An approach to identify functional homologues and suppressors of genes in fission yeast

We have developed a procedure using a bank of temperature-sensitive (ts) mutants of fission yeast to identify mutants which can be rescued by expression of a plasmid-borne gene of interest. The procedure has been used to identify new ts alleles of cdc2 and swi7/pol1, a ts mutant rescued by actin, and to identify a ts allele of cdc11 which can be rescued by combined mammalian Myc and Max expression. The procedure should also be useful as an alternative approach to identify genes in fission yeast which are functionally homologous to genes of interest from other organisms. Received: 28 February 1997     

Simian virus 40 mutants with amino-acid substitutions near the amino terminus of large T antigen

A series of amino-acid substitution mutants has been made with changes in the region of simian virus 40 large tumor antigen (T antigen) that is shared with the small tumor antigen (t antigen). Both single and multiple amino-acid replacements were obtained using the heteroduplex deletion loop method and sodium bisulfite as the mutagen. The mutants could be divided into five phenotypic classes on the basis of their biological properties: a) mutants whose changes did not affect their ability to propagate on permissive monkey cells, nor to transform nonpermissive rodent cells; b) mutants that were not viable, replicated their DNA to 5% or less of wild type, but were positive for transformation; c) mutants that were not viable, replicated their DNA to 5% or less of wild type, and were defective for transformation; and d) mutants that completely lost all three activities coordinately. In addition, one mutant with changes in this region, 5002, replicated its DNA to about 50% of wild type, had an impaired transformation activity, and produced virions at a level of about 4% that of wild type.     

Ofloxacin induces cytoplasmic respiration-deficient mutants in yeast Saccharomyces cerevisiae

Summary Ofloxacin, a new quinolone with potent antibacterial activity, was also found to be effective against yeast. At relatively high concentrations, and at mild alkaline pH, ofloxacin inhibited the growth of yeast cells in medium containing glucose, and prevented growth on glyccrol, as carbon and energy source. The cells growing in the presence of ofloxacin exhibited abberrantly budded forms, lost their viability and many of them converted to cytoplasmic respiration-deficient mutants. Induction of mutants was also observed under non-growing conditions. The petite clones analysed exhibited suppressiveness and contained different fragments of the wild-type mitochondrial genome.     

The mitochondrial genome of the fission yeast Schizosaccharomyces pombe

Summary The three mutator strains ana ^r-8, ana ^r-14, and diu ^r-301 were shown to produce respiratory deficient mutants at different rates. The frequency of respiratory deficient mutants in a culture could be increased by adding ethidium bromide. According to their cytochrome spectra and enzymatic activities they form three classes, namely mutants defective in cytochrome oxidase, in cytochrome b, and in both cytochromes. By restriction enzyme analysis of mitochondrial DNA from about 100 mutants, 22 deletion mutants were identified. The deletions, ranging from 50 to 1,500 base pairs were physically mapped. Deletions were localized in the genes coding for subunit 1 of cytochrome oxidase with its two introns, within the cytochrome b gene and its intron, and within the genes for subunits 2 and 3 of cytochrome oxidase. In several cases, where the physical mapping yielded ambiguous results, pairwise genetic crosses ruled out an overlap between two neighbouring deletions.Using these mitochondrial deletion mutants as tester strains, it was shown that only tetrad analysis and chemical haploidization, but not mitotic segregation analysis, allows a decision between chromosomal and mitochondrial inheritance of respiratory deficiency in Schizosaccharomyces pombe. Abbreviations. MtDNA = mitochondrial DNA; S. pombe = Schizosaccharomyces pombe; cox1, cox2, and cox3 refer to the mt genes coding for the three subunits of cytochrome oxidase; ATPase 6 (oli2), ATPase 8 (aapl in Saccharomyces cerevisiae, urf a61 in HeLa) and ATPase 9 (olil) refer to the three respective subunits of ATP synthase; cob is thegene for apocytochrome b; urf a is the single intergenic unassigned reading frame in S. pombe; 1 rRNA and s rRNA refer to the large and small ribosomal RNA, respectively. Mut^– is a cytoplasmic mutator (the corresponding wild type allele is mut⁺). Mit^– are mitochondrially inherited respiratory deficient mutants with mitochondrial protein synthesis; RC = respiratory competent, RD = respiratory deficient.     

