Biochemical and genetic characterization of -glucosidase mutants in Saccharomyces lactis

Mutants with reduced activity for beta-glucosidase (beta-d-glucoside glucohydrolase EC 3.2.1.21) were isolated from the haploid yeast Saccharomyces lactis. Tetrad analysis indicated that in each mutant a single genetic factor, closely linked or allelic to the structural gene for beta-glucosidase (B locus), is responsible for the decreased activity. beta-Glucosidases produced by wild-type and mutant strains are similar in molecular size and charge but differ in catalytic properties, thermal stability, and serological specificity, indicating that mutants are in the structural gene. All mutants retained their capacity to be induced by either methyl-beta-d-glucoside or glucose. In all cases, the mutant phenotype was dominant in heterozygous diploids.     

Identification of new genes involved in disaccharide fermentation in yeast

Summary Maltose non-fermenting mutants were obtained from strains carrying a MAL4 allele which permits constitutive synthesis of maltase. Cells carrying this allele are able to utilize sucrose in the absence of the classical sucrose genes. All maltose non-fermenting mutants were also sucrose non-fermenters. Eight mutants had become maltase negative; 19 mutants could still form maltase constitutively.In crosses with segregational maltose and sucrose non-fermenting strains, enzyme negative mutants gave diploids unable to ferment maltose and sucrose. Enzyme positive, non-fermenting mutants gave diploids which readily fermented maltose and sucrose. This latter type of mutants was designated dsf (disaccharide fermentation) mutants.The diploids derived from crossing non-fermenting mutants with segregational non-fermenters were subjected to tetrad analysis. Enzyme negative non-fermenters gave only non-fermenting progeny. The dsf mutants segregated both fermenting and non-fermenting progeny, some of which showed the dsf phenotype. This indicated that none of the dsf mutants had a defect in a gene closely linked to MAL4. Crosses between dsf mutants and strains carrying the maltose genes MAL2 and MAL3 showed that the mutations affected maltose fermentation in general. Sucrose fermentation in the presence of the classical sucrose gene SUC3 was not affected, nor were fermentation of glucose, fructose and galactose.The uptake of radioactivity from uniformly labeled maltose appeared to be blocked in mutants of at least four of the dsf genes. Only one non-leaky and a leaky mutant showed a significant uptake.These results suggest that there is an extremely complex transport system for maltose and sucrose or that the utilization of these disaccharides requires a complex series of metabolic reactions.     

The induction of rho- mutants by UV or gamma-rays is independent of the nuclear recombinational repair pathway in Saccharomyces cerevisiae

In order to discover whether the nuclear recombinational repair pathway also acts on lesions induced in mitochondrial DNA (mtDNA), the possible role of the RAD50, -51, -52, -55 and -56 genes on the induction of rho- mutants by radiations was studied. Such induction appeared to be independent of this pathway. Nevertheless, an efficient induction of respiration-deficient mutants was observed in gamma-irradiated rad52 diploids. We demonstrate that these mutants do not result from a lack of mtDNA repair, but from chromosome losses induced by gamma-rays. Such an impairment of the respiratory ability of diploids by chromosome losses was effectively observed in the aneuploid progeny of unirradiated RAD+ cdc6 diploids incubated at the restrictive temperature.     

Assembly of the mitochondrial membrane system. Genetic complementation of mit- mutations in mitochondrial DNA of Saccharomyces cerevisiae.

A method has been devised to test intergenic complementation of mutations in the mitochondrial DNA of Saccharomyces cerevisiae. The test is based on the observation that diploids issued from pairwise crosses of certain mit- mutants with deficiencies in cytochrome oxidase, or coenzyme QH2-cytochrome c reductase, acquire high levels of respiratory activity shortly after zygote formation. Under our experimental conditions neither biochemical complementation, interallelic complementation, nor recombination has been found to contribute to any significant extent toward the respiration measured in the diploids at early times. The test has been used to study the number of complementation groups represented by a large number of mit- mutants. Results of pairwise crosses of mutants in the oxi 1, oxi 2, oxi 3, cob 1, and cob 2 loci indicate that complementation occurs between the oxi and cob loci between different oxi loci but not between the two cob loci. The five loci have, therefore, been assigned to four different complementation groups.     

