High guanine plus cytosine content in the third letter of codons of an extreme thermophile. DNA sequence of the isopropylmalate dehydrogenase of Thermus thermophilus.
(65/111)
In studies on the cause of the extreme stability of the macromolecules of Thermus thermophilus HB8, the leuB gene coding for 3-isopropylmalate dehydrogenase of the leucine synthesis pathway and its flanking regions were cloned and sequenced. The leuB gene of T. thermophilus was expressed in a leuB-less mutant of Escherichia coli, and thermostable dehydrogenase was purified from an extract of the cells. The primary structure of the thermophilic isopropylmalate dehydrogenase was deduced from the nucleotide sequence leuB gene (1017 base pairs) and the amino acid sequence of the peptides isolated from the purified dehydrogenase. The thermophilic dehydrogenase has Mr = 35,968, and the value was close to that determined for the monomer of dehydrogenase (36,000) by gel electrophoresis. The molecular weight of active dimeric dehydrogenase was found to be 73,000 by high speed liquid chromatography. The primary structure of dehydrogenase was consistent with the amino acid composition of the dehydrogenase. In contrast to the isopropylmalate dehydrogenase of E. coli which contains 8 cysteine residues, there was no cysteine in thermophilic isopropylmalate dehydrogenase. The 5'-noncoding region contained a typical Shine-Dalgarno sequence. The guanine plus cytosine content of the coding region was 70.1%, and that of the third letter of the codons was extremely high (89.4%). (+info)
Yeast LEU2. Repression of mRNA levels by leucine and primary structure of the gene product.
(66/111)
It has been known that enzyme activity associated with the yeast LEU1 and LEU2 gene product (beta-isopropylmalate dehydrogenase) drops sharply when yeast is grown in the presence of leucine. RNA blot hybridizations with LEU2-specific probes establish that this is accompanied by a 5-fold repression in LEU2 mRNA levels. A similar repression was noted recently for LEU1 mRNA levels (Hsu, Y.-P., and Schimmel, P. (1984) J. Biol. Chem. 259, 3714-3719). Nuclease mapping of the 5'-end of the LEU2 mRNA shows a major start at approximately 16 nucleotides upstream of the AUG initiation codon. This initiation site in the gene is retained in an extensive LEU2 5'-noncoding region deletion which still expresses the LEU2 gene product (Erhart, E., and Hollenberg, C. P. (1983) J. Bacteriol. 156, 625-635). The primary structure of the LEU2 gene product was established from the nucleotide sequence of the gene-coding region and from fitting amino acid sequences of scattered internal peptides to the nucleotide sequence. The 364-amino acid protein has a 13-amino acid stretch which is highly homologous to the partially sequenced yeast LEU1 gene product (isopropylmalate isomerase). The homology occurs about 290 amino acids from the respective NH2 termini of the two proteins. The homology may represent residues which interact with beta-isopropylmalate, a common ligand for the enzymes. (+info)
Fusion of the Saccharomyces cerevisiae leu2 gene to an Escherichia coli beta-galactosidase gene.
(67/111)
The promoter and translation initiation region of the Saccharomyces cerevisiae leu2 gene was fused to the Escherichia coli beta-galactosidase gene. This fusion located the control region of the leu gene and orientated its direction of expression. When the fusion was placed into yeast cells, beta-galactosidase was expressed under the same regulatory pattern as the original leu2 gene product: its synthesis was repressed in the presence of leucine and threonine. Sensitive chromogenic substrates for beta-galactosidase were used to detect expression in isolated colonies growing on agar medium. Mutant yeast cells with increased beta-galactosidase activity were identified by the color of the colonies they formed. One class of mutants obtained appeared to affect ars1 plasmid maintenance, and another class appeared to affect beta-galactoside uptake. (+info)
Overproduction and control of the LEU2 gene product, beta-isopropylmalate dehydrogenase, in transformed yeast strains.
