Biomedical subjects
Publications and source records attributed to S Pestka.
A fluorescent erythromycin derivative was used to determine the binding of erythromycin derivatives to ribosomes. The assay is rapid, sensitive, and convenient. Because measurements are made in solution, they represent equilibrium conditions, unlike filter binding assays which perturb the equilibrium. Results correlate well with measurements made by other techniques.
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The mRNA for the mouse MOPC-46B K chain, a glycorportein, was prepared translated in Ehrlich ascites cell-free extract. A polypeptide product similar to the MOPC-46B kappa chain was identified by its antigenic properties and by analysis of the products of tryptic digestion. In addition, the cell-free product was glycosylated after translation. It appeared that glycosylation of the cell-free product occurred on the same tryptic peptide glycosylated by intact cells. The characteristics and requirements of glycosylation are described. The presence of Mn2+ was a major requirement.
The effects of chloramphenical on the binding kinetics of a fluorescein isothiocyanate derivative of 9(S)-erythromycylamine with 70S and 50S ribosomes have been studied by direct fluorimetric measurements. While chloramphenicol had little effect on the second-order 70S binding rate of the erythromycin analogue, it substantially reduced the dissociation rate of the fluorescent antibiotic-70S ribosome complex. This could be explained by simultaneous binding of both antibiotics to the 70S ribosome. The kinetic results suggest that chloramphenicol-saturated 70S particles bind the erythromycin analogue four times stronger and this was confirmed by direct binding studies. In additon, chloramphenicol causes a twofold increase in the intrinsic fluorescence of the 70S-bound analogue. This increase in fluorescence was used to study the kinetics of chloramphenicol binding to 70S ribosomes containing the fluorescent derivative. The fluorescence change followed first-order kinetics, suggesting that chloramphenicol induces a conformational change in the 70S particle. This could explain both its effect on erythromycin binding and on the fluorescence of bound analogue. Less detailed results with the 50S particle indicate a qualitively similar picture of erythromycin-chloramphenicol interactions.
Biologically active human interferon was synthesized de novo in a cell-free mouse extract stimulated with messenger RNA from induced human fibroblasts. The identity of the antiviral activity as human interferon was demonstrated by its antigenic and species specificity. Some characteristics of the cell-free synthesis were described.
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Polyadenylylated interferon mRNA, obtained from induced human fibroblasts, was quantitatively assayed by synthesis of biologically active human interferon in Xenopus laevis oocytes. The assay for interferon mRNA was used to distinguish between various hypotheses relating to interferon induction and biosynthesis. The data demonstrate that on induction with poly(I-poly(C) human fibroblasts accumulate interferon mRNA for 1-1.5 hr, after which time the mRNA is rapidly degraded with a half-life (t 1/2) of 18 min. Treatment of cells with cycloheximide prolongs the period of accumulation to 3 hr and decreases the rate of mRNA inactivation (t 1/2 = 49 min). Treatment with actinomycin D decreases the rate of inactivation still further (t 1/2 = 68 min). A comparison of cellular interferon synthesis with the relative amounts of interferon m RNA after simple induction or inductionin the presence of the inhibitors (superinduction) indicated a general correlation. Thus, on induction, the genes for interferon are activated to produce a transcript for a short time. The superinducing treatments prolong the period of accumulation and decrease the rate of degradation of this transcript.
Partially purified mRNA coding for the MOPC-315 heavy immunoglobulin alpha chain was translated in a reticulocyte lysate containing 20 labeled amino acids. Radiolabeled MOPC-315 heavy chain precursor protein, purified by preparative gel electrophoresis and immunoprecipitation, was sequenced by Edman degradation. The labeled phenylthiohydantoin amino acid obtained in each cycle was identified and quantitated by high-pressure liquid chromatography. The precursor sequence of 18 amino acids, Met-Lys-Val-Leu-Ser-Leu-Leu-Tyr-Leu-Leu-Thr-Ala-Ile-Pro-His-Ile-Met-Ser, preceded the sequence corresponding to the NH2 terminus of the mature secreted heavy chain.
Human fibroblasts and leukocytes produce interferons which may be distinguished by their antigenic and species specificity as well as by their molecular weight distributions. To elucidate the basis for these differences, we isolated mRNA from induced human fibroblasts and lymphoblastoid (Namalva) cells and studied the products of translation in Xenopus laevis oocytes. The mRNA from the respective cells yielded translation products, in oocytes, that were characteristic of the cells from which the mRNA was derived. We conclude that human cells contain at least two structural genes for interferon, coding for polypeptides differing in primary sequence. Fibroblasts synthesize a single species of interferon; lymphoblastoid cells synthesize two species, the fibroblast and leukocyte types.
Escherichia coli mutants resistant to streptomycin exhibited differences in countercurrent distribution from the parental strains. The degree of difference from the parental strain correlated with the degree of restriction of translation and thus the particular strA allele. The changes in countercurrent distribution in the phase systems used probably resulted predominantly from surface charge alterations. The differences in countercurrent distribution in these and other mutants may be a useful selective technique to obtain different types of mutants for which specific selective techniques may not be available. In addition, it appears that the surface properties of cells, which determine their position in countercurrent distribution, are a function of the translational efficiency and fidelity, and that the surface of cells consists of a mosaic that is an expression of this translational fidelity.
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The 50S subunit is the site of action of the antibiotic micrococcin. In addition, B. subtilis strain mic-1, which is resistant to micrococcin, contains altered 50S subunits.
A number of thiostrepton-resistant mutants of Bacillus subtilis were obtained. The thi mutations map proximally to strA. Effects of thiostrepton on polyphenylalanine synthesis with ribosomes of S-100 fractions from parent and mutant strains indicated that resistance was localized to the ribosomes. Furthermore, effects of thiostrepton on binding of [3H]GTP to ribosomes and 50S subunits from thiostrepton-sensitive and -resistant strains localized the site of resistance to the 50S subunit. In addition, revertants from thiostrepton-resistance to thiostrepton-sensitivity were obtained. Ribosomes and 50S subunits from these thiostrepton-sensitive revertants were sensitive to thiostrepton similar to parental sensitive B. subtilis.
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