Factors affecting the mitotic stability of high-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae

Yeast vectors suitable for high-level expression of heterologous proteins should combine a high copy number with high mitotic stability. The pMIRY integrative vector system, based upon targeted integration into the yeast rDNA locus, developed in our laboratory satisfies these criteria. However, insertion of a (foreign) gene drastically reduced its mitotic stability of the resulting vector in comparison to its parent. In this paper we have investigated a number of possible reasons for this reduction in stability. The results demonstrate that plasmid size is an important, but not the only, determinant of mitotic stability. Stable maintenance is only observed when the complete plasmid has a size no larger than that of the rDNA unit (9·1 kb). In addition stability depends upon the nature of the rDNA fragment present in the plasmid, required for targeting its integration. On the other hand, it turned out to be irrelevant for mitotic stability whether the heterologous gene was expressed or not. These findings will be important in the design of a pMIRY vector suitable for industrial production of heterologous proteins.     

Identification of durum wheat cultivars and monovarietal semolinas by analysis of DNA microsatellites

 Microsatellites isolated in bread wheat were used to identify 20 Italian durum wheat (Triticum turgidum var. durum L.) cultivars. A total of 15 microsatellite primer pairs were tested against DNA extracted from leaf tissue, single seeds and semolina. Twelve markers showed allelic polymorphism among the 20 cultivars, providing a total of 41 different alleles. Firstly, an analysis of microsatellite informativeness in the chosen set of durum wheat cultivars was made and a set of highly polymorphic microsatellites was established. Secondly, among the most polymorphic cultivars, the minimum number of microsatellites able to distinguish all cultivars was determined. A set of five microsatellites was found sufficient to differentiate the durum wheat cultivars examined. The method is directly applicable to seeds and semolina, and is suitable for detecting seed mixtures in the same seed lot. Received: 22 February 1999 / Revised version: 19 April 1999     

Sequence of a 17·1 kb DNA fragment from chromosome X of Saccharomyces cerevisiae includes the mitochondrial ribosomal protein L8

We have sequenced a continuous segment of 17 137 bp on chromosome X. Sequence analysis of this stretch revealed 14 open reading frames (ORFs) at least 100 amino acids long. One gene, encoding the mitochondrial 60S ribosomal protein L8, had already been sequenced. Four ORF products show weak homologies with known protein sequences. The nine remaining ORF products have no homologies with sequences in data banks. The nucleotide sequence of the 17·1 kb fragment is available through the EMBL data library under Accession Number Z34288.     

Analysis of DNA sequences homologous with the ARS core consensus in Saccharomyces cerevisiae

We have previously identified an autonomously replicating segment (ARS) near the 3' end of the histone H4 gene at the copy-I H3-H4 locus. We have now searched for additional autonomously replicating segments and sequences homologous with the ARS core consensus sequence near the copy-II histone H4 gene and both of the histone H3 genes. No new ARS elements were identified by functional cloning assays. However, several matches to the ARS core consensus element were found within the DNA sequences of the copy-I and copy-II genes. An exact match to the ARS core consensus was identified in the region downstream from the copy-I histone H3 gene and a set of sequences with weak homology was also located within the copy-II region. However, restriction fragments including these sequences did not demonstrate ARS activity on a plasmid in transformed cells.     

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.     

Recovery and characterization of residual DNA from beer as a prerequisite for the detection of genetically modified ingredients

 In an attempt to recover polymerase chain reaction (PCR)-amplifiable DNA from beer, the performance of three different extraction methods was compared. Efficient DNA enrichment, by selective binding to either a membrane or a silica matrix, proved an essential requirement for the procedure to be successful. To identify its origin, the extracted DNA was subjected to PCR using primer pairs specific for eukaryotes, plants or yeast, respectively. It was shown that samples of six beer brands tested positive for yeast, whereas the PCR assay for plant DNA was negative in these cases. The data demonstrate that residual DNA can be recovered from beer, thus opening up the possibility of monitoring the presence of transgenic genomic sequences. Received: 30 September 1998 / Revised version: 23 November 1998     

Genome-wide analysis of coding DNA and amino acid variation in Saccharomyces cerevisiae

The possible causes of variation on amino acid composition in the yeast Saccharomyces cerevisiae were investigated genome-wide. The results indicated that: (a) the base composition of coding DNA and amino acid composition was similar among all the chromosomes, which was in sharp contrast with the great varies of the composition of the individual's coding DNA and amino acid; (b) some amino acids (e.g. Cys and Trp) were not present in all the proteins; and (c) amino acid bias was associated with a base bias (in terms of A-, G-, C- and T-rich codons). Based on the third rule and a proposed universal trend of amino acid gain and loss in protein evolution, the changing pattern of coding DNA was predicted to be T- and C-accruing, whereas A and G were consistently reducing. All these results held the potential to reveal precisely how DNA ongoing change has a major effect on the composition of proteins.     

