酵母细胞分裂周期突变株中磷酸化蛋白的变化

最近对酵母细胞腺苷酸环化酶和依赖于cAMP的蛋白激酶基因的分子生物学研究,说明磷酸化蛋白在细胞周期中有重要作用。本文用聚丙烯酰胺凝胶电泳研究了酵母细胞分裂周期突变株中蛋白质的磷酸化。依赖cAMP的突变株AM18生长在无cAMP的培基中时,发生了几种磷酸化蛋白的变化,最明显的是分子量为72K Da的蛋白的积聚。细胞分裂周期温度敏感突变株cdc35生长在高于允许温度对,以及野生株生长在稳定期时,也出现类似现象。在这三种情况下,72K Da磷酸化蛋白同时还具有相同的pI值(pI=4.7),磷酸化都发生在苏氨酸残基上,用蛋白酶部分水解法证明它们有相似的肽谱。这些结果说明它们为同一蛋白。     

Isolation and Characterization of Chromosome-Gain and Increase-in-Ploidy Mutants in Yeast

We have developed a colony papillation assay for monitoring the copy number of genetically marked chromosomes II and III in Saccharomyces cerevisiae. The unique feature of this assay is that it allows detection of a gain of the marked chromosomes even if there is a gain of the entire set of chromosomes (increase-in-ploidy). This assay was used to screen for chromosome-gain or increase-in-ploidy mutants. Five complementation groups have been defined for recessive mutations that confer an increase-in-ploidy (ipl) phenotype, which, in each case, cosegregates with a temperature-sensitive growth phenotype. Four new alleles of CDC31, which is required for spindle pole body duplication, were also recovered from this screen. Temperature-shift experiments with ipl1 cells show that they suffer severe nondisjunction at 37°. Similar experiments with ipl2 cells show that they gain entire sets of chromosomes and become arrested as unbudded cells at 37°. Molecular cloning and genetic mapping show that IPL1 is a newly identified gene, whereas IPL2 is allelic to BEM2, which is required for normal bud growth.     

Regulatory Mutations of Inositol Biosynthesis in Yeast: Isolation of Inositol-Excreting Mutants

The enzyme inositol-1-phosphate synthase (I-1-P synthase), product of the INO1 locus, catalyzes the synthesis of inositol-1-phosphate from the substrate glucose-6-phosphate. The activity of this enzyme is dramatically repressed in the presence of inositol. By selecting for mutants which overproduce and excrete inositol, we have identified mutants constitutive for inositol-1-phosphate synthase as well as a mutation in phospholipid biosynthesis. Genetic analysis of the mutants indicates that at least three loci (designated OPI1, OPI2 and OPI4) direct inositol-mediated repression of I-1-P synthase. Mutants of these loci synthesize I-1-P synthase constitutively. Three loci are unlinked to each other and to INO1, the structural gene for the enzyme. A mutant of a fourth locus, OPI3, does not synthesize I-1-P synthase constitutively, despite its inositol excretion phenotype. This mutant is preliminarily identified as having a defect in phospholipid synthesis.     

乳酸抗性酵母的筛选及其生长特性的研究

以酿酒酵母 (saccharomycesceevisiae)单倍体YNN -2 7(αtep ura )为亲株 ,在含有 4 %乳酸的梯度平板上直接进行紫外线诱变处理 ,筛选到突变株YNN -2 7-2 4。通过对该突变株乳酸抗性产生原因分析、在含有不同浓度的乳酸和潮霉素B(hygromycinB)的YPDL和YPDLH培养基中的重复特性的研究发现 ,该突变株对乳酸和潮霉素B产生的抗性 ,不是因对环境条件的适应而产生 ,而是由基因突变所引起。与突变株YNN2 7-2 4相比 ,乳酸对亲株生长的影响在于延长了其生长的延迟期 ,而其生长速率没有发生改变。用Mini-photo 51 8测定供试菌株在生长过程中的吸光度 ( 660nm)以研究酵母菌的生长特性 ,是一种行之有效的方法 ,具有较高的灵敏度和较好的再现性。     

Genetic Analysis of Cold-Sensitive Ribosome Maturation Mutants of Escherichia coli

