Combined application of methods to taxonomic identification of Saccharomyces strains in fermenting botrytized grape must

AIMS: Although numerous physiological and molecular methods have been proposed for yeast taxonomy, the unambiguous separation of Saccharomyces sensu stricto species in natural samples is still an incompletely resolved issue. In this study the power of various methods was compared in the identification of strains isolated from fermenting botrytized grape musts. METHODS AND RESULTS: Conventional taxonomic and physiological tests and molecular methods developed for rapid identification were used. CONCLUSIONS: None of the methods tested was sufficiently powerful. However, the combination of electrophoretic karyotyping and the PCR-RFLP of MET2 with growth tests at 10 and 37 degrees C provided results sufficient for species identification of Saccharomyces wine strains which were not interspecific hybrids or recombinants. SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed combination of molecular and physiological methods allows specific taxonomic identification and separation of Saccharomyces wine strains without extensive genetic and molecular analysis. The proposed combined approach can also identify hybrids and recombinants.     

Mating types in the ciliate Dileptus anser. Genetical instability in the mating type system

In F1 and F2 from a cross between two clones of Dileptus anser isolated from natural sources (MT 1 x x MT III; MT = mating type), along with "normal" clones, many clones were observed demonstrating abnormal phenotype with respect to the MT-character. Irregular features of the latter were as follows: a) a delay in maturation; b) temporary reversion to immature or adolescent state, which means instability of maturity state; c) expression of MT I and MT III, rather than MT II as in properly matured clones; d) changes in MT (i.e., MT instability); e) appearance of totally unexpected MTs in terms of the scheme of genetic control of MTs in D. anser previously suggested by Afon'kin and Yudin (1987)--e.g., of all three MTs in F1 from the initial (analysing!) cross. Amazingly, these abnormal D. anser clones closely resembled some selfer-clones of Tetrahymena pigmentosa, previously reported as an example of genetic instability in the ciliate MT system (Simon, 1980; Simon, Orias, 1987).     

Identification and characterization of Saccharomyces cerevisiae and Saccharomyces paradoxus strains isolated from Croatian vineyards

AIMS: The identification, differentiation and characterization of indigenous Saccharomyces sensu stricto strains isolated from Croatian vineyards and the evaluation of their oenological potential. METHODS AND RESULTS: A total of 47 Saccharomyces sensu stricto strains were isolated from Chardonnay grapes and identified by physiological and molecular genetic methods. By using the standard physiological and biochemical tests, six isolates were identified as Saccharomyces cerevisiae and 41 as Saccharomyces paradoxus. However, PCR-RFLP analyses of the internal transcribed spacer (ITS1) region of the 18S ribosomal DNA identified 12 of the isolates as S.cerevisiae and 35 as S. paradoxus. Fermentation trials in a grape juice medium showed that these isolates ferment vigorously at 18 degrees C and display tolerance to high levels of ethanol. None of these isolates appeared to produce either hydrogen sulphide or killer toxins. CONCLUSION: Saccharomyces paradoxus, possessing potentially important oenological characteristics, occurs in much higher numbers than S. cerevisiae in the indigenous population of Saccharomyces sensu stricto strains in Croatian vineyards. SIGNIFICANCE AND IMPACT OF THE STUDY: This study forms an essential step towards the preservation and exploitation of the hidden oenological potential of the untapped wealth of yeast biodiversity in the Croatian grape-growing regions. The results obtained demonstrate the value of using molecular genetic methods, such as PCR-RFLP analyses, in conjunction with the traditional taxonomic methods based on phenotypic characteristics in such ecotaxonomic surveys. The results also shed some light on the ecology and oenological potential of S.paradoxus, which is considered to be the natural parent species of the domesticated species of the Saccharomyces sensu stricto group.     

