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E A Craig

Publications and source records attributed to E A Craig.

At least 109 records · Page 6Linked to original sources
PubMed1988

Molecular and genetic characterization of the Drosophila melanogaster 87E actin gene region.

A combined molecular and genetic analysis of the 87E actin gene (Act87E) in Drosophila melanogaster was undertaken. A clone of Act87E was isolated and characterized. The Act87E transcription unit is 1.57 kb and includes a 556-base intervening sequence in the 5' leader of the gene. The protein-coding region is contiguous and encodes a protein that is greater than 93% identical to the other Drosophila actins. By in situ hybridization with a series of deficiencies that break in 87E, Act87E was localized to a region encompassing one to three faint, polytene chromosome bands. The region between the deficiency endpoints that flank the actin gene was isolated and measures approximately 24-30 kb. The closest proximal deficiency endpoint lies 8-10 kb 5' to the actin gene; the closest distal deficiency endpoint lies 16-20 kb 3' to the actin gene. A single, recessive lethal complementation group lies between the deficiency endpoints that flank the actin gene. An EMS mutagenesis screen produced four additional members of this recessive lethal complementation group. Molecular analysis of the members of this complementation group indicated that two of the newly induced mutations have deletions of approximately 1 kb in a transcribed region 4-5 kb 3' (distal) to the actin gene. This result suggests that the recessive lethal complementation group represents a gene separate from and distal to the actin gene. The mutagenesis screen failed to identify additional recessive lethal complementation groups in the actin gene-containing region. The implications of the failure to identify recessive lethal mutations in the actin gene are discussed in reference to studies of other conserved multigene families and other muscle protein mutations.

Actins↗
PubMed1988

Ancient heat shock gene is dispensable.

Hsp83 is a major eucaryotic heat shock protein and one of the most conserved proteins known. We have isolated an Escherichia coli gene homologous to eucaryotic Hsp83 and used it to construct a deletion mutation. The E. coli mutant was viable but had a slight growth disadvantage that increased with temperature.

Chromosome Deletion↗
PubMed1987

SSC1, a member of the 70-kDa heat shock protein multigene family of Saccharomyces cerevisiae, is essential for growth.

The genome of the yeast Saccharomyces cerevisiae contains a family of genes related to the HSP70 genes (encoding the 70-kDa heat shock protein) of other eukaryotes. Mutations in two of these yeast genes (SSC1 and SSD1), whose expression is increased a few fold after temperature upshift, were constructed in vitro and substituted into the yeast genome in place of the wild-type alleles. No phenotypic effects of the mutation in SSD1 were detected. However, a functional SSC1 gene is essential for vegetative growth. This result, in conjunction with experiments involving mutations in other members of this multigene family, indicates that at least three distinct functions are carried out by genes of the HSP70 family.

Chromosome Mapping↗
PubMed1987

Eukaryotic Mr 83,000 heat shock protein has a homologue in Escherichia coli.

We have isolated a gene from Escherichia coli homologous to the gene encoding the Mr 83,000 Drosophila heat shock protein (hsp83). In E. coli the protein homologous to hsp83 is a heat shock protein called C62.5. The predicted amino acid sequence of C62.5 is 41% and 42% identical to the Drosophila and human hsp83 proteins, respectively. Selected regions of the protein have conservation as high as 90%. The gene encoding C62.5 (named htpG) is located between the dnaZ and adk genes at 11.1 minutes on the E. coli chromosome. The htpG gene appears to be a newly identified locus. The isolation of an E. coli homologue of hsp83 illustrates the remarkable conservation of heat shock proteins in evolution and will facilitate genetic and biochemical experiments aimed at determining the function of hsp83.

Amino Acid Sequence↗
PubMed1987

Transcriptional regulation of an hsp70 heat shock gene in the yeast Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae contains three heat-inducible hsp70 genes. We have characterized the promoter region of the hsp70 heat shock gene YG100, that also displays a basal level of expression. Deletion of the distal region of the promoter resulted in an 80% drop in the basal level of expression without affecting expression after heat shock. Progressive-deletion analysis suggested that sequences necessary for heat-inducible expression are more proximal, within 233 base pairs of the initiation region. The promoter region of YG100 contains multiple elements related to the Drosophila melanogaster heat shock element (HSE; CnnGAAnnT TCnnG). Deletion of a proximal promoter region containing one element, HSE2, eliminated most of the heat-inducible expression of YG100. The upstream activation site (UAS) of the yeast cytochrome c gene (CYC1) can be substituted by a single copy of HSE2 plus its adjoining nucleotides (UASHS). This hybrid promoter displayed a substantial level of expression before heat shock, and the level of expression was elevated eightfold by heat shock. YG100 sequences that flank UASHS inhibited basal expression of UASHS in the hybrid promoter but not its heat-inducible expression. This inhibition of basal UASHS activity suggests that negative regulation is involved in modulating expression of this yeast heat shock gene.

