Osmotic pumps containing Escherichia coliderived recombinant human granulocyte colony-stimulating factor (rhG-CSF) were attached to indwelling jugular vein catheters and implanted subcutaneously into Golden Syrian hamsters. Within 3 days, peripheral granulocyte counts had increased >10-fold with a concomitant 4-fold increase in total leukocytes. Microscopic examination of Wright-Giemsastained blood smears from rhG-CSF hamsters showed that only the neutrophil subpopulation of granulocytes had increased. No significant changes in lymphocyte or monocyte counts were observed during the course of continuous rhG-CSF treatment.After subcutaneous injection at rhG-CSF doses of up to 10 ,ug-kg'l day'1 only granulocyte counts were affected. However, at higher dose levels, a transient thrombocytopenia was noted. Erythrocyte and lymphocyte/monocyte counts remained unaffected by rhG-CSF over the entire dose range (0.3-300 ug-kg-lday-1) studied. Total leukocyte counts increased 3-fold within 12 hr after a single s.c. injection of rhG-CSF. This early effect was associated with an increase in the total number of colony-forming cells and the percent of active cycling cells in the marrow. A sustained elevation of peripheral leukocyte and marrow progenitor counts was observed following seven daily s.c. inJections of rhG-CSF. The ability of rhG-CSF to increase the production and release of granulocytes from the marrow may underlie the beneficial effect it produced on the restoration of peripheral leukocyte counts in hamsters made leukopenic by treatment with 5-fluorouracil.

We have investigated the renaturation kinetics of a single family of cloned interspersed repeated sequences isolated from human DNA. Cross-renaturation studies of individual cloned sequences reveal heterogeneity in both the renaturation rate and the thermal stability of heteroduplexes formed from members of this family of sequences. However, cloned members of this family all renature with approximately the same number of copies in the human genome, demonstrating that they are a single family of sequences by the criterion of DNA renaturation kinetics. When a single cloned member of the family is renatured with total human DNA as a function of temperature, the thermal stability of the renatured heteroduplexes is found to be independent of the renaturation temperature over the range 25 degrees C below to 4 degrees C above the melting temperature. Further, the number of genomic copies with which this cloned family member reacts is also independent of the renaturation temperature from 25 degrees C below up to the melting temperatures. These observations demonstrate a remarkable degree of homogeneity in the evolutionary sequence divergence of members of this family. These results also demonstrate that renaturation kinetics can accurately measure the number of genomic copies of interspersed repeated DNA sequences.

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Conditions for high-cell-density fermentations of Saccharomyces cerevisiae strains producing recombinant-DNA-derived proteins were established. Strains producing human immune interferon (IFN-gamma) from the constitutive PGK promoter failed to grow to high cell densities and exhibited low plasmid stability. Regulated expression of IFN-gamma was obtained in similar strains by employing a hybrid yeast GPD promoter that was subject to carbon source regulation due to the presence of regulatory DNA sequences from the yeast GAL 1,10 intergenic region. IFN-gamma expression programmed by this vector was low during growth on glucose and was induced by galactose. Previously defined fermentation conditions employing glucose as a carbon source were applied to this strain, resulting in high ceil densities with higher plasmid stability. Various methods of galactose induction of IFN-gamma expression in high-cell-density fermentations were investigated. Optimal conditions resulted in a 2000-fold induction and production of 2 g IFN-gamma/L fermentation culture.