Transformation of yeast by a replicating hybrid plasmid.

Chimaeric plasmids have been constructed containing a yeast plasmid and fragments of yeast nuclear DNA linked to pMB9, a derivative of the ColEl plasmid from E. coli. Two plasmids were isolated which complement leuB mutations in E. coli. These plasmids have been used to develop a method for transforming a leu2 strain of S. cerevisiae to Leu+ with high frequency. The yeast transformants contained multiple plasmid copies which were recovered by transformation in E. coli. The yeast plasmid sequence recombined intramolecularly during propagation in yeast.     

Kluyveromyces lactis killer toxin inhibits adenylate cyclase of sensitive yeast cells

K1 killer toxin secreted by the K1 strain of Saccharomyces cerevisiae, has been well characterized. It is a simple protein of molecular weight (MW) 11,470 (ref. 3), encoded by a double-stranded, linear RNA plasmid, called M RNA, of MW 1.1-1.7 x 10(6) (refs 4-6). It is lethal to sensitive Saccharomyces cerevisiae which does not carry M RNA. Leakage of K+ and ATP is the first distinct response in sensitive cells, and the toxic action is thought to be due to its action as a protonophore or K+ ionophore. Recently, a further killer toxin has been found in Kluyveromyces lactis IFO 1267, and it is associated with the presence of the double-stranded linear DNA plasmids, pGK1-1 (MW 5.4 x 10(6)) and pGK1-2 (MW 8.4 x 10(6)). It has been shown, by curing pGK1-1 or deletion mapping, that the structural gene for the killer toxin and immunity-determining gene reside on the smaller plasmid. Moreover, the plasmids could be transferred from K. lactis to S. cerevisiae by protoplast fusion and protoplast transformation. As the K. lactis toxin is encoded by a DNA plasmid and has a relatively wider action spectrum than K1 killer toxin, the mode of action of the toxin is highly interesting. Here we report that K. lactis toxin inhibits adenylate cyclase in sensitive yeast cells and brings about arrest of the cells at the G1 stage.     

Identification of a G1-type cyclin puc1+ in the fission yeast Schizosaccharomyces pombe

In rapidly growing cells of the budding yeast Saccharomyces cerevisiae, the cell cycle is regulated chiefly at Start, just before the G1-S boundary, whereas in the fission yeast Schizosaccharomyces pombe, the cycle is predominantly regulated at G2-M. Both control points are present in both yeasts, and both require the p34cdc2 protein kinase. At G2-M, p34cdc2 kinase activity in S. pombe requires a B-type cyclin in a complex with p34cdc2; this complex is the same as MPF (maturation promoting factor). The p34cdc2 activity at the G1-S transition in S. cerevisiae may be regulated by a similar cyclin complex, using one of the products of a new class of cyclin genes (CLN1, CLN2 and WHI1 (DAF1/CLN3)). At least one is required for progression through the G1-S phase, and deletion of all three leads to G1 arrest. WHI1 was isolated as a dominant allele causing budding yeast cells to divide at a reduced size and was later independently identified as DAF1, a dominant allele of which rendered the cells refractory to the G1-arrest induced by the mating pheromone alpha-factor. The dominant alleles are truncations thought to yield proteins of increased stability, and the cells are accelerated through G1. Without WHI1 function, the cells are hypersensitive to alpha-factor, enlarged and delayed in G1. Heretofore, this G1-class of cyclins has not been identified in other organisms. We have isolated a G1-type cyclin gene called puc1+ from S. pombe, using a functional assay in S. cerevisiae. Expression of puc1+ in S. pombe indicates that it has a cyclin-like role in the fission yeast distinct from the role of the B-type mitotic cyclin.     

Need for DNA topoisomerase activity as a swivel for DNA replication for transcription of ribosomal RNA

