Isolation, characterization, and genetic analysis of monosomic, aneuploid mutants of Candida albicans

A white, prototrophic Candida albicans strain, heterozygous for the ADE2 gene (ade2/ADE2), was treated with the antimitotic agent methyl benzimidazole carbamate, and yielded red, adenine-requiring colonies at a rate of 4 x 10(-3), an order of magnitude higher than the spontaneous rate of Ade- colony formation. These red Ade- colonies were small, growing at approximately half the rate of the parent strain, and gave rise to large red colonies spontaneously. When the chromosomes of the small red colonies were separated by pulsed-field gel electrophoresis, the band hybridizing with the ADE2 gene was diminished in staining intensity by half relative to the parent and large red-colony strains. Restriction fragment-length polymorphism analysis and auxotrophic mutant spectra after mutagenesis suggested that the small red Ade- strains were monosomic aneuploids lacking one of a pair of chromosome homologues, while the large red strains had regained a homologue, presumably via a second non-disjunction event. Parasexual genetic analysis of two of the auxotrophs isolated from a putative aneuploid suggested that both mutations were linked to the ADE2 gene. These experiments suggest that targeted chromosome loss and monosomic, aneuploid strains have the potential to extend the scope of genetic analysis in this diploid, asexual organism.     

Use of Streptomyces sp. 26-115 protoplasts inactivated by heating in fusion experiments

When heated at 55 degrees C for 30 or 60 minutes protoplasts of auxotrophic mutants of Streptomyces sp. 26-115 producer of actinomycin C (active and inactive variants) lost their capacity for regeneration. The protoplasts heated at at 55 degrees C for 30 minutes and not for 60 minutes maintained some ability to yield recombinants on fusion under the effect of PEG 6000. Unlike the parent active strain, the colonies formed by the spores of the prototrophs yielding on fusion of the intact protoplasts showed wide ranges of antibiotic activity against M. flavus while a significant part of the colonies was inactive. The use of the inactive variant protoplasts heated at 55 degrees C for 30 minutes in the fusion procedure increased the proportion of the inactive variants.     

Detection of the loss of 1 chromosome from pair III in Saccharomyces cerevisiae yeasts

A diploid strain of Saccharomyces cerevisiae, T6 is described which monitors both mitotic crossing over and induction of aneuploidy. The chromosome III carries recessive markers: rgh12 of "rough colony" phenotype closely linked to centromere, the left arm is marked with his4, the right arm is marked both with thr4 and the locus of mating type alpha. Expression of all the markers on chromosome III leads to formation of colonies which are rough, require histidine and threonine, and are of alpha mating type. These colonies arise as a result of the loss of a chromosome during mitosis, which makes the strain allow detection of monosomic cells formation. Chromosome XV carries two phenotypically distinguishable and recessive alleles of the gene ade2: ade2-192 (causes red coloration of colonies) and ade2-G45 (causes pink coloration of colonies). Mitotic crossing over generates two reciprocal products which can be revealed together in colonies as pink and red sectors in double-spotted colonies. Both double-spotted and monosomic colonies have been obtained after treatment with gamma-rays. The frequency of mitotic crossing over after irradiation by 1000-3000 Gray increased up to 2-3.2% (the spontaneous level was 0.006%), the frequency of aneuploidy was 0.12 to 0.57% at plating immediately after irradiation (the spontaneous monosomics were not observed among 1.5 X 10(5) cells scored). Induction of mitotic crossing over and aneuploidy by benomyl was rather slight (up to 0.05 and 0.006%, respectively).     

