Construction of a single-copy integration vector and its use in analysis of regulation of the trp operon of Bacillus subtilis

A single-copy integration vector was used for the in vitro construction of translational fusions to the lacZ gene of Escherichia coli. Insertion of a single copy of the lacZ fusion into the B. subtilis chromosome leads to an easily detected Amy- phenotype. A trpE-lacZ fusion was constructed in which the trp promoter directs hybrid beta-galactosidase (beta Gal) synthesis. The level of beta Gal in a wild-type strain carrying the trpE-lacZ fusion in the chromosome is regulated by exogenous tryptophan, while a 5-methyltryptophan-resistant mutant constitutively synthesizes betaGal. A trpF-lacZ fusion was constructed and used to determine the effect of a frameshift mutation in the trpE gene on expression of the trpF-lacZ fusion. The frameshift mutation in trpE led to a three-fold reduction in the levels of the trpF-lacZ fusion. The levels of the betaGal activity of these integrated lacZ fusions appear to provide a quantitative measure of the expression of B. subtilis genes under single-copy conditions.     

GAL2 codes for a membrane-bound subunit of the galactose permease in Saccharomyces cerevisiae.

The gene encoding the galactose permease of Saccharomyces cerevisiae (GAL2) was cloned. The clone restores galactose permease activity to gal2 yeasts and is regulated by galactose in a manner similar to other GAL gene products (GAL1, -7, and -10). Experiments with temperature-conditional secretory mutants indicated that transport of the GAL2 gene product to the cell surface requires a functional secretory pathway. In addition, gene fusions were constructed between the GAL2 gene and the Escherichia coli lacZ gene. The GAL2-lacZ gene fusions code for galactose-regulated beta-galactosidase activity in yeasts. The beta-galactosidase activity was found to be membrane bound.     

Regulation of RAD54- and RAD52-lacZ gene fusions in Saccharomyces cerevisiae in response to DNA damage.

The RAD52 and RAD54 genes in the yeast Saccharomyces cerevisiae are involved in both DNA repair and DNA recombination. RAD54 has recently been shown to be inducible by X-rays, while RAD52 is not. To further investigate the regulation of these genes, we constructed gene fusions using 5' regions upstream of the RAD52 and RAD54 genes and a 3'-terminal fragment of the Escherichia coli beta-galactosidase gene. Yeast transformants with either an integrated or an autonomously replicating plasmid containing these fusions expressed beta-galactosidase activity constitutively. In addition, the RAD54 gene fusion was inducible in both haploid and diploid cells in response to the DNA-damaging agents X-rays, UV light, and methyl methanesulfonate, but not in response to heat shock. The RAD52-lacZ gene fusion showed little or no induction in response to X-ray or UV radiation nor methyl methanesulfonate. Typical induction levels for RAD54 in cells exposed to such agents were from 3- to 12-fold, in good agreement with previous mRNA analyses. When MATa cells were arrested in G1 with alpha-factor, RAD54 was still inducible after DNA damage, indicating that the observed induction is independent of the cell cycle. Using a yeast vector containing the EcoRI structural gene fused to the GAL1 promoter, we showed that double-strand breaks alone are sufficient in vivo for induction of RAD54.     

Analysis of the galactose signal transduction pathway in Saccharomyces cerevisiae: interaction between Gal3p and Gal80p.

The GAL3 gene plays a critical role in galactose induction of the GAL genes that encode galactose- metabolizing enzymes in Saccharomyces cerevisiae. Defects in GAL3 result in a long delay in GAL gene induction, and overproduction of Gal3p causes constitutive expression of GAL. Here we demonstrate that concomitant overproduction of the negative regulator, Gal80p, and Gal3p suppresses this constitutive GAL expression. This interplay between Gal80p and Gal3p is direct, as tagged Gal3p coimmunoprecipitated with Gal80p. The amount of coprecipitated Gal80p increased when GAL80 yeast cells were grown in the presence of galactose. When both GAL80 and GAL3 were overexpressed, the amount of coprecipitated Gal80p was not affected by galactose. Tagged gal3 mutant proteins bound to purified Gal80p, but only poorly in comparison with the wild type, suggesting that formation of the Gal80p-Gal3p complex depends on the normal function of Gal3p. Gal3p appeared larger in Western blots (immunoblots) than predicted by the published nucleic acid sequence. Reexamination of the DNA sequence of GAL3 revealed several mistakes, including an extension at the 3' end of another predicted 97 amino acids.     

