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P A Lowe

Publications and source records attributed to P A Lowe.

At least 37 records · Page 2Linked to original sources
PubMed1985

Molecular cloning and nucleotide sequence of rat lingual lipase cDNA.

Purified rat lingual lipase (EC3113), a glycoprotein of approximate molecular weight 52,000, was used to generate polyclonal antibodies which were able to recognise the denatured and deglycosylated enzyme. These immunoglobulins were used to screen a cDNA library prepared from mRNA isolated from the serous glands of rat tongue cloned in E. coli expression vectors. An almost full length cDNA clone was isolated and the nucleotide and predicted amino acid sequence obtained. Comparison with the N-terminal amino acid sequence of the purified enzyme confirmed the identity of the cDNA and indicated that there was a hydrophobic signal sequence of 18 residues. The amino acid sequence of mature rat lingual lipase consists of 377 residues and shares little homology with porcine pancreatic lipase apart from a short region containing a serine residue at an analogous position to the ser 152 of the porcine enzyme.

Amino Acid Sequence↗
PubMed1983

Efficient synthesis of enzymatically active calf chymosin in Saccharomyces cerevisiae.

We have constructed a high-efficiency expression vector to direct the synthesis of heterologous polypeptides in yeast. The vector is termed a sandwich expression vector as the heterologous gene is inserted between the 5' and 3' control regions of the efficiently expressed yeast PGK gene. We have used this vector to direct the expression of three derivatives of the calf chymosin cDNA gene; preprochymosin, prochymosin and chymosin. Prochymosin is synthesised to at least 5% of total yeast-cell protein and furthermore, it can be readily activated to produce an enzyme which has milk-clotting activity.

Animals↗
PubMed1983

Synthesis of calf prochymosin (prorennin) in Escherichia coli.

A gene for calf prochymosin (prorennin) has been reconstructed from chemically synthesized oligodeoxyribonucleotides and cloned DNA copies of preprochymosin mRNA. This gene has been inserted into a bacterial expression plasmid containing the Escherichia coli tryptophan promoter and a bacterial ribosome binding site. Induction of transcription from the tryptophan promoter results in prochymosin synthesis at a level of up to 5% of total protein. The enzyme has been purified from bacteria by extraction with urea and chromatography on DEAE-cellulose and converted to enzymatically active chymosin by acidification and neutralization. Bacterially produced chymosin is as effective in clotting milk as the natural enzyme isolated from calf stomach.

Animals↗
PubMed1982

Molecular cloning and nucleotide sequence of cDNA coding for calf preprochymosin.

DNA complementary to calf stomach mRNA has been synthesised and inserted into the Pst1 site of pAT153 by G-C tailing. Clones containing sequences coding for prochymosin were recognised by colony hybridisation with cDNA extended from a chemically synthesised oligodeoxynucleotide primer, the sequence of which was predicted from the published amino acid sequence of calf prochymosin. Two clones were identified which together contained a complete copy of prochymosin mRNA. The nucleotide sequence is in substantial agreement with the reported amino acid sequence of prochymosin and shows that this protein has a mol.wt. of 40431 and chymosin a mol.wt. of 35612. The sequence also indicates that prochymosin is synthesised as a precursor molecule, preprochymosin, having a 16 amino acid hydrophobic leader sequence analogous to that reported for other secreted proteins.

Amino Acid Sequence↗
PubMed1982

The nucleotide sequence of cDNA coding for the structural proteins of foot-and-mouth disease virus.

The complete nucleotide sequence of cDNA coding for the structural capsid polypeptides of foot-and-mouth disease virus (FMDV) (strain A(10)61) has been determined. Portions of the flanking sequence coding for the nonstructural proteins p20a and p52 are also provided. The three larger structural polypeptides VP1, VP2 and VP3 have unmodified Mrs of 23248, 24649 and 24213, respectively. The size of the smaller polypeptide, VP4, can only be estimated at 7360 because the 5'-limit of its coding region is not yet known with certainty. The sequence data for VP1 (the major immunising antigen) and the amino-terminal quarter of p52 are compared with the data of Kurz et al. (Nucl. Acids Res. 9 (1981) 1919-1931) for a different serotype (O1K). This shows that variation is much greater in the region coding for VP1 than in that coding for p52. This is reflected in the level of amino acid sequence variation predicted for the two proteins. Analysis of relative codon usage reveals a strong bias in favour of C and G over U and A in the third base position. The dinucleotide frequencies show a bias against A-U and U-A, and for A-C and C-A.

