The calculation of cellular parameters from the turbidity of yeast cultures may give rise to important errors

Abstract It is shown that cellular parameters of the yeast cultures (e.g. intracellular volume, cellular dry weight, protein content, number of cells, and turbidity) are differently influenced by metabolic changes. Therefore, the cellular parameters change independently of each other. It is hence concluded that whenever quantitation is required, the values of these parameters should be measured independently and not calculated from the turbidity of the cultures or other parameters, as is often done.     

Lipase production from an alkalophilic yeast sp. by solid state fermentation

The present investigation has been carried out with the aim to produce lipase from an alkalophilic yeast species by solid state fermentation using rice and wheat brans as alternative cheap solid substrates.     

Construction of an efficient amylolytic industrial yeast strain containing DNA exclusively derived from yeast

An amylolytic industrial yeast strain of Saccharomyces cerevisiae containing the Schwanniomyces occidentalis SWA2 amylase gene was generated. The new strain contains DNA derived exclusively from yeast and expresses a high starch hydrolyzing activity. Yeast transformation was carried out by an integrative process targeted to a dispensable upstream region of the ILV2 locus, which determines sulfometuron resistance. The SWA2 enzyme was constitutively expressed under the ADH1 promoter. The growth, substrate utilization and fermentative capacity of this organism are described.     

啤酒酵母工业菌株单倍体的诱导、分离和鉴定

【目的】探索适宜的方法进行啤酒酵母工业菌株单倍体的诱导、分离和鉴定,为啤酒酵母改良和遗传学研究提供便利。【方法】首先,选择产孢效果最好的培养基进行产孢诱导,诱导产生的孢子在YPD培养基上形成菌落后,用流式细胞技术检测其DNA含量,进而判断其倍性;单倍体菌株的交配型通过MAT-PCR和杂交实验确定。【结果】啤酒酵母工业菌G-03通过产孢诱导和孢子分离、富集后得到26株菌,最终通过流式细胞技术确定了其中4株为单倍体,MATa和MATα型各2株。通过扫描电镜法观察4株单倍体菌株及出发菌G-03的细胞形态,发现单倍体菌株的形态和出发菌有较大区别,单倍体菌株长期培养没有假丝生长的现象发生。【结论】啤酒酵母工业菌单倍体育种较为困难,严格的单倍体筛选、鉴定尤其具有挑战性。     

Complete genome searches for quantitative trait loci controlling blood pressure and related traits in four segregating populations derived from Dahl hypertensive rats

The Dahl salt-sensitive rat is one of the principal animal models of hereditary hypertension. Genome-wide searches were undertaken to detect quantitative trait loci (QTLs) that influence blood pressure, cardiac mass, and body weight in four F₂ populations derived from Dahl salt-sensitive rats and different inbred normotensive control strains of rat. We detected three QTLs associated with one or more of the phenotypes, using a stringent statistical criterion for linkage (p < 0.00003). These included a novel QTL linked to blood pressure on rat Chromosome (Chr) 12, and another QTL on rat Chr 3 linked to body weight. A QTL on rat Chr 10 for which linkage to blood pressure has been described in other crosses was found to be a principal determinant of blood pressure and cardiac mass in some but not all of the crosses examined here. Three other regions showed evidence of linkage to these phenotypes with a less stringent statistical criterion of linkage at QTLs previously reported in other studies. As part of our study, microsatellite markers have been developed for three candidate genes for investigation in hypertension, and the genes have been localized by linkage mapping. These are: the rat Gs alpha subunit (Gnas) gene, the alpha-1B adrenergic receptor (Adra1b), and the Na⁺, K⁺-ATPase beta2 subunit (Atp1b2) gene. Received: 29 June 1998 / Accepted: 30 October 1998     

Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation.

The loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that uses only one type of enzyme. One of the characteristics of the LAMP method is its ability to synthesize extremely large amount of DNA. Accordingly, a large amount of by-product, pyrophosphate ion, is produced, yielding white precipitate of magnesium pyrophosphate in the reaction mixture. Judging the presence or absence of this white precipitate allows easy distinction of whether nucleic acid was amplified by the LAMP method. Since an increase in the turbidity of the reaction mixture according to the production of precipitate correlates with the amount of DNA synthesized, real-time monitoring of the LAMP reaction was achieved by real-time measurement of turbidity.     