Preferential recovery of uniparental streptomycin resistant mutants from diploid Chlamydomonas reinhardtii

Summary Analysis of the streptomycin resistant mutants recovered from control and N-methyl-N-nitro-N-nitrosoguanadine (MNNG) treated haploid cultures of C. reinhardtii reveal that approximately 60% of the mutants are of the sr-1 type known to show Mendelian inheritance while 40% are of the sr-2 and sm-3 types known to be inherited in a uniparental (UP) manner. In contrast, most if not all streptomycin mutants recovered from similarly treated diploid cultures of C. reinhardtii are of the UP variety. Failure to recover sr-1 mutants from the diploid stock is explained by our findings that diploids heterozygous for Mendelian streptomycin resistance (sr-1/sr-1 ⁺) are both stable and sensitive to streptomycin. Efficient recovery of UP streptomycin resistant mutants from diploids can be explained by the observations of Gillham (1963a, 1969) which demonstrate that diploids heterozygous for an sr-2 mutation (sr-2/sr-2 ⁺) segregate resistant and sensitive progeny during mitotic cell division.The utility of diploids for isolating new UP mutant genotypes, for establishing the cellular localization of the UP genome(s), and for characterizing the rules governing UP gene segregation is discussed.Supported by NIH postdoctoral fellowship GM 52359 to R.W.L., NIH predoctoral traineeship GM 02007 to K.P.V., and NSF grant GB-22769 to N.W.G. and J.E.B.     

Properties of heterozygous diploids between strains ofPenicillium chrysogenum selected for high penicillin yield

Three strains ofPenicillium chrysogenum selected for high penicillin yield and of independent lineage were marked with suitable genetical characters prior to the synthesis of several heterozygous diploids. These parental strains had domestic codes, C, D and Y. Two diploids, between differently labelled mutants of strain C and Y, produced similar amounts of penicillin to strain C, which was less than that produced by strain Y. Previous work had indicated that genes responsible for increased penicillin yield were recessive and the present results suggested that such genes in strains C and Y were allelic, apart from the presence of one or more additional recessive mutations leading to greater penicillin production in the higher yielding parent. Three diploids made between mutants of strains D and Y were lower in penicillin yield than either original parent and only in the case of one diploid compared with one of the parental strains was this difference not significant. In strains D and Y, therefore, there may have been some recessive genes concerned with increasing penicillin yield which were non-allelic. However, no first order segregants arising spontaneously or subsequent to X-ray treatment produced higher levels of penicillin than the better yielding original parent in any cross.     

Mutants of Saccharomyces cerevisiae and Candida utilis with increased susceptibility to digestive enzymes.

Mutants of Candida utilis and a haploid strain of Saccharomyces cerevisiae were isolated, after ultraviolet light mutagenesis, which had increased sensitivities to snail gut enzymes (ses). Three of the five S. cerevisiae mutants tested had increased sensitivities to porcine pepsin, all were more susceptible to a sequential treatment with pepsin, lipase, peptidase, and trypsin, four were sensitive to osmotic shock, and two had increased glucan/mannan ratios in their cell walls. All combinations of mutants showed positive complementation in heterozygous diploids, although complementation between one pair, which had the same phenotype, was incomplete, indicating that four to five different cistrons were involved. All mutations were found to be recessive. Haploid strains bearing pairs of ses mutations were not markedly more sensitive to mammalian digestive enzymes than strains with single mutations. Rat-feeding experiments with three mutants and the parental strains indicated that the protein was efficiently utilized in all cases. Net protein ratios for the two mutants of S. cerevisiae tested were slightly higher than that for their parent, but the differences were of marginal significance.     

