complementation testing of lethal mutations, 45 of which identify 18 new, essential genes. This analysis brings the number of essential genes defined by the sDp2 rescue of lethal mutants to 97; 64 of these map between dpy-5 and unc-13. 61% of these essential genes are identified by more than one allele. Positioning of the mutations was done using the breakpoints of six duplications. The mutant phenotypes of 14 loci essential for fertility were characterized by Nomarski microscopy and DAPI staining. None of the mutants were rescued by wild-type male sperm. The cytological data showed that four genes produced mutants with defects in gonadogenesis, let-395, let-603, let-605 and let-610. Mutations in seven genes, let-355, let-367, let-384, let-513, let-544, let-545 and let-606, affected germ cell proliferation or gametogenesis. Mutants for the remaining three genes, let-370, let-599 and let-604, produced eggs that failed to develop or hatch, thereby acting as maternal effect lethals. We observed a nonrandom distribution of arrest phenotypes with regard to map position. Received: 8 May 1996 / Accepted : 27 January 1997     

Genetic analysis of artificial pigmentation mutants in Porphyra yezoensis Ueda (Bangiales, Rhodophyta)

Porphyra yezoensis Ueda artificial pigmentation mutants, yel (green), fre (red‐orange) and bop (pink), obtained by treatment with /V‐methyl‐/V′‐nitro‐N‐nitrosoguanidine, were genetically analysed. The mutations associated with color phenotypes are recessive because all of the heterozygous conchocelis resembled the wild type color when they were crossed with the wild type (wt). In the reciprocal crosses of yel × wt, both parental colors and eight types of blades appeared in the F₁ gametophytic blades from the heterozygous conchocelis. Both colors segregated in the sectored F₁ blades in a 1:1 ratio, indicating that the color pheno‐type of yel resulted from a single mutation in the nuclear gene. In the reciprocal crosses of fre × wt, however, four colors and more than 40 types of blades appeared in the F₁ blades from the heterozygous conchocelis, indicating that the color phenotype of fre resulted from two mutations in different genes. In the reciprocal crosses of bop×wt, three colors and 12 types of blades were observed in the F₁ blades from the heterozygous conchocelis. Both parental colors appeared far more frequently than the third new color. These results indicated that the color phenotype of bop resulted from two closely linked mutations in different genes, and the epistasis occurred in the F₁ blades. The mutants, yel, fre and bop, differ from the spontaneous green (C‐O), the red (H‐25) and the violet (V‐O) mutants of P. yezoensis, respectively.     

Nuclear mutations in Saccharomyces cerevisiae which increase the spontaneous mutation frequency in mitochondrial DNA.

Summary Fourteen mutants have been identified in which the frequency of spontaneous mutations in mitochondrial DNA is increased. As well as increasing the frequency of mutations to resistance to erythromycin, oligomycin and spiramycin, all the mutants also show changes in the frequency of spontaneous petite induction. None of the mutants has any effect on the frequency of spontaneous nuclear mutations. Nine of the mutants are in one complementation group and five are in another. The phenotype of both groups is caused by a single nuclear mutation.     

Sometimes the result is not the answer: the truths and the lies that come from using the complementation test

It is standard genetic practice to determine whether or not two independently obtained mutants define the same or different genes by performing the complementation test. While the complementation test is highly effective and accurate in most cases, there are a number of instances in which the complementation test provides misleading answers, either as a result of the failure of two mutations that are located in different genes to complement each other or by exhibiting complementation between two mutations that lie within the same gene. We are primarily concerned here with those cases in which two mutations lie in different genes, but nonetheless fail to complement each other. This phenomenon is often referred to as second-site noncomplementation (SSNC). The discovery of SSNC led to a large number of screens designed to search for genes that encode interacting proteins. However, screens for dominant enhancer mutations of semidominant alleles of a given gene have proved far more effective at identifying interacting genes whose products interact physically or functionally with the initial gene of interest than have SSNC-based screens.     

