啤酒酵母DNA修复基因RAD24温度敏感突变株的分离及其特性的初步研究

以ＹＣｐｌａｃ系列带Ｔｒｐ、Ｈｉｓ和Ｕｒａ标志基因的载体为骨架构建含野生型和经羟胺处理的突变型的啤酒酵母ＲＡＤ２４基因质粒,用质粒替换方法分离ＲＡＤ２４基因温度敏感突变株（ｒａｄ２４－ｔｓ３）．紫外生存试验发现,ｒａｄ２４－ｔｓ３对紫外线敏感;同位素（３Ｈ－ＴｄＲ,３Ｈ－ＵＲ,３Ｈ－Ｌｅｕ）参入试验表明,该突变株ＤＮＡ、ＲＮＡ及蛋白质合成均较野生型明显降低．     

Nuclear Mutations in Saccharomyces Cerevisiae That Affect the Escape of DNA from Mitochondria to the Nucleus

We have inserted a yeast nuclear DNA fragment bearing the TRP1 gene and its associated origin of DNA replication, ARS1, into the functional mitochondrial chromosome of a strain carrying a chromosomal trp1 deletion. TRP1 was not phenotypically expressed within the organelle. However, this Trp(-) strain readily gave rise to respiratory competent Trp(+) clones that contained the TRP1/ARS1 fragment, associated with portions of mitochondrial DNA (mtDNA), replicating in their nuclei. Thus the Trp(+) clones arose as a result of DNA escaping from mitochondria and migrating to the nucleus. We have isolated 21 nuclear mutants in which the rate of mtDNA escape is increased by screening for increased rates of papillation to Trp(+). All 21 mutations were recessive and fell into six complementation groups, termed YME1-YME6. In addition to increasing the rate of mtDNA escape, yme1 mutations also caused a heat-sensitive respiratory deficient phenotype at 37° and a cold-sensitive growth defect on complete glucose medium at 14°. While the other yme mutations had no detectable growth phenotypes, synergistic interactions were observed in two double mutant combinations: a yme1, yme2 double mutant failed to respire at 30° and a yme4, yme6 double mutant failed to respire at all temperatures tested. None of the respiratory defects were caused by loss of functional mtDNA. These findings suggest that yme1, yme2, yme4 and yme6 mutations alter mitochondrial functions and thereby lead to an increased rate of DNA escape from the organelle.     

产甘油假丝酵母TRP1基因的克隆与功能分析

产甘油假丝酵母(Candida glycerinogenes) WL2002-5 是我国发酵甘油生产菌种, 具有高产甘油和耐高渗透压的优良性能。本文采用遗传互补的方法从产甘油假丝酵母基因文库中克隆了TRP1基因(CgTRP1)。序列分析显示, 该基因编码区全长735 bp, 编码的磷酸核糖氨基苯甲酸同分异构酶(CgPRAI)氨基酸序列与其他酵母来源的PRAI蛋白同源性在32.9%~49.2%之间。功能互补实验显示, CgTRP1基因在高拷贝情况下可以互补酿酒酵母trp1基因功能但在低拷贝情况下只能部分互补酿酒酵母trp1基因功能, 是一条功能明确、结构完整的酵母新基因。在CgTRP1 基因下游发现另一蛋白编码基因, 编码的氨基酸序列与酵母无机焦磷酸酶有很高的相似性。     

酵母菌色氨酸合成酶基因的克隆与表达

用RemHI酶切酿酒酵母(Saceharomyces cercuisiae) 1412-4D染色体DNA,通过蔗糖梯度分离2-4kb DNA片段并插入穿棱质粒pCN60,构成1412-4D基因文库。从基因文库中提取重组质粒,转化受体菌C9(a,trp5,adcl,ade6),用直接功能互补法,分离到9株重组质粒,它们都含有3．2kb的TRP5 DNA片段,分别命名为pCN60(trps)1-90转化体中色氨酸合成酶的酶活水平比原始菌株1412-4D高3倍。     