Characterization of Saccharomyces cerevisiae strains isolated from must of grape grown in experimental vineyard

AIMS: Isolation and characterization of indigenous Saccharomyces cerevisiae strains from 12 grape varieties grown in an experimental vineyard of Apulia. METHODS AND RESULTS: Thirty to 40 colonies from each of the 12 fermentations were obtained at the end stage of spontaneous fermentation. By using morphological and physiological methods and by the PCR analysis of internal transcribed ITS1-5,8S-ITS2, the isolates belonging to Saccharomyces genus were identified. These isolates were further characterized by amplification with S. cerevisiae species- and delta element-specific primers, thus allowing the identification of S. cerevisiae strains selected from each of the 12 fermentations. By means of RFLP analysis of mtDNA, each S. cerevisiae population isolated from a single fermentation appeared to constitute a genetically homogenous group. The comparison of the 12 cultivar-specific mtDNA RFLP patterns, allowed classifying the 12 S. cerevisiae populations into three genetically homogenous groups. The isolated strains fermented vigorously in synthetic and grape juice medium and showed high alcohol and sulphur dioxide (SO(2)) resistance and low hydrogen sulphite (H(2)S) production. CONCLUSIONS: The molecular analysis, in conjunction with the traditional morphological and physiological methods, was useful in discriminating at strain level the indigenous population of S. cerevisiae present in a vineyard of Apulia. The dominant S. cerevisiae strains identified in the 12 fermented musts showed potentially important oenological characteristics. SIGNIFICANCE AND IMPACT OF THE STUDY: The characterization of natural S. cerevisiae strains from several typical Italian grapes grown in a restricted experimental vineyard is an important step towards the preservation and exploitation of yeast biodiversity of Apulia, a relevant wine-producing region. The close relationship between the S. cerevisiae strains from different grapes grown in the same vineyard indicated that the occurrence of native strains is representative of the area rather than of the variety of grapes.     

Stability of isoenzyme and kinetoplast DNA (k-DNA) patterns in successively cloned Trypanosoma cruzi populations.

Four Trypanosoma cruzi strains from zymodemes A, B, C and D were successively cloned on BHI-LIT-agar-blood (BLAB). Twenty clones from the first generation (F1), 10 from the second (F2) and 4 from the third (F3) from the strains A138, B147 and C231 were isolated. The D150 strain provided 29 F1 and 23 F2 clones. The strains and clones had their isoenzyme and k-DNA patterns determined. The clones from A138, B147 and C231 strains presented isoenzyme and k-DNA patterns identical between themselves and their respective parental strains. Therefore showing the homogeneity and stability of isoenzyme and k-DNA patterns after successive cloning. The D150 strain from zymodeme D (ZD) showed heterogeneity. Twenty-eight out of 29 clones of the first generation were of zymodeme A and only one was of zymodeme C, confirming previous reports that ZD strains consisted of ZA and ZC parasite populations. The only D150 strain clone of zymodeme C showed a k-DNA pattern identical to its parental strain. The remaining clones although similar among themselves were different from the parental strain. Thus the T. cruzi strains had either homonogeneus or heterogeneous populations. The clones produced by successive cloning provided genetically homogeneous populations. Their experimental use will make future results more reliable and reproducible.     

Use of genetic analysis to test the potential role of serotonin in alcohol preference.

The hypothesis that alcohol preference in mice is influenced by brain serotonin levels was tested using genetic analysis. Alcohol preference and static serotonin content were assessed in C57BL/Ibg (alcohol-preferring) and DBA/2 (alcohol-avoiding) mice, as well as in Fl and F2 generations obtained by crossbreeding. The two parental strains showed dissimilar alcohol preferences but identical concentrations of brain serotonin. Serotonin concentration segregated independently of alcohol preference in the F1 and F2 generations. These data provides strong evidence against the hypothesis that brain serotonin content influences alcohol preference. However, they do not preclude the possibility that differential alcohol influences on serotonin metabolism or turnover rate may result in differing preference for a alcohol.     

