Isolation and sequence analysis of a K. lactis chromosomal DNA element able to autonomously replicate in S. cerevisiae and K. lactis

We have undertaken a search for autonomously replicating (ARSs) from Kluyveromyces lactis chromosomal DNA able to sustain plasmid replication in K. lactis and in Saccharomyces cerevisiae. The discovery of such sequences might be interesting for the comparison of ARSs from different sources and possibly useful for the construction of multivalent vectors. HindIII fragments from K. lactis chromosomal DNA were inserted in the YIp5 plasmid (lacking an origin of replication) and the resulting chimaeric plasmids were selected for the ability to transform S. cerevisiae. Four plasmids were identified and further analysed. Two contained the same 1.8 kb K. lactis fragment and transformed both K. lactis and S. cerevisiae with the same efficiency and stability, whereas the third transformed only S. cerevisiae and the fourth transformed K. lactis with a higher efficiency than S. cerevisiae. A detailed study was performed on the 1.8 kb fragment which exhibited ARS function in both yeasts. The fragment was subcloned using different restriction enzymes and Bal31 exonuclease. Subclones were tested for ARS function. ARS activities in the two yeasts were localized in the same 100 bp region. Sequencing demonstrated the presence in this region of the dodecanucleotide 5'ATTTATTGTTTT3' differing from the ARS core consensus of S. cerevisiae only by a T insertion. A similar nucleotide sequence is present in the putative replication origin of the 2 mu-like plasmid pKD1 which stably replicates in K. lactis. Homologies with ARSs from S. cerevisiae were also found in the regions flanking the above-mentioned dodecanucleotide.     

Linguistic analysis of chromosome III DNA sequence of Saccharomyces cerevisiae

The analysis of the Saccharomyces cerevisiae chromosome III DNA sequence by computer (‘in silico’) permits the definition of its linguistic characteristics. These characteristics include the designation of non-randomly occurring oligonucleotides, their distribution along the chromosome, and the distribution of some particular homopolymers. All these elements may contribute to the understanding of the organization of information on the chromosome.     

DNA sequence analysis of the YCN2 region of chromosome XI in Saccharomyces cerevisiae

A 6·8 kbp DNA fragment localized to the left arm of chromosome XI from Saccharomyces cerevisiae was sequenced and analysed (EMBL accession no. X69765). Two genes involved in protein phosphatase activity were identified: YCN2 and an open reading frame encoding a protein that shares 46% amino acid identity with the sds22⁺ protein from Schizosaccharomyces pombe. A comparison of the genomic YCN2 sequence with the published cDNA sequence suggests the presence of an intron near the 5′ end of the gene. Further sequence analysis suggests the presence of three additional genes near YCN2: a mitochondrial acyl-carrier protein, a gene encoding a putative hydrophobic protein, and a new gene coding for a tRNA^Leu (UAA) isoacceptor located near a delta sequence.     

Genetic and molecular analysis of hybrids in the genus Saccharomyces involving S. cerevisiae,S. uvarum and a new species,S. douglasii

We have studied the phenomenon of infertility of yeast hybrids obtained with physiological conditions under the control of compatible mating systems. The yeasts investigated are three Saccharomyces species: S. cerevisiae, S. uvarum and a new species, S. douglasii. The diploid hybrids from crosses between these species sporulate well but are essentially infertile. The rare viable spores, one per 10⁴ to 10⁵ asci, that have been examined carry a complete genome comprised of chromosomes contributed by both parents but invariably have extra chromosomes, i.e. they are generally disomic for at least two or three chromosomes. This observation is consistent with a failure, in meiosis I, of the pairing and disjunction of homologous chromosomes which in most cases results in spores with an incomplete set of chromosomes. This apparent lack of pairing of ‘homeologous’ chromosomes in meiosis I was analysed in most detail with S. cerevisiae/S. douglasii hybrids. As a genetic tool we studied frequencies of recombination, taking advantage of an S. douglasii breeding stock of some 50 identified mutations in non-switching haploids. Recombination, although markedly reduced, could be observed at both the chromosomal and allelic levels, implying a sporadic pairing in meiosis to allow genetic exchange. Meiotic recombination frequencies were studied for 14 gene pairs and generally found to be reduced ten-fold. Heteroallelic recombination (gene conversion) frequencies were measured at 22 loci and were judged to be reduced at least two- to 100-fold. DNA hybridization experiments with S. cerevisiae gene probes gave results consistent with low DNA sequence homologies between S. cerevisiae and S. douglasii. Moreover, by chance, our experiments disclosed another Saccharomyces strain (CBS2908, originally classified as S. cerevisiae) with hybridization patterns identical to S. douglasii except for the hybridization with the Ty transposon probes. Crosses between CBS2908 and S. douglasii yielded diploid hybrids with 80–90% spore viability, thus establishing a second member of the S. douglasii species.     