In vitro construction and characterization of phoA-lacZ gene fusions in Escherichia coli.

Using recombinant DNA techniques, we have constructed phoA-lacZ gene fusions. Two of the fusions encode hybrid proteins containing approximately half of alkaline phosphatase at the amino terminus joined to beta-galactosidase. For the one fusion strain analyzed in detail, it was shown that the hybrid protein is found in the membrane fraction of cells. In its membrane location, the beta-galactosidase activity of the hybrid is not sufficient to support cell growth on lactose. Unexpectedly, fusions containing phoA and lacZ joined in the wrong translational reading frame were also obtained. These fusions direct the phosphate-regulated synthesis of beta-galactosidase, apparently via a translation restart mechanism. Thus, when gene fusions are constructed, the presence of properly regulated beta-galactosidase activity does not necessarily indicate that a hybrid protein is being produced.     

Invertase beta-galactosidase hybrid proteins fail to be transported from the endoplasmic reticulum in Saccharomyces cerevisiae.

The yeast SUC2 gene codes for the secreted enzyme invertase. A series of 16 different-sized gene fusions have been constructed between this yeast gene and the Escherichia coli lacZ gene, which codes for the cytoplasmic enzyme beta-galactosidase. Various amounts of SUC2 NH2-terminal coding sequence have been fused in frame to a constant COOH-terminal coding segment of the lacZ gene, resulting in the synthesis of hybrid invertase-beta-galactosidase proteins in Saccharomyces cerevisiae. The hybrid proteins exhibit beta-galactosidase activity, and they are recognized specifically by antisera directed against either invertase or beta-galactosidase. Expression of beta-galactosidase activity is regulated in a manner similar to that observed for invertase activity expressed from a wild-type SUC2 gene: repressed in high-glucose medium and derepressed in low-glucose medium. Unlike wild-type invertase, however, the invertase-beta-galactosidase hybrid proteins are not secreted. Rather, they appear to remain trapped at a very early stage of secretory protein transit: insertion into the endoplasmic reticulum (ER). The hybrid proteins appear only to have undergone core glycosylation, an ER process, and do not receive the additional glycosyl modifications that take place in the Golgi complex. Even those hybrid proteins containing only a short segment of invertase sequences at the NH2 terminus are glycosylated, suggesting that no extensive folding of the invertase polypeptide is required before initiation of transmembrane transfer. beta-Galactosidase activity expressed by the SUC2-lacZ gene fusions cofractionates on Percoll density gradients with ER marker enzymes and not with other organelles. In addition, the hybrid proteins are not accessible to cell-surface labeling by 125I. Accumulation of the invertase-beta-galactosidase hybrid proteins within the ER does not appear to confer a growth-defective phenotype to yeast cells. In this location, however, the hybrid proteins and the beta-galactosidase activity they exhibit could provide a useful biochemical tag for yeast ER membranes.     

HeLa细胞cDNA文库及酵母双杂交诱饵载体pGBKT7-CPn0308的构建

旨在利用酵母双杂交系统构建HeLa细胞的cDNA文库,并构建酵母双杂交诱饵载体pGBKT7-CPn0308。从HeLa细胞中提取总RNA,应用SMARTTM技术,构建以pGADT7-Rec为载体的HeLa细胞酵母GAL4 AD融合cDNA文库。应用PCR技术扩增目的片段CPn0308,并成功克隆该基因到诱饵载体pGBKT7中,将重组质粒转化酵母菌株AH109后,检测诱饵载体有无自激活和细胞毒性作用。结果显示,文库展现良好的多态性,重组诱饵载体pGBKT7-CPn0308不具有毒性且未自主激活报告基因,说明该文库和诱饵质粒可用于酵母双杂交系统。