Purification of the E. coli ogt gene product to homogeneity and its rate of action on O6-methylguanine, O6-ethylguanine and O4-methylthymine in dodecadeoxyribonucleotides.

The E. coli gene ogt encodes the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (O6-AlkG ATase). The protein coding region of the gene was cloned into a multicopy expression vector to obtain high yields of the enzyme (approximately 0.2% of total protein) which was purified to apparent homogeneity by affinity, molecular exclusion and reverse-phase chromatography. Good correlation was found between the determined and predicted amino acid compositions. The ability of the purified protein to act on O6-methylguanine (O6-MeG), O6-ethylguanine (O6-EtG) and O4-methylthymine (O4-MeT) in self-complementary dodecadeoxyribonucleotides was compared to that of 19 kDa fragment of the related ada-protein. With both proteins the rate order was O6-MeG greater than O6-EtG greater than O4-MeT, however, the ogt protein was found to repair O6-MeG, O6-EtG and O4-Met, 1.1, 173 and 84 times, respectively, faster than the ada protein.     

The nucleotide sequence of the uvrD gene of E. coli.

The nucleotide sequence of a cloned section of the E. coli chromosome containing the uvrD gene has been determined. The coding region for the UvrD protein consists of 2,160 nucleotides which would direct the synthesis of a polypeptide 720 amino acids long with a calculated molecular weight of 82 kd. The predicted amino acid sequence of the UvrD protein has been compared with the amino acid sequences of other known adenine nucleotide binding proteins and a common sequence has been identified, thought to contribute towards adenine nucleotide binding.     

Expression of the ogt gene in wild-type and ada mutants of E. coli.

O6-alkylguanine (O6-AlkG) DNA alkyltransferase (ATase) and alkylphosphotriester (AlkP) ATase activity have been quantitated individually in extracts of various E. coli strains by means of ATase specific DNA substrates. O6-AlkG ATase activity was higher than AlkP ATase activity in the wild-type strains F26, AB1157 and SB229 and in the ada- mutants PJ1, PJ3, PJ5 and PJ6 indicating a 5-70 times higher level of expression of the ogt gene than the ada gene. The ada- mutant strains BS23, BS73 and GW5352 expressed O6-AlkG ATase but not AlkP ATase activity indicating expression only of the ogt gene. Southern analysis of DNA from F26, BS23, BS73, PJ1 and GW5352 showed a consistent pattern of hybridisation to an ogt probe but not to an ada probe. Exposure of E. coli to adaptive doses of N-methyl-N-nitro-N-nitroso-guanidine (MeNNG) caused an increase in AlkP ATase activity in F26, AB1156, SB229, PJ1, PJ3, PJ5 and PJ6. O6-AlkG ATase activity also increased in F26, AB1157 and SB229 but decreased to almost undetectable levels in all other strains examined except PJ3 where it remained constant. MeNNG increased ada mRNA abundance in F26 but no ada mRNA was detected in BS23, BS73 or GW5352: there was no evidence for increased ogt mRNA in any of the strains examined. In a limited survey, other bacterial strains have been shown to possess an ogt-like ATase activity.     

Site directed mutagenesis of two cysteine residues in the E. coli ogt O6-alkylguanine DNA alkyltransferase protein.

The E. coli ogt O6-alkylguanine-DNA alkyltransferase has two cysteine residues positioned identically with respect to cysteines in the E. coli ada O6-alkylguanine-DNA alkyltransferase. In order to assess their function, these residues were each substituted by a glycine to generate altered forms of the ogt protein. Mutagenesis of cysteine-139, located within a 'PCHRV' region of homology, eliminated functional activity confirming that this residue is the methyl-accepting cysteine in the active site of the protein. Substitution of cysteine 102 within the sequence 'LRTIPCG' had little effect on the ogt protein activity demonstrating that this cysteine is not directly involved with the transfer of O6-methylguanine adducts.     

