Control of tryptophan synthetase amplified by varying the numbers of composite plasmids in Escherichia coli cells.

Using pSC101, RSF1010, RSF2124 and RP4 plasmids as vectors and bacteriophage lambdatrpD-A60-3 DNA as a source of the Escherichia coli whole tryptophan operon, composite plasmids of pSC101-trp, RSF1010-trp, RSF2124-trp and RP4-trp were constructed in vitro with EcoRI restriction endonuclease and DNA ligase. Each composite plasmid could be maintained stably in E. coli cells. The copy number of pSC101-trp, RSF1010-trp, RSF2124-trp and RP4-trp were 4.2, 11.2, 11.9 and 1.6 per chromosome respectively. The tryptophan synthetase activities in cells containing pSC101-trp, RSF1010-trp, RSF2124-trp aand RP4-trp plasmid were found to be 2.1, 6.0, 5.0 and 2.5 times compared with the level in chromosomal trp+ cells when they were grown in a minimal medium. By partial derepression with indolylacrylic acid, the enzyme levels were elevated to 10.1, 16.3, 15.3, 12.3 times, respectively, that of the control cells. The tryptophan synthetase activities did not increase in proportion to the copy number of the plasmids, but were strongly affected by the repression system of host cells.     

RSF1010 plasmid as a potentially useful vector in Pseudomonas species.

RSF1010 plasmid DNA was introduced into Pseudomonas putida and P. aeruginosa cells and maintained stably, suggesting the potential usefulness of this plasmid as a vector in Pseudomonas species. The number of copies of RSF1010 was 43 per chromosome equivalent in P. putida cells.     

Cloning of Bacillus subtilis leucina A, B and C genes with Escherichia coli plasmids and expression of the leuC gene in E. coli.

Summary The leucine genes of Bacillus subtilis have been cloned directly from the chromosomal DNA into Escherichia coli leuB cells by selection for the Leu⁺ phenotype using RSF2124 as a vector plasmid. The hybrid plasmid designated RSF2124-B·leu contained a 4.2 megadalton fragment derived from B. subtilis DNA, including the leu genes. The fragment had one site susceptible to EcoRI^* and another site susceptible to BamNI endonuclease. Among the three fragments produced by EcoRI^* and BamNI endonucleases, the 1.2 megadalton fragment had the ability to transform B. subtilis leuA, leuB and leuC auxotrophs to leu ⁺. However, B. subtilis ilvB and ilvC auxotrophs were not rescued even by the whole 4.2 megadalton fragment present in the hybrid plasmid. -Isopropylmalate dehydrogenase (leuB gene product) activity found in E. coli cells containing the hybrid plasmid was about 60% of that in E. coli wild type cells, despite the high copy number (7.8) of the plasmid per chromosome observed.     

Activation of a phosphatidylcholine-specific phospholipase C by colony stimulating factor 1 receptor requires tyrosine phosphorylation and a guanine nucleotide-binding protein.

Receptor tyrosine kinases couple to multiple intracellular effector molecules that are crucial for normal cell growth and transformation. Stimulation of membrane phospholipid hydrolysis by receptor tyrosine kinases is one such pathway for generating intracellular second messengers that may be important for mitogenesis. Certain receptor tyrosine kinases tyrosine phosphorylate a phosphoinositide-specific phospholipase C that hydrolyses the membrane phospholipid phosphatidylinositol 4,5-bisphosphate. In contrast, the glycoprotein receptor for colony stimulating factor 1, a transmembrane tyrosine kinase, does not utilize this pathway, but rather stimulates the hydrolysis of phosphatidylcholine. Here we show that eluates of antiphosphotyrosine affinity purified lysates of colony-stimulating factor 1-stimulated cells contain elevated levels of phosphatidylcholine-specific phospholipase C activity. The affinity-purified activity is sensitive to tyrosine-specific T-cell phosphatase, and is detected in the membrane fraction of stimulated cells. Recovery of phospholipase C activity in the antiphosphotyrosine protein fraction is reduced by pertussis toxin pretreatment of cells. The phosphatidylcholine phospholipase C activity in isolated membranes of colony-stimulating factor 1-treated cells was also reduced by pertussis toxin treatment and stimulated by guanosine 5'-3-O-(thio)triphosphate. These results indicate that colony stimulating factor 1 receptor-mediated stimulation of phosphatidylcholine-specific phospholipase C requires tyrosine phosphorylation, and might be affected by a G-protein coupled pathway.     

Xylose (glucose) isomerase gene from the thermophile Thermus thermophilus: cloning, sequencing, and comparison with other thermostable xylose isomerases.

The xylose isomerase gene from the thermophile Thermus thermophilus was cloned by using a fragment of the Streptomyces griseofuscus gene as a probe. The complete nucleotide sequence of the gene was determined. T. thermophilus is the most thermophilic organism from which a xylose isomerase gene has been cloned and characterized. The gene codes for a polypeptide of 387 amino acids with a molecular weight of 44,000. The Thermus xylose isomerase is considerably more thermostable than other described xylose isomerases. Production of the enzyme in Escherichia coli, by using the tac promoter, increases the xylose isomerase yield 45-fold compared with production in T. thermophilus. Moreover, the enzyme from E. coli can be purified 20-fold by simply heating the cell extract at 85 degrees C for 10 min. The characteristics of the enzyme made in E. coli are the same as those of enzyme made in T. thermophilus. Comparison of the Thermus xylose isomerase amino acid sequence with xylose isomerase sequences from other organisms showed that amino acids involved in substrate binding and isomerization are well conserved. Analysis of amino acid substitutions that distinguish the Thermus xylose isomerase from other thermostable xylose isomerases suggests that the further increase in thermostability in T. thermophilus is due to substitution of amino acids which react during irreversible inactivation and results also from increased hydrophobicity.     