The organization of repeating units in mitochondrial DNA from yeast petite mutants

Summary We have reinvestigated the linkage orientation of repeating units in mtDNAs of yeast ^– petite mutants containing an inverted duplication. All five petite mtDNAs studied contain a continuous segment of wild-type mtDNA, part of which is duplicated and present in inverted form in the repeat. We show by restriction enzyme analysis that the non-duplicated segments between the inverted duplications are present in random orientation in all five petite mtDNAs. There is no segregation of sub-types with unique orientation. We attribute this to the high rate of intramolecular recombination between the inverted duplications. The results provide additional evidence for the high rate of recombination of yeast mtDNA even in haploid ^– petite cells.We conclude that only two types of stable sequence organization exist in petite mtDNA: petites without an inverted duplication have repeats linked in straight head-to-tail arrangement (abcabc); petites with an inverted duplication have repeats in which the non-duplicated segments are present in random orientation.     

Isolation and characterization of yeast mutants with thermoconditional sensitivity to the bifunctional alkylating agent nitrogen mustard

Summary Selection of mutants of Saccharomyces cerevisiae sensitive to the DNA cross-linking agent nitrogen mustard (HN2) at two temperatures (23 °C and 36 °C) yielded two isolates with thermoconditionally enhanced (ts) sensitivity to the mutagen. Both were due to single recessive nuclear genes. Mutant allele snm1–2 ^ts showed mainly ts-sensitivity to HN2, whereas mutant allele snm2-1 ^ts conferred ts-sensitivity to HN2, half mustard (HN1) and UV. In temperature-shift experiments it was determined that the functions of SNM1 and SNM2 are needed for recovery within 6 to 7 h. after mutagen exposure during incubation at 23 °C on YEPD when HN2 and UV are applied. After HN1 treatment the SNM2 coded function is required for recovery for about 14 hrs. This possibly indicates a handling of UV- and HN2-induced lesions different from that of HN1-induced lesions.     

Trans-acting factors and properly positioned DNA elements repress mating-type genes in fission yeast

Summary Repression of the mating-type P genes at the silent mat2-P locus in fission yeast is dependent on four cis-acting DNA elements, two on each side of the coding sequences. The mechanism by which these elements exert their influence on the mating-type promoter is studied here by insertion of a bacterial antibiotic resistance gene at several positions in the silent region. The behavior of the resistance gene itself, and the changes its insertion causes in mating-type expression, reveal that the repressive elements have a limited range of action and that the four elements have unequal effects on gene expression. Repression of the antibiotic resistance gene inside the silent region leads to an antibiotic-sensitive phenotype and facilitates the selection of resistant mutants. These mutants can de-repress the resistance gene at other positions than the one used for their selection. Strong antibiotic resistance correlates with derepression of the plasmid-borne mating-type cassette. These data argue that mat2-P repression is dependent on trans-acting factors and the positioning of the repressive DNA elements, but less dependent on the nature of the affected promoter.     

Schistosoma mansoni: effect of maintenance in vitro on the uptake and incorporation of leucine by adult worms

The rates of leucine uptake and incorporation into protein by adult male and female Schistosoma mansoni were not affected by maintenance in vitro for up to 10 days' duration, despite the decline in the protein content of male and female worms of approximately 35 and 55%, respectively, during this period. The effect of maintenance in vitro was obscured in paired female schistosomes by the apparent shielding of the female tegument within the gynaecophoral canal of the male. Incorporation rates were reduced by 50% in the presence of 2 mM cycloheximide whereas uptake rates were unaffected. Adult schistosomes are unable to maintain their in vivo protein levels purely by recourse to exogenous amino acids absorbed across the tegument in vitro, and the rates of uptake and incorporation of leucine appear to reflect the changing somatic requirements of the worms and are probably not correlated with the reproductive activity of adult worms in vitro. The possible role of alimentary rather than tegumental nutrition in egg production in vivo is discussed.     

