Gary Wilson

Isolation of a sequence-specific endonuclease (BamI) from Bacillus amyloliquefaciens H

A restriction endonuclease has been isolated from Bacillus amyloliquefaciens H (strain RUB500). The enzyme, Bam I, cleaves adenovirus-2 DNA at three sites, phase λ DNA at five sites, λ p lac DNA at four sites, 80 pt DNA at 14 sites, and 3T + DNA at four sites. However, it does not cleave DNA from bacteriophage SPO2, 105 or 29.

Vibrational Raman optical activity of α-helical and unordered poly(L-lysine)

Measurements of the vibrational Raman optical activity (ROA) spectra of poly(L-lysine) in H2O and D2O solutions, under conditions said to generate model α-helical and unordered conformations, are reported. These provide new insights into the conformational elements actually present. In addition to several signatures characteristic of α-helix, the α-helical state (pH 11.0, 2 °C) of a sample with text-decoration:overlineMw= 26 000 shows a strong sharp positive ROA band at ca. 1340 cm–1, previously assigned to rigid loop structure with local order possibly that of 310-helix, which suggests that the α-helical sections are connected by segments of the corresponding loop. On increasing text-decoration:overlineMw to 268 000 this loop signature grows to dominate the ROA spectrum with a concomitant decrease in the α-helix bands. The unordered state (pH 3.0, 20 °C) shows clear signatures of several distinct conformational elements, including α-helix, β-strand and, possibly, left-handed helix. Under conditions which tend to disrupt secondary structure (5 mol 1–1 NaCl solution heated to 50 °C) the ROA spectrum of the unordered state shows a decrease in the intensity of the signature of the putative left-handed helix and an increase in bands assigned to α-helix.

Transformation of Bacillus subtilis and Escherichia coli by a hybrid plasmid pCD1.

The gene thyP3 from Bacillus subtilis bacteriophage phi 3T was cloned in the plasmid pMB9. The resulting chimeric plasmid, pCD1, is effective in transforming both Escherichia coli and Bacillus subtilis to thymine prototrophy. The activity of the thyP3 gene product, thymidylate synthetase, was assayed and found to be 9 times greater in a transformed strain of Escherichia coli than in a phi 3T lysogen of Bacillus subtilis. The physical location of restriction sites has been determined for two related plasmids pCD1 and pCD2. Hybridization studies clearly indicate that the plasmid gene responsible for Thy+ transformation is the gene from the bacteriophage phi 3T. The lack of restriction in this transformation process is consistent with our previous studies using bacterial DNA in heterospecific exchanges indicating that the nucleotide sequence surrounding the gene is the dominant factor in determining interspecific transformation.

Vibrational Raman optical activity of lysozyme: hydrogen–deuterium exchange, unfolding and ligand binding

Measurements of the vibrational Raman optical activity (ROA) spectra of hen egg white lysozyme are reported which show that ROA is a useful new probe of protein secondary and tertiary structure and dynamics. ROA spectra can be measured just as easily in D2O as in H2O and a comparison of the two gives information about the relative exchange rates of the amide hydrogens in the peptide backbone for the various types of secondary and tertiary structure in lysozyme. Unfolded lysozyme shows a large conservative ROA couplet in the amide III region which might facilitate the identification of signatures in the ROA spectra of native proteins from irregular structures with the same type of conformational heterogeneity as that of an unfolded protein. The ROA spectrum of lysozyme bound to a saccharide inhibitor shows evidence for an increase in rigid loop content.

Vibrational Raman optical activity of glycoproteins

This paper reports the first vibrational Raman optical activity (ROA) spectrum of a glycoprotein. The sample, orosomucoid (alpha 1-acid glycoprotein), shows ROA bands characteristic of a high beta-sheet content together with new bands which could be specific for the carbohydrate and its association with the protein. Our results suggest that ROA spectra of intact glycoproteins may contain information about both protein and carbohydrate conformation and the mutual influence on each others stability and conformation.

Expression of thymidylate synthetase activity in Bacillus subtilis upon integration of a cloned gene from Escherichia coli

The gene from Escherichia coli encoding thymidylate synthetase was cloned in the plasmid pBR322. The resulting chimeric plasmid, pER2, was effective in transforming both E. coli and Bacillus subtilis to thymine prototrophy. Uncloned linear E. coli chromosomal DNA was unable to transform thymine-requiring strains of B. subtilis to thymine independence. Linearization of the chimeric plasmid, pER2, with restriction enzymes markedly diminished its ability to transform B. subtilis auxotrophs. The Thy+ transformants derived from the transformation of B. subtilis with pER2 DNA did not contain detectable extrachromosomal DNA as demonstrated by Southern hybridization patterns and centrifugation in CsCl gradients of DNA isolated from B. subtilis colonies transformed with the chimeric plasmid. We conclude that the DNA from the chimeric plasmid was integrated into the chromosome of B. subtilis, demonstrating that extensive homology is not required for the integration of foreign DNA. This is the first reported case of a gene from a Gram-negative bacterium functioning in a Gram-positive organism.

Vibrational Raman optical activity of biopolymers

Abstract Vibrational Raman optical activity measurements can now provide a wealth of new information about the structure and conformation of biopolymers in aqueous solution. Typical results are exemplified here for proteins by bovine serum albumin in H 2 O and D 2 O solution, and for polysaccharides by laminarin.