hTCF4酵母双杂交载体的构建

目的　研究Wnt Frizzled信号传导通路上的主要信号分子T细胞因子 (TCF4)在细胞核内转导的辅激活物和辅阻遏物。方法　通过PCR法以真核表达载体pCDNA TCF4为模板 ,扩增获得了15 0 0bp的TCF4基因的末端及DNA结合域片段 ,经过SalI ,NotI酶切后 ,基因重组的方法定向克隆于酵母双杂交载体pPC86和pDBleu中 ,构建形成pDBleu TCF4和pPC86 TCF4真核表达载体。结果　经转化 ,提取质粒酶切鉴定表明构建成功。结论　hTCF4酵母双杂交载体的构建成功为Wnt Frizzled信号通路的深入研究奠定了基础。     

The Hansenula polymorpha per6 mutant is affected in two adjacent genes which encode dihydroxyacetone kinase and a novel protein, Pak1p, involved in peroxisome integrity

The Hansenula polymorpha per6-210 mutant is impaired in respect of growth on methanol (Mut^–) and is characterized by aberrant peroxisome formation. The functionally complementing DNA fragment contains two open reading frames. The first encodes dihydroxyacetone kinase (DAK), a cytosolic enzyme essential for formaldehyde assimilation; the second ORF codes for a novel protein (Pak1p). We have demonstrated that per6-210 cells lack DAK activity, causing the Mut^– phenotype, and have strongly reduced levels of Pak1p, resulting in peroxisomal defects. Sequence analysis revealed that per6-210 contains a mutation in the 3′ end of the DAK coding region, which overlaps with the promoter region of PAK1. Possibly this mutation also negatively affects PAK1 expression. Received: 25 February / 12 May 1998     

Evaluation of estrogenic activity of organic biocides using ER-binding and YES assay.

Biocides have been used not only in everyday items such as clothes, kitchenware, daily necessities, and infant utensils, but also in cosmetics and wrapping papers for foodstuffs. Since there is a high possibility of exposure to biocides from such materials, their safety must be assessed adequately using a range of methods. We investigated the estrogenic activity of 20 organic biocides using two in vitro screen assays: estrogen receptor (ER) binding assay and yeast estrogen screen (YES) assay. Twelve of the biocides were positive in the ER-binding assay. Regardless of the presence or absence of rat S9Mix for metabolic activation, 4-chloro-3-methylphenol was positive in both ER-binding and YES assay. 4-Chloro-3,5-dimethylphenol was positive in the ER-binding assay and showed a pseudopositive manner in the YES assay that was observed the dose-independent estrogenic activity at only one dose point. Hiba oil was positive in the ER-binding assay but was positive in the YES assay only in the presence of rat S9Mix. These results suggest that ER-binding and YES assay could be adapted for evaluation of the endocrine-disrupting activity of biocides. The biocides found to be positive in vitro now require assessment by in vivo screening methods.     

Time-dependent mitotic recombination in Saccharomyces cerevisiae

The time-dependent appearance of prototrophic recombinants between heterologously located artificial repeats has been studied in Saccharomyces cerevisiae. While initial prototrophic colony numbers from independent cultures were highly variable, additional recombinants were found to arise daily at roughly constant rates irrespective of culture. These late-appearing recombinants could be accounted for neither by detectable growth on the selective media nor by delayed appearance of recombinants present at the time of selective plating. Significantly, at no time did the distributions of recombinants fully match those expected according to the Luria-Delbruck model and, in fact, after the first day, the distributions much more closely approximated a Poisson distribution. Prototrophic recombinants accumulated not only on the relevant selective medium, but also on media unrelated to the acquired prototrophy.     

Refined mapping and characterization of the recessive familial amyotrophic lateral sclerosis locus (ALS2) on chromosome 2q33

Amyotrophic lateral sclerosis (ALS) is a progressive degenerative neuromuscular disease that shows familial, autosomal dominant inheritance in 10%–15% of cases. Previous genetic analysis of one large family linked a recessive form of familial ALS (FALS-AR type 3) to the chromosome 2q33–35 region. Using additional polymorphic markers, we have narrowed the size of the linked region to approximately 1.7 cM by linkage and haplotype analysis. We have also established a yeast artificial chromosome contig across the locus that covers an approximate physical distance of 3 million bases. Based on this contig, genes and expressed sequences that map near the 2q33 region have been examined to determine whether they are located within this ALS2 candidate locus. Five identified genes and 34 expressed sequence tags map within the region defined by crossover analysis and merit further consideration as candidate genes for this disease. Accepted: July 15, 1998 / Published online: October 28, 1998     

