Colette Simone Digneffe

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.

Why is ribosomal protein L11 of Escherichia coli methylated

Escherichia coli spends a significant amount of energy to synthesize one (or more than one) enzyme which further spends energy in transferring nine methyl groups from S-adenosyl-methionine (SAM) to ribosomal protein L11. Our current opinion is that spending this energy is meaningless, because an E. coli mutant (prmA1) completely lacks methylation of L11 while exhibiting no detectable deleterious phenotype (Colson and Smith, 1977). We summarize here some of the efforts made, using mutant prmA1, to unveil a function of L11 methylation. In addition we present preliminary results of a cloning project to further characterize the methylating enzyme(s) of L11.