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Biomedical subjects

B E Davidson

Publications and source records attributed to B E Davidson.

At least 73 records · Page 4Linked to original sources
PubMed1989

The Escherichia coli regulatory protein MetJ binds to a tandemly repeated 8 bp palindrome.

Site-directed oligonucleotide mutagenesis has been used to isolate thirty four new mutants in the regulatory region of the Escherichia coli K12 gene, metF. The mutants include single base pair (bp) substitutions and insertions, double bp substitutions and one 7bp deletion. The effects of these and another five previously described mutants on the transcriptional regulation of metF have been analysed by using a metF'-lac'Z fusion in a low copy-number plasmid. These data, and those obtained from DNAse protection studies using pure MetJ with wild-type and mutant metF operator DNA, show that the metF operator is comprised of five tandem 8 bp repeat units that overlap the -10 region of the metF promoter. In the presence of the co-repressor S-adenosylmethionine, the DNAse protection studies yielded dissociation constants of 150 nM and 300 nM for the interaction of MetJ with repeat units 1 to 4 and repeat unit 5, respectively. In the absence of co-repressor, the dissociation constants obtained for these interactions are four to five times greater. It is proposed that regulation at the metF operator requires four molecules of MetJ dimer to bind to the five 8 bp repeat units to form a tandem, overlapping array. Interactions between MetJ molecules make an essential contribution to the stability of this protein-DNA complex.

Base Sequence↗
PubMed1988

Circular Permutation of the Genome of a Temperate Bacteriophage from Streptococcus cremoris BK5.

The temperate bacteriophage BK5-T was isolated from Streptococcus cremoris BK5 by induction with mitomycin C. Electron microscopy revealed that BK5-T DNA consists of linear molecules, ranging in size from 39.7 to 46 kilobase pairs. Restriction analysis of self-ligated BK5-T DNA showed that the ends of the DNA were not cohesive. The EcoRI restriction fragments of the phage genome were cloned into pACYC184. Restriction enzyme analysis of both the phage DNA and the cloned EcoRI fragments with EcoRI, BstEII, PstI, ClaI, and XbaI yielded a 37.6-kilobase-pair-long circular restriction map for the phage genome. It was concluded that the BK5-T DNA molecules in the population differ in their sequence by a circular permutation and that individual DNA molecules are terminally redundant. The map location of the sites at which packaging of BK5-T DNA into phage heads is initiated (pac) and at which the phage integrates into the bacterial chromosome (att) were established.

Journal Article↗
PubMed1986

Structure of the Escherichia coli K12 regulatory gene tyrR. Nucleotide sequence and sites of initiation of transcription and translation.

The nucleotide sequence of 1964 base pairs of the Escherichia coli K12 chromosome containing the autogenously regulated regulatory gene tyrR has been determined. The site of initiation of transcription of tyrR has been mapped by primer-extension analysis, and the initiation codon has been identified by site-specific deletion mutagenesis. The nucleotide sequence predicts a subunit molecular weight of 53,099 for the TyrR protein. Codon usage in the tyrR structural gene shows a bias toward those synonymic codons which are used rarely in efficiently expressed E. coli genes. The nucleotide sequence of a 22-base pair region adjacent to the promoter and distal to the structural gene exhibits considerable identity with corresponding regions of other genes regulated by tyrR. It is proposed that this is a site for repression by the TyrR protein.

Amino Acid Sequence↗
PubMed1986

Construction of plasmid vectors for the detection of streptococcal promoters.

Plasmid vectors have been constructed for detecting DNA fragments that exhibit promoter activity in Streptococcus sanguis. The plasmids are able to replicate in both S. sanguis and Escherichia coli, and contain an erythromycin resistance marker which is expressed in both hosts. Selection for promoter activity is dependent upon the insertion of appropriate DNA fragments upstream from a promoterless chloramphenicol acetyl transferase gene (cat) from Staphylococcus aureus. To facilitate this insertion, a pair of vectors, pMU1327 and pMU1328, were constructed with the polylinker from M13mp 18 in either orientation. The to transcriptional terminator of phage lambda is present downstream from cat. Translation stop codons in all reading frames are located between the polylinker and the initiation codon of cat. These plasmids have been used to isolate DNA fragments from S. sanguis, S. lactis and S. cremoris that exhibit promoter activity in S. sanguis.

Base Sequence↗
PubMed1986

Resistance to In Vitro Restriction of DNA from Lactic Streptococcal Bacteriophage c6A.

DNA isolated from streptococcal bacteriophage c6A was cut only infrequently by many restriction endonucleases. Fragments of c6A DNA cloned in Escherichia coli plasmids were similarly resistant to cleavage. We conclude that the low frequency of cleavage is due to an unusually low number of restriction enzyme recognition sequences in c6A DNA.

Journal Article↗
PubMed1986

Nucleotide sequence of the transcription unit containing the aroL and aroM genes from Escherichia coli K-12.

