Nancy S. Petersen

Induced thermal tolerance and heat shock protein synthesis in Chinese hamster ovary cells.

Abstract We have performed experiments to determine the kinetics of induction of thermal tolerance in Chinese hamster HA-1 cells, and the effects of heat treatments on the recovery of protein synthesis, with particular attention to whether heat induces specific proteins, perhaps the heat shock proteins (HSP). The kinetics of the development of thermal tolerance were measured by increases in cellular survival. In parallel experiments, the effects of heat treatment on the recovery of protein synthesis in HA-1 cells were examined. After heating (45°, 20 minutes), some of these cells were immediately labeled with 35 S-methionine (10 μCi/ml) for 1 hour at 37°, while the others were incubated at 37° for 1–8 hours and then labeled. The cell samples were prepared for electrophoresis on a gradient SDS gel. The incorporation of label into HA-1 cell proteins was drastically inhibited by the 45° heat treatment, but recovered gradually during the 8-hour incubation period at 37°C. A comparison of the proteins synthesized following heat shock with those synthesized by non-heated cells showed that the levels of synthesis of certain proteins were greatly enhanced following the 45° treatment. By 8 hours, it was qualitatively apparent that three proteins, with molecular weights of 59K, 70K and 87K, were synthesized in greater amounts than in untreated cells. The kinetics of HSP synthesis were compared to the kinetics of thermal tolerance; these showed good correlation. Overall protein synthesis also increased during this time, although at a rate slower than the synthesis of the HSP. The question of whether the HSP play a causative role in the development of thermal tolerance and if so, what that role might be, has not been answered.

The morphogenesis of cell hairs on Drosophila wings

We describe in this paper details of morphogenesis of wing hairs in Drosophila pupae. The ultimate objective is to relate specific protein components used in hair construction to specific components produced in the rapidly changing patterns of gene expression that are characteristic for the period of hair differentiation in wing cells (H. K. Mitchell and N. S. Petersen, 1981, Dev. Biol. 85, 233-242). Hair extrusion to essentially full size occurs quite suddenly at about 34 hr (postpupariation) and this is followed by deposition of a double-layer of cuticulin during the next 4 to 5 hr. Extreme changes in shape of cells and hairs, probably related to actin synthesis, then occur for the next 5 to 6 hr. Deposition of fibers within the hairs and on hair pedestals follows. Formation of cuticle on the cell surface begins and continues until some time in the 60-hr range. It appears that cuticle is formed only on the cell surface and not in hairs or on the top of hair pedestals. The protein synthesis patterns associated with these events are described.

Rapid changes in gene expression in differentiating tissues of Drosophila.

Abstract In this report we present evidence to show that the process of differentiation of wing cells to produce hairs in Drosophila pupae is accompanied by rapid changes in patterns of synthesis of all of the most abundant proteins. Additional evidence from in vitro translation of mRNA extracted from the differentiating wing tissue shows that most if not all of the rapid changes result from regulation at the transcriptional level. We have also made use of heat shock-induced phenocopies and the mutant mwh to show that different cell types have similar patterns of change in protein synthesis which are related to phenocopy sensitivity on a temporal basis. It appears that regulation in the system as evaluated by the phenocopies and the mutant may involve transcription concerned with structural materials involved in morphogenesis but it may just as well involve cytoskeleton components responsible for cell movements and/or cell surface receptors.

A comparison of the multiple Drosophila heat shock proteins in cell lines and larval salivary glands by two-dimensional gel electrophoresis☆

Two-dimensional gel electrophoresis of Drosophila proteins from heat-shocked salivary glands resolves the major heat shock proteins into over 14 different molecular weight species. Most of these proteins are in one of the six molecular weight classes previously reported for heat shock proteins: 83,000, 70,000, 68,000, 34,000, 26,000 to 28,000 and 21,000 to 23,000 Mr. A few minor proteins, which range in molecular weight from 44,000 to 66,000, are also observed. The 70,000 Mr heat shock proteins are resolved into seven slightly different molecular weight groups; within each group, there are several isoelectric point variants. The 83,000, 34,000, and 68,000 Mr proteins are single molecular weight species, although there are three isoelectric point variants of the 68,000 Mr protein. The 26,000 to 28,000 Mr proteins separate into eight major components in two different molecular weight groups plus a run of basic proteins that probably are a result of post-translational modifications of the major proteins. The 21,000 to 23,000 Mr proteins consist of three components in two different molecular weight groups. Separation of heat shock proteins from two Drosophila cell lines shows a similar pattern of multiple components, although some of the minor variants visible in salivary glands are not synthesized in the cell lines. In vitro translation of mRNA from heat-shocked salivary glands in a reticulocyte lysate results in a pattern of multiple heat shock proteins nearly identical to that observed in vivo.

Gradients of differentiation in wild-type and bithorax mutants of Drosophila

We present evidence to show that differentiation in wing cells to produce hairs is synchronous over the distal 90% of the wing surface (approximately 28,000 cells). In spite of this synchrony within such a large area a temporal gradient exists between zones (in general anterior to posterior) on the animal surface with rather sharp boundaries in between. In order to evaluate the basis for the gradient we studied two mutants which carry different combinations of the genes of the bithorax complex. These were examined with respect to the temporal aspects of sensitivity to heat shock induction of the multihair phenocopy on wings and the time of initiation of the program of protein synthesis that is related to hair formation. Results show that the gradient observed is based on predetermined properties within specific areas of tissue rather than on the position of the cells in the animal.