Biomedical subjects

J E Mott

Publications and source records attributed to J E Mott.

3 recordsLinked to original sources

PubMed1985

Maturation of Escherichia coli tryptophan operon mRNA: evidence for 3' exonucleolytic processing after rho-dependent termination.

The mature 3' end of Escherichia coli tryptophan operon mRNA in vivo coincides with a site (trp t) having features commonly associated with rho-independent terminators in bacteria. Efficient generation of this 3' end in vivo is nevertheless affected by a distal rho-dependent site (trP-t'), though these two sites behave independently in vitro. We have cloned these sites upstream of the galactokinase gene (galK), and galactokinase levels in vivo indicate that, as terminators per se, their efficiencies (37% for trp t, and 79% for trp t') do not differ significantly from those observed in vitro. However, when the trp t hairpin is placed between galK and a downstream copy of trp t', galactokinase levels are enhanced 2- to 3-fold. This suggests the involvement of a post-transcriptional event, such as RNA processing, in determining the level of gene activity. Indeed, in the presence of the 3' exonuclease RNase II, mRNA terminated by rho factor in vitro at the trp t' site is processed back to the trp t site. The remote trp t' region appears to be the major termination site for trp mRNA, and the trp t hairpin serves a dual function-as a minor terminator, and as a protective barrier to 3' exonucleolytic degradation. We infer that the tandem terminators, rho factor, and RNA processing are all required to generate the mature 3' end of this bacterial mRNA.

Base Sequence↗

PubMed1985

Maximizing gene expression from plasmid vectors containing the lambda PL promoter: strategies for overproducing transcription termination factor rho.

We have constructed two plasmids in which transcription of the rho gene from Escherichia coli K-12 is under the control of the lambda phage PL promoter. In p31-356, the normal rho promoter is deleted, but the remainder of the rho leader region, including the ribosome binding site, is present. In p39-AS, the rho leader is completely absent, and the lambda cII ribosome binding site replaces that of rho. Under noninducing conditions, expression of rho protein from these plasmids is repressed by the lambda cI protein in hosts carrying lambda cryptic prophage. Induction using mitomycin C or nalidixic acid in a cryptic lysogen carrying the cI+ repressor resulted in the overproduction of rho protein to levels of 3%-5% of the total cellular protein with p31-356, and to levels of approximately equal to 40% with p39-AS. The overproduced protein is functionally indistinguishable from the rho protein isolated from the K-12 strain W3110, and it can be obtained from cells harboring p39-AS in yields of up to 25 mg of rho per g of cells. In contrast to chemical induction, heat induction in four cryptic lambda lysogens carrying the thermolabile cI857 repressor failed to yield the same high levels of rho protein (with either plasmid). Our results show that chemical induction of PL-containing plasmid expression vectors can serve as a convenient and useful alternative to the commonly used method of heat induction.

Bacteriophage lambda↗

PubMed1984

Targeted mutagenesis in vitro: lac repressor mutations generated using AMV reverse transcriptase and dBrUTP.

We have cloned the gene for the lac operon repressor (lacI) of Escherichia coli into the M13 related phage f1. Mutagenesis of the lacI gene was performed in vitro by filling dsDNA molecules gapped over the lacI gene with Avian Myeloblastosis Virus (AMV) reverse transcriptase. LacI mutants are found at a frequency of 1 in 10(4) using a genetic screen in vivo. For two-thirds of the 60 mutants, lesions were identified within the first 400 bases of lacI, by dideoxy sequencing. An unexpectedly wide range of different lesions were observed, including transitions, transversions, and deletions (of which the most common were the removal of single base pairs). The replacement of dTTP by dBrUTP in the filling reaction resulted in a doubling of deletions in the sample population as well as the anticipated T to C and C to T transitions. Although the lacI gene has been extensively studied in vivo, the power of this technique for mutagenesis in vitro is demonstrated by the generation of three previously undescribed lacI mutations.

Amino Acid Sequence↗