The nucleotide sequence of 2,021 base pairs (bp) of DNA containing the Escherichia coli aroLM operon was determined, and the coding regions of both aroL and aroM were identified. The 501-bp intercistronic region between aroL and aroM contains an open reading frame which might encode a 63-residue protein. Northern blots with RNA from strains carrying multicopy aroL+ plasmids detected one longer (2,000-base) and two shorter (950- and 1,100-base) transcripts which contained aroL. It was concluded that the longest transcript, which was not abundant, spanned the entire operon and that the shorter transcripts resulted from either termination or posttranscriptional processing in the intercistronic region. The DNA upstream of aroL contains a number of imperfect palindromes which are closely homologous to known sites of regulation by the TyrR protein in other operons.

Bacterial Proteins↗
PubMed1986

Molecular analysis of the promoter operator region of the Escherichia coli K-12 tyrP gene.

The nucleotide sequence of the tyrP promoter region from Escherichia coli has been determined. Two TYR R boxes have been identified, and one of these was shown to overlap the -35 region of a major tyrP promoter (p1). S1 nuclease mapping of in vivo transcripts revealed that transcription from p1 is stimulated by phenylalanine and to a lesser extent by leucine. The demonstration that mutants in which TyrR-tyrosine-mediated repression of tyrP has been abolished have single base changes in the TYR R box which overlaps p1 suggests that TyrR-tyrosine-mediated repression of tyrP also involves p1. TyrR-independent stimulation of tyrP expression by Casamino Acids involves a second promoter 140 bases upstream of p1. There are no TYR R boxes in this region. The sequences of 10 TYR R boxes preceding the genes tyrP, tyrR, and aroG and the operons aroF tyrA and aroL aroM are compared and discussed.

Bacterial Proteins↗
PubMed1985

The reactivity of the sulphydryl groups of chorismate mutase/prephenate dehydratase--a bifunctional enzyme of phenylalanine biosynthesis in Escherichia coli K12.

The reaction of N-ethylmaleimide with chorismate mutase/prephenate dehydratase (chorismate pyruvatemutase/prephenate hydrolyase (decarboxylating) EC 5.4.99.5/EC 4.2.1.51) from Escherichia coli K12, which leads to the preferential inactivation of the prephenate dehydratase activity (Gething, M-J.H. and Davidson, B.E. (1977) Eur. J. Biochem. 78, 111-117), was found to involve only the sulphydryl groups of the enzyme. Determination of the reactivities of the four different cysteine residues indicated that the reaction was not specific for a single residue, although two residues (Cys-216 and Cys-374) were more reactive than the others. The amount of inhibition of the prephenate dehydratase activity approximated in extent to the sum of the stoichiometries of the individual reactions of N-ethylmaleimide with these two cysteine residues. In the presence of either phenylpyruvate, the product of the prephenate dehydratase activity, or cis-aconitate, a competitive inhibitor with respect to prephenate, the prephenate dehydratase activity was substantially protected from inactivation. This protection was concomitant with a significant decline in the reactivities of both Cys-216 and Cys-374. These results are interpreted as indicating that both of these cysteine residues are at, or near to, the prephenate dehydratase active site and are possibly essential for the prephenate dehydratase activity of the enzyme.

Amino Acid Sequence↗
PubMed1985

Cloning of aroG, the gene coding for phospho-2-keto-3-deoxy-heptonate aldolase(phe), in Escherichia coli K-12, and subcloning of the aroG promoter and operator in a promoter-detecting plasmid.

Defective transducing phages carrying aroG, the structural gene for phenylalanine (phe)-inhibitable phospho-2-keto-heptonate aldolase (EC 4.1.2.15; previously known as 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase[phe]), have been isolated, and DNA from two of these phages has been used to construct a restriction map of the region from att lambda to aroG. A 7.6-kb PstI-HindIII fragment from one of these phages was cloned into pBR322 and shown to contain aroG. The location of aroG within the 7.6 kb was established by subcloning and Tn3 transpositional mutagenesis. A fragment carrying the aroG promoter and operator has been cloned into a high copy number promoter-cloning vector (pMC489), and the resulting aroGpo-LacZ' (alpha) fusion subcloned in a low copy number vector. Strains with this fusion on the low copy number vector exhibit negative regulation of beta-galactosidase expression by both phenylalanine and tryptophan and positive regulation by tyrosine in a tyrR+ background.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed1985

Characterization of streptococcal bacteriophage c6A.

Bacteriophage c6A is a lytic phage that infects strains of Streptococcus lactis. Infection of S. lactis C6 under standard conditions yielded 124 +/- 8 p.f.u. per infected cell after a latent period of 25 min at 30 degrees C. The virion of c6A was shown to contain at least 12 polypeptides and a 21.9 kilobase double-stranded, linear DNA genome with complementary 5'-protruding single-stranded termini. The (G + C) content of this DNA was estimated to be 36.7%. A restriction map was constructed which indicates that a number of restriction endonucleases did not digest the DNA and that others cleaved with a much lower frequency than expected.

Bacteriophages↗
PubMed1984

Nucleotide sequence and transcription of the phenylalanine and tyrosine operons of Escherichia coli K12.

