Quantification of juvenile hormone III, vitellogenin, and vitellogenin-mRNA during the oviposition cycle of the lubber grasshopper.

The vitellogenic cycle of the lubber grasshopper (Romalea microptera) was studied by measuring levels of juvenile hormone (JH III), vitellogenin, and vitellogenin-mRNA through the first oviposition cycle. JH III and vitellogenin were measured by radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), respectively. To measure vitellogenin-mRNA, a partial (753 bp) cDNA fragment of vitellogenin was isolated from the fat body of vitellogenic animals. The sequence of this cDNA was related to vitellogenin sequences in other insect species. Using these sequence data, an RT-PCR (reverse transcriptase polymerase chain reaction) assay was developed to quantify vitellogenin-mRNA levels during the oviposition cycle. Vitellogenin-mRNA levels in the fat body tissue from virgin females were measured on specific days after eclosion and compared to hemolymph levels of JH III and vitellogenin from the same individuals. The levels of all three compounds (JH III, vitellogenin, and vitellogenin-mRNA) showed similar changes throughout the oviposition cycle, being undetectable or nearly undetectable initially (day 3), rising to maximum levels on days 23 and 28, and then dropped to lower or undetectable levels on the day of oviposition. The ability to measure these characteristics will be useful for studying the effects of hormonal and nutritional manipulations on reproduction.

Analysis of the herpes simplex virus type 1 promoter controlling the expression of UL38, a true late gene involved in capsid assembly.

The cistrons encoding the herpes simplex virus type 1 (HSV-1) UL37 and UL38 genes are adjacent to one another but are transcribed from opposite strands of the viral DNA. The UL37 gene encodes a 1,123-amino-acid protein of unknown function, while the 465-amino-acid UL38 protein is involved in capsid assembly. Previous work from our laboratory indicated that the transcripts encoding these proteins are expressed with significantly different kinetics in productive infection. In the present communication we confirm the kinetic classes and precisely map the cap sites of the UL37 and UL38 mRNAs. A bifunctional reporter gene vector was used to demonstrate that divergent promoters control the expression of these reporter genes in trans-activation assays. The UL38 promoter is functionally separable from that controlling UL37 in a recombinant virus. We used deletion analysis to demonstrate that as few as 29 bases 5' of the mRNA cap site are adequate for full activity of the UL38 promoter in trans-activation assays. Finally, we analyzed the protein-binding properties of the UL38 promoter; several sites that form complexes containing ICP4, with clear homology to those identified in the HSV-1 gamma 42 promoter, are present. Thus, in general, the properties of this promoter are quite similar to those of other gamma promoters.

Insulin-stimulated protein kinase activity in rat skeletal muscle that phosphorylates ribosomal protein S6.

Treatment of rats with a single high dose of insulin leads to rapid stimulation of cytosolic protein kinase activity in skeletal muscle that phosphorylates ribosomal protein S6. This stimulation is maximal within 15 minutes after insulin treatment, and the activity remains elevated for at least 90 minutes. The insulin-stimulated protein kinase activity elutes as two peaks from DEAE-Sepharose. Peak I elutes at 0.04-0.06 M KCl and is stimulated by insulin approximately 1.4-fold above the control. Peak II elutes at 0.09-0.11 M KCl and is stimulated 2.8-fold above the control. The peak II activity, which is most strongly stimulated by insulin, is resolved from cyclic AMP-dependent protein kinase on DEAE-Sepharose and appears to be distinct from protein kinase C. These results represent a novel finding of the stimulation of S6 kinase activity by insulin in skeletal muscle tissue in vivo.

Insulin-sensitive, serum-sensitive protein kinase activity that phosphorylates ribosomal protein S6 in cultured fibroblast-melanoma hybrid cells.

A protein kinase activity (S6PK) that phosphorylates ribosomal protein S6 has been detected in cytosolic extracts prepared from an insulin-sensitive mouse fibroblast-melanoma hybrid cell line. The activity of this enzyme is greatly increased in cells that have been stimulated with insulin or serum for 30 min before preparation of the extract. In the parental melanoma cells, which are insensitive to the growth-stimulatory action of insulin, the activity of the enzyme is lower than in the hybrid cells and is not increased in response to insulin. The insulin-sensitive, serum-sensitive S6PK from the hybrid cells is eluted as a single peak from diethylaminoethyl (DEAE)-cellulose between 0.15 and 0.2 M KCl. The apparent mol wt of the enzyme, as determined by gel permeation chromatography, is approximately 105,000. A second S6 kinase activity from the hybrid cells is trypsin dependent and elutes from DEAE-cellulose at a lower salt concentration than S6PK. In contrast to S6PK, the trypsin-dependent S6 kinase activity does not vary in a consistent manner in response to insulin or serum. Fractions obtained from DEAE-cellulose chromatography of extracts of the hybrid cells have also been assayed for ability to phosphorylate the synthetic octapeptide Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala (S6-1), the structure of which is based on a phosphorylated region of the S6 protein. Two trypsin-dependent peaks of protein kinase activity have been found to phosphorylate this peptide, one eluting at 0.05 M KCl and the other at 0.10-0.15 M KCl. The first peak elutes at the same salt concentration as the trypsin-dependent protein kinase(s) that phosphorylate ribosomal protein S6, while the second elutes slightly, but reproducibly ahead of S6PK. Several properties of the second peak of S6-1 phosphorylating activity suggest that it is not S6PK.