Showing papers by "George M. Weinstock published in 1983"

Open reading frame expression vectors: a general method for antigen production in Escherichia coli using protein fusions to beta-galactosidase

Abstract: We have developed an Escherichia coli plasmid vector for the identification and expression of foreign DNA segments that are open reading frames (ORFs). The 5' end of ompF, an E. coli gene encoding an abundant outer membrane protein, is used to provide a strong, regulated promoter, translation initiation site, and signal sequence for export from the cytoplasm. This sequence is coupled to the lacZ gene of E. coli so that expression of beta-galactosidase requires ompF transcription and translation signals. However, this hybrid gene is LacZ- because lacZ is out of frame with respect to ompF. Restriction enzyme recognition sites are located between ompF and lacZ to allow convenient insertion of DNA fragments. If an insert is an ORF of the correct length, ompF and lacZ become realigned in frame, resulting in a LacZ+ gene that produces a tribrid protein with the translation product of the insert sandwiched between OmpF and beta-galactosidase. The LacZ+ phenotype thus identifies clones containing an expressed ORF. To demonstrate the vector's utility we inserted a fragment from the herpes virus thymidine kinase gene and used the resulting tribrid protein to raise antibodies that precipitate thymidine kinase from herpes virus-infected cells. We also inserted a fragment from the E. coli lexA gene to produce a tribrid protein that is precipitated by antiserum raised with LexA protein. Thus, tribrid fusion proteins can be used to produce or detect antibodies and also to identify the product of a cloned gene.

Stimulation of Reca Protein Dependent Strand Assimilation and DNA Complex Formation by Single-Stranded DNA Binding Proteins

Abstract: RecA protein promotes the homology dependent hybridization of single-stranded DNA (ssDNA) with linear or circular duplex DNA (dsDNA) [strand-assimilatin]. Single-stranded DNA binding protein of E. coli (SSB) or the gene 32 protein of bacteriophage T4 stimulates the rate and extent of strand assimilation by a mechanism that does not appear to involve a recA protein. SSB complex. Furthermore, SSB stimulates pairing of circular ssDNA with RFII DNA indicating that free ends on the ssDNA partner are not necessary for SSB action. SSB also stimulates formation of recA protein-DNA complexes under conditions where it enhances strand assimilation, suggesting that these complexes are intermediates in the assimilation reaction. Finally, the recA629 mutant protein, which is cold labile for ssDNA annealing, promotes complex formation at 37°C, but not at 30°C, indicating that the mechanisms of strand pairing in assimilation and ssDNA annealing are closely related.