Identification of clinically relevant yeasts by PCR/RFLP

For molecular diagnosis of fungal disease using DNA amplification procedures in the routine laboratory, choice of appropriate target structures and rapid and inexpensive identification of amplification products are important prerequisites. Most diagnostic procedures described thus far are characterized by limited applicability, considerable cost for laboratory equipment or low power of discrimination between species. This study aimed at identification of a PCR target appropriate for diagnosis of clinically relevant yeasts and an affordable procedure for characterization of the PCR products to the species level. Here, we describe a PCR-based system using amplification of intergenic spacers ITS1 and ITS2 and restriction length polymorphism of PCR products after sequence-specific enzymatic cleavage. We show the evaluation of the system for clinically relevant Candida species. The simple and inexpensive procedure should be instrumental for rapid identification of medically important yeasts.     

Enhancing the performance of brewing yeasts

Beer production is one of the oldest known traditional biotechnological processes, but is nowadays facing increasing demands not only for enhanced product quality, but also for improved production economics. Targeted genetic modification of a yeast strain is one way to increase beer quality and to improve the economics of beer production. In this review we will present current knowledge on traditional approaches for improving brewing strains and for rational metabolic engineering. These research efforts will, in the near future, lead to the development of a wider range of industrial strains that should increase the diversity of commercial beers.     

Flocculation in brewing yeasts: a computer simulation study

A new simulator, INDISIM-FLOC, based on the individual-based simulator INDISIM, is used to examine the predictions of two different models of yeast flocculation. The first, proposed by Calleja is known as the "addition" model. The second, proposed by Stratford is known as the "cascade" model. The simulations show that the latter exhibits a better qualitative agreement with available experimental data.     

用PCR及RFLP方法分析支原体与植原体的同源性

将猪鼻支原体和泡桐丛枝病植原体的16S rDNA进行PCR扩增,分别得到一条1 kb左右的扩增片段。PCR扩增产物用限制性内切酶EcoRⅠ、HindⅢ、BamHⅠ、SalⅠ和SmaⅠ进行RFLP(限制性片段长度多态性)分析,发现用RFLP分析猪鼻支原体和泡桐丛枝病植原体16S rDNA序列同源性的相关系数为0.72。     

Differentiation of the yeasts Williopsis, Zygowilliopsis and Komagataea by karyotypic and PCR analyses

We used polymerase chain reaction with universal and microsatellite primers, and molecular karyotyping to evaluate the extent of divergence between the genomes of the yeasts currently assigned to the heterogeneous genus Williopsis. Pulsed-field gel electrophoresis of chromosomal DNAs indicates that Zygowilliopsis californica, Komagataea pratensis, Williopsis mucosa, Williopsis salicorniae species and Williopsis sensu stricto complex have clearly different karyotypes. In contrast, the latter six species, Williopsis saturnus, W. beijerinckii, W mrakii, W. suaveolens, W. subsufficiens and W. sargentensis, show similar banding patterns and practically cannot be differentiated on the basis of their karyotypes. The data revealed that a PCR method employing the universal primer N21 is appropriate for the distinction of Williopsis, Zygowilliopsis and Komagataea yeasts. Unique fingerprints were generated with this primer for all 10 species studied while strains of the same species showed nearly identical profiles. The data of UP-PCR are in good agreement with genetic classification and provide support for the species status of the yeasts composing the Williopsis sensu stricto complex. Microsatellite primer (GTG)5 allowing molecular typing of individual strains of the same species may be useful for investigating population structure of the saturn-spored yeasts.     

Mixed-species biofilm formation by direct cell-cell contact between brewing yeasts and lactic acid bacteria

Mixed-species biofilm was remarkably formed in a static co-culture of Lactobacillus plantarum ML11-11 and Saccharomyces cerevisiae Y11-43 isolated from brewing samples of Fukuyama pot vinegar. Mixed-species biofilm is probably formed by direct cell-cell contact between ML11-11 and S. cerevisiae including Y11-43 and laboratory yeast strains. Scanning electron microscopic observation suggested that the mixed-species biofilm had a thick, bi-layer structure.     