High levels of the mitochondrial large ribosomal subunit protein 40 prevent loss of mitochondrial DNA in null mmf1 Saccharomyces cerevisiae cells

Members of the YERO57c/YJGFc/UK114 protein family have been identified in bacteria and eukaryotes. The budding yeast Saccharomyces cerevisiae contains two different proteins of this family, Hmf1p and Mmf1p. We have previously shown that Mmf1p is a mitochondrial protein functionally related to its human homologue and able to influence the maintenance of mitochondrial DNA. Deletion of Mmf1 results in loss of the mitochondrial genome. Using a multicopy suppression approach, we have identified a protein of the mitochondrial large ribosomal subunit, MRPL40, which stabilizes mtDNA in Deltammf1 cells. Overexpression of MRPL40 did not prevent loss of mtDNA in a mutant strain lacking the mitochondrial protein Abf2p. Thus, MRPL40 does not have a general effect on mtDNA stability, but it may be specific for the mmf1-null strain. We also show that the Deltamrpl40 cells present a similar phenotype to the mmf1-null strain, having reduced mtDNA stability and growth rate. Furthermore, we observed that rho(+)Deltamrpl40 haploid cells can be obtained when tetrads are directly dissected on medium containing a non-fermentable carbon source. Thus, replication and segregation of the mtDNA can occur in the absence of MRPL40. We also show that another mitochondrial ribosomal protein, MRPL38, is able to overcome the Deltammf1-associated defect. Together, our results suggest a link between Mmf1p and the two mitochondrial ribosomal proteins.     

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.     

Discrimination between fortuitous and biologically constrained open reading frames in DNA sequences of Saccharomyces cerevisiae

The systematic sequencing of the yeast genome has raised the problem of the biological significance of the open reading frames (ORFs) revealed: it is possible that some of these are fortuitous. To avoid the analysis of such fortuitous ORFs, a minimum length of 100 sense codons was adopted. Nevertheless, the presence of fortuitous ORFs of more than 100 codons cannot be excluded. Thus, in the context of functional analysis, a method for discrimination between fortuitous and biologically active ORFs may be useful. The discrimination method described here is based on multiple criteria: ORF length, codon bias, and both amino-acid and dipeptide composition of the corresponding polypeptide. The thresholds for each criterion are based on the comparison between two learning sets: one drawn from random DNA sequences and the second from known genes. The method was validated by two test sets (one random and one biological) and then applied to the ORFs of chromosomes I, II, III, V, VIII, IX and XI. This method predicts 123 fortuitous ORFs among the 1773 identified on these chromosomes.     

Identification of microbial flora in kefir grains produced in Turkey using PCR

Kefir grains might have different ratios and/or content of microflora according to their origin. The purpose of this study was to determine microbial flora of kefir grains produced in three regional universities in Turkey using polymerase chain reaction (PCR) and consensus sequence primers. According to the results of PCR products with the specific primers, the following were identified as the natural inhabitants of the kefir grains: Lactobacillus acidophilus, Lactobacillus helveticus, Streptococcus thermophilus, Bifidobacterium bifidum and Kluyveromyces marxianus kefir. The results of this study revealed that traditional kefir produced by using kefir grains as natural starter cultures contains lactic acid bacteria especially Lactobacillus spp. One of the sources also contained B. bifidum. This is the first record on the presence of B. bifidum in kefir grains.     

Ecology of Saccharomyces cerevisiae in Spontaneous Fermentations at a Rías Baixas Appelation Controlé Winery

PCR techniques and pulse field electrophoresis karyotyping showed wide genetic diversity in Saccharomyces cerevisiae strains isolated from spontaneous fermentations in progress at a wine cellar in the Rías Baixas appelation controlé region (Spain). The karyotyping method showed greater discriminating power than PCR profiling, making it better suited for the detection of genetic diversity in wine strains, and for the monitoring of selected strains in controlled fermentations.     

