Pierre Emile Cornelis

Cloning and expression of a Bacillus coagulans amylase gene in Escherichia coli.

SummaryA partial EcoRI fragment of Bacillus coagulans DNA cloned in an Escherichia coli K12 bacteriophage λ host-vector system was shown to direct the synthesis of a thermostable α-amylase whose activity could be detected in situ on petri plates using the iodine staining method. A 3.31 kb EcoRI fragment containing the active gene with its own promoter was subcloned in pBR322; in the new clone, called pAMY2, the amylase was shown to accumulate in the periplasmic space. The molecular weight of the enzyme, confirmed by in vivo labelling of plasmid products in minicells, was estimated to be 60000.The restriction map of the plasmid was determined for five restriction enzymes and two new plasmids with smaller DNA inserts were constructed, both directing the synthesis of amylase; one of them with a 2.2 kb PstI insert was shown to be responsible for the synthesis of a fused β-lactamase-α-amylase protein with amylase activity.

Purification of Escherichia coli amplifiable plasmids by high-salt Sepharose chromatography

Abstract This new method allows an easy and rapid purification of amplifiable Escherichia coli plasmids such as pBR 322 without the use of cesium chloride centrifugation. After gentle lysis, centrifugation, and phenol extraction, the material is reextracted with acid phenol to remove the bacterial DNA. The high-molecular-weight ribosomal RNA is removed by precipitation with 2 m ammonium sulfate and the tRNA by passage through a small column of Sepharose CL 4B in the presence of 2 m ammonium sulfate.

Characterization of the tRNA and aminoacyl-tRNA synthetases of healthy and tumorous callus tissues from Nicotiana tabacum

Abstract Aminoacyl-tRNA synthetases extracted from healthy and crown gall tumor tissues (induced by Agrobacterium tumefaciens strain B6) from Nicotiana tabacum (strain Wisconsin 38) grown in vitro, showed the same ability to charge Phaseolus vulgaris tRNA, for all the 15 amino acids tested. For each amino acid, optimal charging conditions (enzyme concentration, Mg2+/ATP ratios, K+ ion effects) have been determined with Phaseolus vulgaris tRNA and were found to be the same whether aminoacyl-tRNA synthetases from healthy or tumor tissues were used. In each case, valyl- and glutamyl-tRNA synthetases were very sensitive to an excess of Mg2+ and K+ ions. Although tRNAs extracted from healthy and tumor tissues gave the same electrophoretic patterns, charging levels obtained with turner tRNAs were generally 45% higher than those obtained with tRNAs from healthy tissues.