Regulation of IgA secretion by T cell clones derived from the human gastrointestinal tract

The role of T cells in Ig isotype regulation is still unclear. To address this question, we generated mitogen-stimulated T cell clones from normal human lymphoid follicles of the gut-associated lymphoid tissue (appendix). Both the T cell clones and clonal supernatants provided preferential help for IgA secretion by PWM-stimulated B cells. Many of these CD3+, CD4+, 4B4+, DR+ helper clones co-expressed Fc-gamma and Fc-alpha R, but there was poor correlation between the expression of Fc-alpha R and IgA help (p = 0.31). Most of the T cell clones helped both IgM+A- and IgM-A+ B cell populations to secrete IgA, suggesting that they mediate switch of isotype-uncommitted B cells as well as post-switch expansion of IgA-committed B cells; however, some of the T cell clones helped IgM+A- B cell populations much more than IgM-A+ B cell populations, suggesting that, in this case, the regulatory effect is predominantly at the level of B cell switch. In all, these results show that the mucosal immune system contains individual T cells which are capable of positively regulating IgA-specific isotype differentiation at two levels of B cell development, thus allowing for efficient generation of IgA-secreting B cells.     

Specific repression of the yeast silent mating locus HMR by an adjacent telomere.

The yeast silent mating loci HML and HMR are located at opposite ends of chromosome III adjacent to the telomeres. Mutations in the N terminus of histone H4 have been previously found to derepress the yeast silent mating locus HML to a much greater extent than HMR. Although differences in the a and alpha mating-type regulatory genes and in the cis-acting silencer elements do not appear to strongly influence the level of derepression at HMR, we have found that the differential between the two silent cassettes is largely due to the position of the HMR cassette relative to the telomere on chromosome III. While HML is derepressed to roughly the same extent by mutations in histone H4 regardless of its chromosomal location, HMR is affected to different extends depending upon its chromosomal positioning. We have found that HMR is more severely derepressed by histone H4 mutations when positioned far from the telomere (cdc14 locus on chromosome VI) but is only minimally affected by the same mutations when integrated immediately adjacent to another telomere (ADH4 locus on chromosome VII). These data indicate that the degree of silencing at HMR is regulated in part by its neighboring telomere over a distance of at least 23 kb and that this form of regulation is unique for HMR and not present at HML. These data also indicate that histone H4 plays an important role in regulating the silenced state at both HML and HMR.     

Field performance of lines derived from haploid and diploid tissues of Hordeum vulgare

Summary Plant tissue culture technology is of increasing interest to plant breeders. As part of a continuing investigation into breeding methods with spring barley two studies were conducted to assess the field performance of the progenies of material regenerated in tissue culture. The first study involved two spring barley cultivars, Golden Promise and Mazurka and compared lines produced from immature embryo (IE) derived callus with those from embryos developed by the Hordeum bulbosum (Hb) technique of chromosome elimination. In general the mean values for the seven characters scored were lower for the IE than the Hb material. In the second study F₁ hybrid material (Golden PromisexMazurka) was used and doubled haploid lines produced by the H. bulbosum and microspore culture (M) techniques were compared with single seed descent (SSD) material. Analysis of these F samples indicated that the mean values for the M lines were significantly lower than those of the Hb and SSD lines. Furthermore, data from the M lines showed significant evidence of variation created during the culture phase. The implications of these findings for barley breeding are discussed.     