Mitochondrial DNA enrichment for species identification and evolutionary analysis

 Nucleic acid-based species identification often targets the mitochondrial encoded cytb gene. However, polymerase chain reaction (PCR)- restriction fragment length polymorphism (RFLP) analysis using universal primers sometimes leads to ambiguous results, which are due to the presence of nuclear encoded pseudo-cytb genes. Such ambiguities were succesfully avoided using a newly developed method for the enrichment of mitochondrial DNA. In addition, a mitochondrial cytb-specific PCR system was designed allowing the unambiguous identification of game meat. Received: 24 February 1998     

Mitochondrial DNA enrichment for species identification and evolutionary analysis

 Nucleic acid-based species identification often targets the mitochondrial encoded cytb gene. However, polymerase chain reaction (PCR)- restriction fragment length polymorphism (RFLP) analysis using universal primers sometimes leads to ambiguous results, which are due to the presence of nuclear encoded pseudo-cytb genes. Such ambiguities were succesfully avoided using a newly developed method for the enrichment of mitochondrial DNA. In addition, a mitochondrial cytb-specific PCR system was designed allowing the unambiguous identification of game meat. Received: 24 February 1998     

The fate of recombinant DNA in thermally treated fermented sausages

 The fate of recombinant DNA (rDNA) from genetically modified starter organisms in thermally treated fermented sausages (Kochsalami) was monitored by a polymerase chain reaction (PCR)-based estimation of the concentration of the rDNA. We defined an rDNA titre as the smallest volume of the DNA solution which still results in a detectable amplicon on PCR. In sausages a degradation of the rDNA was observed during storage. However, this did not proceed so far that rDNA was no longer detectable after 9 weeks of incubation. It was observed that the rDNA is strongly protected against the activity of DNase I in the meat matrix. To include the aspect of release of rDNA from genetically modified starter organisms into the meat matrix, Kochsalami-type sausage was produced with the aid of recombinant strains of Lactobacillus curvatus as starter organisms. It was shown that only a minute part of the rDNA contained in the starter organisms can be recovered from the meat matrix by extraction as "free" DNA. To recover the total rDNA, cell wall lytic enzymes were necessary. No significant effect on the detectability of the rDNA was observed on variation of the ecological factors prevailing during the production and storage of the sausages, such as the temperature of thermal treatment, pH and fat content. The concomitant presence of Staphylococcus aureus (>10⁶ cfu/g) producing heat-stable nuclease did not affect the detection of the rDNA. Received: 29 March 1999     

酿酒酵母GY-1的固定化及其对魔芋寡糖中单糖的利用

以酿酒酵母（Saccharomyces cerevisiae）GY-1作为实验菌株,利用魔芋粉和海藻酸钠搭配作为固化交联剂进行固定化酵母制备,并通过固定化酵母去除魔芋水解产物中的还原单糖。结果表明,2%的魔芋粉和1%的海藻酸钠组合作为固定化剂,并混合5%的粉状酵母GY-1添加到40 ℃热水中,搅拌混合形成多糖胶体,通过微量泵加入到4%的CaCl2溶液中,在低温条件下交联反应10 h,形成固定化酵母颗粒均匀,平均直径为4～5 mm,包埋的酵母死亡率低于10%;采用薄层层析（TLC）法检测,发现固定化酵母GY-1能够利用魔芋胶中还原单糖（葡萄糖、甘露糖）,从而达到纯化魔芋寡糖的目的。     

酿酒酵母乙醇脱氢酶Ⅱ基因反义干扰及对乙醇发酵的影响

通过构建酿酒酵母沉默表达载体PURH-ADH2,使ADH2基因在PGK强启动子、CYC1终止子在特定区域内进行干扰和表达。采用Bam HI和Xmal I限制性内切酶对SADH2基因和PURH质粒进行双酶切,构建反义重组表达质粒PURH-SADH2,通过高效酵母转化法和电转法将重组质粒组件转化至酿酒酵母SY01细胞中,获得阳性克隆菌株JY01。酿酒酵母JY01突变菌株与出发菌株SY01和Y01发酵试验结果相比,JY01甘油脱氢酶酶活比出发菌株Y01与SY01分别下降了16.31%和13.5%;当酿酒酵母Y01、SY01和JY01菌株发酵36~60 h时,JY01菌株甘油含量相比Y01分别下降了16.34%、14.25%、14.89%;当酿酒酵母突变菌株发酵48 h时,Y01、SY01和JY01的乙醇浓度分别为6.243 g/100 m L、7.145 g/100 m L和7.288 g/100 m L,酿酒酵母JY01发酵液乙醇量比比原始菌株Y01乙醇含量提高了14.33%。结果表明通过反义干扰ADH2基因5’UTR序列,能有效干扰酵母工程菌株ADH2转录与表达,削弱甘油积累途径,促进乙醇代谢途径。