Base Sequence↗
PubMed1987

Complex interactions among members of an essential subfamily of hsp70 genes in Saccharomyces cerevisiae.

Saccharomyces cerevisiae contains a large family of genes related to hsp70, the major heat shock-inducible gene of Drosophila melanogaster. One subfamily, identified by sequence homology, contains four genes, SSA1, SSA2, SSA3, and SSA4 (formerly YG100, YG102, YG106, and YG107, respectively). Previous studies showed that strains containing mutations in SSA1 and SSA2 are temperature sensitive for growth. SSA4, which is normally heat inducible and not expressed during vegetative growth, is expressed at high levels in ssa1 ssa2 strains at 23 degrees C. We constructed mutations in SSA3 and SSA4 and analyzed strains carrying mutations in the four genes. Strains carrying mutations in SSA3 SSA4 or SSA3 and SSA4 were indistinguishable from the wild type. However, ssa1 ssa2 ssa4 strains were inviable. SSA3, like SSA4, is a heat-inducible gene that is not normally expressed at 23 degrees C. Nevertheless, an intact copy of SSA3 regulated by the constitutive SSA2 promoter was capable of rescuing a ssa1 ssa2 ssa4 strain. This indicates that SSA3 encodes a functional protein and that the SSA1, SSA2, SSA3, and SSA4 gene products are functionally similar.

Genes, Fungal↗
PubMed1986

Expression and localization of Drosophila melanogaster hsp70 cognate proteins.

Monoclonal antibodies have been used to identify three proteins in Drosophila melanogaster that share antigenic determinants with the major heat shock proteins hsp70 and hsp68. While two of the proteins are major proteins at all developmental stages, one heat shock cognate protein, hsc70, is especially enriched in embryos. hsc70 is shown to be the product of a previously identified gene, Hsc4. We have examined the levels of hsp70-related proteins in adult flies and larvae during heat shock and recovery. At maximal induction in vivo, hsp70 and hsp68 never reach the basal levels of the major heat shock cognate proteins. Monoclonal antibodies to hsc70 have been used to localize it to a meshwork of cytoplasmic fibers that are heavily concentrated around the nucleus.

Animals↗
PubMed1985

Consensus sequence for Escherichia coli heat shock gene promoters.

We have identified promoters for the Escherichia coli heat shock operons dnaK and groE and the gene encoding heat shock protein C62.5. Transcription from each promoter is heat-inducible in vivo, and each is recognized in vitro by RNA polymerase containing sigma 32, the sigma factor encoded by rpoH (htpR) but not by RNA polymerase containing sigma 70. We compared the sequences of the heat shock promoters and propose a consensus promoter sequence, having T-N-t-C-N-C-c-C-T-T-G-A-A in the -35 region and C-C-C-C-A-T-t-T-a in the -10 region. These sequences differ from the consensus sequence recognized by holoenzyme containing sigma 70, the major sigma in E. coli. We suggest that the accumulated consensus sequences of promoters recognized by alternate forms of holoenzyme are compatible with a model in which sigma recognizes only the -10 region of the promoter.

Base Sequence↗
PubMed1985

Mutations in cognate genes of Saccharomyces cerevisiae hsp70 result in reduced growth rates at low temperatures.