草地植物群落物种多样性取样强度的研究

本文以羊草(Leymus chinensis)-杂类草群落和贝加尔针茅(Stipa baicalensis)-线叶菊(Filifolium sibiricum)群落为代表,在东北松嫩平原研究了草地植物群落物种多样性的取样强度。巢式样方种-面积曲线结果表明：两个群落的最小面积均为1/4～1/2 m²。 Pielou积累样方多样性指数-取样数曲线结果表明：羊草-杂类草群落1、1/4、1/6 m²正方形样方最小取样数分别在12、22、28个左右; 贝加尔针茅-线叶菊群落分别在10、18、25个左右。群落水平结构的复杂性导致取样数目的增加。当积累样方数超过最小取样数后,多样性指数-取样数曲线进入平衡状态,上述3种取样面积的结果趋于一致,并在概率95%水平差异不显著。赞成小面积大数目的取样策略。     

Storage of brewing yeasts by liquid nitrogen refrigeration

Many yeast strains are difficult to maintain in culture in a stable state, and long-term preservation by lyophilization, which has proved useful for other fungi, has given poor results with brewing yeasts. As an alternative to continuous subculture, which maximizes strain variability, various methods of cryogenic storage were investigated. Yeast strains were frozen with or without cryoprotectants (such as glycerol or inositol) and stored at -196 C. Recovery after warming was estimated from plate counts, and survivors were screened to detect changes in the frequency of morphological types, respiratory-deficient mutants, and glycerol-sensitive mutants. Strains varied in their sensitivity to freezing, and survival was modified by the growth medium, the freezing munstrua, and the freezing conditions. Suspension of cells in 10% (vol/vol) glycerol, cooled at 1 C/min, warmed rapidly and plated on malt-yeast extract-glucose-peptone agar produced the highest percentage of viable colonies with a minimal change in metabolic characteristics. In two of the strains tested, no significant increase in mutation rate was detected under any of the treatments; the strains were maintained in a stable state and were metabolically comparable to unfrozen strains. In one strain of Saccharomyces uvarum after some freezing treatments, the percentage of respiratory-deficient mutants increased markedly, the fermentation rate declined, and a loss of flocculation occurred. The freezing parameters which increased the level of respiratory-deficient cells should be avoided in maintaining this strain. Maintenance of cultures of brewing yeasts by cryogenic storage has several advantages over other preservation techniques: the method is simple and reproducible, the cultures have remained stable over a 3-year test period, and the viability is high.     

Study of the intra-cultivar variability of grape DNA using RFLP and PCR methods

Evaluation of DNA polymorphism among Vitis vinifera varieties using RFLP and PCR methods has been performed to choose a DNA technology for detection of grape intracultivar variation. DNA polymorphism of clones of the varieties Riparia Gluar, Riparia x Rupestris 101-14, Cabernet Sauvignon, Riestling reinskiy has been studied using Southern hybridization and amplification techniques. It has been shown that grape intracultivar variability of rDNA in Riparia x Rupestris 101-14 and Cabernet Sauvignon clones was caused by the modification in Alul restriction sites of rDNA. DNA variability of the randomly amplified and inter-SSR sequences of the Riparia Gluar, Riparia x Rupestris 101-14, Cabernet Sauvignon clones was also detected. A set of molecular DNA loci which can be used for grape clone identification has been obtained.     

26S rDNA-RFLP分析在非酿酒酵母菌分类研究中的应用

使用26S rDNA-RFLP分析,分析了分离自甘肃莫高葡萄酒厂的29株非酿酒酵母菌,被测菌株被分为10个类型。通过26S rDNA D1/D2区序列分析验证,证明此方法在葡萄酿酒酵母菌种多样性研究中良好的应用价值。     

Aberrations in sexual behavior in some brewing and other industrial yeasts

Summary The mating behavior of a number of brewer's and distiller's yeasts was determined with a and haploid and aa and diploid tester strains. Mating frequencies were not high, ranging from one to (rarely) 2,000/10⁸ cells in the mating mixture. Sporulating hybrids were obtained in most matings, though the percentage spore viability initially obtained was often low. Notable the spore viability obtained in hybrids with the haploid tester strains and the brewing strains DIB and DICH was much higher than from the a haploid tester strain, and higher in hybrids between these strains and the aa diploid tester than in those from the tester strain. With the brewing strain NBA, the spore viability in hybrids with the a haploid tester strain was higher than in the case of strains DIB and DICH, but the spore viability in the hybrid of NBA x the haploid strain was higher still. The data are consistent with the hypothesis that with the a and aa tester strains, most of the industrial yeasts tested mate as diploids, and with the and testers, they mate as haploids, an hypothesis which is supported by the segregation of adenine markers in the progeny of these hybrids.Presented in part at the 6th International Specialized Symposium on Yeasts, Montpellier, France, 2–8 July, 1978     

Interaction between lactic acid bacteria and yeasts in sour-dough using a rheofermentometer

