Nucleotide sequence of the ARGRII regulatory gene and amino acid sequence homologies between ARGRII PPRI and GAL4 regulatory proteins

We report here the DNA sequence of the ARGRII gene, one of the three regulatory genes involved in controlling the anabolism and catabolism of arginine in yeast. This gene encodes a protein of 880 amino acids with a deduced molecular mass of about 100 kDa. The ARGRII protein shows significant homology with two other regulatory proteins of yeast, PPRI and GAL4.     

Dissection of the bifunctional ARGRII protein involved in the regulation of arginine anabolic and catabolic pathways.

ARGRII is a regulatory protein which regulates the arginine anabolic and catabolic pathways in combination with ARGRI and ARGRIII. We have investigated, by deletion analysis and fusion to LexA protein, the different domains of ARGRII protein. In contrast to other yeast regulatory proteins, 92% of ARGRII is necessary for its anabolic repression function and 80% is necessary for its catabolic activator function. We can define three domains in this protein: a putative DNA-binding domain containing a zinc finger motif, a region more involved in the repression activity located around the RNase-like sequence, and a large activation domain.     

Regulation of arginine metabolism in Saccharomyces cerevisiae: expression of the three ARGR regulatory genes and cellular localization of their products

Three regulatory proteins are involved in the control of arginine metabolism in yeast: ARGRI, ARGRII and ARGRIII. The control region and part of the coding sequence of the ARGR genes were fused to the Escherichia coli lacZ gene. These chimeras were used to study the expression of the regulatory genes as well as the cellular compartmentalization of the regulatory products. Our results show that the three ARGR proteins are localized in the nucleus and that their synthesis is not regulated by arginine nor by any of the other ARGR products. However, some data suggest that the ARGRIII protein could control ARGRI activity.     

Structure and myofiber-specific expression of the rat muscle regulatory gene MRF4.

We have cloned an 11.3-kb rat genomic DNA fragment encompassing the muscle regulatory factor 4 (MRF4) protein-coding sequence, 8.5 kb of 5'-flanking sequence, and 1.0 kb of 3'-flanking sequence. In order to study MRF4 gene expression, the rat myogenic cell line, L6J1-C, which expresses the endogenous MRF4 gene only in differentiated myofibers, was transfected stably with the full-length genomic clone and various 5' deletions. RNase protection assays demonstrated that MRF4 genes containing as little as 430 bp of 5'-flanking sequence exhibited an increase in expression as the cells differentiated into myofibers, indicating that elements responsible for fiber-specific expression are contained within this cloned DNA fragment. Similar up-regulation was observed with genes containing 1.5 kb of 5'-flanking sequence. Interestingly, MRF4 genes containing 5.0 kb and 8.5 kb of 5'-flanking sequence were up-regulated to even higher levels, suggesting that additional myofiber-specific regulatory elements located between 1.5 and 5.0 kb upstream from the coding region play a role in regulating the expression of this muscle-specific gene.     

Isolation of the ERG12 gene of Saccharomyces cerevisiae encoding mevalonate kinase

The ERG12 gene of Saccharomyces cerevisiae has been cloned by complementation of an erg12-1 mutation affecting mevalonate kinase. From the 2.8-kb insert isolated, the functional gene has been localized on a DNA fragment of 2.1 kb. The mRNA is 1.45 kb long. Gene disruption shows that the ERG12 gene is essential in yeast, both for spore germination and vegetative growth.     

Molecular cloning of eucaryotic genes required for excision repair of UV-irradiated DNA: isolation and partial characterization of the RAD3 gene of Saccharomyces cerevisiae.

We describe the molecular cloning of a 6-kilobase (kb) fragment of yeast chromosomal DNA containing the RAD3 gene of Saccharomyces cerevisiae. When present in the autonomously replicating yeast cloning vector YEp24, this fragment transformed two different UV-sensitive, excision repair-defective rad3 mutants of S. cerevisiae to UV resistance. The same result was obtained with a variety of other plasmids containing a 4.5-kb subclone of the 6-kb fragment. The UV sensitivity of mutants defective in the RAD1, RAD2, RAD4, and RAD14 loci was not affected by transformation with these plasmids. The 4.5-kb fragment was subcloned into the integrating yeast vector YIp5, and the resultant plasmid was used to transform the rad3-1 mutant to UV resistance. Both genetic and physical studies showed that this plasmid integrated by homologous recombination into the rad3 site uniquely. We conclude from these studies that the cloned DNA that transforms the rad3-1 mutant to UV resistance contains the yeast chromosomal RAD3 gene. The 4.5-kb fragment was mapped by restriction analysis, and studies on some of the subclones generated from this fragment indicate that the RAD3 gene is at least 1.5 kb in size.     