The isolation of strains of Saccharomyces cerevisiae showing altered plasmid stability characteristics by means of selective continuous culture.

A recombinant strain of Saccharomyces cerevisiae containing a plasmid-encoded lacZ gene from Escherichia coli was grown for 420 generations under selective conditions in glucose-limited continuous culture. A ura3-based auxotrophic system was used to apply selection in favour of plasmid-containing organisms. A similar strategy had previously proved successful at evolving clones of Bacillus subtilis, showing improved plasmid stability characteristics. In this study a series of clones were isolated which exhibited large variation in their ability to retain the recombinant plasmid. Clones showed both significantly increased and reduced capacity to maintain the recombinant plasmid. The probabilities of obtaining clones in either category were essentially equal so that selection was not seen to enrich for more stable clones. Periodic selection events appeared to exert a greater influence on the distribution of stability characteristics amongst clones than did the applied selective pressure. Alterations in plasmid retention characteristics could be associated with host or plasmid. The most stable clone isolated exhibited a approximately 30% improvement of its overall stability (sigma(N+)) and an 80% improvement in productivity, when compared to the parental strain CGpLG. This improved stability was associated with alterations in the plasmid genome.     

Progressive Myoclonus Epilepsy EPM1 Locus Maps to a 175-kb Interval in Distal 21q

The EPM1 locus responsible for progressive myoclonus epilepsy of Unverricht-Lundborg type (MIM 254800) maps to a region in distal chromosome 21q where positional cloning has been hampered by the lack of physical and genetic mapping resolution. We here report the use of a recently constituted contig of cosmid, BAC, and P1 clones that allowed new polymorphic markers to be positioned. These were typed in 53 unrelated disease families from an isolated Finnish population in which a putative single ancestral EPM1 mutation has segregated for an estimated 100 generations. By thus exploiting historical recombinations in haplotype analysis, EPM1 could be assigned to the ~175-kb interval between the markers D21S2040 and D21S1259.     

基因枪转化的bar基因表达盒在水稻中遗传行为复杂

基因枪介导基因表达盒(仅包括启动子、编码区和终止子)转化是基因枪转化植物的新趋势,它能消除质粒载体主干序列对转基因植物的不利影响。本文研究了基因枪转化的bar基因表达盒在转基因水稻T1～T3世代中的遗传行为。结果发现:作为筛选标记的bar基因表达盒在水稻基因组中多拷贝整合,遗传分离行为复杂,还出现了Basta抗感分离比在35:1～144:1之间的"假纯合体",但50%转基因株系中(5/10)bar基因可作为一个显性基因按孟德尔方式稳定遗传至自交T2代。虽然bar基因为多拷贝整合,30%的转基因株系(3/10)在自交低世代(T1)能获得纯合体。Southern杂交分析发现,多拷贝的bar基因表达盒倾向于连接成转基因串联子整合在水稻基因组内。我们发现在Basta抗性正常分离的株系后代中bar基因表达盒Southern杂交模式能稳定遗传,但异常分离的株系后代中bar基因表达盒的一些拷贝发生了丢失。我们推测,bar基因表达盒在水稻中遗传分离行为的复杂原因可能是bar基因表达盒多拷贝整合、基因丢失和基因表达互作。     

Gene transfer using fusion of isolated cell nuclei with protoplasts of the yeast Saccharomyces cerevisiae

Hybrid clones of Saccharomyces cerevisiae with different genotypes have been obtained by polyethyleneglycol induced fusion of isolated cellular nuclei with protoplasts. The genetic instability of complete nuclei after fusion results in formation of different genotypes.     