Yeast strains with mutations in the genes for DNA topoisomerases I and II have been identified previously in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. The topoisomerase II mutants (top2) are conditional-lethal temperature-sensitive (ts) mutants. They are defective in the termination of DNA replication and the segregation of daughter chromosomes, but otherwise appear to replicate and transcribe DNA normally. Topoisomerase I mutants (top1), including strains with null mutations are viable and exhibit no obvious growth defects, demonstrating that DNA topoisomerase I is not essential for viability in yeast. In contrast to the single mutants, top1 top2 ts double mutants from both Schizosaccharomyces pombe and Saccharomyces cerevisiae grow poorly at the permissive temperature and stop growth rapidly at the non-permissive temperature. Here we report that DNA and ribosomal RNA synthesis are drastically inhibited in an S. cerevisiae top1 top2 ts double mutant at the restrictive temperature, but that the rate of poly(A)+ RNA synthesis is reduced only about threefold and transfer DNA synthesis remains relatively normal. The results suggest that DNA replication and at least ribosomal RNA synthesis require an active topoisomerase, presumably to act as a swivel to relieve torsional stress, and that either topoisomerase can perform the required function (except in termination of DNA replication where topoisomerase II is required).     

Plasmids from Staphylococcus aureus replicate in yeast Saccharomyces cerevisiae

It is known that some plasmids, such as RP4, can replicate in many Gram-negative bacteria. Certain small Staphylococcus aureus plasmids have an even broader host range, being able to replicate in not only phylogenetically distant Gram-positive bacteria such as Bacillus subtilis or Streptococcus pneumoniae, but also in the Gram-negative bacterium Escherichia coli. Here we have examined whether these plasmids can also replicate in a lower eukaryote, the yeast Saccharomyces cerevisiae. For this purpose we constructed hybrids between a S. aureus plasmid pC194 and an E. coli plasmid YIp5, which carries a ura-3 gene easy to select for in yeast but cannot replicate in this host. We found that the hybrids transformed yeast with high efficiency (as did hybrids between YIp5 and three other S. aureus plasmids); were maintained extrachromosomally in yeast; and were not modified during residence in yeast. We conclude from this evidence that S. aureus plasmids can replicate in yeast, which raises the questions of whether the replication signals used by prokaryotes and eukaryotes are similar, and how far up the phylogenetic tree the organisms still able to be hosts to S. aureus plasmids may be.     

S-phase feedback control in budding yeast independent of tyrosine phosphorylation of p34cdc28.

In somatic cells, entry into mitosis depends on the completion of DNA synthesis. This dependency is established by S-phase feedback controls that arrest cell division when damaged or unreplicated DNA is present. In the fission yeast Schizosaccharomyces pombe, mutations that interfere with the phosphorylation of tyrosine 15 (Y15) of p34cdc2, the protein kinase subunit of maturation promoting factor, accelerate the entry into mitosis and abolish the ability of unreplicated DNA to arrest cells in G2. Because the tyrosine phosphorylation of p34cdc2 is conserved in S. pombe, Xenopus, chicken and human cells, the regulation of p34cdc2-Y15 phosphorylation could be a universal mechanism mediating the S-phase feedback control and regulating the initiation of mitosis. We have investigated these phenomena in the budding yeast Saccharomyces cerevisiae. We report here that the CDC28 gene product (the S. cerevisiae homologue of cdc2) is phosphorylated on the equivalent tyrosine (Y19) during S phase but that mutations that prevent tyrosine phosphorylation do not lead to premature mitosis and do not abolish feedback controls. We have therefore demonstrated a mechanism that does not involve tyrosine phosphorylation of p34 by which cells arrest their division in response to the presence of unreplicated or damaged DNA. We speculate that this mechanism may not involve the inactivation of p34 catalytic activity.     

Nucleotide sequence of the yeast plasmid

The nucleotide sequence of the yeast DNA plasmid (2 mu circle) from Saccharomyces cerevisiae strain A364A D5 has been determined. The plasmid contains 6,318 base pairs, including two identical inverted repeats of 599 base pairs. Possible functions are suggested, and attributes of an improved vector for cloning foreign DNAs in yeast are discussed.     

Primary structure homology between the product of yeast cell division control gene CDC28 and vertebrate oncogenes

In the budding yeast, Saccharomyces cerevisiae, division is controlled in response to nutrient limitation and in preparation for conjugation. Cells deprived of an essential nutrient or responding to mating pheromones cease division and become synchronous in the G1 interval, apparently constrained from completing a critical event. This event has been given the operational designation of 'start'. We have isolated a large number of start mutations which confer on S. cerevisiae cells a conditional inability to complete start (Fig. 1) presumably because they define genes which must be expressed for the start event to be successfully completed. We have described the isolation on plasmids of one of the start genes, CDC28, by genetic complementation and initial characterization of its product. We now describe the DNA sequence of the gene CDC28.     