Isolation and characterization of xyl mutants in a xylose-utilizing yeast,Pichia stipitis

Summary Mutants of the xylose-utilizing yeast, Pichia stipitis, unable to grow on xylose as the sole carbon source were isolated and characterized. The mutants were deficient in either xylose reductase or xylitol dehydrogenase. By immunological means and upon analysis of revertants, both mutant types could be attributed to the structural genes XYL1 and XYL2, which code for xylose reductase and xylitol dehydrogenase, respectively. These data support previous assumptions that both NADH- and NADPH-dependent xylose reductase activity are due to one protein or gene, respectively. Revertant analysis of xyl1 mutants has revealed the existence of a second xylose reductase gene in P. stipitis. This gene is very likely cryptic. Its corresponding xylose reductase activity is NADPH-dependent.     

Protein secretion in yeast: Two chromosomal mutants that oversecrete killer toxin in Saccharomyces cerevisiae

Summary Two chromosomal mutations in yeast that result in oversecretion of the K₁ killer toxin protein were examined. A recessive mutation in gene ski5 appears to lead to toxin oversecretion through a defect in a cell surface, PMSF-inhibited protease. A wild type killer strain degraded toxin following synthesis, and degradation could be partially prevented by addition of PMSF to the growth medium. The ski5 mutation caused an approximate ten fold oversecretion of toxin, similar to that seen in a PMSF-treated wild type culture, and no increased oversecretion in the presence of PMSF. The ski5 mutation caused oversecretion of other low molecular weight secreted proteins and appeared to oversecrete the -factor pheromone, as judged by activity tests. The ski5 mutation was complemented by mutations in ski genes 1–4, and the mutant was not supersensitive to mating pheromones or K₂ killer toxin.We also examined killer strains with a mutation in the nuclear gene krel which results in a defective (16)--D-glucan cell wall receptor for killer toxin. Such strains oversecrete toxin into the growth medium, but also, unexpectedly, oversecrete most other secreted proteins. The defect in (16)--D-glucan in these mutants appears to perturb the partitioning of secreted proteins between the cell wall and the medium.     

Lack of correlation between mms-toxicity in larvae and in adults of mutagen-sensitive mutants of drosophila melanogaster

Adult males of 20 different stocks of Drosophila melanogaster, including 16 with various X-linked mutagen-sensitive mutations, were tested for sensitivity to the lethal action of methyl methane sulfonate (MMS) in a continuous feeding experiment. It was impossible to establish a correlation between MMS-toxicity in adults and MMS-toxicity in larvae. In addition, experiments to examine the fertility of MMS-treated flies of four mutagen-sensitive strains and one wild-type strain were performed. These experiments demonstrated that the spermatogonia of mei-9^L1 and mei-41^D5 in larvae and in adults are more sensitive to MMS than the spermatogonia of the wild-type strain or the other two mutagen-sensitive strains.     

Use of reporter genes for the isolation and characterisation of different classes of sporulation mutants in the yeast Saccharomyces cerevisiae

Reporter genes consisting of sporulation-specific promoters fused to lacZ were used as markers to monitor the sporulation pathway of the yeast Saccharomyces cerevisiae. Strains transformed with these lacZ gene fusions expressed -galactosidase (assayable on plates using the substrate 5-bromo-4-chloro-3-indolyl--D-galactopyranoside, X-gal) in a sporulation-dependent manner. Mutagenesis experiments performed on transformed strains resulted in the recovery of a number of novel sporulation mutants. Three classes of mutants were obtained: those which overexpressed the reporter gene under sporulation conditions, those which did not express the gene under any conditions, and those which expressed the gene in vegetative cells not undergoing sporulation. On the basis of the blue colony-colour produced in the presence of X-gal these have been described as superblue, white, and blue vegetative mutants, respectively. These were further characterised using earlier reporter genes and other marker systems. This study established that the multicopy reporter plasmids chosen do not interfere with sporulation; they are valid tools for monitoring the pathway and they provide a way to isolate mutations not readily selected by other markers.     