Rheofermentometer assays were used to characterize the leavening of sour-doughs produced using species of lactic acid bacteria (LAB) and yeasts, alone or in combination. Saccharomyces cerevisiae 141 produced the most CO₂ and ethanol whereas S. exiguus M14 and Lactobacillus brevis subsp. lindneri CB1 contributed poorly to leavening and gave sour-doughs without porosity. In comparison with that seen in sour-dough produced with yeast alone, yeast fermentation with heterofermentative LAB present was faster whereas that with homofermentative LAB (L. plantarum DC400, L. farciminis CF3) present was slower and produced more CO₂. Combining L. brevis subsp. lindneri CB1 with S. cerevisiae 141 decreased bacterial cell numbers and souring activity. However, addition of fructose to the sour-dough overcame these problems as well as activating S. cerevisiae 141.The authors are with the Institute of Dairy Microbiology, Faculty of Agriculture, University of Perugia, S. Costanzo, 06126 Perugia, Italy     

Selective antimicrobial activity of chitosan on beer spoilage bacteria and brewing yeasts

Chitosan (0.1 g l(-1)), assayed in a simple medium, reduced the viability of four lactic acid bacteria isolated during the beer production process by 5 logarithmic cycles, whereas activity against seven commercial brewing yeasts required up to 1 g chitosan l(-1). Antimicrobial activity was inversely affected by the pH of the assay medium. In brewery wort, chitosan (0.1 g l(-1)) selectively inhibited bacterial growth without altering yeast viability or fermenting performance.     

乙型肝炎病毒基因亚型检测意义

乙型肝炎病毒基因亚型检测意义曹洪林张显忠陈禹保乙型肝炎是危害我国人民身体健康的重要疾病,患者和病毒携带者约占人群的１０％～１５％,感染人群约５０％～７０％。乙型肝炎病毒有四种重要的血清学亚型（ａｄｒ、ａｄｗ、ａｙｒ、ａｙｗ）,血清学亚型检测方法为蛋白质水平的检测技术,所观察的亚型属蛋白质水平的表型结果,不能反映决定其亚型的基因和其变异情况。乙型肝炎病毒血清学亚型的决定基因位于Ｓ基因区,该基因...     

Pyramiding of bacterial blight resistance genes in rice: marker-assisted selection using RFLP and PCR

 DNA marker-assisted selection was used to pyramid four bacterial blight resistance genes, Xa-4, xa-5, xa-13 and Xa-21. Breeding lines with two, three and four resistance genes were developed and tested for resistance to the bacterial blight pathogen (Xanthomonas oryzae pv. oryzae). The pyramid lines showed a wider spectrum and a higher level of resistance than lines with only a single gene. To speed up the gene pyramiding process and to facilitate future marker-aided selection, we developed PCR markers for the two recessive genes, xa-5 and xa-13, and used these to survey a range of rice germplasm. The results of the germplasm survey will be useful for the selection of parents in breeding programs aimed at transferring these bacterial blight resistance genes from one varietal background to another. Received: 6 December 1996/Accepted: 20 December 1996     

Characterisation of wheat-rye recombinants with RFLP and PCR probes

Summary The introgression of genetic material from alien species into wheat has become an important tool in modern wheat breeding. Ideally, only the trait of interest and no flanking material should be transferred. Random recombination between the genetic material is therefore of paramount importance. In a model system, we examined 17 recombinants putatively between chromosome 1D of wheat and 1R of rye with 60 random RFLP and three PCR markers. The recombinants had been generated by removing the normal effect of the Ph1 gene in the wheat background. Amongst the nine short-arm recombinants, three breakpoints were identified but no differentiation could be made between the five proximal recombinants. For the eight long-arm recombinants analysed only two breakpoints were identified with 36 markers. However, only a single RFLP marker was able to differentiate between the recombinants. Indeed the long-arm results are consistent with the possibility that only the rye telomeric region had been transferred. These results indicate either a strong clustering of the RFLP markers near the centromere or else imply that recombination induced between wheat and rye in the absence of the normal effect of the Ph1 gene occurs at only restricted sites. The results allow new primary recombinants to be selected for intercrossing to generate secondary recombinants which are expected to have a smaller interstitial rye segment than that present in DR-A1.     

The potential of genetic engineering for improving brewing, wine-making and baking yeasts

The end of the twentieth century was marked by major advances in life technology, particularly in areas related to genetics and more recently genomics. Considerable progress was made in the development of genetically improved yeast strains for the wine, brewing and baking industries. In the last decade, recombinant DNA technology widened the possibilities for introducing new properties. The most remarkable advances, which are discussed in this Mini-Review, are improved process performance, off-flavor elimination, increased formation of by-products, improved hygienic properties or extension of substrate utilization. Although the introduction of this technology into traditional industries is currently limited by public perception, the number of potential applications of genetically modified industrial yeast is likely to increase in the coming years, as our knowledge derived from genomic analyses increases.