Genetic characterization of the 44D-45B region of the Drosophila melanogaster genome based on an F2 lethal screen

We have performed an F₂ genetic screen to identify lethal mutations that map to the 44D-45B region of the Drosophila melanogaster genome. By screening 8500 mutagenized chromosomes for lethality over Df(2R)Np3, a deficiency which encompasses nearly 1% of the D. melanogaster euchromatic genome, we recovered 125 lines with lethal mutations that represent 38 complementation groups. The lethal mutations have been mapped to deficiencies that span the 44D-45B region, producing an approximate map position for each complementation group. Lethal mutations were analyzed to determine the phase of development at which lethality occurred. In addition, we have linked some of the complementation groups to P element-induced lethals that map to 44D-45B, thus possibly providing new alleles of a previously tagged gene. Some of the complementation groups represent potentially novel alleles of previously identified genes that map to the region. Several genes have been mapped by molecular means to the 44D-45B region, but do not have any reported mutant alleles. This screen may have uncovered mutant alleles of these genes. The results of complementation tests with previously identified genes in 44D-45B suggests that over half of the complementation groups identified in this screen may be novel. Received: 13 July 1999 / Accepted: 4 November 1999     

What is a bona fide mating-type gene? Internuclear complementation of mat mutants in Podospora anserina

In the heterothallic ascomycete Podospora anserina, the mating-type locus is occupied by two mutually exclusive sequences termed mat+ and mat–. The mat+ sequence contains only one gene, FPR1, while the mat– sequence contains three genes: FMR1, SMR1 and SMR2. Previous studies have demonstrated that FPR1 and FMR1 are required for fertilization. Further analyses have led to the hypothesis that mat+ and mat– genes establish a mat+ and mat– nuclear identity, allowing recognition between nuclei of opposite mating type within the syncytial cells formed after fertilization. This hypothesis was based on the phenotypes of strains bearing mutations in ectopic mat genes. Here we present an analysis of mutations in resident mat– genes which suggests that, unlike FMR1 and SMR2, SMR1 is not involved in establishing nuclear identity. In fact, mutations in these two genes impair nuclear recognition, leading to uniparental progeny, while mutations in SMR1 block the sexual process, probably at a step after nuclear recognition. The nuclear identity hypothesis has also been tested through internuclear complementation tests. In these experiments, the mat– mutants were crossed with a mat+ strain carrying the wild-type mat– genes. Our rationale was that internuclear complementation should not be possible for nuclear identity genes: the relevant genes should show nucleus-restricted expression, and diffusion of their products to other nuclei should not occur. This test confirmed that SMR1 is not a bona fide mat gene since it can fulfill its function whatever its location, in either a mat− or a mat+ nucleus, and even when present in both nuclei. SMR2, but not FMR1, behaves like a nuclear identity gene with respect to internuclear complementation tests. A model is proposed that tentatively explains the ambiguous behaviour of the FMR1 gene and clarifies the respective functions of the three mat– proteins. Received: 15 October 1996 / Accepted: 25 April 1997     

Mutagenic activities of aflatoxin B 1 and G 1 in Neurospora crassa

Summary The mutagenicity and mutagenic specificity of aflatoxin B₁ and G₁ were studied with the adenine-3 (ad-3) test system of Neurospora crassa. Aflatoxin B₁ and G₁ failed to show mutagenicity in resting conidia, but both agents were mutagenic in growing vegetative cultures. The frequencies of ad-3 mutants induced by aflatoxin B₁ and G₁ (40g/ml) were 70.7x10^-6 survivors and 9.6x10^-6 survivors, respectively. Since aflatoxin B₁ gave a 177-fold increase over the spontaneous mutation frequency it is a rather potent mutagen, whereas aflatoxin G₁ gave only a 24-fold increase and so is only moderately mutagenic.Genetic analyses of ad-3 mutants induced by aflatoxin B₁ and G₁ indicate that both agents induce a low frequency of multilocus deletions. The spectra of point mutations at the ad-3A and ad-3B loci induced by aflatoxin B₁ and G₁ are not distinguishable from each other. Hence both agents probably induce the same relative frequencies of genetic alterations. The frequencies of leakiness, allelic complementation, and classes of complementation patterns among the ad-3 mutants induced by both agents are higher than the frequencies among ICR-170-induced mutants and somewhat lower than those among NA- and AP-induced mutants. The results of reversion tests with NA, MNNG, and ICR-170 indicate that in addition to multilocus deletion, aflatoxin B₁-induced ad-3 mutants consist of frameshifts, base-pair transitions, and possibly other types of intragenic alterations.     