Four cold-sensitive mutants of Escherichia coli, which have defects in the maturation of the 50S ribosomal subunit, were isolated. Each of the mutations was shown to map at a different locus. The loci were assigned the name rim (ribosome maturation) and were shown to map as follows: rimA is co-transduced with ilvD and with pyrE; rimB is co-transduced with aroD; conjugation experiments limited rimD to a region between ilv and malB, and conjugation experiments limited rimC to the 22 to 30 min region of the chromosome. In merodiploids heterozygous for rimA, rimB, or rimD, the wild-type allele was shown to be dominant to the mutant allele. The observation that the rim loci lie outside the strA region and separate from each other, as well as the recessive character of the rim loci, suggests that the mutants may be defective in ribosome maturation factors rather than being defective in ribosomal structural proteins.     

Cold-Sensitive Mutants of DROSOPHILA MELANOGASTER Defective in Ribosome Assembly

Thirteen X-linked, cold-sensitive lethal, female-sterile mutants of Drosophila melanogaster located at eight separate loci were screened for their ability to assemble ribosomes at the restrictive temperature of 17°. Females were labelled with ³H-uridine for either 2 or 20 hours at 17°. A mitochondria-free extract was prepared and analyzed by means of sucrose gradient centrifugation. Four of the mutants, l(1)TW-2 ^cs, l(1)HM16^cs, l(1)HM23^cs, and l(1)HM20 ^cs, had a lower ratio of cpm in the 40S subunit to cpm in the 60S subunit (40S:60S ratio) than wild type with a 2-hour label. The same was true of a 20-hour label of l(1)TW-2^cs, l(1)HM16^cs, and l(1)HM23^cs, which are allelic, resulted in a 40S:60S ratio higher than wild type. Four other cs mutants were found to have less drastic effects on ribosome assembly. The ribosomal subunits of mutants l(1)HM16^sc and l(1)HM20^cs sediment at the same rate as their wild-type counterparts. The same is true for the RNA in their ribosomal particles. Sucrose gradient analysis of ribosomes from cold-sensitive lethal, female-sterile mutants appears to be an effective method for finding mutants that affect ribosome assembly.     

Mutants of Escherichia coli with Cold-Sensitive Deoxyribonucleic Acid Synthesis

Ten cold-sensitive mutants defective in deoxyribonucleic acid (DNA) synthesis at 20 C have been identified among 218 cold-sensitive mutants isolated from a mutagenized population of Escherichia coli K-12. Four of the ten mutant alleles, dna-339 dna-340, dna-341, and dna-342, cotransduce with serB(+) and hence may be dnaC mutants. Two of these, dna-340 and dna-341, are recessive to their wild-type allele. The gene product of their wild-type allele is trans acting. Complementation tests have demonstrated that dna-340 and dna-341 are in the same cistron. The mapping of the remaining six mutations is in progress. In an attempt to determine whether LW4 and LW21 were initiator mutants, cultures of these strains were starved of an essential amino acid at 37 C and then incubated at 15 C with the essential amino acid. The amount of DNA synthesis observed under these circumstances was insignificant. These data are consistent with the idea that LW4 and LW21 are initiator mutants. However, attempts to integratively suppress LW4 and LW21 with F' factors were unsuccessful. To resolve the question of whether or not LW4 and LW21 are initiator mutants, more specific tests and criteria are required. Cultures of LW4 and LW21 were toluene treated and used to measure in vitro DNA synthesis. If the cells were incubated either at 15 or 20 C before toluene treatment, they were capable of markedly less DNA synthesis than if preincubation had not occurred. The amount of in vitro DNA synthesis is directly proportional to the amount of DNA synthesis occurring during preincubation in vivo; i.e., more DNA synthesis is observed at 20 than at 15 C. The fact that the cold-sensitive mutants are unable to synthesize DNA when supplied with deoxyribonucleoside triphosphates, DNA precursors, is evidence they are not defective in precursor synthesis.     