Improvement of Enzyme Productivities through Mutation or Haploidization of Heterozygous Diploids Obtained by Protoplast-fusion of Aspergillus sojae

A new breeding process for Aspergillus sojae involving protoplast fusion was studied to obtain more desirable koji-molds for soy-sauce production, especially as to their enzyme productivities. A pair of double-marker mutants with characteristic enzyme-productivities, derived from genealogically unrelated A. sojae cultures, were fused by means of the protoplast fusion technique. The fusants were UV-treated, and stable heterozygous diploids were obtained. Further improvement of their enzyme-productivities was attempted using two approaches. Through the usual mutation of the heterozygous diploids, the activities of protease and glutaminase were simultaneously improved to a certain extent. Through the haploidization of the heterozygous diploids with benomyl or p-fluorophenylalanine (FPA), some haploids considered phenotypically to be recombinants were segregated, in addition to a large number of haploids that had reverted to the original double marker mutants. On screening these haploid-recombinants, the authors obtained a few more excellent recombinants, as to their enzyme productivities, that is, strains producing as much protease as the hyper-protease producer and producing almost as much glutaminase as the hyper- glutaminase producing parent. It has been shown that mutation or especially haploidization of heterozygous diploids produced by cell-fusion could be a very useful technique for the breeding of new industrial koji-molds.     

Intracellular cAMP in Dictyostelium discoideum: Characterization of an aggregation-deficient mutant with elevated cAMP pools

Forty aggregation-deficient mutants of Dictyostelium discoideum were screened for changes in intracellular cAMP during the first 10 hr of starvation. The pools in 39 of the mutants remained low and relatively static during this period. However, amoebae of one mutant, strain HC151, exhibited significantly elevated levels of intracellular cAMP during vegetative growth and for several hours after starvation. A more detailed analysis of this mutant indicated that the elevated cAMP pools in these cells are a consequence of the premature appearance and partial activation of an adenylate cyclase. The mutation(s) altering adenylate cyclase regulation in this strain appears to map in linkage group IV. Complementation tests between strain HC151 and another mutant, HH201, which has recently been shown to produce an adenylate cyclase activity precociously [1], indicated that the mutations affecting adenylate cyclase activity in these strains map at different loci. Although both of these mutations behave recessively in heterozygous diploids with respect to gross development, an examination of early cAMP metabolism and terminal spore differentiation in these diploids suggest that these mutations are at least partially expressed during some stage(s) of the developmental cycle.     

Isolation and Characterization of Mms-Sensitive Mutants of SACCHAROMYCES CEREVISIAE

We have isolated mutants sensitive to methyl methanesulfonate (MMS) in Saccharomyces cerevisiae. Alleles of rad1, rad4, rad52, rad55 and rad57 were found amoung these mms mutants. Twenty-nine of the mms mutants which complement the existing radiation-sensitive (rad and rev) mutants belong to 22 new complementation groups. Mutants from five complementation groups are sensitive only to MMS. Mutants of 11 complementation groups are sensitive to UV or X rays in addition to MMS, mutants of six complementation groups are sensitive to all three agents. The cross-sensitivities of these mms mutants to UV and X rays are discussed in terms of their possible involvement in DNA repair. Sporulation is reduced or absent in homozygous diploids of mms mutants from nine complementation groups.     

Dominant mutations affecting expression of pH-regulated genes inYarrowia lipolytica

In the yeastYarrowia lipolytica the levels of the alkaline extracellular protease (AEP) and acid extracellular protease (AXP) are controlled by the pH of the growth medium. When the pH of growth medium is kept close to 4.0, levels of AXP are high and those of AEP are low, whereas at pH above 6.0 the opposite is true. Mutations which mimic the effects on the protease system of growth at alkaline pH have been identified in two genes,RPH1 andRPH2, inY. lipolytica. Detailed genetic studies showed that mutations in these two genes are dominant in heterozygous diploids, and that their effects are additive in haploid double mutants. These mutants show that pH regulates AEP expression independently from other metabolic signals. These mutants are not detectably affected in their growth rates, nor in internal pH homeostasis.     

Dominant mutations affecting expression of pH-regulated genes inYarrowia lipolytica

In the yeastYarrowia lipolytica the levels of the alkaline extracellular protease (AEP) and acid extracellular protease (AXP) are controlled by the pH of the growth medium. When the pH of growth medium is kept close to 4.0, levels of AXP are high and those of AEP are low, whereas at pH above 6.0 the opposite is true. Mutations which mimic the effects on the protease system of growth at alkaline pH have been identified in two genes,RPH1 andRPH2, inY. lipolytica. Detailed genetic studies showed that mutations in these two genes are dominant in heterozygous diploids, and that their effects are additive in haploid double mutants. These mutants show that pH regulates AEP expression independently from other metabolic signals. These mutants are not detectably affected in their growth rates, nor in internal pH homeostasis.     

