Biomedical subjects

C S Tomich

Publications and source records attributed to C S Tomich.

25 records · Page 2Linked to original sources

PubMed1988

Use of lacZ expression to monitor transcription.

For use in monitoring transcription in operon fusion, we have constructed a lacZ sequence with the initiation codon ATG and the Escherichia coli consensus Shine-Dalgarno site. There are unique restriction endonuclease sites flanking the sequence to allow easy isolation of the lacZ sequence with or without the Shine-Dalgarno site. We have placed this lacZ sequence behind the bovine growth hormone (BGH) gene and found that the lacZ product beta-galactosidase synthesized reflects the level of BGH-specific mRNA.

Animals↗

PubMed1988

Stabilization of an associated folding intermediate of bovine growth hormone by site-directed mutagenesis.

By using oligonucleotide-directed mutagenesis, Lys-112 of bovine growth hormone (bGH) was changed to leucine, and its resulting effect on folding was studied. Equilibrium denaturation curves for the mutant protein exhibit biphasic or nonsymmetrical transitions by a variety of spectroscopic and hydrodynamic techniques, whereas the wild-type protein at the same concentration exhibits symmetrical transitions. The mutant protein refolds slower (by a factor of 30) and more readily precipitates upon refolding than the wild-type protein. These folding characteristics of the mutant protein are demonstrated to be a result of stabilization of an associated folding intermediate. A 38-amino acid fragment (96-133) derived from the mutant protein is helical, likely amphipathic, and more stabilized by increasing peptide concentration than is the corresponding helical peptide from the wild-type protein. The increased stability of the associated intermediate and the increased helicity of the peptide from the mutant protein are explained by preferential intermolecular interactions between helices due to enhanced hydrophobic attraction by their amphipathic surfaces.

Animals↗

PubMed1988

Effect of ribosome binding site on gene expression in Escherichia coli.

Using the expression of human renin gene in Escherichia coli as a model, we have observed that a distance of 7-10 nucleotides between the Shine-Dalgarno site to the initiation codon ATG is optimal for translation initiation. The sequence ATA as the triplet preceding the ATG gives the best expression among those tested. These observations agree with the statistical bias observed for the genes of E. coli and its phages. We have also compared gene expression with three different Shine-Dalgarno sites. Expression of a given gene can be increased by as much as 1000-fold with slight modifications in the Shine-Dalgarno sequence.

Base Sequence↗

PubMed1987

Folding and activation of recombinant human prorenin.

In vitro folding of mature renin, prorenin, and fused prorenin, all produced in denatured form in inclusion bodies in recombinant Escherichia coli, has been studied in order to evaluate the importance of prosequence in the folding of human renin. These studies have been compared with the in vivo folding and subsequent in vitro activation of recombinant human prorenin secreted by a nonbacterial expression system, namely Chinese hamster ovary (CHO) cells grown in serum-free medium. It is concluded that prosequence is essential in the folding of human renin and, therefore, the DNA coding for this sequence cannot be removed without affecting the recovery of active human renin from recombinant bacterial and nonbacterial systems.

Cell Line↗

PubMed1984

The nusA recognition site. Alteration in its sequence or position relative to upstream translation interferes with the action of the N antitermination function of phage lambda.

The phage lambda transcription antitermination protein, pN, acts with host factors, Nus, at sites on the phage genome, nut, to render RNA polymerase resistant to subsequent downstream termination signals. The NusA protein appears to recognize a seven to eight base-pair consensus sequence (5'Py-G-C-T-C-T-T(T)3') called boxA that is found in the promoter-proximal part of the nut region. Two types of change within or near the boxA sequence in the nutR region are shown to interfere with pN-mediated antitermination of transcription that has initiated at the upstream pR promoter. (1) A change of one base-pair (from G to T at the second position) in the boxA sequence significantly reduces pN action. (2) We prove that a frameshift mutation, cro delta 62, at the end of the gene promoter-proximal to the lambda nutR region, interferes with the pN antitermination reaction by allowing translation to proceed beyond cro into the nutR region. Using a series of plasmid constructions, we now show that the inhibition of antitermination caused by the cro delta 62 mutation can be suppressed when translation is terminated upstream from this mutation.

Bacterial Proteins↗

PubMedDate not supplied

Simple and efficient oligonucleotide-directed mutagenesis using one primer and circular plasmid DNA template.

A rapid and simple procedure for site-directed mutagenesis is described. This method uses only a single oligonucleotide primer with the double-stranded circular plasmid DNA as the template for mutagenesis. The phage T4 gene 32 product is included during primer extension in vitro to increase efficiency. Single and multiple changes as well as deletions have been obtained at an efficiency of 1-2%.

Animals↗

PubMedDate not supplied

A two step purification of recombinant human interleukin-1 beta expressed in E. coli.

Recombinant human interleukin-1 beta has been expressed in high yield using E. coli with a cDNA clone obtained from SKhep1RNA. The rIL-1 beta is purified to apparent homogeneity using freeze-thaw extractions followed by hydrophobic interaction chromatography over phenyl Sepharose. The procedure can provide pure rIL-1 beta (up to 15 mg per liter of E. coli culture) without the use of denaturants and if desired, in the absence of column chromatographic steps. Purity is defined by the presence of a single band on 1-D polyacrylamide gels and a single spot on 2-D polyacrylamide gels. The purified protein exhibits a biological activity of 1 x 10(7) units/mg in a fibroblast proliferation assay and is shown to cross-react with rabbit anti-human IL-1 beta sera.

Animals↗