Molecular cloning and analysis of autonomous replicating sequence of Candida maltosa.

A Candida maltosa chromosomal DNA fragment which confers high frequency transformation of C. maltosa and autonomous replication of recombinant plasmids was cloned and sequenced. Analysis of the nucleotide sequence of the cloned DNA revealed a sequence homologous for C. maltosa autonomously replicating sequence (ARS) elements. Vector pRJ1 for C. maltosa was constructed, which contained a 1.3 kb ARS sequence, pICEM-19H and the ADE1 gene of C. maltosa. Southern blot analysis suggested that the copy number of pRJ1 in C. maltosa was approximately 20 per genome. The sequence analysis also revealed an open reading frame, encoding a polypeptide with high homology (70%) to the RS15 protein of Brugia pagangi. This open reading frame has an intron with canonical sites for correct splicing in Saccharomyces cerevisiae.     

几种乳酸菌S-层蛋白的普查以及slp基因的克隆与序列分析

从内蒙古传统发酵乳制品中分离的几种乳酸菌,包括干酪乳杆菌、乳酸乳球菌、发酵乳杆菌、植物乳杆菌、嗜酸乳杆菌、双歧乳杆菌和嗜酸乳杆菌标准株(L.acidophilus ATCC4356)等为实验材料,应用SDS-PAGE方法和PCK方法分别进行S-层蛋白(S-layers pro-tein,SLP)的普查和slp基因的检测.结果表明:在各种乳酸茵中,经SDS-PAGE电泳方法检测,只有植物乳杆菌、嗜酸乳杆菌和其标准菌株的样品中出现膜外蛋白的可疑条带,大小为44～66ku之间,与文献报道的slp基因表达产物大小范围是一致;经PCR方法检测,只有嗜酸乳杆菌和其标准菌株L.acidoilus ATCC4356中扩增出slp基因可疑条带,其大小约为1 300 bp.并对嗜酸乳杆菌slp基因进行克隆、基因序列测序和分析.     

Specificity of DNA uptake during whole cell transformation of S. cerevisiae

We have studied the mechanism of DNA transformation of whole yeast cells in Saccharomyces cerevisiae with particular emphasis on the role of the cell wall complex in DNA uptake. Two new aspects of the process have been investigated in order to evaluate its specificity. Such aspects are: (i) effect of monovalent vs. divalent cations during incubation with the transforming DNA and (ii) timing of DNA adsorption and uptake. We found that the specificity for cation requirement is a strain-dependent characteristic influenced by the presence of transforming DNA in the cell suspension. This finding is supported by reports from several laboratories that some yeast strains show mutually exclusive transformability with monovalent vs. divalent cations. While irreversible adsorption of plasmid DNA molecules is induced by both heat shock and polyethylene-glycol (PEG), DNA uptake seems to occur only after the removal of PEG. In the course of this study we have developed a new, alternative method of whole cell DNA transformation with CaCl2 able to transform strains that do not respond to other methods.     