生长相关分子标记在翘嘴鳜五代中的富集

为进一步了解人工选育对翘嘴鳜生长相关遗传标记的影响作用,研究以翘嘴鳜华康1号的5代选育群体为实验材料,对具有生长相关优势基因型的5个标记的6个位点进行扩增,通过直接测序和聚丙烯酰胺凝胶电泳两种方法分型后,统计其优势基因型个体数目在翘嘴鳜5代中的变化。结果显示,在5代群体中,2个单核苷酸多态性位点和4个微卫星位点优势基因型的数目的分布范围为0-4,从F1到F5代,这6个位点优势基因型的平均值分别为0.36、0.71、0.68、0.77和0.94,优势基因型的平均含量随选育世代的增加呈现递增趋势,从侧面反映了人工选育在一定程度上富集了优良基因。此外,对微卫星位点进行了遗传相似性和遗传距离分析,结果显示,随着选育的进行,后续世代与F1的遗传距离有明显的增大趋势,遗传相似性减小,这符合育种的客观规律。但相邻世代间的遗传距离则逐代减小,遗传相似性逐代增大,说明人工选育将遗传相似性较大的群体保留下来了,这种相似性表现在表型上包括生长快、体重大、体长增加等。F1到F5代处于中度遗传多样性的稳定状态,说明群体还存在选育空间。     

Double sterility barrier between Saccharomyces species and its breakdown in allopolyploid hybrids by chromosome loss

The analysis of 57 synthetic interspecies hybrids revealed that Saccharomyces cerevisiae and Saccharomyces uvarum ( Saccharomyces bayanus var. uvarum) are isolated by a double sterility barrier: by hybrid sterility (hybrid cells cannot produce viable spores) operating in allodiploids and by F1 sterility (F1 cells cannot produce viable spores) operating in allopolyploids. F1-sterility is caused by mating-type heterozygosity. It can be overcome by eliminating chromosome 2 of the S.?uvarum subgenome that carries a MAT locus. The loss of this MAT gene abolishes the repression of mating activity. In cultures of the resulting fertile alloaneuploid F1 segregants, the cells can conjugate with each other like haploids and form zygotes capable of performing meiotic divisions producing viable and fertile F2 spores. To the best of our knowledge, this is the first report on breaking down interspecies hybrid sterility by chromosome loss in eukaryotic organisms. The filial generations are genetically unstable and can undergo additional changes mainly in the S.?uvarum subgenome (directional changes). It is proposed that regaining fertility and subsequent preferential reduction in one of the subgenomes may account for the formation of chimerical ('natural hybrid') genomes found among wine and brewery strains and may also play roles in speciation of hybrid taxa in the Saccharomyces genus.     

Genetic instability of heterozygous, hybrid, natural wine yeasts

We describe a genetic instability found in natural wine yeasts but not in the common laboratory strains of Saccharomyces cerevisiae. Spontaneous cyh2(R)/cyh2(R) mutants resistant to high levels of cycloheximide can be directly isolated from cyh2(S)/cyh2(S) wine yeasts. Heterozygous cyh2(R)/cyh2(S) hybrid clones vary in genetic instability as measured by loss of heterozygosity at cyh2. There were two main classes of hybrids. The lawn hybrids have high genetic instability and generally become cyh2(R)/cyh2(R) homozygotes and lose the killer phenotype under nonselective conditions. The papilla hybrids have a much lower rate of loss of heterozygosity and maintain the killer phenotype. The genetic instability in lawn hybrids is 3 to 5 orders of magnitude greater than the highest loss-of-heterozygosity rates previously reported. Molecular mechanisms such as DNA repair by break-induced replication might account for the asymmetrical loss of heterozygosity. This loss-of-heterozygosity phenomenon could be economically important if it causes sudden phenotype changes in industrial or pathogenic yeasts and of more basic importance to the degree that it influences the evolution of naturally occurring yeast populations.     

Preferential deletion of a specific region of mitochondrial DNA in Saccharomyces cerevisiae by ethidium bromide and 3-carbethoxy-psoralen: directional retention of DNA sequence.