A tRNA(TRP) gene mediates the suppression of cbs2-223 previously attributed to ABC1/COQ8

The Saccharomyces cerevisiae gene ABC1 was originally isolated as a multicopy suppressor of a yeast strain harboring a mutation in a cytochrome b translational activator (cbs2-223). Based on this identification, Abc1p was postulated to activate the bc1 complex and function as a chaperone of cytochrome b. ABC1 was subsequently identified as COQ8 and found to be necessary for yeast coenzyme Q synthesis. In this work we show that a segment of yeast genomic DNA containing ABC1/COQ8 and neighboring genes suppresses the respiratory and Q-deficient phenotypes of the coq6 mutant, coq6-1. COQ6 is essential for yeast coenzyme Q biosynthesis. We show that a tRNA(TRP) gene located downstream of ABC1/COQ8 mediates suppression of the cbs2-223 and coq6-1 mutations, and each is identified here as containing UGA nonsense codons. The inability of ABC1/COQ8 to suppress the cbs2-223 allele in multicopy indicates it may not be a chaperone as previously reported.     

Construction, replication, and chromatin structure of TRP1 RI circle, a multiple-copy synthetic plasmid derived from Saccharomyces cerevisiae chromosomal DNA.

Transformation studies with Saccharomyces cerevisiae (bakers' yeast) have identified DNA sequences which permit extrachromosomal maintenance of recombinant DNA plasmids in transformed cells. It has been hypothesized that such sequences (called ARS for autonomously replicating sequence) serve as initiation sites for DNA replication in recombinant DNA plasmids and that they represent the normal sites for initiation of replication in yeast chromosomal DNA. We have constructed a novel plasmid called TRP1 R1 Circle which consists solely of 1,453 base pairs of yeast chromosomal DNA. TRP1 RI Circle contains both the TRP1 gene and a sequence called ARS1. This plasmid is found in 100 to 200 copies per cell and is relatively stable during both mitotic and meiotic cell cycles. Replication of TRP1 RI Circle requires the products of the same genes (CDC28, CDC4, CDC7, and CDC8) required for replication of chromosomaL DNA. Like chromosomal DNA, its replication does not occur in cells arrested in the B1 phase of the cell cycle by incubation with the yeast pheromone alpha-factor. In addition, TRP1 RI Circle DNA is organized into nucleosomes whose size and spacing are indistinguishable from that of bulk yeast chromatin. These results indicate that TRP1 RI Circle has the replicative and structural properties expected for an origin of replication from yeast chromosomal DNA. Thus, this plasmid is a suitable model for further studies of yeast DNA replication in both cells and cell-free extracts.     

低双乙酰抗老化啤酒酵母工程菌的构建

用来源于啤酒酵母的γ-谷氨酰半胱氨酸合成酶基因（GSH1）和铜抗性基因（CUP1）取代质粒pLZ-2中α-乙酰乳酸合成酶基因（ILV2）内部约2.3kb的DNA片段,构建成重组质粒pICG。限制酶酶切质粒pICG后获得在基因GSH1和CUP1两端含有ILV2序列的6.0kb的DNA片段。用此片段转化啤酒酵母YSF31,得到铜抗性高的转化子。并通过PCR和α-乙酰乳酸合成酶（AHAS）活性测定筛选到酵母工程菌。小试实验结果表明酵母工程菌谷胱甘肽含量比受体高34％,而双乙酰含量是受体的75％。其他发酵指标并没有发生改变。中试实验表明酵母工程菌发酵周期缩短3d,而且成品啤酒的保鲜时间延长50％。由于DNA操作过程中没有外源基因介入,因此啤酒酵母工程菌为生物安全的自克隆菌株,具有重要的实际应用价值。     

Construction of an Arxula adeninivorans host-vector system based on trp1 complementation