可降解淀粉酿酒酵母基因工程菌的构建及其生产应用

将酿酒酵母的rDNA片段,黑曲霉葡萄糖淀粉酶基因表达盒及G418抗性基因表达盒重组进经过改造的质粒pSP72,构建酿酒酵母整合型质粒YIp4RGAn及YIp19RGAn,转化酿酒酵母实验室菌株GRF18、生产菌株JL108、SD和JM,获得能高效表达葡萄糖淀粉酶和分解淀粉的酿酒酵母基因工菌。Southern印迹分析证明,葡萄糖淀粉酶基因已整合进工程菌染色体。这些工程菌在含有20%淀粉的培养基中培养,产酒率都在11%以上。     

Telomeric repeat from T. thermophila cross hybridizes with human telomeres

The ends (telomeres) of eukaryotic chromosomes must have special features to ensure their stability and complete replication. Studies in yeast, protozoa, slime moulds and flagellates show that telomeres are tandem repeats of simple sequences that have a G-rich and a C-rich strand. Mammalian telomeres have yet to be isolated and characterized, although a DNA fragment within 20 kilobases of the telomeres of the short arms of the human sex chromosomes has been isolated. Recently we showed that a chromosome from the fission yeast Schizosaccharomyces pombe could, in some cases, replicate as an autonomous mini-chromosome in mouse cells. By extrapolation from other systems, we reasoned that mouse telomeres could be added to the S. pombe chromosome ends in the mouse cells. On setting out to test this hypothesis we found to our surprise that the telomeric probe used (containing both the S. pombe and Tetrahymena thermophila repeats) hybridized to a series of discrete fragments in normal mouse DNA and DNA from a wide range of eukaryotes. We show here that the sequences hybridizing to this probe are located at the telomeres of most, if not all, human chromosomes and are similar to the Tetrahymena telomeric-repeat component of the probe.     

DNA self-recognition in the structure of Pot1 bound to telomeric single-stranded DNA

Telomeres, specialized protein-DNA complexes that cap the ends of linear chromosomes, are essential for protecting chromosomes from degradation and end-to-end fusions. The Pot1 (protection of telomeres 1) protein is a widely distributed eukaryotic end-capping protein, having been identified in fission yeast, microsporidia, plants and animals. Schizosaccharomyces pombe Pot1p is essential for telomere maintenance, and human POT1 has been implicated in telomerase regulation. Pot1 binds telomeric single-stranded DNA (ssDNA) with exceptionally high sequence specificity, the molecular basis of which has been unknown. Here we describe the 1.9-A-resolution crystal structure of the amino-terminal DNA-binding domain of S. pombe Pot1p complexed with ssDNA. The protein adopts an oligonucleotide/oligosaccharide-binding (OB) fold with two loops that protrude to form a clamp for ssDNA binding. The structure explains the sequence specificity of binding: in the context of the Pot1 protein, DNA self-recognition involving base-stacking and unusual G-T base pairs compacts the DNA. Any sequence change disrupts the ability of the DNA to form this structure, preventing it from contacting the array of protein hydrogen-bonding groups. The structure also explains how Pot1p avoids binding the vast excess of RNA in the nucleus.     

Ca~(2 )对酿酒酵母与粟酒裂殖酵母细胞增殖的影响

SD培养基中外加不同浓度的Ca2 可促进酿酒酵母与粟酒裂殖酵母细胞的增殖 ,但在促进增殖方式上存在明显差异 .随SD培养基中外Ca2 浓度增加 ,酿酒酵母到达稳定期的细胞终浓度也越高 ;而粟酒裂殖酵母生长到达稳定期细胞终浓度随Ca2 浓度增加而增加的效应不明显 .同时SD培养基中外加Ca2 对酿酒酵母的促进作用主要是通过加快生长对数期细胞分裂速度 ;对粟酒裂殖酵母主要是靠缩短生长延滞期来促进增殖 .     

