Redox interconversion of Escherichia coli glutathione reductase. A study with permeabilized and intact cells

Summary The redox interconversion of Escherichia coli glutathione reductase has been studied both in situ, with permeabilized cells treated with different reductants, and in vivo, with intact cells incubated with compounds known to alter their intracellular redox state.The enzyme from toulene-permeabilized cells was inactivated in situ by NADPH, NADH, dithionite, dithiothreitol, or GSH. The enzyme remained, however, fully active upon incubation with the oxidized forms of such compounds. The inactivation was time-, temperature-, and concentration-dependent; a 50% inactivation was promoted by just 2 M NADPH, while 700 M NADH was required for a similar effect. The enzyme from permeabilized cells was completely protected against redox inactivation by GSSG, and to a lesser extent by dithiothreitol, GSH, and NAD(P)⁺. The inactive enzyme was efficiently reactivated in situ by physiological GSSG concentrations. A significant reactivation was promoted also by GSH, although at concentrations two orders of magnitude below its physiological concentrations. The glutathione reductase from intact E. coli cells was inactivated in vivo by incubation with DL-malate, DL-isocitrate, or higher L-lactate concentrations. The enzyme was protected against redox inactivation and fully reactivated by diamide in a concentration-dependent fashion. Diamide reactivation was not dependent on the synthesis of new protein, thus suggesting that the effect was really a true reactivation and not due to de novo synthesis of active enzyme. The glutathione reductase activity increased significantly after incubation of intact cells with tert-butyl or cumene hydroperoxides, suggesting that the enzyme was partially inactive within such cells. In conclusion, the above results show that both in situ and in vivo the glutathione reductase of Escherichia coli is subjected to a redox interconversion mechanism probably controlled by the intracellular NADPH and GSSG concentrations.     

Sequence analysis of a Dictyostelium discoideum gene coding for an active dihydroorotate dehydrogenase in yeast

A Dictyostelium discoideum DNA fragment isolated on the basis of its ability to complement the ural mutation of yeast, codes for a dihydroorotate dehydrogenase activity. The complete nucleotide sequence of this 1898 bp fragment has been determined and reveals an open reading frame capable of coding for a 369 amino acid polypeptide of molecular mass 47.000. The gene shows preferential use of codons with weak pairing forces. Eleven codons, mainly those with a G in the third position, are absent. The flanking sequences are unusually rich in A + T (80%). Several direct and inverted repeats exist in the 5' flanking sequence.     

Development of a high-frequency transforming vector for Aspergillus nidulans

The pyr4 gene of Neurospora crassa, which codes for orotidine-5'-phosphate decarboxylase, is capable of transforming an Aspergillus nidulans pyrG mutant by chromosomal integration, despite low homology between the transforming DNA and the recipient genome. Integration of pFB6, a plasmid carrying pyr4 and capable of replication in Escherichia coli, was not observed at the pyrG locus. The efficiency of transformation was considerably enhanced (50-100 fold) by inclusion in the transforming vector of a 3.5-kb A.nidulans chromosomal sequence, ans1. Although this sequence was isolated on the basis of replicating activity in Saccharomyces cerevisiae, there was no evidence for such activity in A.nidulans. Part of the ans1 fragment appears to be reiterated in the A.nidulans genome, though it is not yet clear whether this is directly responsible for the high transformation frequency. The efficiency of transformation of A.nidulans by plasmids bearing ans1, using an improved protocol, was approx. 5 X 10(3) stable transformants per microgram of plasmid DNA.     

Analysis of the inducible MEL1 gene of Saccharomyces carlsbergensis and its secreted product, alpha-galactosidase (melibiase)

We have determined both the nucleotide sequence of the MEL1 gene of Saccharomyces carlsbergensis and the N-terminal amino acid (aa) sequence of its extracellular gene product, alpha-galactosidase (melibiase) (alpha-Gal). The predicted translation product of MEL1 is a pre-alpha-Gal protein containing an 18 aa N-terminal signal sequence for secretion. The purified enzyme is a dimer consisting of two 50-kDal polypeptides, each of which is glycosylated with no more than eight side chains. The 5'-flank of the MEL1 gene contains a region (UASm) having certain areas of sequence homology to similar sites found upstream of the structural genes GAL1, GAL7 and GAL10, which are also regulated by the action of the products of genes GAL4 and GAL80. There are three TATA boxes between UASm and the initiation codon of pre-alpha-Gal, as well as a typical yeast cleavage/polyadenylation sequence in the 3'-flank of the gene.     