Purification to apparent homogeneity and partial amino acid sequence of rat liver O6-alkylguanine-DNA-alkyltransferase.

O6-alkylguanine-DNA-alkyltransferase (ATase) activity was increased in rat liver from 80 to 320 fmoles/mg total protein 48 h after administration of 2-acetylaminofluorene at 60 mg/kg body weight. This tissue was used as a source of ATase which was purified by ammonium sulphate precipitation and DNA-cellulose, molecular exclusion and ion exchange chromatography (IEC). IEC purified material showed a major 24 kDa band after polyacrylamide gel electrophoresis (PAGE) with silver staining. Fluorography of purified ATase following incubation with [3H]-methylated substrate DNA and PAGE showed a single band at 24 kDa suggesting that, as with bacterial ATases, the protein itself accepts the alkyl group from O6-alkylguanine in substrate DNA during the repair reaction. Further purification of the protein using reverse phase HPLC resulted in a single peak representing approximately 125,000 fold purification. This was subjected to amino-terminal sequencing and it was found that the protein was blocked at the amino-terminal end: it was cleaved using trypsin or cyanogen bromide and the amino acid sequence of several reverse phase HPLC purified fragments was determined.     

Complete nucleotide sequence of the Escherichia coli recB gene.

The complete nucleotide sequence of the Escherichia coli recB gene which encodes a subunit of the ATP-dependent DNase, Exonuclease V, has been determined. The proposed coding region for the RecB protein is 3543 nucleotides long and would encode a polypeptide of 1180 amino acids with a calculated molecular weight of 133,973. The start of the recB coding sequence overlaps the 3' end of the upstream ptr gene, and the recB termination codon overlaps the initiation codon of the downstream recD gene, suggesting that these genes may form an operon. No sequences which reasonably fit the consensus for an E. coli promoter could be identified upstream of the proposed recB translational start. The predicted RecB amino acid sequence contains regions of homology with ATPases, DNA binding proteins and DNA repair enzymes.     

大肠杆菌青霉素G酰化酶基因及其邻近区域的核苷酸全序列

Some of microorganisms have been known to possess penicillin G acylase activity. The E. coli derived penicillin G acylase (PGA) can catalyze the conversion of penicillin G into phenylacetic acid and 6-amino-penicillanic acid, the latter is used as the starting compound for the industrial formation of semi-synthetic penicillins. Apart from its industrial importance, the enzyme PGA displays a number of interesting properties. Catalytically active enzyme is localized in the periplasmic space of E. coli cells and composed of two dissimilar subunits. The two subunits are apparently produced from a precursor protein, via a processing pathway hitherto unique in its features for a prokaryotic enzyme. The studies on processing of the precursor and on the relationship between structure and function of the mature enzyme are important theoretically. Previously we cloned a 3.5 kb DNA fragment from a strain (E. coli AS 1.76), which displays PGA activity. In this paper, we report a nucleotide sequence of the 3.5 kb DNA fragment containing PGA gene. After insertion of the DNA fragment into EcoR I and Hind III sites in pWR 13, pPGA 20 had been obtained. We subcloned the Hind III and Bg1 II treated fragment of 1.6 kb in length from pPGA 20 into Hind III and BamH I sites of pWR 13 to get a pPGA 1.6, and Bg1 II and EcoR I treated fragment of 1.9 kb in length into BamH I and EcoR I sites of pWR 13 to get a pPGA 1.9. The linearized pPGA 1.9 which were digested with appropriate restriction enzymes were progressively shortened from both ends respectively by digestion with Bal 31 nuclease, followed by cleavage of shortened target DNA off vector DNA molecules with appropriate restriction enzymes. The series of the DNA fragments shortened from EcoR I end were then cloned into plasmid pWR 13 which had previously digested with Hind III and Sma I enzymes (Fig. 1). The DNA fragment cloned in pWR 13 were directly sequenced on the resulted plasmids by using primer I and primer II. Thus we have obtained the complete nucleotide sequence of the 3.5 kb DNA fragment. The 3.5 kb fragment contains an intact PGA gene which is 2.6 kb.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chromosomal location and nucleotide sequence of the Escherichia coli dapA gene.