IX. Yeast sequencing reports. Cloning and sequence of REV7, a gene whose function is required for DNA damage-induced mutagenesis in Saccharomyces cerevisiae

The function of the REV7 gene is required for DNA damage-induced mutagenesis in budding yeast, Saccharomyces cerevisiae, and is therefore thought to promote replication past sites of mutagen damage in the DNA template. We have cloned this gene by complementation of the rev7-2 mutant defect, and determined its sequence. REV7 encodes a predicted protein of M_r 28 759 which is unlike any other protein in the NCBI non-redundant protein sequence data base, and which is inessential for viability. The sequence of the 3·88 kb yeast genomic fragment containing REV7 has been deposited in Genbank accession number U07228.     

Isolation, DNA sequence and regulation of a new cell division cycle gene from the yeast Saccharomyces cerevisiae

A new complementation group of temperature-sensitive mutants of the yeast Saccharomyces cerevisiae (ts26-1 and ts26-2) has been isolated and characterized. This mutation maps at 40.7 cM from arg8 and 48.9 cM from arg1 on the left arm of chromosome XV of yeast, providing that it is a newly identified gene. The dumbbell-shape terminal morphology of the mutant cells at the restrictive temperatures is a characteristic of mutants defective in DNA replication. To study the defect of macromolecule synthesis in the mutant cells, DNA, RNA, and protein synthesis were measured at both permissive and restrictive temperatures. The data suggest that the primary defect of this mutation is at the initiation step of DNA synthesis. The gene has been cloned from an S. cerevisiae genomic library by rescue of the conditional lethality of the mutants. It is present as a single copy in the haploid genome. DNA-RNA hybridization of the gene has identified 1 kb RNA, which is under cell-division-cycle control. DNA sequence analysis of the gene has identified an open reading frame capable of encoding a protein of molecular weight 25,055 (214 amino acids).     

Microfluidics device for single cell gene expression analysis in Saccharomyces cerevisiae

We have measured single-cell gene expression over time using a microfluidics-based flow cell which physically traps individual yeast using microm-sized structures (yeast jails). Our goal was to determine variability of gene expression within a cell over time, as well as variability between individual cells. In our flow cell system, yeast jails are fabricated out of PDMS and gene expression is visualized using fluorescently-tagged proteins of interest. Previously, single-cell yeast work has been done using micromanipulation on agar, or FACS. In the present device agar is eliminated, resulting in a superior optical system. The flow of media through the flow cell washes daughter cells away, eliminating the need for micromanipulation. Unlike FACS, the described device can track individual yeast over a time course of many hours. The flow cells are compatible with the needs of quantitative fluorescence microscopy, and allow simultaneous measurements to be done on a large number of individual yeast. We used these flow cells to determine the expression of HSP104-GFPand RAS2-YFP, genes known to affect yeast life span. The results demonstrate inter-cell variation in expression of both genes that could not have been detected without this single-cell analysis.     

Molecular cloning,characterizing, and expression analysis of CTR1 genes in harvested papaya fruit

Fruit softening is an ethylene-dependent ripening event. 1-Methylcyclopropene (1-MCP), a synthetic plant growth regulator structurally related to the natural plant hormone ethylene, is used to slow down the fruit ripening. However, inappropriate 1-MCP treatment tends to cause the elastic texture in papaya fruit. Constitutive triple response 1 (CTR1), a downstream protein of the ethylene receptors, acts as a negative regulator of ethylene signaling. To elucidate the signal transduction pathway involved in the ethylene regulation during papaya ripening and softening, four genes homologous to Arabidopsis CTR1 were isolated from papaya fruit and designated as CpCTR1–4. Their molecular and biochemical properties were characterized, and their expression patterns in papaya fruit after treatments with 1-MCP were investigated. Four CTR1-like genes differ in sequence length and molecular size, and have a relative distance relationship in the evolutionary tree analysis. However, sequences analysis showed that the C-terminus of CpCTR1–4 proteins contained the highly conserved kinase domains, including a protein kinase ATP-binding signature (IGAGSFGTVH) and a serine/threonine protein kinase active site signature (IVHRDLKSPNLLV). Their N-terminus contains the conserved motifs (CN box) that exist in all CTR1-like proteins. CpCTR1–4 proteins were predicted to be located differently in either chloroplast or nuclei or both. Gene expression analysis showed that 1-MCP treatment significantly repressed the expression of CpCTRs and high concentration of 1-MCP treatment had a more significant effect. These results suggested that CpCTR1–4 genes may play different roles in papaya fruit ripening and softening and that the rubbery fruit might be relate to the expression of CpCTRs genes.     