Aphthovirus↗
PubMed1981

Molecular cloning of foot and mouth disease virus genome and nucleotide sequences in the structural protein genes.

Foot and mouth disease virus (FMDV), of the family Picornaviridae, consists of a single-stranded RNA (approximately 8,000 nucleotides), the translation of which is initiated on the 3' side of a 150-nucleotide poly(C) tract and yields a single polyprotein which is processed by host cell proteases into four primary products (Fig. 1). One or more virus-specified proteases further cleave these into the final products, the capsid proteins (VP1-4) being derived from the precursor p88 (for review see ref. 5). There are seven serotypes of the virus and as it has been shown that the immunizing activity of FMDV particles is associated primarily with VP1 (refs 6, 7), it seems likely that antigenic variation in FMDV is a result of changes in the structure of this protein. To further our understanding of this variation and as a first step in the possible development of FMDV vaccines from genetically manipulated microorganisms, we report here the construction and analysis of recombinant plasmids containing cDNA copies of the RNA. Comparison of the deduced amino acid sequence with the known polypeptide sequences shows that the NH2-termini of VP2 and VP3 are conserved between the A and O serotypes whereas that of VP1 (the immunizing antigen) varies by as much as 42% between serotypes.

Amino Acid Sequence↗
PubMed1981

In vitro thermal inactivation of a temperature-sensitive sigma subunit mutant (rpoD800) of Escherichia coli RNA polymerase proceeds by aggregation.

A temperature-sensitive mutant sigma subunit (rpoD800) purified from Escherichia coli was inactivated in vitro by temperatures in excess of 37 degrees C whereas wild type sigma remained stable up to 49 degrees C. Both temperature-sensitive and wild type sigma formed multimeric aggregates upon thermal inactivation which were visualized by electron microscopy as polymeric chains. Conditions favoring sigma monomer (low sigma concentration and binding to core polymerase) protected temperature-sensitive sigma from heat inactivation. Full activity was recovered from inactivated temperature-sensitive sigma aggregates by incubation in a buffer containing 6 M guanidine HCl and subsequent removal of denaturant by dilution. Both wild type and temperature-sensitive sigma recovered full activity levels, retaining their characteristic thermal inactivation temperatures after denaturation in 6 M guanidine HCl and renaturation. Transcription of T4 DNA by RNA polymerase containing the rpoD800 mutant sigma subunit remained undiminished for 10 min after shift up to 46 degrees C but was almost completely inhibited within the following 10 to 15 min.

DNA-Directed RNA Polymerases↗
PubMed1981

Temperature-sensitive RNA polymerase mutants of a picornavirus.

Temperature-sensitive (ts) RNA polymerase mutants of a picornavirus are reported. Two foot-and-mouth disease virus (FMDV) mutants designated ts 22 and ts 115 have been characterized. As judged by isoelectric focusing, both have charge alterations in P56a, the FMDV RNA polymerase protein. Virus RNA synthesis in cells infected with the mutants is severely impaired at the nonpermissive temperature. RNA polymerase purified from baby hamster kidney cells infected with these mutants exhibits a marked ts transcribing activity in vitro. Spontaneous revertants of both mutants have P56a polypeptides that are indistinguishable from the parental proteins on the basis of charge. The revertants regain the ability to synthesize virus RNA in vivo at the nonpermissive temperature. RNA polymerase purified from the revertants remains transcriptionally active at the nonpermissive temperature.

Animals↗
PubMed1979

Altered chemical properties in three mutants of E. coli RNA polymerase sigma subunit.