Molecular characterization and genetic mapping of two clusters of genes encoding chlorophyll a/b-binding proteins in Lycopersicon esculentum (tomato)

We have constructed a tomato genomic library in the λ Charon 4 phage vector. The library was screened with a pea cDNA probe encoding a chlorophyll a/b-binding protein (CAB), and several recombinant phages containing tomato CAB genes were isolated and characterized by restriction mapping, heteroduplex analysis and nucleotide sequencing. Two phages with overlapping segments of the tomato genome contain a total of four CAB genes, all arranged in tandem. A third phage contains three CAB genes, two arranged in tandem and one in opposite orientation, and an additional, truncated CAB gene. Genetic mapping experiments showed that the four CAB genes on the first two phages belong to a locus, previously designated Cab-1, on chromosome 2. The CAB genes from the third phage belong to the Cab-3 locus on chromosome 3. Complete sequence determination of two CAB genes, one from each locus, and additional sequence determination of about 50% of each of the other five CAB genes showed that each gene within a CAB locus is more similar to other CAB genes in the same locus than it is to the CAB genes from the second locus. Furthermore, the polypeptides encoded by Cab-1 genes diverge significantly from those encoded by Cab-3 genes in the domains of transit peptide and the N terminus of the mature polypeptide but are essentially identical in the rest of the sequence.     

Versatile shuttle cloning vectors for the unicellular cyanobacterium Anacystis nidulans R2

Abstract 3 new shuttle cloning vectors for gene transfer into Escherichia coli and Anacystis nidulans have been constructed by utilizing the cyanobacterial origin of replication of the small plasmid pANS from A. nidulans . 2 of these new vectors, pXB7 (pDPL13 derivative) and pECAN8 (pUC8 derivative), convey ampicillin resistance, and transform A. nidulans with relatively high frequencies. Vector pXB7 has 10 unique cloning sites; pECAN8 contains 4 cloning sites within the lacZ gene permitting rapid detection of DNA inserts in the presence of Xgal. The third vector, pKBX, has a lower transformation frequency but adds kanamycin resistance as a selectable gene for shuttle vectors of cyanobacteria.     

Role of verocytotoxigenic Escherichia coli in cattle and buffalo calf diarrhoea

Abstract 273 Strains of Escherichia coli isolated from diarrhoeic and healthy control cattle and buffalo calves in Sri Lanka, were tested for Verocytotoxin (VT) and for heat-stable (ST) and heat-labile (LT) enterotoxins. VT and ST toxigenic E. coli were significantly associated with diarrhoea, accounting for 28% and 18% of diarrhoeic episodes, respectively. LT toxigenic E. coli were not significantly associated with diarrhoea.     

Molecular cloning and nucleotide sequences of cDNAs specific for rat liver ribosomal proteins S17 and L30

cDNA clones coding for rat liver ribosomal proteins S17 and L30 have been isolated by positive hybridization-translation assay from a cDNA library prepared from 8-9S poly(A)+RNA from free polysomes of regenerating rat liver. The cDNA clone specific for S17 protein (pRS17-2) has a 466-bp insert with the poly(A) tail. The complete amino acid (aa) sequence of S17 protein was deduced from the nucleotide sequence of the cDNA. S17 protein consists of 134 aa residues with an Mr of 15 377. The N-terminal aa sequence of S17 protein determined by automatic Edman degradation is consistent with the sequence data. The aa sequence of S17 shows strong homology (76.9%) to that of yeast ribosomal protein 51 [Teem and Rosbash, Proc. Natl. Acad. Sci. USA 80 (1983) 4403-4407] in the two-thirds N-terminal region. The cDNA clone specific for L30 protein (pRL30) has a 394-bp insert. The aa sequence of L30 protein was deduced from the nucleotide sequence of the cDNA. The protein consists of 114 aa residues with an Mr of 12 652. When compared with the N-terminal aa sequence of rat liver L30 protein [Wool, Annu. Rev. Biochem. 48 (1979) 719-754], pRL30 was found not to contain the initiation codon and 5'-noncoding region. The cDNA showed twelve silent changes in the coding region, one point mutation and one base deletion in the 3'-noncoding region, compared with mouse genomic DNA for L30 protein [Wiedemann and Perry, Mol. Cell Biol. 4 (1984) 2518-2528].     

Isolation and characterization of the yeast aspartyl-tRNA synthetase gene

A yeast genomic library in Escherichia coli, constructed by insertion of Sau3A restriction fragments into the hybrid Saccharomyces cerevisiae-E. coli plasmid pFL1, was screened by a radioimmunoassay (RIA) for colonies expressing yeast aspartyl-tRNA synthetase (AspRS). Four clones were isolated by this technique. Data obtained by Southern and restriction analysis of the inserts showed a common 3.8-kb BamHI restriction fragment which, when inserted into the plasmid pFLl, gave a positive RIA. Several controls showed that this 3.8-kb insert codes for the entire AspRS : (i) S. cerevisiae transformed by the PFL1 plasmid carrying the 3.8-kb fragment overproduces AspRS activity by a factor of ten compared to the wild-type yeast strain; and (ii) a new protein with electrophoretic behaviour similar to AspRS and immuno-reactive toward anti-AspRS appears in crude extracts of transformed yeast and E. coli.