Jon Ira Williams

Cloning and expression of a collagen-analog-encoding synthetic gene in Escherichia coli

A family of totally synthetic genes coding for multiple tandem repeats of the amino acid sequence (Gly-Pro-Pro) has been prepared and inserted into the ClaI cloning site of the expression vector pJL6. A representative recombinant plasmid, pAC1, with an insert of about 340 bp was established in an Escherichia coli strain bearing a defective lambda prophage, to study expression of the CII-collagen analog fusion protein produced from pAC1 upon heat induction. Authentic fusion protein production was demonstrated by nucleotide sequencing, Northern-blot analysis, and in vivo synthesis. Conversion of a wild-type rpoH allele to the rpoH165 mutation was shown to suppress proteolysis of the unstable fusion protein.

A novel diagnostic method based on DNA strand displacement

A novel approach for the detection of specific DNA or RNA sequences based on branch migration and DNA strand displacement is described. A partially double-stranded probe complex is prepared with a detectable label on one of the two strands and incubated with analyte molecules under hybridization conditions. The analyte molecules hybridize to the single-stranded portion of the probe complex and undergo branch migration to release the labelled DNA strand from the complex. Initial characterization of the assay indicates that both qualitative and quantitative information about analytes present in a sample can be obtained. The strand displacement assay is more sensitive to sequence alterations in the analyte than is a hybridization assay and can be promoted by rec A protein at 37 degrees C. Finally, a method for preparing probe complexes by cloning in a single-stranded DNA vector is also described.