Characterization of blasticidin S — resistant mutants of Saccharomyces cerevisiae

Summary Blasticidin S-resistant mutants of S. cerevisiae were isolated and characterized. Resistant mutations were found to fall into two complementation groups. A single recessive nuclear gene was responsible for each group, donated as bls1 and bls2, respectively. A gene bls1 was linked to an ilv3 gene located on the right arm of chromosome X. The resistant phenotypes from both genes were not associated with ribosomes known to be target sites of Blasticidin S, when analyzed by poly(U)-directed polyphenylalanine synthesis. The resistant mechanisms of the mutations are discussed in this paper.     

Six new amino acid-auxotrophic markers for targeted gene integration and disruption in fission yeast

Fission yeast Schizosaccharomyces pombe is amenable to genetics and is an excellent model system for studying eukaryotic cell biology. However, auxotrophic markers that can be used for both targeted gene integration and disruption are very limited. Here we performed a forward genetic screen in an effort to develop a new set of selectable markers for use in this yeast. Mutants that were auxotrophic for arginine, asparagine, cysteine, lysine, methionine and phenylalanine were isolated. Six genes were analyzed in detail and the mutations in the genes were identified. Among these six are three new genes: asn1 ⁺, cys2 ⁺ and pha2 ⁺ were required for biosynthesis of asparagine, cysteine and phenylalanine, respectively. New alleles of arg1 ⁺, lys3 ⁺ and met6 ⁺ were also identified. All of these genes proved to be suitable as selectable markers for targeted gene integration and disruption. We also showed that in Schizosaccharomyces pombe there are two apparent homologues of Saccharomyces cerevisiae MET2: the previously known met6 ⁺, and SPBC106.17c (named cys2 ⁺). The cys2 mutation required cysteine rather than methionine. These new tools, specifically, new selectable markers, will be useful in further genetic and biological studies in fission yeast.     

Characterization of cytoplasmic mutants of Nicotiana tabacum with altered photosynthetic function

Summary A series of cytoplasmic mutants of tobacco (Nicotiana tabacum) were generated and characterized. Compared to wild type tobacco, they were found to have diminished levels of photosynthetic pigments and a range of functional impairments including modified chlorophyll fluorescence properties, loss of Photosystem I and/or II electron transport activity, and aberrant ultrastructure. Although the loss of defined functional activities was correlated with the depletion of specific thylakoid membrane proteins, no simple rules governed the relationship between structural defects and photosynthetic deficiencies. All of these mutants exhibited pleiotropic losses of polypeptides, including those known to be nuclear-encoded; this is consistent with the concept that loss of one component of a multi-subunit membrane protein complex results in unstable complex assembly. The phenotype of two mutants was developmentally regulated, in one case with slow chloroplast developments and in the other by premature senescence of Photosystem II centers as a function of leaf development. These mutants should be especially useful in studying membrane protein assembly.Abbreviations CF₁ coupling factor (chloroplast ATPase) - Chl chlorophyll - CP chlorophyll protein - Diuron 3-(3,4-dichlorophenyl)-1,1-dimethyl urea - DPIP 2,6-dichlorophenolindolphenol - EDTA ethylenediaminetetraacetic acid - F₀ initial fluorescence - F_p peak fluorescence - F_t terminal fluorescence - F_v variable fluorescence - kDa kilodalton(s) - LDS lithium dodecyl sulfate - LHC light harvesting complex - MV methyl viologen - OEC oxygen evolving complex - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonylfluoride - PS photosystem - Q_A the primary quinone acceptor of Photosystem II - RC reaction center - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - TMQH₂ tetramethyl-p-benzohydroquinone - TP tobacco plastome - Tricine N-tris (hydroxymethyl) methylglycine - WT wild type     

An efficient method for the isolation of interaction‐null/impaired mutants using the yeast two‐hybrid technique