Rugosity in Grimontia hollisae

Grimontia hollisae, formerly Vibrio hollisae, produces both smooth and rugose colonial variants. The rugose colony phenotype is characterized by wrinkled colonies producing copious amounts of exopolysaccharide. Cells from a rugose colony grown at 30 degrees C form rugose colonies, while the same cells grown at 37 degrees C form smooth colonies, which are characterized by a nonwrinkled, uncrannied appearance. Stress response studies revealed that after exposure to bleach for 30 min, rugose survivors outnumbered smooth survivors. Light scatter information obtained by flow cytometry indicated that rugose cells clumped into clusters of three or more cells (average, five cells) and formed two major clusters, while smooth cells formed only one cluster of single cells or doublets. Fluorescent lectin-binding flow cytometry studies revealed that the percentages of rugose cells that bound either wheat germ agglutinin (WGA) or Galanthus nivalis lectin (GNL) were greater than the percentages of smooth cells that bound the same lectins (WGA, 35% versus 3.5%; GNL, 67% versus 0.21%). These results indicate that the rugose exopolysaccharide consists partially of N-acetylglucosamine and mannose. Rugose colonies produced significantly more biofilm material than did smooth colonies, and rugose colonies grown at 30 degrees C produced more biofilm material than rugose colonies grown at 37 degrees C. Ultrastructurally, rugose colonies show regional cellular differentiation, with apical and lateral colonial regions containing cells embedded in a matrix stained by Alcian Blue. The cells touching the agar surface are packed tightly together in a palisade-like manner. The central region of the colony contains irregularly arranged, fluid-filled spaces and loosely packed chains or arrays of coccoid and vibrioid cells. Smooth colonies, in contrast, are flattened, composed of vibrioid cells, and lack distinct regional cellular differences. Results from suckling mouse studies showed that both orally fed rugose and smooth variants elicited significant, but similar, amounts of fluid accumulated in the stomach and intestines. These observations comprise the first report of expression and characterization of rugosity by G. hollisae and raise the possibility that expression of rugose exopolysaccharide in this organism is regulated at least in part by growth temperature.     

Isolation of heat- and cold-sensitive mutants of chinese hamster lung cells affected in their ability to express the transformed state.

A clone of spontaneously transformed Chinese hamster lung cells was exposed to N-methyl-N'-nitro-N-nitroso-guanidine (MNNG), and six heat-sensitive and three cold-sensitive mutants were isolated after selection for inability to form colonies in soft agar at 39.5 degrees C and 34.5 degrees C, respectively. The heat-sensitive mutants had growth characteristics of transformed cells at 34.5 degrees C, but exhibited a normal phenotype at 39.5 degrees C. By contrast, cold-sensitive mutants displayed the characteristics of the normal cells at 34.5 degrees C and converted to a transformed phenotype at 39.5 degrees C. Transformed parent cells exhibited no obvious temperature-dependent properties. Temperature shift experiments showed that the colony-forming ability of both types of mutants was fully reversible. All of the mutants were able to grow well at both permissive and nonpermissive temperatures when grown on the surface of plastic dishes. Such mutants will be useful in analysis of factors involved in the expression of the transformed state or the maintenance of the nontransformed state.     

Antimutagenic effects of chemicals on mutagenesis resulting from excision of a transposon in Salmonella typhimurium

We have found that a temperature-sensitive mutation in the polA gene of Salmonella typhimurium strain LT2 causes precise excision of transposon Tn10 to occur at significantly increased frequencies in cells incubated at the restrictive temperature. In our experiments, precise excision from a site in the tryptophan operon was measured by determining the frequency of reversion of the auxotrophic trp1014::Tn10 polA7 strain to prototrophy on defined medium containing a trace amount of broth. Because the yields of revertants at 37 degrees C were of the order of 200 colonies per plate, it was possible to measure the effects of chemical inhibitors on the processes involved in precise excision. We now report that all of the DNA-repair inhibitors we have studied (caffeine, ethionine, acriflavine, procaine and cinnamaldehyde) are effective inhibitors of precise excision of Tn10, and can therefore be defined as antimutagens.     

Transfer of isolated nuclei into protoplasts ofSaccharomyces cerevisiae

Cell nuclei were prepared from protoplasts of an adenine-requiring strain ofSaccharomyces cerevisiae, then purified in a discontinuous sucrose gradient, and applied to protoplasts of a recipient strain auxotrophic for uracil, leucine, and histidine. The transfer of the isolated nuclei into protoplasts was induced with polyethylene glycol. The main products of nuclear transfer in young complemented colonies were heterokaryons giving rise to parental type spontaneuos segregants on nutritionally complete medium. After several passages in minimal medium, however, the prototrophic colonies consisted exclusively of stable heterozygous diploid cells.     

Protein Arp and protein H from group A streptococci. Ig binding and dimerization are regulated by temperature.