Cloning and characterization of the gene for the yeast cytoplasmic threonyl-tRNA synthetase.

A fragment of DNA from the yeast nuclear gene MST1 that codes for the mitochondrial tRNAThr1 synthetase was used as a probe to screen for other yeast threonyl-tRNA synthetase genes. At low stringency, the MST1 probe hybridizes strongly to a 6.6 kb EcoRI fragment of yeast genomic DNA with the homologous gene and in addition hybridizes more weakly to a smaller 3.6 kb EcoRI fragment with a second threonyl-tRNA synthetase gene (THS1). To clone THS1, a library was constructed by ligation to pUC18 of size selected (3-4.5 kb) EcoRI fragments of genomic DNA. Several clones containing the 3.6 kb EcoRI fragment were isolated. A 2,202 nucleotide long open reading frame corresponding to THS1 has been identified in the cloned fragment of DNA. The predicted protein encoded by THS1 is 38% identical to the E. coli threonyl-tRNA synthetase over the latter's length (642 amino acids) and is 42% identical to the predicted MST1 product over its 462 residues. In situ disruption of the chromosomal copy of THS1 is lethal to the cell, indicating that this gene codes for the cytoplasmic threonyl-tRNA synthetase.     

圈卷产色链霉菌7100分化相关基因——sawE的结构与功能

以天蓝色链霉菌的whiB基因为探针,从圈卷产色链霉菌7100的总DNA部分文库中克隆了含有whiB同源序列的28kb DNA片段,并对其中的14kb片段进行了序列测定。序列分析表明,该片段含有一个完整的开放阅读框—sawE。预测的蛋白质结构及同源性分析显示,sawE与天蓝色链霉菌孢子形成早期的关键基因whiB高度同源,编码产物为一个调控蛋白。sawE的破坏使圈卷产色链霉菌7100的分化终止在气生菌丝阶段,在延长培养时间的情况下仍保持白色的表型,菌丝不能分隔,不能形成成熟的灰色孢子,结果表明sawE基因是一个与圈卷产色链霉菌分化有关的重要基因。     

野油菜黄单胞菌野油菜致病型胞外多糖合成有关的DNA序列的克隆

以从野油菜黄单胞菌野油菜致病型（Xanthomonascampestrispv.campestris）胞外多糖突变体T117中克隆到的含有突变序列的DNA片段作探针,从野生型菌株中鉴定和克隆了含有相应序列的9．4kbHindⅢ片段。该片段可反式互补突变体T117,完全恢复其胞外多糖的合成,说明该片段至少含有一个完整的与该菌胞外多糖合成有关的基因。     

The ntr genes of Escherichia coli activate the hut and nif operons of Klebsiella pneumoniae

Regulation of the synthesis of glutamine synthetase and of the arginine and glutamine transport systems (Ntr phenotype) in Salmonella have been shown to require two regulatory genes on the C-terminal side of the glnA gene (McFarland et al., 1981). We have cloned a HindIII-EcoRI DNA fragment from Escherichia coli coding for analogous properties with respect to the Ntr phenotype in E. coli. A plasmid containing this E. coli DNA fragment joined to another fragment carrying a cyanobacterial glnA gene (but no functional regulatory genes) was introduced into a Klebsiella pneumoniae mutant with a Gln-Ntr- phenotype, i.e., which could not derepress nitrogenase. The cyanobacterial gene made the Klebsiella strain Gln+ and the E. coli DNA fragment made the strain Ntr+, including the ability to derepress nitrogenase fully. Thus the products of the glnA-linked ntr genes of E. coli can regulate expression of the Ntr-dependent genes of Klebsiella.     

The study of binding of the virG gene product with the virD gene promotor region of the Agrobacterium tumefaciens Ti-plasmid C58

The 1.5 kb EcoRI--HindIII fragment of the pTiC58 containing the virD regulatory sequence demonstrates a constitutive promoter activity in E. coli background and an inducible one in agrobacterium. The virG gene was cloned in pTZ19R plasmid. To reveal the virG product--virD regulatory sequence interaction a few protein fractions of E. coli harbouring the obtained recombinant plasmid pTZ19G lysate were used. PAGE-retardation assay revealed the specific binding between the 1.5 kb DNA fragment containing 5'-end of virD and a separate protein fraction of the bacterial lysate.