Germline transformation with Drosophila mutant actin genes induces constitutive expression of heat shock genes

Two Drosophila mutants KM75 and HH5, which are mutated in the act88F actin gene specific for the indirect flight muscles (IFM), synthesize heat shock proteins (hsps) constitutively in a tissue-specific manner. We have introduced cloned mutant act88F genes into a strain containing the wild-type act88F allele by P-element-mediated transformation. Flies transformed with a 4.05 kb KM75 act88F gene fragment encoding the p42 actin variant express both p42 and hsps specifically in the IFM. Using normal/mutant chimeric genes, the mutation sites of KM75 and HH5 were mapped within the sequence encoding the last 72 amino acids of actin. An in vitro mutated gene encoding a protein that lacks the 72 carboxy-terminal amino acids also induces constitutive hsp synthesis.     

In vitro transcription of Drosophila ribosomal genes using Drosophila RNA polymerases

Drosophila RNA polymerases I &; II were used to transcribe a recombinant bacterial plasmid containing one copy of Drosophila ribosomal DNA. Both supercoiled and relaxed, closed circular plasmids were used. With Mg⁺² as the divalent cation, enzyme I is much more active on both forms of the plasmid; the relaxed form in particular supports almost no RNA synthesis by enzyme II. When Mn⁺² is present, differences in template efficiencies are minimal. The differences observed in the absence of Mn⁺² seem to depend only on different preferences for the physical state of the template and not on recognition of specific promotor sequences, since enzyme I shows no strand selection when transcribing these plasmids.     

In vitro effect of the Escherichia coli heat shock regulatory protein on expression of heat shock genes.

In Escherichia coli, the ability to elicit a heat shock response depends on the htpR gene product. Previous work has shown that the HtpR protein serves as a sigma factor (sigma 32) for RNA polymerase that specifically recognizes heat shock promoters (A.D. Grossman, J.W. Erickson, and C.A. Gross Cell 38:383-390, 1984). In the present study we showed that sigma 32 synthesized in vitro could stimulate the expression of heat shock genes. The in vitro-synthesized sigma 32 was found to be associated with RNA polymerase. In vivo-synthesized sigma 32 was also associated with RNA polymerase, and this polymerase (E sigma 32) could be isolated free of the standard polymerase (E sigma 70). E sigma 32 was more active than E sigma 70 with heat shock genes; however, non-heat-shock genes were not transcribed by E sigma 32. The in vitro expression of the htpR gene required E sigma 70 but did not require E sigma 32.     

Phosphorylation of Drosophila heat shock transcription factor.

The role that phosphorylation plays in regulating heat shock factor (HSF) function and activity has been the subject of several studies. Here, we demonstrate that Drosophila melanogaster HSF (DmHSF) is a phosphoprotein that is multiply phosphorylated at some sites and is dephosphorylated at others upon heat shock. However, the steady-state level of phosphorylation of Drosophila HSF remains unchanged after heat shock. Phosphoamino-acid analysis reveals that predominantly serine residues are phosphorylated for both the non-shocked and heat shocked molecules. Gel mobility shift assays using extracts from SL2 cells treated with a variety of phosphatase and kinase inhibitors show little or no effect on the heat shock induced DNA binding activity of HSF or on its recovery. We conclude that phosphorylation plays no significant role in regulating the heat induced DNA binding activity of Drosophila HSF.     

Characterization and purification of the small 28,000-dalton mammalian heat shock protein

We describe the biochemical characterization and purification of the small 28,000-dalton heat shock protein (28-kDa protein) of mammalian cells. Metabolic pulse labeling of heat shock-treated cells with either [3H]leucine or H3 32PO4 and analysis of the labeled proteins by two-dimensional gel electrophoresis revealed increased levels of three 28-kDa proteins differing only in their relative isoelectric point. Using both peptide mapping and immunological analysis, we demonstrate that all three proteins are related isoforms, with two of the isoforms containing phosphate. Cell fractionation studies revealed that the 28-kDa protein localizes predominantly within the nuclear pellet very shortly after the heat shock treatment. With increasing times of recovery of the heat-treated cells back at 37 degrees C, the majority of the 28-kDa protein was now observed to fractionate within the soluble fraction of the cells. Both gel filtration and velocity sedimentation studies revealed that the 28-kDA protein exists as a higher order structure with an approximate S20,w value of 10-18 S, a Stokes radius of about 60-70 A, and an estimated native molecular mass of at least 500,000 daltons. We describe a relatively simple and rapid purification of the proteins employing both ion-exchange and gel filtration chromatography.     

Structural changes in chromatin of heat shock protein 70 gene of Drosophila during transcription

Using "protein-image" hybridization technique combined with various crosslinking methods, for formaldehyde-prefixed nuclei we have analysed changes induced by activation in the chromatin structure of HSP-70 genes. From the crosslinking data it follows that chromatin of actively transcribed genes undergoes some structural rearrangements resulting in certain weakening of the contacts between DNA and the globular parts of histones so that the histones remain bound to DNA through their N-terminal regions. In addition, there have been found two specific regions with a reduced content of histones: the 5'-promoter of HSP-70 gene and a region distanced by approximately 1 k.b. from the 3'-end of the HSP-70 gene.     

Different induction of 70,000-Da heat shock protein and metallothionein in HeLa cells by copper.

To clarify the physiological roles of heat shock proteins induced by copper, we studied the synthesis of these proteins and metallothionein, as well as the level and nature of copper incorporated into HeLa cells. Incubation in medium containing 200 microM cupric sulfate and above induced the synthesis of 70,000-Da heat shock protein (hsp70) in these cells. However, the synthesis of hsp70 did not increase in the presence of less than 200 microM cupric sulfate. On the other hand, the synthesis of metallothionein increased due to 100 microM cupric sulfate. The uptake of copper into the cells depended on the cupric sulfate concentration in the medium. To analyze the nature of the intracellular copper, cell extracts were separated by gel filtration chromatography into three fractions: the high molecular weight, metallothionein, and low molecular weight fractions. No copper was found in the low molecular weight fraction of control cells, but appeared distinctly at 200 microM cupric sulfate and above. Copper in the high molecular weight fraction also began to increase at 200 microM cupric sulfate and above, whereas in the metallothionein fraction it began to increase even at 50 to 100 microM cupric sulfate. Furthermore, inhibition of cell growth was also observed at 200 microM cupric sulfate and above but not at 100 microM and below.(ABSTRACT TRUNCATED AT 250 WORDS)