Cell surface proteins that bind to the Fc part of Ig are expressed by many strains of group A streptococci, an important human pathogen. Two such bacterial strains, AP4 and AP1, were shown to bind IgA and IgG, respectively, in a temperature-dependent manner. The binding of radiolabeled Ig to the bacterial cells was lower at 37 degrees C than at 22 and 4 degrees C. Similarly, protein Arp, the IgA-binding protein isolated from strain AP4, and protein H, the IgG-binding protein isolated from strain AP1, displayed a strong Ig-binding at 22 degrees C and lower temperatures, and virtually no binding at all at 37 degrees C. The effect was reversible: lowering of the temperature restored the binding and vice versa. A gradual shift between binding and nonbinding took place between 27 and 37 degrees C. Gel chromatography and velocity sedimentation centrifugation showed that protein Arp and protein H appeared as noncovalently associated dimers at 10 and 22 degrees C, and as monomers at 37 degrees C. These results strongly suggest that the dimerization of protein Arp and protein H, rather than the low temperature itself, yielded the strong Ig-binding of the proteins at 10 and 22 degrees C. Indeed, after covalent cross-linking of the dimers at 10 degrees C by incubation with low concentrations of glutaraldehyde, full Ig-binding was achieved even at 37 degrees C. A carboxyl-terminal proteolytic fragment of protein Arp, which completely lacked the IgA-binding capacity at any temperature, showed the same temperature-dependent dimerization as intact protein Arp, suggesting that the Ig-binding part of the protein is not required for dimerization. The implications of these results for the function of Ig-binding group A streptococcal proteins, and their role in the host-parasite relationship are discussed.     

Preferred secondary attachment site of prophage phi80 on Escherichia coli K-12 chromosome]

The integration frequency of phage att80 immlambdac1857 into the chromosome of a mutant strain H47 Escherichia coli K-12 deleted for the normal prophage insertion site is found to be about 20-fold decreased as compared with its integration into the wild type strain. The most of the resulting lysogens contain the prophage at the secondary attachment site of the mutant bacterial chromosome which is preferentially utilized for prophage insertion. This attachment site (att80-II) is located close to his-genes on the chromosome of H47 strain. Prophage curing procedure of such abnormal lysogens results in the appearance of rare auxotrophic heat-resistant survivors with the His- phenotype. In some cases the prophage insertion can induce an inversion of a neighbouring genetic region. Such lysogens contain the purC gene near prophage located at the att80-II site, and after curing they segregate the heat-resistant His- and Pur- colonies.     

Distribution of Tn551 insertion sites responsible for auxotrophy on the Staphylococcus aureus chromosome

A method was devised to efficiently select isolates of Staphylococcus aureus 8325 in which Tn551, a transposon originating on the pI258 plasmid responsible for erythromycin resistance (Emr), had translocated to the host chromosome. This method consisted of selecting for Emr at 43 degrees C with a strain in which the pI258 plasmid was unable to replicate at 43 degrees C because of a temperature-sensitive plasmid mutation. By selecting isolates that were Emr at 43 degrees C and auxotrophic for nutrients not required by the parent strain. Tn551-induced auxotrophic mutants were readily isolated. The incidence of auxotrophic classes was not random; 80% of the isolates in one experiment were Trp-, whereas only a single example of each of some of the other classes was isolated. Among the Trp- mutants, the distribution of trp genes affected and the frequency of precise excision of Tn551 from individual sites varied. When analyzed by transformation, the Tn551-induced ala, his, ilv, lys, rib, thrA, thrB, and trp mutations were shown to occupy sites previously defined by nitrosoguanidine-induced mutations. Tn551-induced mutagenesis provided three previously unrecognized classes of auxotrophs (tyr, met, and thrC), and the Tn551 integration sites resulting in these mutations have been identified. In addition, a chromosomal region (uraB) was identified by Tn551 mutagenesis that is distinct from uraA (previously defined by chemical mutagenesis). Some Tn551-induced mutations (most notably pur) could not be linked to the known linkage groups of the chromosome by transformation. With the exception of two pur mutations, all of the Tn551-induced auxotrophic mutational sites cotransformed at unity with Tn551 and, in cases in which they were selected, prototrophic transformants were always Ems. Thus, the Tn551 and auxotrophic sites are identical.     

Identification and characterization of a new Escherichia coli gene that is a dosage-dependent suppressor of a dnaK deletion mutation.