A conditional antimutator in E. coli.

Summary A pleiotropic mutation in thepurB gene ofE. coli is described which lowers the spontaneous mutation frequency of other genes. The antimutator effect is very large for some genetic loci, but is absent at other sites. Both forward and reverse mutations are affected. This mutation inpurB is temperature sensitive for both adenine auxotrophy and the antimutator action. Adenine, or adenosine, or low temperature growth abolish the antimutator effect. The mutagenicity of base analogs and nitrosoguanidine at several loci was found to be reduced by thispurB mutation. The antimutator effect is recessive in strains merodiploid for thepurB region. The frequency of reversion of mutation on F episomes is affected by the chromosomal antimutator, which therefore acts in trans. Xray and UV sensitivity are normal in this mutant, which is the first antimutator characterized inE. coli.     

Isolation of synthetic lethal mutations in the rsm1-null mutant of fission yeast

To identify mutations in genes that are genetically linked to rsm1, we performed a synthetic lethal genetic screen in the fission yeast, Schizosaccharomyces pombe. Four mutations that showed synthetic lethality in combination with the rsm1null allele were isolated from approximately 320,000 colonies and defined in three complementation groups. One mutant (SLrsm1) exhibited a significant accumulation of poly(A)⁺ RNA in the nucleus under synthetic lethal conditions, while the rest had no mRNA export defects. In addition, some genes (spmex67, rae1, or mlo3) required for mRNA export complemented the growth defects of the identified mutants. These results suggest that the isolated mutants contain mutations in genes that are involved in mRNA export and/or pre-mRNA retention.     

Genetic fine structure and complementation at the albino locus in spider mites (Tetranychus species: Acarina)

Two mutations inTetranychus urticae and nine inTetranychus pacificus, all originating spontaneously, block the production of red and yellow carotenoid pigments in these spider mite species. Inter-mutant crosses were carried out to study complementation and recombination relationships between the mutations. InT. urticae, the two albino mutants complement one another completely, i.e., crosses between them produce wild-type hybrid females; while they recombine with a frequency of 0.05%. Of the nine mutants inT. pacificus, fivep mutants in general are complementary to a high degree with foura mutants.p mutants fail to complement one another, while somea mutants are mutually complementary to a slight degree. Scoring the degree of complementation produced by all possible combinations of mutants permits the construction of a linear complementation map. Certain combinations, however, are exceptional to such a representation. Moreover, marked reciprocal differences in complementation indicate that maternal effects are involved, implying that the albino locus may control more than one enzymatic step. Attempts to derive a genetic map were impeded by the absence of suitable linked markers, by a pronounced maternal effect (high pigmentation) in the haploid F₂ males, and by the appearance of pseudowild type F₂ males. The given genetic sequence, although comparable in a limited fashion to the complementation map, is considered tentative. Pink types appeared in crosses with certainp mutants. These were due to mutation at a separate locus, called rose, and seem to involve the production of pink pigments in an alternative or substitute pathway. A scheme attempting to orientate the present state of understanding of pigmentation in spider mites is presented.This work forms part of a thesis submitted in fulfillment of the requirements for the Ph. D. degree at the University of Amsterdam (1969).     

Genetic and physiological analysis of UV-sensitive mutants of Saccharomyces cerevisiae

The use of tetrad analysis and complementation tests indicates that the groups of UV-sensitive mutants assigned the labels radI and rad3 are alleles of two single genes involved in the process of cellular repair of UV-induced damage in the yeast Saccharomyces cerevisiae.