Protein–protein interactions are one of the most basic and critical processes underlying biological functions. Thus, identification of the interacting proteins of a protein of interest and further elucidation of the roles of the interactions is critical for understanding the related biological processes. The yeast two‐hybrid (Y2H) method is a popular approach for identifying protein–protein interactions. Once interacting proteins are identified, a comparison of the phenotypes of mutants lacking the specific protein–protein interaction with those of the wild‐type strain is a powerful tool for uncovering the former interactions’ biological significance. However, isolation of such interaction‐defective mutants is often laborious. Here, I describe a novel and efficient approach for isolating such mutants that uses the Y2H technique with a modified Y2H vector, and provide an example of how this approach can be used to screen interaction‐null/impaired mutants. Because the strategy is simple and the modification of a pre‐existing Y2H vector is sufficient for the screening purpose, the same strategy can be applied to any existing two‐hybrid system.     

Phenotypes of major immediate-early gene mutants of mouse cytomegalovirus

Immediate-early (IE) genes are the first genes to be transcribed during the lytic replication cycle of cytomegaloviruses (CMV), and encode nonstructural proteins, which are assumed to have mainly regulatory functions. The IE proteins may play important roles in the pathogenesis of CMV in vivo, for instance during the establishment of latency and during reactivation. We constructed mouse CMV mutants with disruptions in the major IE genes, ie1 and ie3, to study the roles of these genes in the context of the viral infection. Here we summarize the current results on the characterization of these mutants and give a perspective of the future research in this field.     

Glucosamine-resistant mutations in yeast affecting the glucose repression sensitivity of electron transport enzymes

Summary We have isolated two mutant strains, GSA^r-4 and GSA^r-5, which are able to grow on lactate in the presence of D-glucosamine. The glucosamine-resistant phenotype results from the cooperative effects of mutations in three loci, GAR1, GAR2 and GAR3. Both glucosamine resistant mutant strains were doubly mutant at gar1 gar2 (GSA^r-4) or gar1 gar3 (GSA^r-5). The mutants were also shown to exhibit glucose repression insensitive synthesis of NADH-cytochrome c reductase and cytochrome c oxidase. Glucose-repressible synthesis of the following enzymes was seen: succinic dehydrogenase, succinic: cytochrome c reductase, maltase (PNPGase), galactokinase, -galactokinase. The glucose-repression insensitivity segregates in association with the glucosamine resistance.     

Benomyl resistant mutants of Schizosaccharomyces pombe cold-sensitive for mitosis

Summary We have isolated 150 benomyl resistant mutants of the fission yeast Schizosaccharomyces pombe. Seven of these mutants were found to be cold sensitive for mitosis. These mutants were the subject of physiological, cytological and genetical characterisation. Growth and division of the seven mutants were similar to the wild type strain at 35 °C. After shift from the permissive (35 °C) to the restrictive temperature (20 °C) the mutants became blocked in mitosis whilst cellular growth continued. Consequently, elongate cells were formed. Six of the seven benomyl resistant mutants became blocked in mitosis at 20 °C with a single aberrant nucleus. In every case the benomyl resistant and cold sensitive phenotype was due to a mutation in a single nuclear gene. These mutants were found to comprise a single genetic linkage group (ben4) and were unlinked to existing TBZ/MBC resistant mutants of S. pombe. Whilst no cross resistance was found in our mutants to TBZ, six of the seven mutants were super sensitive to the spindle poison CIPC. We believe that the phenotype exhibited by these mutants is consistent with a defective tubulin subunit.     

Detection of hepatitis B virus core mutants by PCR-RFLP in chronically infected patients

HBV chronic infection is an important health problem. The HBV core antigen carries several epitopes for T and B cell recognition and the immune response is crucial for determining the outcome of viral infection. Using PCR-RFLP several point mutations were detected in the HBV core ORF of HBV extracted from the serum of 140 chronically infected patients and 86 samples from another 37 patients followed-up in a longitudinal study. Mutations at position 2248 and 2147 (A3) and at 2038 (M2) were found most frequently. The wild type core genotype was found in about 50% of the samples. PCR-RFLP results were confirmed by direct sequencing of amplified products from HBV DNA present in chronically infected patients. The method is rapid and reliable and may be particularly useful for a rapid detection of viral mutants in a large number of patients.