酵母菌细胞自溶突变株的研究

细胞壁是酵母菌的重要细胞器 ,参与细胞内外多方面的生理生化过程 ,如细胞絮凝、信号转导、致病性等 ,在决定细胞结构完整性方面起着重要的作用。酵母细胞壁是由 β - 1 ,3-葡聚糖、β - 1 ,6-葡聚糖、甘露聚糖蛋白及几丁质等相互交链构成的复杂的双层网状结构[1] 。细胞壁组成或结构的改变会使细胞产生对温度或低渗透压的敏感性 ,在相应的条件下发生自溶 ,使细胞内容物释放到胞外。产生上述效应的突变株称为温度敏感自溶突变株和低渗透敏感自溶突变株[2～ 4 ] 。对此类突变株的研究一方面有利于进一步阐明酵母菌细胞壁代谢及组装的调控机制…     

Isolation and Properties of Selenomethionine-Resistant Mutants of NEUROSPORA CRASSA

Mutants resistant to selenomethionine were isolated, and their properties studied. Mapping studies indicate that the mutation sites are located near the eth-1(r) locus in linkage group I, about ten map units away from the mating type locus. The sites of new mutation are either allelic to or very close to eth-1(r). They are resistant not only to selenomethionine but also to ethionine, while the ethionine-resistant mutant, eth-1(r), is sensitive to selenomethionine. The selenomethionine-resistant mutants are also temperature-sensitive mutants. However, they can grow at higher temperatures in medium containing 1 M glycerol.-It is very unlikely that the resistance is due to a change in the permeability of the membrane. Aryl sulfatase of se-met(r) mutants is not repressed by a high concentration of methionine (5 mM), although inorganic sulfate (2 mM) still can cause total repression. The gamma-cystathionase levels of the mutants are normal, but the S-adenosylmethionine synthetase levels are only one-tenth of that observed in the wild-type strain. The heat-stability of this enzyme in the mutant is also different from that of the wild-type enzyme suggesting that the mutation might affect the structural gene of S-adenosylmethionine synthetase.     

Isolation and Primary Identification of Methylotrophic Yeast Hansenula polymorpha Mutants for Peroxisome Biogenesis

After exposure of cells of the methylotrophic yeast Hansenula polymorphaHF246leu1-1 to N-nitro-N-nitrosoguanidine, a collection of 227 mutants unable to grow on methanol at elevated temperature (45°C) was obtained. Ninety four ts mutants (35% of the total number of mutants), which were unable to grow on methanol only at 45°C but could grow at optimal temperature (37°C), were isolated. Complementation analysis of mutants using 12 deletion mutants for genes of peroxisome biogenesis (PEX) (available in this yeast species by the beginning of our work) allowed to assign 51 mutants (including 16 ts) to the separate group of mutants unable to complement deletion mutants with defects in eight PEX genes. These mutants were classified into three groups: group 1 contained 10pex10 mutants (4ts mutants among them); group 2 included 19 mutants that failed to complement otherpex testers: 1 pex1; 2 pex4(1ts); 6 pex5(5ts); 3 pex8; 1 pex13; 6 (3ts) pex19; group 3 contained 22 multiple mutants. In mutants of group 3, hybrids with several testers do not grow on methanol. All mutants (51) carried recessive mutations, except for mutant 108, in which the mutation was dominant only at 30°C, which suggests that it is ts-dominant. Recombination analysis of mutants belonging to group 2 revealed that only five mutants (two pex5 and three pex8) carried mutations for the corresponding PEX genes. Analysis of the spore population from the hybrids of remaining 14 mutants with the pex tester demonstrated the presence of methanol-utilizing segregants, which indicates mutation localization in other genes. In 19 mutants, random analysis of ascospores from hybrids obtained upon crossing mutants of group 3 with a strain lacking peroxisomal disorders (ade11) revealed a single mutation causing the appearance of a multiple phenotype. A more detailed study of two mutants from this group allowed us to localize this mutation in the only PEX gene (PEX1 or PEX2). The revealed disorder of complementation interactions between nonallelic genes is under debate.     