酿酒酵母的基因改良

酿酒酵母的发酵性能直接影响到酒的质量与生产成本。利用基因工程改良酿酒酵母可提高其生产性能。基因改良酿酒酵母的研究和应用有：①增加酿酒酵母发酵性能的基因改良，如：构建含α-乙酰乳酸脱羧酶基因的低双乙酰工程酵母；含乙醇乙酰酶基因的高生香工程酵母；含糖化酶、葡聚糖酶等基因的高发酵度工程酵母；高絮凝性工程酵母和嗜杀酵母。②增强或缺失酵母自身基因的菌种改良，如构建高级醇低生成量的工程酵母和构建双乙酰低生成量的工程酵母。     

Genetic and molecular analysis of DNA43 and DNA52: two new cell-cycle genes in Saccharomyces cerevisiae.

Two Saccharomyces cerevisiae genes previously unknown to be required for DNA synthesis have been identified by screening a collection of temperature-sensitive mutants. The effects of mutations in DNA43 and DNA52 on the rate of S phase DNA synthesis were detected by monitoring DNA synthesis in synchronous populations that were obtained by isopycnic density centrifugation. dna43-1 and dna52-1 cells undergo cell-cycle arrest at the restrictive temperature (37 degrees C), exhibiting a large-budded terminal phenotype; the nuclei of arrested cells are located at the neck of the bud and have failed to undergo DNA replication. These phenotypes suggest that DNA43 and DNA52 are required for entry into or completion of S phase. DNA43 and DNA52 were cloned by their abilities to suppress the temperature-sensitive lethal phenotypes of dna43-1 and dna52-1 cells, respectively. DNA sequence analysis suggested that DNA43 and DNA52 encode proteins of 59.6 and 80.6 kDa, respectively. Both DNA43 and DNA52 are essential for viability and genetic mapping experiments indicate that they represent previously unidentified genes: DNA43 is located on chromosome IX, 32 cM distal from his5 and DNA52 is located on chromosome IV, 0.9 cM from cdc34.     

一种酿酒酵母表达cDNA文库分离纤维质降解酶系基因的方法

目的:建立一种筛选自然界产纤维质降解酶系基因的新方法。方法:对酿酒酵母表达载体pYES2多克隆位点加入真核生物稀有限制性酶切位点SfiI进行改造,利用SMART技术,以大肠杆菌文库为转导,构建了拟青霉(Paecilomyces sp.H28)的酿酒酵母全长cDNA表达文库。结果:利用纤维质-刚果红染色法从文库筛选到多种纤维素和半纤维素降解酶系基因。结论:成功构建了拟青霉的酿酒酵母表达cDNA文库,加快了纤维质降解酶系基因的快速分离,也为其它相关基因的快速分离提供了有益的借鉴。     

Molecular cloning and sequence analysis of Zygosaccharomyces rouxii ADE2 gene encoding a phosphoribosyl-aminoimidazole carboxylase.

The nucleotide sequence of a 2.8 kb fragment containing the ADE2 gene of the osmotolerant yeast Zygosaccharomyces rouxii has been determined. The gene was cloned from a Z. rouxii genomic DNA library by complementation of the Saccharomyces cerevisae ade2 mutant strain. The sequenced DNA fragment contains a 1710 bp open reading frame predicting a protein of 570 amino acids. The deduced amino acid sequence shares a high degree of homology with Ade2p homologues in five other yeast species.     