We have analyzed some chemical properties of the sigma subunit of RNA polymerase from the sigma mutants: rpoD1 (Gross et al., 1978), rpoD2 (formerly known as alt-1) (Silverstone et al., 1972; Travers et al., 1978), and rpoD800 (Gross et al., 1979). Each of the three mutants is located at about 66 min on the E. coli genetic map and exhibits an alteration in the enzymatic properties of its sigma subunit. The tryptic peptides and isoelectric focusing behavior were analyzed for mutant and wild type sigma. A single, but different altered lysine tryptic peptide was observed for each mutant. No altered arginine tryptic peptides were observed. The rpoD800 mutant sigma showed an altered isoelectric point. These studies provide chemical evidence that the sigma polypeptide in all three mutants is altered and strongly support the conclusion that the mutations are in the structural gene for sigma.

Alleles↗
PubMed1979

Purification and properties of the sigma subunit of Escherichia coli DNA-dependent RNA polymerase.

An improved purification procedure is described for the sigma subunit of escherichia coli DNA-dependent RNA polymerase [ribonucleoside triphosphate:RNA nucleotidyl-transferase, EC 2.7.7.6]. The method involves chromatography of purified RNA polymerase on single-stranded DNA-agarose, Bio-Rex 70, and finally Ultragel AcA44. The sigma factor obtained is electrophoretically pure with a yield of about 40%. A number of the chemical--physical properties of sigma are presented. A molecular weight of 82,000 was determined by phosphate buffered sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Ultraviolet absorption spectra were used to determine an E280nm 1% of 8.4. The amino acid composition and 12-residue N-terminal sequence (Met-Glx-Glx-Asx-Pro-Glx-(Ser or Cys)-Glx-Leu-Lys-Leu-Leu) of sigma have been determined. The isoelectric focusing properties of sigma are presented. Denaturation--renaturation studies indicate that sigma is capable of an unusually rapid and complete recovery of activity after being subjected to denaturing conditions. A stable, 40,000-dalton fragment is generated from sigma by mild trypsin treatment.

Amino Acid Sequence↗
PubMed1979

Isolation and characterization of transducing phage coding for sigma subunit of Escherichia coli RNA polymerase.

A transducing phage has been isolated with codes for the sigma subunit of Escherichia coli RNA polymerase. Transducing phage were selected from E. coli shotgun collections of HindIII or Sac I fragments cloned into Charon 25, a new bacteriophage lambda vector that is capble of forming lyosogens at high temperature. Transduction of an E. coli strain carrying a temperature-sensitive mutation in the sigma gene was used for the selection. The positions of restriction sites for Sac I, HindIII, Xho I, Bgl II, and Kpn I in the cloned bacterial DNA segments were determined. Phage containing the HindIII fragment complement both primase (dnaG) and sigma (rpoD) whereas those containing the Sac I fragment complement only sigma. Results of analyses of the proteins made both in vivo after infection of UV-irradiated cells and in vitro in a coupled transcription/translation system suggest that a Sac I site separates the promoter for sigma from the sigma structural gene. The direction of transcription of sigma was determined to be clockwise with respect to the E. coli genetic map.

Bacterial Proteins↗
PubMed1977

The dissociation of sigma-factor from ribonucleic acid polymerase.

The sigma-factor of Escherichia coli RNA polymerase was shown to dissociate from the core enzyme as a function of absolute concentration. The association constant is in the range 10(6)-10(8) litre/mol. This implies that the amount of holoenzyme, core enzyme and sigma-factor in RNA polymerase assays may vary according to the absolute concentration of the enzyme.

DNA-Directed RNA Polymerases↗
PubMed1976

Rifampicin binding as a probe for subunit interactions in Escherchia coli RNA polymerase.

The binding of the inhibitor rifampicin to RNA polymerase (alpha2betabeta') and its deficient subunit mixtures was investigated. The ability of beta to bind stoichiometric amounts of rifampicin was restored by formation of the alpha2beta subassembly. beta,beta' alpha, betabeta' and alpha2beta' were unable to bind rifampicin. RNA polymerase denatured with 6 M guanidine hydrochloride and dialysed against a renaturing buffer at 0degrees C ("renatured inactive enzyme") bound stoichiometric amounts of rifampicin but had lost the ability of bind dna. compared with native RNA polymerase "renatured inactive" enzyme possessed a markedly different tertiary structure as judged by limited proteolysis.

Binding Sites↗