A host/vector expression system based on an Arxula adeninivorans Delta atrp1 gene disruption mutant has been constructed. For this purpose the ATRP1 gene encoding a phosphoribosyl anthranilate isomerase was isolated from the yeast A. adeninivorans and its genome locus was characterized. The Delta atrp1 mutant was generated applying an amplified DNA fragment containing the ALEU2m gene flanked by ATRP1 gene sequences of some 750 bp. The generated auxotrophic host strain was transformed with the plasmid pAL-ATRP1-amyA, which contains the ATRP1 gene as selection marker and the 25S rDNA for targeting. For expression assessment, the plasmid was equipped with an expression cassette consisting of the Bacillus amyloliquefaciens-derived amyA gene fused to the constitutive A. adeninivorans-derived TEF1 promoter and Saccharomyces cerevisiae-derived PHO5 terminator. Transformants contained a single chromosomal copy of the heterologous DNA and were found to be mitotically stable. In initial fermentation trials on a 200 ml shake flask scale maximal alpha-amylase product levels of ca. 300 nkat ml(-1) were observed after 72 h of cultivation with more than 95% of the recombinant alpha-amylase accumulated in the culture medium.     

酿酒酵母(Saccharomyces cerevisiae)半乳糖可诱导表达系统特性的研究

把大肠杆菌β-半乳糖苷酶基因克隆到带有酵母半乳糖可诱导启动子GAL1的穿梭表达质粒pYESZ中,并把得到的重组质粒分别转化到两种不同遗传性状的宿主菌中,其中一株菌为蛋白酶活性缺失90％以上的pep4－3突变菌株。通过比较两株重组菌产生的β-半乳糖苷酶活性水平发现在所述实验条件下,蛋白酶缺失突变菌株中产生的β-卜半乳糖苷酶活水平不仅均要高于另一对照菌株,并且pep4-3突变菌株表现出受葡萄糖阻遏的严紧程度高及对诱导反应迅速等特点。此外,带有重组质粒的pep4－3突变菌株在葡萄糖阻遏培养基中最大生长量和重组对照菌株基本相同,但β-半乳糖苷酶在pep4－3突变菌株中的表达对细胞生长的影响明显小于对照菌株。     

Cloning and characterization of a gene which determines osmotic stability in Saccharomyces cerevisiae.

The srb1-1 mutation of Saccharomyces cerevisiae is an ochre allele which renders the yeast dependent on an osmotic stabilizer for growth and gives the cells the ability to lyse on transfer to hypotonic conditions. A DNA fragment which complements both of these phenotypic effects has been cloned. This clone contains a functional gene which is transcribed into a 2.3-kb polyadenylated mRNA molecule. Transformation of yeast strains carrying defined suppressible alleles demonstrated that the cloned fragment does not contain a nonsense suppressor. Integrative transformation and gene disruption experiments, when combined with classical genetic analysis, confirmed that the cloned fragment contained the wild-type SRB1 gene. The integrated marker was used to map SRB1 to chromosome XV by Southern hybridization and pulsed-field gel electrophoresis. A disruption mutant created by the insertion of a TRP1 marker into SRB1 displayed only the lysis ability phenotype and was not dependent on an osmotic stabilizer for growth. Lysis ability was acquired by growth in (or transfer to) an osmotically stabilized environment, but only under conditions which permitted budding. It is inferred that budding cells lyse with a higher probability and that weak points in the wall at the site of budding are involved in the process. The biotechnological potential of the cloned gene and the disruption mutant is discussed.     

Transformation of Kluyveromyces fragilis

For the transformation of the yeast species Kluyveromyces fragilis, we have constructed a vector containing a bacterial kanamycin resistance (Kmr) gene, the TRP1 gene of Saccharomyces cerevisiae, and an autonomously replicating sequence of Kluyveromyces lactis called KARS2 . By utilizing the method based on treatment by alkali cations and with the Kmr gene as the selective marker, a wild-type strain of K. fragilis was transformed to resistance against the antibiotic G418 . In the transformed cell the plasmid replicates autonomously. The same plasmid could also be used to transform S. cerevisiae trp1 mutant to Trp+. Thus, KARS2 of K. lactis enables the vector to replicate in K. fragilis, K. lactis, and S. cerevisiae, whereas ARS1 of S. cerevisiae allows autonomous replication only in S. cerevisiae.     