We report the isolation and characterization of a previously unidentified Escherichia coli gene that suppresses the temperature-sensitive growth and filamentation of a dnaK deletion mutant strain. Introduction of a multicopy plasmid carrying this wild-type gene into a dnaK deletion mutant strain rescued the temperature-sensitive growth of the dnaK deletion mutant strain at 40.5 degrees C and the filamentation, fully at 37 degrees C and partially at 40.5 degrees C. However, the inability of dnaK mutant cells to support bacteriophage lambda growth was not suppressed. This gene was also able to suppress the temperature-sensitive growth of a grpE280 mutant strain at 41 degrees C. Filamentation of the grpE280 mutant strain was suppressed at 37 degrees C but not at 41 degrees C. The dnaK suppressor gene, designated dksA, maps near the mrcB gene (3.7 min on the E. coli chromosome). DNA sequence analysis and in vivo experiments showed that dksA encodes a 17,500-Mr polypeptide. Gene disruption experiments indicated that dksA is not an essential gene.     

Effects of growth temperatures on plating efficiencies and stabilities of heterokaryons of Candida albicans

Heterokaryons (hets) of Candida albicans constructed by fusing protoplasts of complementing auxotrophs produce heterogeneous clones on minimal medium consisting of (i) a minority of slow-growing hets, (ii) a preponderance of non-growing, parental-type auxotrophic monokaryons, and (iii) some prototrophic monokaryons bearing hybrid nuclei. Hets grown at a given temperature within the range 25° C to 41° C replate with higher efficiencies at any lower temperature and exhibit progressively declining plating efficiencies as plate temperatures increase beyond that at which they were initially grown. Neither auxotrophic nor prototrophic monokaryons show such responses. Growth of colonies produced by hets, wild-type strains or prototrophic hybrid monokaryons is stimulated by temperatures in the order, 37° C > 30° C > 41° C > 25° C. However, the proportion of hets to auxotrophic monokaryons within individual net clones increases directly from 25° C to 41° C. Though this pattern obtains whether colonies are compared at equivalent sizes or ages, het frequencies decline as colonies age at all temperatures. Appearance of hybrid monokaryons within het clones is unaffected by growth temperature. The relationships of temperatures to plating efficiencies and stabilities of hets are independent of the natures of their complementing auxotrophies or the wild-type backgrounds of their nuclear components and are, therefore, functions of heterokaryosis per se. Modifications of these relationships by selective metabolic antagonists or by growth of hets on different preand post-plating carbon sources indicate that they reflect temperature-dependent properties of mitochondria which are peculiar to hets.     

Recombination analysis of naturally diploid Candida albicans.

A multiply auxotrophic strain, hOG45, was derived from Candida albicans ATCC 10261. Prototrophic revertants of this multiple auxotroph were selected after mutagenesis. These prototrophic revertants were distinguishable from the original prototroph, ATCC 10261, because of their mitotic instability. They gave rise to auxotrophic derivatives which displayed one or more of the auxotrophic requirements characteristic of hOG45. Two of the auxotrophic requirements, those for adenine and methionine, frequently reappeared together in the auxotrophic derivatives of the prototrophic revertants. This apparent linkage of ade and met was confirmed by protoplast fusion analysis of the original auxotroph. These data indicate that C. albicans ATCC 10261 is diploid, the multiple auxotroph h0G45 is homozygous for recessive auxotrophic alleles, the prototrophic revertants are multiple heterozygotes, the auxotrophic derivatives are homozygotes produced by mitotic crossing-over, and the association between the ade and met alleles is due to linkage.     

Isolation and characterization of Schizosaccharomyces pombe mutants defective in cell wall (1-3)beta-D-glucan.

