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Biomedical subjects

S Harford

Publications and source records attributed to S Harford.

14 recordsLinked to original sources
PubMed1990

Regulation of isopenicillin N synthetase (IPNS) gene expression in Acremonium chrysogenum.

Total RNA was extracted daily from the beta-lactam antibiotic producing fungus A. chrysogenum strain CO728 during a 7 day cephalosporin C fermentation. IPNS mRNA species, with a size of about 1.5 kb, were detected by Northern blotting at high levels between days 2 and 4. The rapid appearance of IPNS mRNA in mycelial extracts up to day 2 suggests that IPNS is regulated at the transcriptional level. Primer extension and S1 endonuclease mapping studies indicate the existence of two major and at least two minor transcription initiation start sites. There was no change in the relative levels of the four transcripts during the period they could be detected. A region upstream of the IPNS structural gene (pcbC) has been sequenced and the transcription initiation sites appear as major and minor pairs on either side of one of the pyrimidine-rich blocks that punctuate the promoter sequence.

Acremonium↗
PubMed1990

A comparative study on the inhibitory actions of chloramphenicol, thiamphenicol and some fluorinated derivatives.

Chloramphenicol, thiamphenicol and three fluorinated derivatives, Sch 24893, Sch 25298 and Sch 25393, were studied with respect to inhibition of the growth of selected bacterial strains and cell-free translation systems. Thiamphenicol was the least potent inhibitor in the former experiments, but behaved similarly to chloramphenicol and Sch 25298 in the latter, thereby displaying selective inhibition of prokaryotic protein synthesis. Thiamphenicol and Sch 25298 were shown to be like chloramphenicol in inhibiting peptidyl transferase activity specifically on 70 S ribosomes, but the antibiotics bound to their common ribosomal-receptor site with different efficiencies in the order chloramphenicol greater than thiamphenicol greater than Sch 25298. Selected bacterial strains highly resistant to chloramphenicol and thiamphenicol because of chloramphenicol acetyltransferase production were, in contrast, highly sensitive to inhibition by the fluorinated antibiotics. Thus Sch 24893, Sch 25298 and Sch 25393 may have important uses in veterinary and clinical medicine.

Chloramphenicol↗
PubMed1989

Characterization of a loss-of-function mutation in the isopenicillin N synthetase gene of Acremonium chrysogenum.

The N-2 strain of Acremonium chrysogenum accumulates the beta-lactam precursor tripeptide delta-(L-alpha-amino-adipoyl)-L-cysteinyl-D-valine and has no discernible activity for three of the cephalosporin C (Ce) biosynthetic enzymes. This phenotype is consistent with a mutation either within pcbC [the isopenicillin N synthetase (IPNS)-encoding gene] or in a pathway-regulator gene. To distinguish these possibilities we have cloned and sequenced pcbC from strain N-2. There is a single C----T mutation at nt 854 within the coding sequence, changing aa 285 from proline to leucine. An IPNS-specific monoclonal antibody recognises a catalytically inactive IPNS protein in extracts of N-2 cells. These findings suggest that strain N-2 carries a simple IPNS mutation and that IPNS or its biosynthetic product isopenicillin N is involved in regulation of the later stages of the Ce biosynthetic pathway.

Acremonium↗
PubMed1989

Inactivation of the Escherichia coli heat-labile enterotoxin by in vitro mutagenesis of the A-subunit gene.

In vitro mutagenesis of the LTA gene, encoding the A subunit of the Escherichia coli heat-labile enterotoxin, has been used to obtain A subunits deficient in enzymic activity. One inactive A-subunit mutant which contained two amino acid substitutions, was shown to associate with native B subunits to form a holotoxoid lacking toxin activity. A serine to phenylalanine mutation appears to be responsible for the loss of toxicity.

Amino Acid Sequence↗
PubMed1985

M13 clones carrying point mutations: identification by solution hybridization.

A solution hybridization procedure for the rapid identification of M13 clones carrying a particular sequence is described. The method, which employs a radiolabeled oligonucleotide probe, can discriminate between sequences which differ by only a single base, and can therefore be used for the identification of mutant sequences created by oligonucleotide-directed mutagenesis. Samples of phage-containing supernatant from cultures of M13-infected Escherichia coli are incubated with radiolabeled probe in the presence of sodium dodecyl sulfate. The mixtures are then subjected to agarose gel electrophoresis to separate hybrid molecules from unbound probe and hybridization is detected by autoradiography. This solution hybridization procedure is quicker and more convenient than membrane hybridization and has the added advantage that more than one probe can be used on a given gel.

Antigens, Bacterial↗
PubMed1985

Nucleotide sequences of four variants of the K88 gene of porcine enterotoxigenic Escherichia coli.