Recombinant industrial brewing yeast strains with ADH2 interruption using self-cloning GSH1+CUP1 cassette

A self-cloning module for gene knock-out and knock-in in industrial brewing yeast strain was constructed that contains copper resistance and γ-glutamylcysteine synthetase gene cassette, flanked by alcohol dehydrogenase II gene ( ADH2 ) of Saccharomyces cerevisiae . The module was used to obtain recombined strains RY1 and RY2 by targeting the ADH2 locus of host Y1. RY1 and RY2 were genetically stable. PCR and enzyme activity analysis of RY1 and RY2 cells showed that one copy of ADH2 was deleted by GSH1 + CUP1 insertion, and an additional copy of wild type was still present. The fermentation ability of the recombinants was not changed after genetic modification, and a high level of glutathione (GSH) was secreted, resulting from GSH1 overexpression, which codes for γ-glutamylcysteine synthetase. A pilot-scale brewing test for RY1 and RY2 indicated that acetaldehyde content in fermenting liquor decreased by 21–22%, GSH content increased by 20–22% compared with the host, the antioxidizability of the recombinants was improved, and the sensorial evaluation was also better than that of the host. No heterologous DNA was harbored in the recombinants; therefore, they could be applied in the beer industry in terms of their biosafety.     

小麦吸收土壤磷转运子在酵母突变体中的功能互补分析

以小麦磷转运子全长编码cDMA(TaPT2)为探针与小麦基因组DNA进行Southern杂交,结果表明,在小麦基因组中存在该基因的不同家族成员,另外,将TaPT2基因转入酵母突变体MB192中,以野生型菌株YPH084为对照,分别检测YTaPT2,YPH084和MB192酸性磷酸酶分泌情况,生长情况以及对培养基的磷吸收情况,得到结论：TaPT2的功能与酵母磷转运子编码基因PHO84相似,具有增强酵母吸收磷的作用。     

Multiple genes encode the translation elongation factor EF-1 gamma in Saccharomyces cerevisiae.

A gene encoding a yeast homologue of translation elongation factor 1 gamma (EF-1 gamma), TEF3, was isolated as a gene dosage extragenic suppressor of the cold-sensitive phenotype of the Saccharomyces cerevisiae drs2 mutant. The drs2 mutant is deficient in the assembly of 40S ribosomal subunits. We have identified a second gene, TEF4, that encodes a protein highly related to both the Tef3p protein (Tef3p), and EF-1 gamma isolated from other organisms. In contrast to TEF3, the TEF4 gene contains an intron. Gene disruptions showed that neither gene is required for mitotic growth. Haploid spores containing disruptions of both genes are viable and have no defects in ribosomal subunit composition or polyribosomes. Unlike TEF3, extra copies of TEF4 do not suppress the cold-sensitive 40S ribosomal subunit deficiency of a drs2 strain. Low-stringency genomic Southern hybridization analysis indicates there may be additional yeast genes related to TEF3 and TEF4.     

Molecular Genetics of Cryptopleurine Resistance in Saccharomyces Cerevisiae: Expression of a Ribosomal Protein Gene Family

