用酵母双杂交技术筛选DND1相互作用蛋白

目的:小鼠睾丸生殖细胞瘤易感蛋白DND1是在进化中保守的RNA结合蛋白.为了进一步了解DND1的功能,我们筛选了与DND1相互作用的蛋白.方法:采用酵母双杂交系统,用Dnd1基因编码序列构建诱饵载体筛选小鼠10.5天的胚胎文库.结果:筛选小鼠胚胎cDNA文库得到4个与DND1相互作用蛋白,分别为Tun蛋白、辅肌动蛋白α...     

PDR-like ABC systems in pathogenic fungi

ABC transporters of the Pleiotropic Drug Resistance (PDR) family are the main actors of antifungal resistance in pathogenic fungi. While their involvement in clinical resistant strains has been proven, their transport mechanism remains unclear. Notably, one hallmark of PDR transporters is their asymmetry, with one canonical nucleotide-binding site capable of ATP hydrolysis while the other site is not. Recent publications reviewed here show that the so-called “deviant” site is of crucial importance for drug transport and is a step towards alleviating the mystery around the existence of non-catalytic binding sites.     

Second-site antibiotic resistance mutations in the ribosomal region of yeast mitochondrial DNA

Summary A large proportion of the spontaneous erythromycin resistant mutants isolated from a strain carrying a previously-induced chloramphenicol resistance mutation at cap3 do not map at ery1, the locus most often associated with mitochondrial erythromycin resistance. Most of the new mutations are also nonallelic at spil, spi2, and other known antibiotic resistance loci within the 21S rRNA gene; they are allelic with each other and define the new locus, ery2. Induced second-site erythromycin resistant mutants from the cap ^r3 strain, as well as spontaneous or induced mutants from strains carrying a cap ^r 1 mutation, all tend to map at eryl. The cap ^r3 mutation is apparently necessary for the expression of erythromycin resistance resulting from a second mutation at ery2.     

Conserved sequences upstream of yeast ribosomal protein genes

Summary Computer analysis has previously revealed the presence of a 12-nucleotide common sequence element (AACATC _CA ^TG T _A ^G CA; HOMOL1) in the upstream regions of several yeast ribosomal protein genes. By extending the sequence analysis of the 5-flanking regions of a number of other ribosomal protein genes (including those encoding S10-1, S10-2, S33 and L16-2) we could establish that HOMOL1 occurs upstream of most but not all yeast ribosomal protein genes. Apart from HOMOL1 an additional conserved sequence (ACCCATACATT _A ^T ; RPG-box) was detected in front of nearly all yeast ribosomal protein genes, although in some cases it is present in the opposite orientation in the other strand. There seems to be no correlation between the occurrence of one box and that of the other. However when both boxes are present the RPG-box is always located 3 to the HOMOL1-sequence mostly at a distance of only a few nucleotides. A further one-to-one comparison of the upstream regions of several yeast ribosomal protein genes revealed extensive additional sequence homologies that are suggested to be involved in the coordinate control of ribosomal protein gene expression in yeast.     

Blockage to exonuclease III digestion in the chromatin of Saccharomyces cerevisiae maps to the in vitro — determined binding site of a trans-acting regulatory factor

Summary A series of transposable element-induced mutations at the HIS4 locus in Saccharomyces cerevisiae have been attributed to the transposition of a Ty element into the 5 regulatory region of this gene. Various Ty-containing His⁺ revertants have been isolated and the HIS4/Ty junction region sequenced. The only difference found in this region between a His^- and a weak His⁺ strain was a single point mutation, an AG transition. The position of Ty remained unaltered. Examination of lacZ fusion plasmids further implicated this AG transition as being reponsible for the altered phenotype, the bp transition representing an allele of a cis-acting regulatory element. Subsequent gel retardation and methylation interference experiments revealed that this AG mutation enabled the binding of a trans-acting factor (TyBf) in vitro. In this paper we show that the TyBf binding site is in a region of chromatin hypersensitive to digestion by DNase I. The binding site is protected in vivo from digestion with exonuclease III, suggesting the presence of a bound protein in His⁺ (on) but not His^- (off) Ty-containing strains. We propose that a trans-acting factor binding in vivo, presumably TyBf, is responsible for the activation of HIS4 expression in these insertion mutants.     

Reversible pseudohyphal growth in haploid Saccharomyces cerevisiae is an aerobic process

Pseudohyphal growth in Saccharomyces cerevisiae has been postulated to be an adaptation to foraging for nitrogen during nitrogen starvation. This process was described as a strictly diploid phenomenon which did not occur in haploid yeast cells and was under the genetic control of both the mating-type locus and a group of five genes, the BUD genes, regulating bud formation. We have also observed a dimorphic growth pattern in yeast growing on various nitrogen-limiting synthetic media. However, and in contrast to a previous report, we find that pseudohyphal growth is not precluded in haploid cells. We demonstrate that haploid pseudohyphal growth is strictly oxygen-dependent and is rapidly reversible, defining pseudohyphal growth as a reversible developmental pathway in yeast.     

The Saccharomyces cerevisiae RAD2 gene complements a Schizosaccharomyces pombe repair mutation

Summary Two Saccharomyces cerevisiae genes necessary for excision repair of UV damage in DNA, RAD1 and RAD2, were introduced individually, on a yeast shuttle vector, into seven Schizosaccharomyces pombe mutants — rads1, 2, 5, 13, 15,16 and 17. The presence of the cloned RAD1 gene did not affect survival of any of the S. pombe mutants. The RAD2 gene increased survival of S. pombe rad13 to near the wild-type level after UV irradiation and had no effect on any of the other mutants tested. S. pombe rad13 mutants are somewhat defective in removal of pyrimidine dimers so complementation by the S. cerevisiae RAD2 gene suggests that the genes may code for equivalent proteins in the two yeasts.