(68/111)
Two transformed yeast strains, 21D/pYT14-LEU2 and AH22/CV9-2, were found to produce beta-isopropylmalate dehydrogenase to such an extent that the enzyme constitutes 2 and 1%, respectively, of the total extractable protein. This is 30 and 15 times, respectively, above wild type level. beta-Isopropylmalate dehydrogenase was purified from strain 21D/pYT14-LEU2 to a purity of about 95% in essentially three steps. Strain 21D/pYT14-LEU2 carries the LEU2 gene on a vector that also contains the yeast 2-micrometers plasmid and therefore replicates autonomously, whereas strain AH22/CV9-2 carries multiple copies of the LEU2 gene integrated at its normal chromosomal location. Despite the different genetic arrangements, regulation of LEU2 gene expression by leucine and leucine plus threonine was normal. Immunotitration showed that the decrease in specific activity caused by leucine and threonine corresponded to a decrease in immunoreactive material. (+info)
Transposed LEU2 gene of Saccharomyces cerevisiae is regulated normally.
(69/111)
The repression of beta-isopropylmalate dehydrogenase, the LEU2 gene product, by leucine and leucine plus threonine was unaffected by the transposition of LEU2 from its original locus on chromosome III to a new locus within the ribosomal deoxyribonucleic acid gene cluster on chromosome XII. Since the expression of the LEU2 gene is probably controlled at a pretranslational level, we conclude that the recombinant plasmid used for transformation carries regulatory information in addition to LEU2 structural information. (+info)
Cloning of 3-isopropylmalate dehydrogenase gene of an extreme thermophile and partial purification of the gene product.
(70/111)
The gene of an extreme thermophile, Thermus thermophilus HB8, which codes for a leucine biosynthetic enzyme, 3-isopropylmalate (3-IPM) dehydrogenase [EC 1.1.1.85], was cloned in Escherichia coli using pBR322 as a vector. E. coli cells carrying this recombinant plasmid, pHB2, produced the thermophilic enzyme 7-fold more than did T. thermophilus HB8 cells. When the crude extract of the pHB2-carrying cells was treated at 70 degrees C for 10 min, approximately 75% of the protein in the extract was precipitated with full activity of the thermophilic 3-IPM dehydrogenase being left in the supernatant, indicating that 4-fold purification was achieved during this process. This shows that the thermophilic 3-IPM dehydrogenase was purified 28-fold by these two procedures, cloning and heat treatment, and demonstrates that the extract from the plasmid-harboring cells is a good starting material for purification of the enzyme. Following the heat treatment, 3-IPM dehydrogenase was further purified by ammonium sulfate precipitation and DEAE-cellulose column chromatography. The enzyme preparation thus obtained contained 3-IPM dehydrogenase as a major component with a few minor impurities as shown by polyacrylamide gel electrophoresis, whereas the enzyme preparation from T. thermophilus HB8 cells obtained by the same procedures showed multiple bands on a polyacrylamide gel electrophoresis. (+info)
Evidence that alpha-isopropylmalate synthase of Saccharomyces cerevisiae is under the "general" control of amino acid biosynthesis.
(71/111)
The specific activity and the immunoreactive amount of alpha-isopropylmalate synthase were more than three times above wild-type values in a Saccharomyces cerevisiae mutant (cdr1) with constitutively derepressed levels of enzymes known to be under the "general" control of amino acid biosynthesis. The specific activity was also higher in lysine- and arginine-leaky strains when these were grown under limiting conditions, and in wild-type cells grown in the presence of 5-methyltryptophan. A low specific activity was found in a mutant (ndr1) unable to derepress enzymes of the general control system. Neither isopropylmalate isomerase nor beta-isopropylmalate dehydrogenase responded to general control signals. (+info)
Sequence non-specific double-strand breaks and interhomolog interactions prior to double-strand break formation at a meiotic recombination hot spot in yeast.
(72/111)
The HIS4LEU2 meiotic recombination hot spot specifies two double-strand break (DSB) sites, I and II. Results presented demonstrate that DSBs at site I occur at many positions throughout a region of approximately 150 bp; we infer that breaks occur in a sequence non-specific fashion. Single-strand nicks at sites I and II are not detectable. Analysis of the effects of a 36 bp linker insertion at site I reveals the existence of communication along and between homologs prior to DSB formation. In cis, the insertion allele causes an increase in DSBs at site I but a decrease in DSBs at site II. In trans, two effects are observed. One effect likely reflects very early pre-DSB interhomolog interactions; the second is suggestive of a later, more intimate interaction in which sites I and II on the two homologs all compete for DSBs. The existence of interhomolog interactions in early meiotic prophase can explain how the sites of crossovers come to lie between the homolog axes at pachytene. (+info)