Mutations Affecting Sexual Conjugation and Related Processes in SACCHAROMYCES CEREVISIAE. I. Isolation and Phenotypic Characterization of Nonmating Mutants

Nonmating mutants were also isolated from haploid strains of yeast of both mating types. The mutants were characterized with respect to their ability to produce and respond to specific yeast sex factors, their ability to mate at low frequencies, and the ability of the low-frequency diploids to sporulate. Loss of the ability to mate by either mating type was invariably accompanied by the loss of one or more, and in some cases, all, of the above capabilities. The results strongly indicate that the sex factors are functionally involved in the conjugation process.     

Mutants of Saccharomyces cerevisiae supersensitive to the mutagenic effect of 6-N-hydroxylaminopurine

Yeast mutants hypersensitive to the mutagenic action of 6-N-hydroxylaminopurine (HAP) were obtained by EMS mutagenesis. One of the mutants segregated monogenically and possessed reduced capacity to utilize HAP as a purine source. A set of diploids suitable for parallel study of mutagenesis and induction of recombination, and differing in the trait of mutability after exposure to HAP ("hm" trait or HAP mutability), were constructed. It was shown that a weak recombinogenic effect of HAP is not enhanced in "hm" mutants when HAP mutability increases.     

Mitochondrially encoded resistance to paromomycin in Saccharomyces cerevisiae: Reinvestigation of a controversy

We have reinvestigated the nature of mitochondrially inherited resistance to paromomycin in Saccharomyces cerevisiae. Resistance to this antibiotic can arise by a nucleotide alteration in the gene coding for 15 S ribosomal RNA at a recognition site for the restriction endonuclease ThaI (CGCG), as has been observed by Li (M. Li, K. Lyon, N. Martin and A. Tzagoloff (1981). “Abstracts, Cold Spring Harbor Meeting on Mitochondrial Genes,” p. 56. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.). We have confirmed this finding and document here also a new type of paromomycin resistance that is unrelated to this ThaI restriction site. Certain petite mutants derived from different locations of the mtDNA of S. cerevisiae KL14-4A can elicit resistance to paromomycin when crossed with a wild-type sensitive strain. These petite mutants lack detectable sequence homology with the 15 S ribosomal RNA gene and they have no extensive sequence homology with each other. We have constructed paromomycin-resistant diploids by crossing such KL14-4A petite mutants with a sensitive wild-type strain. The diploids that receive the paromomycin-resistant allele from a petite mutant retaining the 15 S ribosomal RNA gene no longer contained the ThaI site. However, diploids that become resistant after a cross with petite mutants retaining fragments from other mtDNA regions than the 15 S ribosomal RNA, still contain the ThaI site. This shows that paromomycin resistance can occur in the presence of the ThaI site. After sporulation, suitable paromomycin-resistant haploids were crossed with each other and sensitive recombinant diploids were found, indicating the existence of more than one form of paromomycin resistance. Possible explanations for this novel type of paromomycin resistance and the unorthodox way in which it arises, are presented.     

Mechanism of conjugation and recombination in bacteria XVI

Summary Protein synthesis by ribosomes from several cryptopleurine-resistant yeast mutants is also resistant to emetine and tubulosine. These mutants can be classified into two different types: Class I mutants which display high levels of resistance to emetine and tubulosine and Class II mutants that are only weakly resistant to tubulosine and are slightly more sensitive to emetine than those of Class I. Apparently all mutants have similar levels of resistance to cryptopleurine. The distinct phenotypes of Class I and Class II strains are expressed through their 40S ribosomal subunit. Genetic analysis has shown that the mutations to cryptopleurine resistance are allelic and that in a particular case (strain CRY6) the pleiotropic phenotype is a result of the expression of the cryl locus. It is suggested that Class I and Class II mutants arise from two independent mutational events within the cryl allele. in heterozygous (+/cryl) diploids both the sensitive and the resistant genes are expressed as shown by studies of the action of cryptopleurine on polyphenylalanine-synthesizing system derived from each parental sensitive and resistant haploid strain and heterozygous diploid strains. The apparent dominance of sensitivity over resistance which may be observed in vivo in heterozygous (+/cryl) diploids has been explained in terms of the mode of action of the inhibitors.     