Expression of two Saccharomyces cerevisiae genes (YG101 and YG103) that are related to the gene encoding inducible 70K protein (hsp70) is repressed upon heat shock. Mutations of the two genes were constructed in vitro and substituted into the yeast genome in place of the wild-type alleles. No phenotypic effect of single mutations of either gene was detected. However, cells containing both YG101 and YG103 mutations showed altered growth properties; double-mutation cells possess an optimal growth temperature of 37 degrees C rather than 30 degrees C and grow increasingly poorly as the temperature is lowered. Mutations of two other members of this hsp70-related multigene family, YG100 and YG102, have been analyzed (E. A. Craig and K. Jacobsen, Cell 38:841-849, 1984). Cells containing both YG100 and YG102 mutations cannot form colonies at 37 degrees C. Fusions between the YG101 and YG102 promoter regions and the YG100 and YG101 structural genes, respectively, were constructed. The YG101 promoter-YG100 structural gene fusion was not able to restore normal growth properties to the yg101- yg103- mutant. Also, yg100- yg102- cells containing the YG102 promoter-YG101 structural gene fusion were unable to grow at 37 degrees C. Failure of the protein products of related genes to rescue the relative cold sensitivity of growth suggests that members of the hsp70 multigene family are functionally distinct.

Cell Division↗
PubMed1985

The heat shock response.

The response of cells to a heat shock or other stresses is the activation of a small number of genes which were previously inactive or transcribed at low levels. This response has been observed in a wide variety of bacterial, plant, and animal species. Evidence is accumulating that at least some of the proteins found in diverse species are similar, indicating a conservation of the response and the proteins in evolution. In a number of organisms a strong positive correlation has been found between the presence of heat shock proteins and ability of the organism to withstand thermal stress. This review attempts to assess the available data concerning the homology of proteins in different species, the localization of the proteins in cells, and the relationship between heat shock proteins and thermoresistance.

Animals↗
PubMed1984

Mutations of the heat inducible 70 kilodalton genes of yeast confer temperature sensitive growth.

S. cerevisiae contains a family of genes related to the major heat shock induced gene of Drosophila (Hsp70). Two members of this family, YG100 and YG102, are 97% identical to each other in their protein coding regions. RNA transcribed from YG100 increases markedly after a heat shock, while transcripts of YG102 increase minimally. Mutants of the two genes were constructed in vitro and substituted in the yeast genome in place of the wild-type alleles. No phenotypic effect of single mutations of either gene was detected. However, cells containing both the YG100 and YG102 mutations grew slowly at 30 degrees C and could not form colonies at 37 degrees C. The temperature sensitive phenotype can be overcome by inserting either an intact YG100 or YG102 gene into the genome. Pretreatment at 37 degrees C before shift to a normally lethal temperature of 51 degrees C protected the double mutant as well as the wild type, indicating that YG100 and YG102 gene products are not needed for resistance to high temperatures for short periods.

Genes↗
PubMed1984

Major heat shock gene of Drosophila and the Escherichia coli heat-inducible dnaK gene are homologous.

The Escherichia coli dnaK gene is homologous to the major heat shock-induced gene in Drosophila (Hsp70). The primary DNA sequence of the entire protein-coding region of the dnaK gene was determined and compared with that of the Hsp70 gene of Drosophila. The two sequences are homologous; the dnaK gene could encode a 69,121-Da polypeptide, 48% identical to the hsp70 protein of Drosophila. The homology between the Hsp70 gene of Drosophila and the E. coli dnaK gene illustrates the remarkable conservation of the heat shock genes in evolution. In contrast to Drosophila and Saccharomyces cerevisiae, both of which contain multigene families related to the Hsp70 gene, hybridization analyses indicate that E. coli contains only a single Hsp70-related gene, dnaK. Hybridization between the DNA of an archaebacterium Methanosarcina barkeri and the Hsp70 genes of Drosophila, Saccharomyces, and E. coli has been detected, suggesting the existence of Hsp70-related genes in the three "primary kingdoms": eukaryotes, eubacteria, and archaebacteria.

Amino Acid Sequence↗
PubMed1984

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.

Base Sequence↗
PubMed1983

Expression of Drosophila heat-shock cognate genes during heat shock and development.

