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Biomedical subjects

R W Hartley

Publications and source records attributed to R W Hartley.

At least 55 records · Page 3Linked to original sources
PubMed1986

Use of plasmid pTV1 in transposon mutagenesis and gene cloning in Bacillus amyloliquefaciens.

The plasmid pTV1, constructed in Bacillus subtilis as a tool for insertional mutagenesis by the transposon Tn917, has been transferred to Bacillus amyloliquefaciens by transduction with the phage PBS1. Insertional mutants containing Tn917 were observed in the new host. Southern blot analysis of such mutants indicated no preference for insertion sites. The copy numbers of pTV1 in B. amyloliquefaciens and B. subtilis were found to be 1.4 and 14, respectively; the plasmid is less stable against loss in B. amyloliquefaciens. The overall transposition rate in B. amyloliquefaciens is nevertheless comparable to that in B. subtilis and large numbers of mutants are readily obtained. The yield of auxotrophs was about 0.7% of all mutants, but the preponderance of glutamate auxotrophs seen in B. subtilis was not observed. A number of auxotrophs were identified as to nutritional requirements and those tested were found to be stable. Mutants deficient in extracellular proteases, amylase, and ribonuclease (barnase) were also found and the inactivated barnase gene has been cloned in Escherichia coli. It seems likely, therefore, that any B. amyloliquefaciens gene for which there is a functional test could be cloned by this technique.

Bacillus↗
PubMed1985

Cloning, sequencing and transcription of an inactivated copy of Bacillus amyloliquefaciens extracellular ribonuclease (barnase).

The gene for Bacillus amyloliquefaciens extracellular RNase (barnase) has been cloned in an inactive form in Escherichia coli following insertional mutagenesis by transposon Tn917. The nucleotide (nt) sequence of the gene was determined and the deduced amino acid (aa) sequence found to correspond almost precisely to the previously determined sequence. An open reading frame (ORF) of 72 codons precedes the mature sequence. The probable translation start site is 46 or 47 codons before the N-terminal alanine of the mature protein, 11 (or 14) bp from a putative ribosome-binding site (RBS). Within this leader sequence is a hydrophobic 15 aa core preceded by three basic residues which is characteristic of a secretory signal sequence. A pro-barnase protein with four extra aa at the N-terminus has been detected extracellularly indicating that the signal peptidase-cutting site lies before the mature protein. An inverted repeat that may act as a transcription terminator was found at the 3' end of the gene. The gene is maintained in E. coli with a short inverted repeat from the termini of Tn917 inserted into the coding sequence. Northern blot analysis of RNA from B. amyloliquefaciens shows an approx. 780-nt transcript produced during exponential and stationary growth phases. The inactive cloned gene produces an approx. 480-nt transcript in E. coli and two transcripts of approx. 480 and 780 nt in Bacillus subtilis.

Amino Acid Sequence↗
PubMed1980

Homology between prokaryotic and eukaryotic ribonucleases.

There is homology between the amino acid sequences of the extracellular ribonucleases T1 and St, from the eukaryote Aspergillus oryzae and the prokaryote Streptomyces erythreus, respectively. Together with other extracellular ribonucleases homologous to each, these enzymes make up a family of interest to evolutionary biology and useful in studies of protein structure and function.

Amino Acid Sequence↗
PubMed1980

An improved apparatus for intragastric titration in the conscious dog.

An apparatus for automatic intragastric titration with on-line display of results has been described. The apparatus has been validated by titration of exogenous acid injected into the test meal in a reservoir and in vivo by assessing the secretory response of a dog with a gastric fistula to a test meal. A titration display unit provides a record of the secretory response both as a digital printout and a bar chart display. Cimetidine (2 mg kg-1h-1) significantly reduced the secretory response to a test meal.

Animals↗
PubMed1977

Complementation of peptides of barnase, extracellular ribonuclease of Bacillus amyloliquefaciens.

Recovery of ribonuclease activity by complementation of peptides of barnase is reported. Activity is restored to barnase-(1-102), which lacks eight amino acids from its COOH terminus, by combination with peptides-(88-110), -(95-110), or -(99-108), and also with succinylated peptide-(88-110). The dissociation constants are about 8 X 10(-6) M for the first two combinations and little, if any, greater for the other two. Based on barnase-(1-102) concentration, up to 80% of native activity is obtained with peptide-(88-110) but only 5 to 20% with the others. The octapeptide-(103-110), equivalent to the residues missing from barnase-(1-102), does not complement barnase-(1-102), suggesting that an intact sequence about His-102 and Tyr-103 is required for activity. Barstar, the natural inhibitor of barnase, completely inhibits all activity of the complementing peptides.

Amino Acid Sequence↗
PubMed1975

A two-state conformational transition of the extracellular ribonuclease of Bacillus amyloliquefaciens (barnase) induced by sodium dodecyl sulfate.

Barnase, the extracellular ribonuclease of Bacillus amyloliquefaciens, is shown to undergo a reversible two-state conformational transition at 0.65 mM sodium dodecyl sulfate (SDS) AAT 37 DEGREES. The prinicipal evidence is based on the equivalence of two independent values of the SDS-barnase binding ratio; about 14 mol of SDS/mol of barnase. Both were derived from fluorometric titration data, one being based on simple conservation of SDS and the other on the use of Wyman's theory of linked functions. No SDS is bound to barnase at SDS concentrations below the transition region.

Bacillus↗