Grande strains of Saccharomyces cerevisiae were mutagenized either by ethidium bromide or by 3-carbethoxy-psoralen (a monofunctional furocoumarin derivative) activated by 365nm light. 973 primary rho- clones induced were randomly collected and analyzed individually for the presence or absence of fifteen mitochondrial genetic markers. 1. Under mild conditions of mutagenesis, 83% of the primary clones showed single-deletion genotypes; a unique order of 14 markers could be deduced from the patterns of the deletion. The gene order confirmed our previous map constructed from the analysis of established non-random petite clones. From the frequencies of disjunction between markers, the distance separating 14 mitochondrial markers were estimated. 2. One region, carrying oxi-3, pho-1 and mit 175 loci, was preferentially lost in rho- mutants: there is a strong constraint in the frequencies of various genotypes found in rho- clones. On each side of this particular region, a bidirectionally oriented pattern of retention of markers is observed.     

Chromosome polymorphism in the yeast Saccharomyces

The variability of chromosomal band patterns was determined by pulse electrophoresis. The natural strains differed by the quantity and electrophoretic mobility of chromosomal DNA bands. The strains of independent genetic stocks originated from the XII race of Saccharomyces cerevisiae showed less significant difference in band patterns than the strains of different species of the Saccharomyces genus. The progeny of among strains with different karyotypes hybrid showed non-regular segregation of parental bands, the occurrence of new bands and the bands with altered mobility. Reverse crosses of hybrid progeny with strains of Peterhoff genetic stocks of S. cerevisiae led to decrease in chromosomal polymorphism. Homozygotization for ski5 allele and selection for increasing the copy number of killer plasmids was accompanied with repeated splash of polymorphism in 1-2 generations of intratetrad and intrafamily crossed hybrid progeny. Subsequent stabilization of electrophoretic karyotype took place, excluding the mendelian dimorphism of chromosome III, with was a stable trait of the last 6 generations of that progeny.     

RAPD标记技术用于WHBE兔近交系培育中的遗传分析

目的探讨用随机引物扩增多态性DNA技术对大耳白黑眼兔近交系培育中的遗传监测作用。方法选用F4、F5、F6和F7代共70只WHBE兔的皮肤组织样品提取基因组DNA,用60个随机引物对基因组DNA进行PCR扩增,根据电泳结果筛选出其中25个多态性较高的引物进行RAPD-PCR分析,再利用Popgene 3.2统计软件对共检测到的584个扩增片段进行遗传分析,获得实验数据。结果①F4代扩增得到124条片段,F5代扩增得到150条片段,F6扩增得到152条片段,F7代扩增得到158条条带;其中F4代与F5代的共有条带数为105,F5代与F6代的共有条带数为119,F6代与F7代的共有条带数为125。②F4代与F5代的遗传相似度为0.7674,F5代与F6代的遗传相似度为0.7984,F6代与F7代的遗传相似度为0.8092。结论随着WHBE兔近交培育代数的增加,遗传相似度呈上升趋势,说明RAPD技术可以用于WHBE兔近交系培育的遗传检测。     

A single low dose of X-rays induces high frequencies of genetic instability (aneuploidy) and heritable damage (apoptosis), dependent on cell type and p53 status

We harvested and analyzed cells from four different non-transformed cell lines surviving a single X-ray exposure. Evidence of radiation-induced karyotype instability was observed in 100% of C3H 10T1/2 fibroblast clones and 11.3% of V79 fibroblast clones. Heritable damage: predisposition to apoptosis, but not karyotype instability, was induced in TK6 (p53(wt/wt)) and WTK1 (p53(mut/mut)) human B-lymphoblastoid cell clones. The studies indicate: (1) genetic instability and/or heritable damage are induced in cells exposed to radiation at a high frequency, and induction of genetic instability is not limited to morphologically transformed cells [Radiat. Res. 138 (1994) S105; Radiat. Environ. Biophys. 36 (1998) 255]; (2) sensitivity to genetic instability and heritable damage depend on cell type; (3) checkpoint stringency and p53 status significantly influence the frequency of radiation-induced genetic instability and heritable damage; (4) in some cell lines, damage induced by low doses of radiation (below 2 Gy) leads to heritable cytotoxic and genotoxic effects in 100% of cells exposed. The data suggest that mammalian cells misinterpret damage induced by ionizing radiation as if it were a physiological cell signal. This contrasts strongly with the response of mammalian cells to damage induced by other types of DNA-toxic agents where damage-specific repair mechanisms are activated.     