Schizosaccharomyces pombe thermosensitive mutants requiring the presence of an osmotic stabilizer to survive and grow at a nonpermissive temperature were isolated. The mutants were genetically and biochemically characterized. In all of them, the phenotype segregated in Mendelian fashion as a single gene which coded for a recessive character. Fourteen loci were defined by complementation analysis. Studies of cell wall composition showed a reduction in the amount of cell wall beta-glucan in three strains (JCR1, JCR5, and JCR10) when growing at 37 degrees C. Galactomannan was diminished in two others. Strains JCR1 and JCR5, with mutant alleles cwg1-1 and cwg2-1, respectively, were further studied. The cwg1 locus was mapped on the right arm of chromosome III, 18.06 centimorgans (cM) to the left of the ade5 marker; cwg2 was located on the left arm of chromosome I, 34.6 cM away from the aro5 marker. (1-3)beta-D-Glucan synthase activities from cwg1-1 and cwg2-1 mutant strains grown at 37 degrees C were diminished, as measured in vitro, compared with the wild-type strain; however, Km values and activation by GTP were similar to the wild-type values. Mutant synthases behaved like the wild-type enzyme in terms of thermostability. Analyses of round shape, lytic behavior, and low (1-3)beta-D-glucan synthase activity in cultures derived from ascospores of the same tetrad showed cosegregation of all these characters. Detergent dissociation of (1-3)beta-D-glucan synthase into soluble and particulate fractions and subsequent reconstitution demonstrated that the cwg1-1 mutant was affected in the particulate fraction of the enzymatic activity while cwg2-1 was affected in the soluble component. The antifungal agents Papulacandin B and Aculeacin A had similar effects on the enzymatic activities of the wild type and the cwg2-1 mutant strain, whereas the cwg1-1 mutant, when growing at 37 degrees C, had a more inhibitor-resistant (1,3)beta-D-glucan synthase. It is concluded that the cwg1+ and cwg2+ genes are related to (1,3)beta-D-glucan biosynthesis.     

The detection of monosomic colonies produced by mitotic chromosome non-disjunction in the yeast Saccharomyces cerevisiae

A diploid yeast strain, D6, is described which monitors mitotic non-disjunction by the phenotypic expression of a set of coupled and recessive markers flanking the centromere of chromosome VII. These markers are not expressed in the heterozygous condition prevailing in D6. The left arm of chromosome VII carries a tightly centromere linked marker, leu1 (leucine requirement), distal to leu1 in this order: trp5 (trytophan requirement), cyh2 (recessive resistance to cycloheximide) and met 13 (requirement for methionine). The right arm is marked with ade3 (simultaneous requirement for adenine and histidine). D6 is homozygous for ade2 and consequently, forms red rather than the normally white colonies. It shows no requirement for the above amino acids and it is sensitive to cycloheximide.Unmasking of all the markers on chromosome VII leads to colonies that are white because ade3 sets a block preceding the ade2 block (which causes the accumulation of a precursor of the red pigment), they require leucine, tryptophan and methionine, and grow on media with cycloheximide. Cells are plated on a cycloheximide medium where red and white colonies are formed. Colonies of spontaneous origin were tested. The majority of the white colonies expressed all the recessive markers whereas only few of the red colonies expressed all the markers on the left arm of chromosome VII.Basically expression of recessive markers on both sides of the centromere can be explained as a result of two coincident events of mitotic crossing over. However, the frequency of colonies expressing centromere linked leu1 was 14 times higher among the white types than the red ones. This suggested that the white, cycloheximide resistant, leucine requiring colonies arose by mitotic non-disjunction and not only by two coincident mitotic crossing over events.Presumptive spontaneous monosomic segregants were placed on sporulation medium. Only 8 out of 30 isolates sporulated, which showed that these eight segregants were diploid at the time of sporulation. They could have arisen by two coincident crossover events or through restoration of a normal disomic condition after non-disjunction had occurred. The genetic data thus leaves us with only its statistical argument in favour of non-disjunction. Further confirmation of monosomic nature of the white cycloheximide resistant colonies was provided by the estimates of their DNA contents. Compared to the stock wild type diploids the presumptive monosomics showed a reduction in DNA content.We have utilized D6 to investigate the possible induction of mitotic non-disjunction after treatment with gamma rays, heat shock at 52°C and ultraviolet irradiation. In all cases white, cycloheximide resistant colonies were produced at levels significantly higher than that found in untreated cultures. In order to detect the production of monosomic cells, treated cultures were grown for 48 h in non-selective medium after exposure to allow for “expression” of the monosomic condition.     

Characterization of a cycloheximide-resistant Tetrahymena thermophila mutant which also displays altered growth properties.