The Saccharomyces cerevisiae CRY1 gene encodes the 40S ribosomal subunit protein rp59 and confers sensitivity to the protein synthesis inhibitor cryptopleurine. A yeast strain containing the cry1-δ1::URA3 null allele is viable, cryptopleurine sensitive (Cry(S)), and expresses rp59 mRNA, suggesting that there is a second functional CRY gene. The CRY2 gene has been isolated from a yeast genomic library cloned in bacteriophage λ, using a CRY1 DNA probe. The DNA sequence of the CRY2 gene contains an open reading frame encoding ribosomal protein 59 that differs at five residues from rp59 encoded by the CRY1 gene. The CRY2 gene was mapped to the left arm of chromosome X, centromere-proximal to cdc6 and immediately adjacent to ribosomal protein genes RPS24A and RPL46. Ribosomal protein 59 is an essential protein; upon sporulation of a diploid doubly heterozygous for cry1-δ2::TRP1 cry2-δ1::LEU2 null alleles, no spore clones containing both null alleles were recovered. Several results indicate that CRY2 is expressed, but at lower levels than CRY1: (1) Introduction of CRY2 on high copy plasmids into Cry(R) yeast of genotype cry1 CRY2 confers a Cry(S) phenotype. Transformation of these Cry(R) yeast with CRY2 on a low copy CEN plasmid does not confer a Cry(S) phenotype. (2) Haploids containing the cry1-δ2::TRP1 null allele have a deficit of 40S ribosomal subunits, but cry2-δ1::LEU2 strains have wild-type amounts of 40S ribosomal subunits. (3) CRY2 mRNA is present at lower levels than CRY1 mRNA. (4) Higher levels of β-galactosidase are expressed from a CRY1-lacZ gene fusion than from a CRY2-lacZ gene fusion. Mutations that alter or eliminate the last amino acid of rp59 encoded by either CRY1 or CRY2 result in resistance to cryptopleurine. Because CRY2 (and cry2) is expressed at lower levels than CRY1 (and cry1), the Cry(R) phenotype of cry2 mutants is only expressed in strains containing a cry1-δ null allele.     

Cloning of genes related to exo-beta-glucanase production in Saccharomyces cerevisiae: characterization of an exo-beta-glucanase structural gene

The EXG1 gene of Saccharomyces cerevisiae was cloned and identified by complementation of a mutant strain (exg1-2) with highly reduced extracellular exo-beta-1,3-glucanase (EXG) activity. Two recombinant plasmids containing an overlapping region of 5.2 kb were isolated from a genomic DNA library and characterized by restriction mapping. The coding region was located by subcloning the original DNA inserts in a 2.7-kb HindIII-XhoI fragment. Exg+ strains and Exg- mutants transformed with yeast multicopy plasmids containing this DNA fragment showed an EXG activity 5- to 20-fold higher than for the untransformed Exg+ wild-type (wt) strains. The overproduced EXG had the same enzymic activity on different substrates, and showed the same electrophoretic behaviour on polyacrylamide gels and identical properties upon filtration through Sephacryl S-200 as those of the main EXG from Exg+ wt strains. The EXG1 gene transformed Schizosaccharomyces pombe, yielding extracellular EXG activity which showed cross-reactivity with anti-S. cervisiae EXG antibodies. A fragment including only a part of the EXG1 region was subcloned into the integrating vector YIp5, and the resulting plasmid was used to transform an Exg+ strain. Genetic and Southern analysis of several stable Exg- transformants showed that the fragment integrated by homology with the EXG1 locus. The chromosomal DNA fragment into which the plasmid integrated has a restriction pattern identical to that of the fragment on which we had previously identified the putative EXG1 gene. Only one copy of the EXG1 gene per genome was found in several strains tested by Southern analysis. Furthermore, two additional recombinant plasmids sharing a yeast DNA fragment of about 4.1 kb, which partially complements the exg1-2 mutation but which shows no homology with the 2.7-kb fragment containing the EXG1 gene, were also identified in this study. This 4.1-kb DNA fragment does not appear to contain an extragenic suppressor and could be related in some way to EXG production in S. cerevisiae.     

Sequence of a yeast DNA fragment containing a chromosomal replicator and the TRP1 gene

The DNA sequence of a 1.45 kb EcoRI fragment from the yeast (Saccharomyces cerevisiae) TRP1 region has been determined. The fragment contains the TRP1 gene and a yeast chromosomal replicator. The TRP1 gene has been located on the fragment by analysis of potential initiation and termination codons in the DNA sequence. This location has been confirmed by subcloning portions of the fragment. Both the 5' and 3' noncoding regions of the TRP1 gene contain sequence homologies with analogous areas surrounding other yeast genes. The yeast replicator has been localized in a region near the 3' end of the TRP1 gene. The DNA sequence in this region contains several structural features which may be involved in the initiation of DNA replication.