Cloning, sequence analysis and overexpression of a Saccharomyces cerevisiae endopolygalacturonase-encoding gene (PGL1)

Only a few yeast strains produce pectin-degrading enzymes such as pectin esterases and depolymerases (hydrolases and lyases). Strain SCPP is the only known Saccharomyces strain to produce these pectinases. One of these pectolytic enzymes. PGL1-encoded endopolygalacturonase (EC 3.2.1.15), hydrolyses the alpha-1,4-glycosidic bonds within the rhamnogalacturonan chains in pectic substances. This paper presents the cloning and sequencing of the first S. cerevisiae gene involved in pectin degradation. Few differences were found between the two deduced amino acid sequences encoded by PGL1-1 from a pectolytic (PG+) strain (SCPP) and PGL1-2 from a non-pectolytic (PG-) strain (X2180-1B). Similarities were found with other polygalacturonases from plants and other microorganisms. Of the two S. cerevisiae genes, only the one isolated from strain SCPP was able, by overexpression, to confer endopolygalacturonase activity to a laboratory strain of S. cerevisiae. Overexpression of PGL1-1 gene in a non-pectolytic strain resulted in halo formation on polygalacturonic acid-containing agar plates stained with ruthenium red.     

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: Isolation of the MAK16 gene and analysis of an adjacent gene essential for growth at low temperatures

MAK16 is an essential gene on chromosome I defined by the thermosensitive lethal mak16–1 mutation. MAK16 is also necessary for M double-stranded RNA replication at the permissive temperature for cell growth. As part of an effort to clone all the DNA from chromosome I, plasmids that complemented both the temperature-sensitive growth defect, and the M₁ replication defects of mak16–1 strains were isolated from a plasmid YCp50: Saccharomyces cerevisiae recombinant DNA library. The two plasmids analysed contained overlapping inserts that hybridized proportionally to strains carrying different dosages of chromosome I. Furthermore, integration of a fragment of one of these clones occurred at a site linked to ade1, confirming that this clone was derived from the appropriate region of chromosome I. An open reading frame adjacent to MAK16 potentially coding for a 468 amino acid protein was defined by sequence analysis. 185 amino acids of this open reading frame were replaced with a 1·2 kb fragment carrying the S. cerevisiae URA3 gene by a one-step gene disruption. The resulting strains grew at a rate indistinguishable from the wild type at 20°C, 30°C, or 37°C, but could not grow at 8°C. The deleted region is thus essential only at 8°C, and we name this gene LTE1 (low temperature essential).     

安琪酿酒酵母与管囊酵母原生质体制备和再生

通过紫外诱变筛选得到呼吸缺陷型酿酒酵母R40,再以R40和管囊酵母P01为亲本,进行原生质体制备与再生的研究,考察了酶浓度、酶解温度和酶解时间对两亲本原生质体制备和再生的影响。结果表明,当酶解时间为1 h,酶浓度为2%时,35℃下酶解酿酒酵母R40所得原生质体的形成率和再生率分别达到96.1%和12.5%;30℃下酶解管囊酵母P01所得原生质体的形成率和再生率为97.2%和10.1%。     

大豆rbcL基因克隆、序列分析及原核表达

利用叶绿体基因组保守性的特征,根据菜豆、豌豆、烟草的rbcL基因序列设计引物,从大豆叶绿体DNA中克隆rbcL基因,全长序列为1488bp,包括1449bp的开放阅读框,编码482个氨基酸。相似性比较显示,此序列与其它10个物种rbcL基因核苷酸的同源性为85．37％～95．31％,氨基酸的同源性为90．87％-96．47％。将该基因与表达载体pET-30a（＋）连接,转化大肠杆菌Rosseta感受态细胞,PCR和酶切鉴定筛选阳性克隆,阳性菌液IPTG诱导后经10％SDS—PAGE分析,结果显示,诱导表达出分子量约为60kD的特异融合蛋白。