Basidiomycetousras cDNA functionally replaces its homolog genes in yeast

It was shown by a plasmid exchange procedure that the Ras-encoding cDNA of the basidiomyceteLentinus edodes (namedLeras cDNA) can functionally replace its homolog genes (ScRAS1 andScRAS2) in the yeastSaccharomyces cerevisiae to maintain the viability of an yeast strain containing genetic disruptions of bothRAS genes. The strain replaced by aLeras–cDNA-carrying plasmid, however, grew slower than the strains replaced by aScRAS1– or aScRAS2–carrying plasmid. The intracellular level of cAMP in the strain harboring theLeras–cDNA-carrying plasmid was clearly higher than that of a parental strain which maintains a plasmid carrying theS. cerevisiae cAMP-dependent protein kinase catalytic subunit C₁ gene,TPK1, but was lower than that in a strain harboring anScRAS2–carrying plasmid. These results suggest that theLeras cDNA can complement theras1 ^– ras2^– mutation of yeast by virture of the stimulation of adenylate cyclase activity, although the complementation is not as efficient as that obtained by expressing theScRAS2 gene.     

Simultaneous induction of multiple mutations by N-methyl-N′-nitro-N-nitrosoguanidine in the yeast Saccharomyces cerevisiae

Summary Contrary to what happens in bacteria, mutations induced by nitrosoguanidine in yeast are not accompanied by an excess of mutations in nearby genes. We have investigated nitrosoguanidine mutagenesis in three regions of the yeast genome: the contiguous DNA segments HIS4A, HIS4B and HIS4C, located on chromosome III; ADE1 and CDC15 separated by about 3 map units on chromosome I; and CAN1, some 50 map units away from the centromere on chromosome V. Revertants at HIS4C never suffered mutations at HIS4A or HIS4B. Reversion at CDC15 did not affect the frequency of mutation at ADE1. No tsm mutations, leading to thermonsensitivity, were found in the immediate vicinity of the locus CAN1 after selecting for canavanine resistant mutants. However, as expected from nitrosoguanidine mutagenesis of replication points and the fixed pattern of chromosome replication, the induced tsm mutations seem not to map randomly over the yeast genome; in fact, two out of the three groups of such tsm mutations studied are located in the same chromosome arm as CAN1, indicating that these two regions are replicated at the same time as CAN1. Replication synchrony is less than perfect, since the tsm mutations of each group affect many different genes.     

A new killer toxin produced by Saccharomyces cerevisiae

A wine-making Saccharomyces cerevisiae yeast strain isolated in our laboratory produces two different killer toxins, each one encoded by one dsRNA plasmid. One toxin has the same specificity as the one produced by strain M437 described by Naumov, but the dsRNA plasmid which encodes it migrates slightly faster in poly acrylamide gel electrophoresis. The other toxin has not been previously described, and is encoded by a dsRNA fraction which migrates at a lower rate than the × fraction of M437. These two dsRNA plasmids can be maintained separately in different yeast strains.     

Sensitivity of Clinical Yeast Isolates in Kuwait against a Number of Antifungal Agents

Summary: Clinical yeast isolates were taken from 37 patients with presumptive skin candidiasis in Kuwait. In vitro sensitivity tests using amphotericin B, nystatin, miconazole nitrate, 5-fluorocytosine and chlorhexidine were carried out. Amphotericin B was found to be the most effective and 5-fluorocytosine the least.
Zusammenfassung: Von 37 Patienten wurden wegen vermuteter Candida-Mykose Hefen isoliert. In-vitro-Resistenztestungen wurden mit Amphotericin B, Nystatin, Miconazolnitrat, 5-Fluorocytosin und Chlorhexidin ausgeführt. Bei dieser Testung wurde Amphotericin B als die wirksamste und 5-Fluorocytosin als die am wenigsten wirksame Substanz ermittelt.     

Isolation and characterization of mutants as an approach to a transformation system in Kluyveromyces marxianus

 A method to obtain K. marxianus mutants has been developed. Different auxotrophic mutants were isolated by nystatin and snail-enzyme enrichment procedures using an incubation time of 2 h before adding the antibiotic or the enzyme respectively. All his mutants analyzed by complementation tests turned out to belong to the same complementation group. Some of them were transformed and complemented by the S. cerevisiae HIS3 gene. These non-reverting his3 mutants contain no heterologous sequence, which is essential to make them acceptable for application in the food industry. Received: 15 November/21 December 1995