A DBL-homologous region of the yeast CLS4/CDC24 gene product is important for Ca(2+)-modulated bud assembly.

The CLS4/CDC24 is essential for the budding process of the yeast Saccharomyces cerevisiae. Disruption of the CLS4/CDC24 gene is lethal, and expression of the CLS4 product under the control of the GAL1 promoter is sufficient for cellular growth. The CLS4 product is detected in yeast cell lysate with an apparent molecular mass of 93 kD (854 amino acid residues) and shows homology with the human DBL oncogene product. Temperature-sensitive cdc24-1 mutation is located in the N-terminal portion of the protein whereas Ca(2+)-sensitive cls4-1 mutation is present after the DBL-homologous region (amino acid residues 281-518) near the putative Ca(2+)-binding site. Mutations within the DBL-homologous region are responsible for the Ca(2+)-sensitive phenotype. Thus the CLS4 gene product seems to have several functional domains within the molecule essential for bud assembly.     

Mus308 Mutants of Drosophila Exhibit Hypersensitivity to DNA Cross-Linking Agents and Are Defective in a Deoxyribonuclease

Mutagen-sensitive strains that identify 16 different Drosophila genes have been screened for alterations in DNA metabolic enzymes. A characteristic defect in an acid-active deoxyribonuclease was observed in strains carrying the six available mutant alleles of the mus308 gene. Since that enzyme is detected at normal levels in a mutant strain that is deficient in the previously identified enzymes DNase 1 and DNase 2, it represents a new Drosophila nuclease that is designated Nuclease 3. The mus308 mutants were originally distinguished from all other mutagen-sensitive mutants of Drosophila because they exhibit hypersensitivity to the DNA cross-linking agent nitrogen mustard without expressing a concurrent sensitivity to the monofunctional agent methyl methanesulfonate. Further observations of hypersensitivity to the mutagens trimethylpsoralen, diepoxybutane and cis-platinum now establish a more general sensitivity of these mutants to agents capable of generating DNA cross-links. In spite of the hypersensitivity of the mus308 mutants to DNA cross-linking agents, the initial incision step of DNA cross-link repair is normal in mus308 cells as assayed by the alkaline elution procedure. The Drosophila mus308 mutants show promise of providing a useful model for analogous defects in other organisms including man.     

Structural requirements for interruption of protein translocation across rough endoplasmic reticulum membrane

Co-translational translocation of proteins across the membrane of rough endoplasmic reticulum (ER) is interrupted by particular amino acid sequences, which are functionally termed "stop-transfer sequence." We analyzed the structural requirements for the interruption of the peptide translocation. By the manipulation of the cDNA of interleukin 2 (IL2), which passes through ER membrane co-translationally, the middle portion of the IL2 molecule was replaced with systematically altered hydrophobic segments, leucine, alanine, or leucine/alanine mixed clusters. Furthermore, charged amino acid residues were introduced just downstream of the hydrophobic segments. These modified IL2 peptides were synthesized with wheat germ cell-free system in the presence of rough microsomes and the topology of the peptides in the microsomes was assessed by post-translational digestion with proteinase K. We obtained the following results. (i) Each modified protein was processed to the mature form but the extent of stop-translocation varied widely. The ratio of the stopped to the translocated products increased as the length and hydrophobicity of the inserted segment increased. (ii) Shorter hydrophobic segments than naturally occurring native transmembrane segment promoted stop-translocation. (iii) Proteins with hydrophobic segments followed by positive charges were more efficiently stop-translocated than those having negative charges. (iv) If the hydrophobicity of the segment was sufficiently high, the positive charges after the segment were not essential for stop-translocation. We also suggest that the stop-transfer process includes protein-protein interaction between the hydrophobic segment and translocation channel.     

Recovery of uranium by immobilized microorganisms

Summary Some attempts were made to recover uranium from sea and fresh water using immobilized Streptomyces viridochromogenes and Chlorella regularis cells. The cells immobilized in polyacrylamide gel have the most favorable features for uranium recovery; high adsorption ability, good mechanical properties, and applicability in a column system. The adsorption of uranium by the immobilized cells is not affected by the pH values between 4 and 9. These results show that uranium adsorption becomes independent of pH after immobilization. The amounts of uranium adsorbed by the immobilized cells increased linearly with temperature, suggesting that the adsorption of uranium by the immobilized cells is an endothermic reaction. The immobilized cells can recover uranium almost quantitatively from both fresh and sea water containing uranium, and almost all uranium adsorbed is desorbed with a solution of Na₂CO₃. Thus the immobilized cells of Streptomyces and Chlorella can be used repeatedly in adsorption-desorption process.Studies on the Accumulation of Heavy Metal Elements in Biological Systems. XXI