Major Heat Shock Genes Research Articles

A Mutation in the Yeast Heat-Shock Factor Gene Causes Temperature-Sensitive Defects in Both Mitochondrial Protein Import and the Cell Cycle

Yeast cells containing the recessive mas3 mutation display temperature-sensitive defects in both mitochondrial protein import and the cell division cycle. The import defect is characterized by two pools of mitochondrial precursors and a dramatically slower rate of posttranslational import. The effect of mas3 on cell cycle progression occurs within one cell cycle at the nonpermissive temperature and retards progression through the G2 stage. The mas3 mutation maps to the gene encoding yeast heat-shock transcription factor (HSF), and expression of wild-type HSF complements the temperature-sensitive defects. The mas3 lesion has no apparent effect on protein secretion. In mas3 cells, induction of a major heat-shock gene, SSA1, is defective at 37 degrees C. The properties of the mas3 mutant cells indicate that HSF mediates the response to stress of two basic cellular processes: mitochondrial protein import and cell cycle progression.

A mutation in the yeast heat-shock factor gene causes temperature-sensitive defects in both mitochondrial protein import and the cell cycle.

Yeast cells containing the recessive mas3 mutation display temperature-sensitive defects in both mitochondrial protein import and the cell division cycle. The import defect is characterized by two pools of mitochondrial precursors and a dramatically slower rate of posttranslational import. The effect of mas3 on cell cycle progression occurs within one cell cycle at the nonpermissive temperature and retards progression through the G2 stage. The mas3 mutation maps to the gene encoding yeast heat-shock transcription factor (HSF), and expression of wild-type HSF complements the temperature-sensitive defects. The mas3 lesion has no apparent effect on protein secretion. In mas3 cells, induction of a major heat-shock gene, SSA1, is defective at 37 degrees C. The properties of the mas3 mutant cells indicate that HSF mediates the response to stress of two basic cellular processes: mitochondrial protein import and cell cycle progression.

Induction of a heat shock gene (hsp 70) in rabbit retinal ganglion cells detected by in situ hybridization with plastic-embedded tissue

Elevation of body temperature by 2-3 degrees C induces a 2.7 kilobase hsp70 mRNA species in the rabbit retina within 1 hr. In situ hybridization with thin sections derived from plastic-embedded tissue permitted a higher level of resolution of retinal cell types compared to procedures which involved the use of frozen tissue sections. A prominent induction of hsp70 mRNA in retinal ganglion cells was observed when an hsp70 riboprobe was utilized for in situ hybridization. These results indicate that this neuronal cell type responds rapidly to fever-like body temperatures by inducing one of the major heat shock genes.

The RNA polymerase II molecule at the 5′ end of the uninduced hsp70 gene of D. melanogaster is transcriptionally engaged

Protein-DNA cross-linking of cultured Drosophila cells has shown that, in vivo, prior to the induction of heat shock, there is approximately one molecule of RNA polymerase II associated with the promoter region of the major heat shock gene, hsp70. Here, we show that this promoter-associated RNA polymerase II molecule is transcriptionally engaged and has formed a nascent RNA chain of approximately 25 nucleotides in length, but is apparently arrested at that point and unable to penetrate further into the hsp70 gene without heat induction. The detection of a post-initiation RNA polymerase complex on the promoter region of the inactive gene suggests that there is a transcriptional control mechanism that acts at a step early in transcript elongation.

Suppression of rpoH (htpR) mutations of Escherichia coli: heat shock response in suhA revertants.

Temperature-resistant pseudorevertants were isolated from rpoH (htpR) mutants of Escherichia coli K-12 that cannot grow at a high temperature owing to a deficiency in sigma 32 required for the induction of heat shock proteins. Among them was a class of revertants carrying a suppressor mutation, designated suhA, that suppressed all the nonsense and missense rpoH mutations tested. suhA is located at 77 min, about 1 min away from rpoH, on the genetic map. In contrast to the rpoH mutants, the suhA revertants that contained both rpoH (nonsense) and suhA mutations were fully or partially proficient in the induction of heat shock proteins upon exposure to a high temperature. Under these conditions, transcription from two heat shock promoters as determined by operon fusion was transiently activated. In one of the rpoH(Am) suhA revertants studied in detail, an increase in temperature caused the synthesis of significant amounts of sigma 32, accompanied by increased stability and accumulation of rpoH mRNAs. On the other hand, the same mutation (suhA6) only weakly suppressed the rpoH deletion mutant; however, two of the major heat shock genes, dnaK and groE, were apparently induced in the absence of sigma 32. Thus, suhA6 seems to bring about the induction of heat shock genes by at least two mechanisms, one increasing the level of sigma 32 synthesis, and the other activating some transcription factor other than sigma 32.

Detection of low-level gene induction using in vitro transcription heat shock genes

We have recently described a technique of in vitro transcription (or nuclear run-off transcription) that is highly efficient for production of RNA polymerase II transcripts (Love, J., and Minton, K., Anal. Biochem., in press). In the current work we show that this technique may be applied to the evaluation of small transcriptional alterations in genes that are expressed at low levels; such alterations would be missed using traditional in vitro transcription techniques because the levels of transcription would be below limits of detection. We demonstrate this technique by evaluating in vitro transcription of the seven major heat shock genes by Drosophila melanogaster Schneider line 2 tissue culture cells after a variety of cellular stresses. We detected small transcriptional inductions of several of the heat shock genes following exposure of cells to extracellular hydrogen peroxide or γ irradiation. This latter stress has not been appreciated previously to affect heat shock gene transcription because it modulates their activity very weakly. A number of rigorous controls, required to avoid false positive and negative results, are described.