Restriction fragments hybridizing to phage HP1c1 DNA were identified in digests of DNA from lysogenic strains of Haemophilus influenzae. The results showed that the cohesive ends of the mature phage DNA were joined in lysogens and that the phage genome was covalently linked to the host DNA, indicating that lysogeny involves recombination between specific sites on the phage and host chromosomes. The site on the phage chromosome at which this recombination occurred was between 110 and 750 base pairs of the left end on the mature phage genome.     

Investigation of the specificity of O6-alkylguanine-DNA-alkyltransferase

Comparison of the abilities of alkylated RNA and DNA to serve as substrates for the O6-alkylguanine-DNA-alkyltransferase have been carried out. It was found that the O6-methylguanine in tRNA was much less active as a substrate for the protein than O6-methylguanine in double stranded DNA. The difference in rates of repair was such that it is unlikely that the alkyltransferase would act on RNA in vivo and, therefore, the reaction with RNA should not contribute towards the exhaustion of its repair capacity.     

Cloning and nucleotide sequence of the type E staphylococcal enterotoxin gene.

The gene for staphylococcal enterotoxin type E (entE) was cloned from Staphylococcus aureus into plasmid vector pBR322 and introduced into Escherichia coli. A staphylococcal enterotoxin type E-producing E. coli strain was isolated. The complete nucleotide sequence of the cloned structural entE gene and the N-terminal amino acid sequence of mature staphylococcal enterotoxin type E were determined. The entE gene contained 771 base pairs that encoded a protein with a molecular weight of 29,358 which was apparently processed to a mature extracellular form with a molecular weight of 26,425. DNA sequence comparisons indicated that staphylococcal enterotoxins type E and A are closely related. There was 84% nucleotide sequence homology between entE and the gene for staphylococcal enterotoxin type A; these genes encoded protein products that had 214 (83%) homologous amino acid residues (mature forms had 188 [82%] homologous amino acid residues).     

Cloning, nucleotide sequence, and expression of the Escherichia coli gene encoding carnitine dehydratase.

Carnitine dehydratase from Escherichia coli O44 K74 is an inducible enzyme detectable in cells grown anaerobically in the presence of L-(-)-carnitine or crotonobetaine. The purified enzyme catalyzes the dehydration of L-(-)-carnitine to crotonobetaine (H. Jung, K. Jung, and H.-P. Kleber, Biochim. Biophys. Acta 1003:270-276, 1989). The caiB gene, encoding carnitine dehydratase, was isolated by oligonucleotide screening from a genomic library of E. coli O44 K74. The caiB gene is 1,215 bp long, and it encodes a protein of 405 amino acids with a predicted M(r) of 45,074. The identity of the gene product was first assessed by its comigration in sodium dodecyl sulfate-polyacrylamide gels with the purified enzyme after overexpression in the pT7 system and by its enzymatic activity. Moreover, the N-terminal amino acid sequence of the purified protein was found to be identical to that predicted from the gene sequence. Northern (RNA) analysis showed that caiB is likely to be cotranscribed with at least one other gene. This other gene could be the gene encoding a 47-kDa protein, which was overexpressed upstream of caiB.     

Cloning, expression, and nucleotide sequence of the Escherichia coli K-12 ackA gene.

The Escherichia coli K-12 ackA gene, which encodes an acetate kinase, was cloned. The acetate kinase activities of ackA+ plasmid-containing strains were amplified 160- to 180-fold. The complete nucleotide sequence of the ackA gene was determined. It was deduced that the ackA gene coded for a protein of 400 amino acids with an Mr of 43,297. The ackA gene was found to be located about 15 kilobases upstream of the purF-folC-hisT region of the chromosome.