A 4509 base-pair DNA fragment containing the phenylalanine and tyrosine operons of Escherichia coli K12 has been sequenced, and the pattern of transcription of these operons examined by S1 mapping, primer extension and galK fusion analyses. The phe operon consists of promoter, operator, leader region containing the phe attenuator and the pheA gene encoding chorismate mutase/prephenate dehydratase. The tyr operon consists of promoter, operator, a short leader region without an attenuator, and two structural genes aroF and tyrA encoding the tyrosine-sensitive isoenzyme of 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthetase and chorismate mutase/prephenate dehydrogenase, respectively. A bidirectional transcription terminator occurs between the two operons. The predicted amino acid sequences of chorismate mutase/prephenate dehydrogenase and chorismate mutase/prephenate dehydratase are homologous at their N termini, while the tyrosine-sensitive isoenzyme of DAHP synthetase is closely homologous to the phenylalanine-sensitive isoenzyme encoded by aroG.

Amino Acid Sequence↗
PubMed1984

Chorismate mutase/prephenate dehydrogenase from Escherichia coli K12: purification, characterization, and identification of a reactive cysteine.

The bifunctional enzyme involved in tyrosine biosynthesis, chorismate mutase/prephenate dehydrogenase, has been isolated from extracts of a regulatory mutant of Escherichia coli K12. The pure enzyme is a homodimer of total molecular weight 78 000 and displays Michaelis-Menten kinetics for both activities. Fingerprinting and amino acid sequencing of tryptic and thermolytic peptides of the S-[14C]carboxymethylated enzyme allowed the identification of three unique cysteine-containing sequences per subunit. Chemical modification of the native enzyme with 5,5'-dithiobis(2-nitrobenzoate) or iodoacetamide showed that one sulfhydryl group per subunit was particularly reactive, and the integrity of this group was essential for both enzymic activities. This work supports previous proposals for a close spatial relationship between the active sites.

Amino Acid Sequence↗
PubMed1984

Cloning of the tyrP gene and further characterization of the tyrosine-specific transport system in Escherichia coli K-12.

The tyrP gene which codes for a component of the tyrosine-specific transport system of Escherichia coli has been cloned on a 2.8-kilobase insert into plasmid pBR322. Transposon mutagenesis, using Tn1000, indicates that the tyrP+ gene is at least 1.1 kilobase in length. Labeling of the tyrP protein in maxicells with [35S]methionine indicates an apparent molecular weight of ca. 24,500. Sedimentation analysis reveals that the tyrP protein is associated with the cell membrane and is not free in the cytoplasm or periplasm. Strains with many copies of the tyrP+ gene show an enhanced uptake of tyrosine, but the expression of the system is still modulated by tyrosine and phenylalanine in the presence of the tyrR+ regulator protein. Accumulated radioactive tyrosine is rapidly effluxed by the addition either of energy uncouplers or of excess nonradioactive tyrosine, indicating that the transport system is energized by the proton motive force and that the internal pool is readily exchangeable. The effect of increasing expression of the tyrP gene on the steady-state level of tyrosine accumulated by cells indicates that although the transport system may be dependent on the proton motive force to drive uptake, the system never reaches thermodynamic equilibrium with it.

Base Sequence↗
PubMed1983

Kinetic studies on the mechanism of chorismate mutase/prephenate dehydratase from Escherichia coli K12.

The effect of pH on chorismate mutase/prephenate dehydratase (chorismate pyruvate mutase/prephenate hydro-lyase (decarboxylating) EC 5.4.99.5/EC 4.2.1.51) from Escherichia coli K12 has been studied. While the maximum velocity of both activities is independent of pH, Km for chorismate or prephenate shows a complex pH dependence. Differences in mutase activity in acetate/phosphate/borate and citrate/phosphate/borate buffers were traced to inhibition by citrate. When a variety of analogues of citrate were tested as possible inhibitors of the enzyme, several were found to inhibit mutase and dehydratase activities to different extents, and by different mechanisms. Thus citrate competitively inhibits mutase activity, but inhibits dehydratase activity by either a non-competitive or an uncompetitive mechanism. Conversely, cis- and trans-aconitate competitively inhibit dehydratase activity, but are partially competitive inhibitors of mutase activity. The differential effects of these inhibitors on the two activities are consistent with the existence of two distinct active sites, but additionally suggest some degree of interconnection between them. The implications of these results for possible mechanisms of catalysis by chorismate mutase/prephenate dehydratase are discussed.

Drug Stability↗
PubMed1982

The nucleotide sequence of aroG, the gene for 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase (phe) in Escherichia coli K12.

We have determined the nucleotide sequence of aroG, the gene coding for 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe), one of three isoenzymes that catalyse the first step of the biosynthesis of aromatic amino acids and vitamins in Escherichia coli K12. The DNA sequence agrees with previously published data on the N-terminal sequence, amino acid composition, and subunit molecular weight of 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(pne). There is significant identity in the nucleotide sequences of aroG and aroH (the gene for 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase (trp), indicating that these two genes have evolved from a common ancestral gene. There is no attenuator structure in the leader region of aroG.

3-Deoxy-7-Phosphoheptulonate Synthase↗