Expression of heat shock genes in fetal and maternal rabbit brain

Cloned fragments of members of the Drosophila and mouse major heat shock (hsp70) gene family were used to demonstrate that homologous sequences are present in the rabbit genome. After a physiologically relevant increase in body temperature of 3 degrees C, transcription of inducible hsp70 genes is detected in both the fetal and maternal brain and kidney. The induced hsp70 gene transcripts decay rapidly after whole body hyperthermia subsides. Transcripts of constitutively expressed member(s) of the hsp70 gene family, the heat shock cognate genes (hsc70), are detected in unstressed fetal and maternal rabbit tissues.

Nonexpression of a major heat shock gene in mouse plasmacytoma MPC-11.

The major heat shock gene coding for a 68,000-dalton protein was found to be silent in mouse plasmacytoma MPC-11 in both control and stressed cells. The block appears to be at the level of transcription, although RNA processing or instability has not been ruled out. It is not caused by a major deletion or rearrangement of the gene.

Nonexpression of a major heat shock gene in mouse plasmacytoma MPC-11.

The major heat shock gene coding for a 68,000-dalton protein was found to be silent in mouse plasmacytoma MPC-11 in both control and stressed cells. The block appears to be at the level of transcription, although RNA processing or instability has not been ruled out. It is not caused by a major deletion or rearrangement of the gene.

Differential regulation of the 70K heat shock gene and related genes in Saccharomyces cerevisiae.

Saccharomyces cerevisiae contains a family of genes related to Hsp70, the major heat shock gene of Drosophila melanogaster. The transcription of three of these genes, which show no conservation of sequences 5' to the protein-coding region, was analyzed. The 5' flanking regions from the three genes were fused to the Escherichia coli beta-galactosidase structural gene and introduced into yeasts on multicopy plasmids, putting the beta-galactosidase production under yeast promoter control. Analysis of beta-galactosidase mRNA and protein production in these transformed strains revealed that transcription from the three promoters is differentially regulated. The number of transcripts from one promoter is vastly increased for a brief period after heat shock, whereas mRNA from another declines. Transcripts from a third gene are slightly enhanced upon heat shock; however, multiple 5' ends of the mRNA are found, and a minor species increases in amount after heat shock. Transcription of these promoters in their native state on the chromosome appears to be modulated in the same manner.

Differential regulation of the 70K heat shock gene and related genes in Saccharomyces cerevisiae.

Saccharomyces cerevisiae contains a family of genes related to Hsp70, the major heat shock gene of Drosophila melanogaster. The transcription of three of these genes, which show no conservation of sequences 5' to the protein-coding region, was analyzed. The 5' flanking regions from the three genes were fused to the Escherichia coli beta-galactosidase structural gene and introduced into yeasts on multicopy plasmids, putting the beta-galactosidase production under yeast promoter control. Analysis of beta-galactosidase mRNA and protein production in these transformed strains revealed that transcription from the three promoters is differentially regulated. The number of transcripts from one promoter is vastly increased for a brief period after heat shock, whereas mRNA from another declines. Transcripts from a third gene are slightly enhanced upon heat shock; however, multiple 5' ends of the mRNA are found, and a minor species increases in amount after heat shock. Transcription of these promoters in their native state on the chromosome appears to be modulated in the same manner.

Protein component from drosophila larval nuclei showing sequence specificity for a short region near a major heat-shock protein gene

Using a nitrocellulose filter binding assay we have detected a protein component in extracts prepared from the nuclei of D. melanogaster third instar larvae which shows sequence-specific binding to a short region on a cloned D. melanogaster DNA segment containing a copy of the major heat-shock protein gene. The specificity of binding was confirmed by direct visualization of the protein DNA complexes in the electron microscope. Protein blotting experiments demonstrated that three polypeptides in the extract were capable of such sequence-specific binding. The protein binding site detected in this cloned sequence lies 800–1000 bp upstream from the presumptive transcription start.

Sequence of three copies of the gene for the major Drosophila heat shock induced protein and their flanking regions

The primary sequence of the major heat shock gene of D. melanogaster, that for the 70,000 protein, has been determined. One of the reading frames is devoid of stop codons for over 2000 bp. The region between the first ATG and the first stop codon encodes a protein of molecular weight 70,270. The 5′ end of the messenger RNA was localized in the DNA sequence by two independent methods. The 5′ flanking sequences of three distinct 70K genes were also determined. Extensive homology in the primary sequences extends about 500 bp upsteam from the ATG, which is the presumptive initiation of protein synthesis. Each 70K gene has the putative promoter sequence TATAAATA about 325 bp upstream from this ATG. A heptanucleotide sequence identified as the capping site for other messengers is found 24–30 bp downstream from the ends of the A-T-rich sequence. A 12 bp sequence with dyad symmetry begins 23 bp upstream from the beginning of the above A-T-rich sequence.