A cycloheximide-resistant strain of Tetrahymena thermophila, expressing a mutant chx-B gene (Ares and Bruns, Genetics 90:463-474, 1978), displayed very different temperature-dependent growth characteristics than either wild-type cells or another cycloheximide-resistant strain expressing a different mutant gene. Whereas wild-type cells showed an immediate decline in ribosome translocation rates when shifted from 30 to 38 or 40 degrees C, this mutant strain (X-8) showed no such decline. These results directly correlated with the growth rate differences we found for these cells at these temperatures. By genetic analysis, we showed that the phenotype of cycloheximide resistance cosegregated with the ability to grow rapidly at 40 degrees C. Analyses, both direct and indirect, suggested that a number of functional and structural characteristics of the ribosomes from strain X-8 cells are most likely conformationally different from those of wild-type ribosomes.     

Evidence for cooperation between cells during sporulation of the yeast Saccharomyces cerevisiae.

Diploid Saccharomyces cerevisiae cells heterozygous for the mating type locus (MATa/MAT alpha) undergo meiosis and sporulation when starved for nitrogen in the presence of a poor carbon source such as potassium acetate. Diploid yeast adenine auxotrophs sporulated well at high cell density (10(7) cells per ml) under these conditions but failed to differentiate at low cell density (10(5) cells per ml). The conditional sporulation-deficient phenotype of adenine auxotrophs could be complemented by wild-type yeast cells, by medium from cultures that sporulate at high cell density, or by exogenously added adenine (or hypoxanthine with some mutants). Adenine and hypoxanthine in addition to guanine, adenosine, and numerous nucleotides were secreted into the medium, each in its unique temporal pattern, by sporulating auxotrophic and prototrophic yeast strains. The major source of these compounds was degradation of RNA. The data indicated that differentiating yeast cells cooperate during sporulation in maintaining sufficiently high concentrations of extracellular purines which are absolutely required for sporulation of adenine auxotrophs. Yeast prototrophs, which also sporulated less efficiently at low cell density (10(3) cells per ml), reutilized secreted purines in preference to de novo-made purine nucleotides whose synthesis was in fact inhibited during sporulation at high cell density. Adenine enhanced sporulation of yeast prototrophs at low cell density. The behavior of adenine auxotrophs bearing additional mutations in purine salvage pathway genes (ade apt1, ade aah1 apt1, ade hpt1) supports a model in which secretion of degradation products, uptake, and reutilization of these products is a signal between cells synchronizing the sporulation process.     

Induction and selection of the minute-rough (MR) colonial variant of Candida albicans

Summary An adenine requiring strain ofCandida albicans, WC-7, forms large smooth colonies. When grown at 37° C under conditions of severe adenine deprivation, WC-7 cultures accumulate variant cells (MR variants) which produce minute, rough colonies. The variants are stable in that they persist upon repeated selective subculturing. However, they do exhibit high rates of reversion to their large, smooth colony progenitor form. It is shown that cultural conditions which favor the appearance of variants in WC-7 populations create a metabolic stress within the WC-7 cells which leads to their direct transformation into variants. These same conditions also impart a selective growth advantage to variant cells over WC-7 cells. Considerations of the genetic properties of the variants and the factors involved in their induction argue strongly that variant cells originate through alteration of a non-genic, hereditary determinant.     

利用营养缺陷诱变对金顶侧耳进行基因连锁分析研究

利用金顶侧耳的营养缺陷型菌株配制杂交菌株,通过营养缺陷型标记对其后代进行连锁分析,确定不亲和性因子和营养缺陷标记所在的连锁群及其排列顺序。截止目前的试验数据表明,金顶侧耳至少由6条染色体(连锁群)组成。其中A因子存在的第1连锁群上分布着ade2、pab1、ade5、met2、met7营养缺陷型基因位点;B(Bα、Bβ)因子存在的第2连锁群上分布着cho1、ade1营养缺陷型基因位点;第3连锁群上分布着met9、arg1、his1、ade7、his3、met1营养缺陷型基因位点;第4连锁群分布着nic1、nic2、ade4、pdx2营养缺陷型基因位点;第5连锁群分布着pan1、ino1营养缺陷型基因位点;第6连锁群分布着ile1营养缺陷型基因位点。金顶侧耳与其他担子菌的遗传图谱相同,第1连锁群A因子附近均分布着Ade及Pab营养缺陷型基因位点。