The Drosophila genome contains a number of genes related to the major heat-shock-inducible (Hsp70) genes of Drosophila. The structure and expression of three of these heat-shock cognate (Hsc) genes have been studied; Hsc1 (previously described), Hsc2, and Hsc4 are dispersed on chromosome 3 at cytological loci 70C, 87D, and 88E, respectively. RNA homologous to Hsc4 was shown by hybridization selection translation experiments to encode a protein of approximately 70kd, with similar, but distinguishable, mobility to that of the heat-shock-inducible 70K protein. Because of variance in size of the 5' nonprotein coding region, it was possible to identify in adult flies distinct transcripts from each gene. Unlike transcripts from Hsp genes, the abundance of the Hsc transcripts was not increased after heat shock. Transcription of these Hsc genes is regulated during development. While transcripts from Hsc4 were equally abundant in RNA isolated from embryos, larvae and adults, Hsc1 and Hsc2 transcripts were not detected in embryo and larval RNA, and are therefore at least 20 times less abundant in these stages than in adults. The primary DNA sequences of the regions homologous to that encoding the amino terminus of hsp70 were compared. Approximately 76% homology of the base sequence and 78% homology of the deduced amino acid sequence were found among the four genes. The Hsp70 genes contain no intervening sequences; Hsc4 contains no insertions relative to Hsp70 in the region encoding the first 101 aa. However, in the codon specifying aa66 Hsc1 is interrupted by 1.7 kb of DNA; in the codon for aa55 Hsc2 contains an insertion of about 650 b. The sequences at the 5' and 3' junctions of the insertions in both Hsc1 and Hsc2 are similar to those found in intervening sequences of Drosophila and of many other organisms.

Amino Acid Sequence↗
PubMed1982

Drosophila gene related to the major heat shock-induced gene is transcribed at normal temperatures and not induced by heat shock.

A gene related to the major heat shock-induced (hsp70) gene of Drosophila has been isolated from the sibling species D. melanogaster and D. simulans. This heat shock-cognate (hsc70) gene is present at cytological locus 70C. The primary sequence of approximately one-third of the protein-coding region has been compared with that of the hsp70 gene; 72% homology of the base sequence and 74% homology of the deduced amino acid sequence was found. In the codon specifying amino acid 66, the hsc70 gene contains an insertion of 1.7 kilobases; the hsp70 genes contain no intervening sequences. The sequence at the 5' and 3' junctions of the insertion is similar to that found in many intervening sequences. cDNA extension experiments indicate that the hsc70 gene is transcribed at normal temperatures in adult flies and that transcription is not enhanced by heat treatment.

Amino Acid Sequence↗
PubMed1982

Four small Drosophila heat shock proteins are related to each other and to mammalian alpha-crystallin.

The primary base sequence of the protein coding regions of the four small heat shock genes of Drosophila melanogaster present at cytological locus 67B has been determined. A single open reading frame large enough to encode a small heat shock protein is found for each gene. The molecular weights of the predicted proteins are in good agreement with experimentally determined values obtained from gel electrophoresis. The predicted amino acid sequences of the four small heat shock genes show striking homologies over approximately 50% of their lengths. This region of extensive homology extends from about amino acid 85 to amino acid 195 out of a total of approximately 200 amino acids. Comparison of the predicted sequence with the known sequences of other proteins revealed a remarkable similarity between this region of homology and the corresponding region of mammalian alpha-crystallin. The possible functional significance of this structural similarity is discussed.

Amino Acid Sequence↗
PubMed1982

Saccharomyces cerevisiae contains a complex multigene family related to the major heat shock-inducible gene of Drosophila.

Saccharomyces cerevisiae contains a family of genes related to the major heat shock-induced gene of Drosophila (hsp 70). Two members of the multigene family (YG100 and YG101) were isolated. The primary DNA sequences of more than one-half of the protein-encoding regions of YG100 and YG101 were determined and compared with the Drosophila hsp 70 gene sequence; the predicted amino acid sequences were 72 and 64% homologous to the sequence of the Drosophila hsp 70 protein, respectively. The predicted amino acid sequences of the yeast genes were 65% homologous. Our results demonstrate a striking sequence conservation of hsp 70-related sequences in evolution. Hybridization of the S. cerevisiae genes to total S. cerevisiae DNA indicated that the multigene family consists of approximately 10 members. Hybridization of labeled RNAs from heat-shocked and control cells suggested that, like transcription of the Drosophila hsp 70 gene, transcription of YG100 or a closely related gene is enhanced after heat shock. However, the amount of RNA sequences homologous to YG101 was reduced after heat shock. A multigene family related to the hsp 70 gene exists in Drosophila; transcription of some members is induced by heat shock, whereas transcription of others is not. Our results suggest that S. cerevisiae, like Drosophila, contains a multigene family of hsp 70-related sequences under complex transcriptional regulation and that the differential control, as well as the nucleotide sequence, has been highly conserved in evolution.

Amino Acid Sequence↗