Construction of a yeast artificial chromosome library of pepper (Capsicum annuum L.) and identification of clones from the Bs2 resistance locus

A yeast artificial chromosome (YAC) library was constructed using high-molecular-weight DNA isolated from pepper (Capsicum annuum L.) leaf protoplasts. Insert DNA was prepared by partial digestion using EcoRI and subjected to electrophoretic fractionation before in-gel ligation to the pJS97/98 YAC vector. Prior to transformation of yeast spheroplasts, ligation products were subjected to a second electrophoretic size selection. The library consists of about 19 000 clones with an average insert size of 500 kb, thus representing approximately three haploid genome equivalents. Three PCR-based markers tightly linked to the pepper Bs2 resistance gene were used to assess the utility of this library for positional cloning. Three YAC clones containing pepper genomic DNA from the Bs2 resistance locus were isolated from the library. The clones ranged in size from 270 kb to 1.2 Mb and should prove useful for the cloning of the Bs2 gene. Received: 15 January 1999 / Accepted: 11 May 1999     

Conditional ancestral times and related properties of an extant mutant at a locus A given the number of segregating sites observed at a linked locus B

 It is shown how the mean ancestral times at one locus are affected in a two- locus model with recombination when information is given regarding the number of segregating sites at another locus. For samples of n genes, recursive equations are derived that describe precisely the evolution of the time-depth of such a linked genealogy. Exact numerical solutions and Markov chain Monte Carlo simulations are discussed and compared. The dependence of some properties of a singleton mutation on waiting times between events in the two-locus genealogy is quantified and illustrates the effect of recombination on these properties. The following cases are presented: (1) the distribution of the number of mutant genes in a sample arising from a singleton mutation; (2) the probability that an allele observed in a genes of a sample of size n is the ancestral type (the oldest); (3) the expectation and variance of the age of a mutant having b copies in a sample of n genes. Received: 1 September 2000 / Revised version: 1 October 2001 / Published online: 8 May 2002     

Variation in the life-span of clones derived from human diploid cell strains

The doubling potential of several hundred clones derived from WI-38 and WI-26 cell cultures has been determined. Clones were isolated at various population doubling levels (PDLs) during the finite in vitro life-span of the mass (uncloned) cultures. In all cases, there was a large variation in population doubling potential (or life-span) among the clones isolated from a single mass culture. When clones were isolated from mass cultures which had undergone eight or nine population doublings, only about 50% of the clones were capable of more than eight population doublings. This percentage was further reduced when clones were isolated from mass cultures at higher PDLs. Mass cultures appear to be composed of two subpopulation classes: one with a low population doubling potential, and the other with a higher population doubling potential. Nevertheless, the highest doubling potential observed in clones isolated from any single culture was about the same as the doubling potential of the mass culture from which single cells were taken.     

Molecular clones of the mouse t complex derived from microdissected metaphase chromosomes

Fragments of the proximal half of mouse chromosome 17 including the t-complex region were microdissected from metaphase spreads. DNA was isolated from a pool of such fragments, and was cloned on microscale. Individual clones were used to probe genomic digests of DNA from a pair of Chinese hamster cell lines with or without mouse chromosome 17, and livers of congenic inbred lines of mice carrying wild-type and/or t-haplotype forms of chromosome 17. The data obtained indicate that 95% of the low copy number microclone inserts recognize DNA sequences present on mouse chromosome 17. It has been possible to use one-third of these clones to identify restriction-fragment-length polymorphisms between wild-type and t-haplotype DNA on a congenic background. These results demonstrate that these clones have been derived from the t-complex or regions closely linked to it. Clones of this type should provide starting points for a molecular analysis of this region of the mouse genome.     

Association of an isozyme locus and strawbreaker foot rot resistance derived from Aegilops ventricosa in wheat

Summary Thirty lines from a cross between VPM/ Moisson 421 and Selection 101 were used in the study to determine whether strawbreaker foot rot resistance derived from Aegilops ventricosa was associated with an allele for endopeptidase. The progeny examined for foot rot lesions represented F₂ derived F₅ lines and enzyme assays were done on F₆ seedlings. The results indicate that the wheat and VPM/Moisson 421 endopeptidase alleles are distinctly different. The endopeptidase allele frequencies of 30 lines were compared with strawbreaker foot rot resistance as measured by the lesion severity index. The results demonstrate a close association between the gene for strawbreaker foot rot resistance and the endopeptidase allele derived from Ae. ventricosa.