Giant linear plasmids in Streptomyces which code for antibiotic biosynthesis genes

A number of examples of circular plasmids with specific functions are known in both prokaryotes and eukaryotes. Several linear plasmids have also been identified, but these are all relatively small: large linear plasmids cannot be separated from chromosomal DNA by conventional techniques. There are several cases where the genetic evidence suggests that a character is encoded by a plasmid but no plasmid can be physically detected. This has been the case for antibiotic synthesis genes in Streptomyces; in particular a plasmid SCP1 in Streptomyces coelicolor has been shown to be involved in methylenomycin production by genetic evidence. We report here the application of orthogonal-field-alternation gel electrophoresis to the isolation of linear plasmids from Streptomyces. We have discovered a large linear plasmid of around 520 kilobases in Streptomyces lasaliensis and subsequently similar giant linear plasmids in other Streptomyces strains. We have confirmed that genes for methylenomycin biosynthesis are located on a series of giant linear plasmids in S. coelicolor. These observations may bear on the genetic variability and unstable genetic character of Streptomyces species.     

Isolation and characterisation of a yeast chromosomal replicator.

A yeast DNA sequence that behaves as a chromosomal replicator, ars1 (autonomously replicating sequence), has been isolated. On transformation, ars1 allows autonomous replication of all co-linear DNA. The replicator can integrate into other replication units and can function in multimeric form. The 850-base pair ars1 element has no detectable homology to other yeast sequences. Such replicator-containing plasmids can be used for the isolation of DNA sequences in yeast cells as well as for the study of chromosomal DNA replication.     

Telomere-telomere recombination provides an express pathway for telomere acquisition

DNA termini from Tetrahymena and Oxytricha, which bear C4A2 and C4A4 repeats respectively, can support telomere formation in Saccharomyces cerevisiae by serving as substrates for the addition of yeast telomeric C1-3A repeats. Previously, we showed that linear plasmids with 108 base pairs of C4A4 DNA (YLp108CA) efficiently acquired telomeres, whereas plasmids containing 28-64 base pairs of C4A4 DNA also promoted telomere formation, but with reduced efficiency. Although many of the C4A4 termini on these plasmids underwent recombination with a C4A2 terminus, the mechanism of telomere-telomere recombination was not established. We now report the sequence of the C4A4 ends from the linear plasmids. The results provide strong evidence for a novel recombination process involving a gene conversion event that requires little homology, occurs at or near the boundary of telomeric and nontelomeric DNA, and resembles the recombination process involved in bacteriophage T4 DNA replication.     

粟酒裂殖酵母--一种良好的真核模式生物(Ⅱ)

粟酒裂殖酵母（Schizosaccharomyces pombe)虽然与酿酒酵母（Saccharomyces cerevisiae)同属子囊真菌，但许多研究表明两种酵母在系统分类，细胞周期，rRNA的生物合成和基因的组织、结构及基因的表达调控等方面并不相同，在某些方面粟酒裂殖酵母与高等动物却有一定的相似性，因此，粟酒裂殖酵母也是一种良好的研究真核生物的模式生物。     

Regulation of p34CDC28 tyrosine phosphorylation is not required for entry into mitosis in S. cerevisiae.

Progression from G2 to M phase in eukaryotes requires activation of a protein kinase composed of p34cdc2/CDC28 associated with G1-specific cyclins. In some organisms the activation of the kinase at the G2/M boundary is due to dephosphorylation of a highly conserved tyrosine residue at position 15 (Y15) of the cdc2 protein. Here we report that in the budding yeast Saccharomyces cerevisiae, p34CDC28 also undergoes cell-cycle regulated dephosphorylation on an equivalent tyrosine residue (Y19). However, in contrast to previous observations in S. pombe, Xenopus and mammalian cells, dephosphorylation of Y19 is not required for the activation of the CDC28/cyclin kinase. Furthermore, mutation of this tyrosine residue does not affect dependence of mitosis on DNA synthesis nor does it abolish G2 arrest induced by DNA damage. Our data imply that regulated phosphorylation of this tyrosine residue is not the 'universal' means by which the onset of mitosis is determined. We propose that there are other unidentified controls that regulate entry into mitosis.     

粟酒裂殖酵母--一种良好的真核模式生物(Ⅰ)

粟酒裂殖酵母(Schizosaccharomyces pombe)虽然与酿酒酵母(Saccharomyces cerevisiae)同属于子囊真菌,但许多研究表明两种酵母在系统分类、细胞周期、rRNA的生物合成和基因的组织、结构及基因的表达调控等方面并不相同,在某些方面粟酒裂殖酵母与高等动物却有一定的相似性,因此,粟酒裂殖酵母也是一种良好的研究真核生物的模式生物.