The Formation, General Characteristics and Production of α-Amylase in Heterocaryons and Diploids of Aspergillus oryzae

S ummary . Heterocaryons and diploids from Aspergillus oryzae were investigated with respect to nuclear number/conidium and to conidial size. Heterocaryons usually had larger conidia and more nuclei/conidium than diploids and the haploid parent mutants. Diploids contained significantly fewer nuclei/conidium than haploids. However, they could not be distinguished from haploids by measurement of conidial size. The strains were examined for the production of α-amylase. All auxotrophic mutants produced less α-amylase than the prototrophic wild type. Heterocaryons gave yields which were intermediate between that of their parent mutants or the same as the best producing parent. Diploids which produced more α-amylase than the best producing parent strain were synthesized. The highest yield from a diploid was of the same order of magnitude as the yield from the wild type.     

Fine structure of the arg-7 cistron in Chlamydomonas reinhardi

Summary In Chlamydomonas reinhardi, the arg-7 cistron is the structural gene for the enzyme argininosuccinate lyase which catalyzes the last reaction in the biosynthesis of arginine.Fourteen mutants (nine previously analyzed and five new mutants) defective in the lyase have been investigated so far: they all map within a cistron (length: 1.0–1.6 recombination units) of the linkage group I and fall within six groups of complementation. The enzyme activity found in the diploids formed by intragenic complementation was always lower than in wild-type haploid or diploid strains. The study of the denaturation curves obtained by heat treatment of the lyase indicates that in some diploids, several enzyme varieties can be present.These results and those previously obtained with diploids formed by intragenic and intergenic complementation (Matagne and Loppes, 1972; Matagne, 1976) are discussed in relation to the recent data showing that the argininosuccinate lyase is a multimeric enzyme probably composed of five identical polypeptide chains (Matagne and Schlösser, 1977)     

Elucidating polyploidization of bermudagrasses as assessed by organelle and nuclear DNA markers

Clarification of relationships among ploidy series of Cynodon accessions could be beneficial to bermudagrass breeding programs, and would enhance our understanding of the evolutionary biology of this warm season grass species. This study was initiated to elucidate polyploidization among Cynodon accessions with different ploidy series collected from Turkey based on chloroplast and nuclear DNA. Forty Cynodon accessions including 7 diploids, 3 triploids, 10 tetraploids, 11 pentaploids, and 9 hexaploids were analyzed using chloroplast DNA restriction fragment-length polymorphism (cpDNA RFLP), chloroplast DNA simple sequence repeat (cpDNA SSR), and nuclear DNA markers based on neighbor-joining (NJ) and principle component analyses (PCA). All three-marker systems with two statistical algorithms clustered the diploids apart from the other ploidy levels. Assuming autopolyploidy, spontaneous polyploidization followed by rapid diversification among the higher ploidy levels than the diploids is likely in Cynodon's evolution. Few tetraploid and hexaploid accessions were clustered with or closely to the group of diploids, supporting the hypothesis above. Eleven haplotypes as estimated by cpDNA RFLP and SSR markers were detected. This study indicated that the diploids had different organelle genome from the rest of the ploidy series and provided valuable insight into relationships among ploidy series of Cynodon accessions based on cp and nuclear DNAs.     

Non-motile mini-transposon mutants of Bordetella bronchiseptica exhibit altered abilities to invade and survive in eukaryotic cells

Non-motile mutants of Bordetella bronchiseptica were generated after mini-transposon mutagenesis. One non-motile mutant (designated VMM1) was derived from the bvg-positive strain BB7865 and four mutants (designated AMM1–4) were derived from the isogenic bvg-negative strain BB7866. Southern hybridisation analysis indicated that loss of motility was not due to the disruption of the flagellin subunit gene. Western blot and transmission electron microscopic analysis indicated that three of the five mutants expressed neither the flagellin subunit (40 kDa) nor flagella whereas one mutant expressed intact flagella under all conditions tested. One unique bvg-negative mutant, AMM4, exhibited temperature-dependent repression of flagella biosynthesis and motility at 37°C. The ability of AMM4 to invade and survive in HeLa cells was significantly decreased.