Transformation of Kluyveromyces lactis with the kanamycin (G418) resistance gene of Tn903

Direct selection of Kluyveromyces lactis resistant to the antibiotic G418 following transformation with the kanamycin resistance gene of Tn903 required the development of a procedure for producing high yields of viable spheroplasts and for the isolation of autonomous replication sequences (ARS). To obtain high yields of viable spheroplasts, cells were treated with (1) a thiol-reducing agent (L-cysteine), and (2) a high concentration of an osmotic stabilizer, 1.5 M sorbitol. Several ARS-containing plasmids were selected from a K. lactis recombinant DNA library in K. lactis and in Saccharomyces cerevisiae. Two of four ARS clones selected in K. lactis promoted transformation frequencies of 5-10 X 10(2) G418-resistant cells/micrograms of plasmid DNA. This frequency of transformation was at least twice as high as with ARS clones selected in S. cerevisiae. The stability of ARS-containing plasmids varied; after 20 generations of growth in the presence of G418, 16-38% of the cells remained resistant to the drug. In the absence of selection pressure less than 5% of the cells retained the drug-resistance phenotype. Plasmids containing the ARS1 or 2 mu replicon of S. cerevisiae failed to transform K. lactis for G418 resistance. Inclusion of S. cerevisiae centromere, CEN4, in a K. lactis ARS recombinant plasmid did not increase the stability of the plasmid in K. lactis, and marker genes on the vector segregated predominantly 4-:0+ through meiosis. We conclude that neither the ARS sequences or the centromere of S. cerevisiae was functioning in K. lactis.     

Cytogenetic Studies on the Cross generations Between Triticum aestivum and Eremopyrum orientale

The cytogenetics of the backcross generations and self-bred progenies (BC2F1, BC3F1, BC2F2, BC3F2 and BC2F3) of intergenetic hybrid of Triticum aestivum L. x Eremopyrum orientale (kedeb) Jaub. Et Spach were studied. The results showed that the plants BC3F1 (2n = 43 ) isolated from the backcross generations of the plants (2n = 44 ) accounted for 41.09%, but the plants (2n = 44) isolated was only 4. 11%, and the plants BC2F2 (2n = 44) isolated from self-cross generations of the plants BC2F1 (2n = 44) accounted for 13.21%. The number of univalents in pollen mother cells was higher in some plants (BC2F1) and the averages of univalents were negatively related to the backcross seed-setting and self-cross seed-setting with a related coefficient of － 0.6766* and －0.7429* respectively. The results of genomic in situ hybridization (GISH) showed that some plants of BC2Fs (2n=44) contained different number of alien chromosomes. All those indicated that alien chromosomes caused unusual pairing and segregation of wheat homologous chromosomes and also lowed the hereditary stability of wheat chromosomes.     

Production of high levels of acetoin in Saccharomyces cerevisiae wine yeasts is a recessive trait

P. ROMANO, G. SUZZI, R. MORTIMER AND M. POLSINELLI. 1995. A genetic study of acetoin production was performed on wild wine yeast strains of Saccharomyces cerevisiae producing different amounts of the compound. By using differences found in these strains as source of genetic variability, it was found that crosses between high and low acetoin producing strains yielded the low level in the hybrids, indicating the low production as a dominant trait. Tetrad analysis showed that high vs low acetoin production is segregated as a single gene.