Isolation of Auxotrophic Mutants of Diploid Industrial Yeast Strains after UV Mutagenesis

Auxotrophic mutants of the yeast Saccharomyces cerevisiae are usually isolated in haploid strains because the isolation of recessive mutations in diploids is thought to be difficult due to the presence of two sets of genes. We show here that auxotrophic mutants of diploid industrial sake yeast strains were routinely obtained by a standard mutant selection procedure following UV mutagenesis. We isolated His⁻, Met⁻, Lys⁻, Trp⁻, Leu⁻, Arg⁻, and Ura⁻ auxotrophic mutants of five sake strains, Kyokai no. 7, no. 9, no. 10, no. 701, and no. 901, by screening only 1,700 to 3,400 colonies from each treated strain. Wild-type alleles were cloned and used as markers for transformation. With HIS3 as a selectable marker, the yeast TDH3 overexpression promoter was inserted upstream of ATF1, encoding alcohol acetyltransferase, by one-step gene replacement in a his3 mutant of Kyokai no. 7. The resulting strain contained exclusively yeast DNA, making it acceptable for commercial use, and produced a larger amount of isoamyl acetate, a banana-like flavor. We argue that the generally recognized difficulty of isolating auxotrophic mutants of diploid industrial yeast strains is misleading and that genetic techniques used for haploid laboratory strains are applicable for this purpose.     

Histidine Decarboxylaseless Mutants of Lactobacillus 30a: Isolation and Growth Properties

Mutants of Lactobacillus 30a deficient in their ability to form an inducible histidine decarboxylase (EC 4.1.1.22) were selected by plating nitrosoguanidine-treated cultures on a medium containing histidine and methyl red. Wild-type organisms produce histamine, thus raising the pH and forming yellow colonies; mutant colonies remain red. In the presence of added histidine, decarboxylase-producing cultures grow more heavily than mutant cultures when the initial pH of the growth medium is low or when the lactic acid produced lowers the pH to growth-limiting values. Addition of the decarboxylation products, histamine and carbon dioxide, did not favor growth in crude medium.     

Isolation and Properties of Fumarate Reductase Mutants of Escherichia coli

Escherichia coli produces two enzymes which interconvert succinate and fumarate: succinate dehydrogenase, which is adapted to an oxidative role in the tricarboxylic acid cycle, and fumarate reductase, which catalyzes the reductive reaction more effectively and allows fumarate to function as an electron acceptor in anaerobic growth. A glycerol plus fumarate medium was devised for the selection of mutants (frd) lacking a functional fumarate reductase by virtue of their inability to use fumarate as an anaerobic electron acceptor. Most of the mutants isolated contained less than 1% of the parental fumarate reduction activity. Measurements of the fumarate reduction and succinate oxidation activities of parental strains and frd mutants after aerobic and anaerobic growth indicated that succinate dehydrogenase was completely repressed under anaerobic conditions, the assayable succinate oxidation activity being due to fumarate reductase acting reversibly. Fumarate reductase was almost completely repressed under aerobic conditions, although glucose relieved this repression to some extent. The mutations, presumably in the structural gene (frd) for fumarate reductase, were located at approximately 82 min on the E. coli chromosome by conjugation and transduction with phage P1. frd is very close to the ampA locus, and the order of markers in this region was established as ampA-frd-purA.     

Mitotic Recombination in Yeast: Isolation and Characterization of Mutants with Enhanced Spontaneous Mitotic Gene Conversion Rates

Semi-dominant mutants displaying greatly elevated (up to 200-fold above control) levels of spontaneous mitotic recombination have been isolated in a disomic haploid strain of yeast heteroallelic at the arg4 locus. They are designated by the symbol MIC. The mutants variously exhibit associated sensitivity to UV and ionizing radiation and to methyl methanesulfonate, enhanced UV-induced mitotic recombination, and enhanced spontaneous forward mutation rates. Possible enzyme defects and involvement in repair and editing of DNA are discussed. The mutants are expected to simplify the analysis of recombination pathways in yeast.     

Acetate-nonutilizing Mutants of Neurospora crassa I. Mutant Isolation, Complementation Studies, and Linkage Relationships

Sixty mutants of Neurospora crassa unable to grow on acetate as sole source of carbon, but able to utilize sucrose, were isolated. On the basis of complementation tests, they were divided into seven groups, each group representing a different gene. Six of the genes have been mapped; no two are closely linked. These loci have been designated acu-1 to acu-7. Mutations at four of these loci result in poor germination of ascospores.     

Isolation and Properties of Mutants of Zymomonas mobilis Deficient in Sugar Assimilation

An enrichment method using d-cycloserine was designed for the isolation of spontaneous mutants of Zymomonas mobilis deficient in glucose or fructose utilization. The mutants could easily be isolated since they represented 80 to 90% of the population after two and three enrichment cycles. Glucokinase and fructokinase activities in the mutants were affected.