The nucleotide sequences of four variants of the Escherichia coli K88 antigen gene, K88ab1, K88ab2, K88ac, and K88ad, have been determined. The K88ab2 and K88ac sequences have not been reported previously. The K88ab1 sequence is very similar to that determined by other workers, but the K88ad sequence differs considerably from that described in a previous report. Comparison of the amino acid sequences inferred from the gene sequences revealed certain clusters of amino acid substitutions which have been correlated with areas of potential antigenicity in the mature proteins.

Animals↗
PubMed1985

Resistance to chloramphenicol in Proteus mirabilis by expression of a chromosomal gene for chloramphenicol acetyltransferase.

Proteus mirabilis PM13 is a well-characterized chloramphenicol-sensitive isolate which spontaneously gives rise to resistant colonies on solid media containing chloramphenicol (50 micrograms ml-1) at a plating efficiency of 10(-4) to 10(-5). Such chloramphenicol-resistant colonies exhibit a novel phenotype with respect to chloramphenicol resistance. When a single colony grown on chloramphenicol agar is transferred to liquid medium and grown in the absence of antibiotic for 150 generations, a population of predominantly sensitive cells arises. This mutation-reversion phenomenon has been observed in other Proteus species and Providencia strains, wherein resistance has been shown to be mediated in each case by the enzyme chloramphenicol acetyltransferase. The cat gene responsible for the phenomenon is chromosomal and can be cloned from P. mirabilis PM13 with DNA prepared from cells grown in the absence or the presence of chloramphenicol. Recombinant plasmids which confer resistance to chloramphenicol carry an 8.5-kilobase PstI fragment irrespective of the source of host DNA. The location of the cat gene within the PstI fragment was determined by Southern blotting with a cat consensus oligonucleotide corresponding to the expected amino acid sequence of the active site region of chloramphenicol acetyltransferase, and the direction of transcription was deduced from homology with the type I cat variant.

Acetyltransferases↗
PubMed1980

5-epi-Sisomicin and 5-epi-Gentamicin B: substrates for aminoglycoside-modifying enzymes that retain activity against aminoglycoside-resistant bacteria.

A number of bacterial strains, each possessing a different aminoglycoside-modifying enzyme, were examined for susceptibility to sisomicin and gentamicin B and the semisynthetic derivatives 5-epi-sisomicin and 5-epi-gentamicin B. Although strains possessing AAC (6') or APH(3') enzymes were equally resistant to the 5-epi-compounds, those possessing AAC(3)-I, ANT(2"), or AAC(2') enzymes were much more sensitive to the 5-epi derivatives. Analysis of partially purified aminoglycoside-modifying enzymes from the strains showed that the 5-epi compounds were substrates even for those enzymes found in susceptible strains [AAC(3)-I, ANT(2"), and AAC(2')]. However, a more detailed study of the enzymes showed that they had much increased Km values for the 5-epi derivatives; the 5-epi compounds were much less effectively modified than the parent antibiotics. This confirms and extends the notion that enzymatic modification of aminoglycosides is not in itself sufficient to confer resistance to the drugs, but also that the modification must be efficient, as reflected in the Km values.

Aminoglycosides↗
PubMed1979

Studies on a mutant form of Escherichia coli citrate synthase desensitised to allosteric effectors.

Naturally occurring citrate synthases fall into distinct molecular and catalytic types. Gram-negative bacteria produce a 'large' enzyme, allosterically inhibited by NADH and, in the facultative anaerobes such as Escherichia coli, also by 2-oxoglutarate. On the other hand, Gram-positive bacteria and all eukaryotes produce a 'small' citrate synthase which is insensitive to these metabolites. As a complement to structure-function studies we have explored the possibility of genetically altering one type of citrate synthase to the other. By mutagenesis and suitable selection we have succeeded in isolating a mutant of E. coli whose citrate synthase is both 'small' and insensitive to NADH and 2-oxoglutarate. Some characteristics of the enzyme are described. Such mutant enzymes offer a novel approach to the study of citrate synthase, its regulation and its natural diversity.

Allosteric Regulation↗
PubMed1975

Evidence of isosteric and allosteric nucleotide inhibition of citrate synthease from multiple-inhibition studies.

Citrate synthases from diverse organisms are inhibited by ATP and NADH. Evidence is presented, from multiple-inhibition studies on various citrate synthases, that ATP acts in all cases as an isosteric inhibitor at the acetyl-CoA site. On the other hand, NADH also acts isosterically with eukaryotic and Gram-positive bacterial citrate synthases, but behaves as an allosteric inhibitor specifically in the case of the Gram-negative bacterial enzyme. After desensitization to this allosteric inhibition, only the isosteric nucleotide inhibition, as found in other citrate syntheases, is observed.

Adenosine Triphosphate↗