Hsp65 Gene Sequencing Research Articles

Evolution of HSP70 gene and its implications regarding relationships between archaebacteria, eubacteria, and eukaryotes.

The 70-kDa heat-shock protein (HSP70) constitutes the most conserved protein present in all organisms that is known to date. Based on global alignment of HSP70 sequences from organisms representing all three domains, numerous sequence signatures that are specific for prokaryotic and eukaryotic homologs have been identified. HSP70s from the two archaebacterial species examined (viz., Halobacterium marismortui and Methanosarcina mazei) have been found to contain all eubacterial but no eukaryotic signature sequences. Based on several novel features of the HSP70 family of proteins (viz., presence of tandem repeats of a 9-amino-acid [a.a.] polypeptide sequence and structural similarity between the first and second quadrants of HSP70, homology of the N-terminal half of HSP70 to the bacterial MreB protein, presence of a conserved insert of 23-27 a.a. in all HSP70s except those from archaebacteria and gram-positive eubacteria) a model for the evolution of HSP70 gene from an early stage is proposed. The HSP70 homologs from archaebacteria and gram-positive bacteria lacking the insert in the N-terminal quadrants are indicated to be the ancestral form of the protein. Detailed phylogenetic analyses of HSP70 sequence data (viz., by bootstrap analyses, maximum parsimony, and maximum likelihood methods) provide evidence that archaebacteria are not monophyletic and show a close evolutionary linkage with the gram-positive eubacteria. These results do not support the traditional archaebacterial tree, where a close relationship between archaebacterial and eukaryotic homologs is observed. To explain the phylogenies based on HSP70 and other gene sequences, a model for the origin of eukaryotic cells involving fusion between archaebacteria and gram-negative eubacteria is proposed.

Sex-specific control of Drosophila melanogaster yolk protein 1 gene expression is limited to transcription.

The sex of Drosophila melanogaster is determined by a hierarchy of genes. The ultimate targets of this regulatory hierarchy are the genes encoding terminal differentiation products of one sex. For one of the best-characterized target genes, that encoding female-specific yolk protein 1 (YP1), sex-specific transcriptional controls have been clearly demonstrated. In addition, sex-specific posttranscriptional controls were suggested from experiments in which YP1 RNA was induced in males with hormones. To determine whether males can efficiently process and translate a transcript which is normally found only in females, we used a non-sex-specific promoter, the hsp70 gene promoter, to drive YP1 gene transcription in germ line transformed males. The efficiency of expression of the YP1 gene at levels of RNA splicing, translation, and protein secretion in these males was compared with that in wild-type females. These experiments show that there are no sex-specific posttranscriptional controls operating to limit the production of secreted YP1 in males. Promoters containing different numbers of heat shock elements were tested for their ability to drive YP1 gene transcription in males. These results show that incompatibility between the hsp70 gene heat shock elements and the YP1 gene promoter can be overcome by increasing the amount of hsp70 gene sequence up or downstream of the TATA box. In the course of this study, two vectors useful for placing genes under heat shock regulation were constructed. One of these vectors is designed so that the heat-induced transcript produced is the "authentic" primary transcript; it should be useful for studies of posttranscriptional regulation.

Sex-specific control of Drosophila melanogaster yolk protein 1 gene expression is limited to transcription.

The sex of Drosophila melanogaster is determined by a hierarchy of genes. The ultimate targets of this regulatory hierarchy are the genes encoding terminal differentiation products of one sex. For one of the best-characterized target genes, that encoding female-specific yolk protein 1 (YP1), sex-specific transcriptional controls have been clearly demonstrated. In addition, sex-specific posttranscriptional controls were suggested from experiments in which YP1 RNA was induced in males with hormones. To determine whether males can efficiently process and translate a transcript which is normally found only in females, we used a non-sex-specific promoter, the hsp70 gene promoter, to drive YP1 gene transcription in germ line transformed males. The efficiency of expression of the YP1 gene at levels of RNA splicing, translation, and protein secretion in these males was compared with that in wild-type females. These experiments show that there are no sex-specific posttranscriptional controls operating to limit the production of secreted YP1 in males. Promoters containing different numbers of heat shock elements were tested for their ability to drive YP1 gene transcription in males. These results show that incompatibility between the hsp70 gene heat shock elements and the YP1 gene promoter can be overcome by increasing the amount of hsp70 gene sequence up or downstream of the TATA box. In the course of this study, two vectors useful for placing genes under heat shock regulation were constructed. One of these vectors is designed so that the heat-induced transcript produced is the "authentic" primary transcript; it should be useful for studies of posttranscriptional regulation.

A hsp70-related gene is constitutively highly expressed in testis of rat and mouse

The expression pattern of major heat shock related genes (hsp70 gene family) in various organs of mouse and rat was investigated using Northern blot analysis. Heat shock gene related transcripts were detected in total RNA by hybridization with cloned mouse hsp70 gene sequences. Cells of various organs of intact mouse and rat constitutively synthesize a 2.2 kb and a 2.5 kb RNA. Exceptionally high levels of the 2.5 kb RNA, 50-250 fold higher than in other tissues are found in testis of both rodents. The 2.5 kb RNA hybridizes strongly to an extended region of mouse hsp70 gene; it also hybridizes poorly to the Drosophila hsp70 gene. The data suggest that the 2.5 kb RNA is transcribed from a hsp70-related gene in mouse and rat.

Organization, nucleotide sequence, and transcription of the chicken HSP70 gene.

We have isolated a gene encoding a 70,000-Da heat shock protein (HSP70) from a chicken genomic library. The recombinant clone C411 was isolated by hybridization to a radioactively labeled plasmid containing Drosophila HSP70 gene sequences. The identity was established by hybrid selected translation using a subcloned restriction fragment, pC1.8, and total cytoplasmic RNA from chicken cells. The selected mRNA translated in vitro a product which co-migrates with in vivo synthesized chicken HSP70 by SDS-polyacrylamide gel electrophoresis. By Northern blot analysis of poly(A)+ mRNA from heat-shocked cells, we detected a 2.6-kilobase pair mRNA homologous to pC1.8. The nucleotide sequences within pC1.8 and a flanking 2.0-kilobase pair HindIII fragment contain a single contiguous open reading frame of 1905 nucleotides that corresponds to a protein of predicted size 70,000 daltons. Upstream of the initiator methionine codon, ATG, lie sequences homologous to the canonical transcription elements TATA, CCAAT, and SP1 binding site, and two overlapping heat shock elements. The order and spacing of these sequences share many features in common with the promoter for the human HSP70 gene.

Structure and expression of the human gene encoding major heat shock protein HSP70.

We have cloned a human gene encoding the 70,000-dalton heat shock protein (HSP70) from a human genomic library, using the Drosophila HSP70 gene as a heterologous hybridization probe. The human recombinant clone hybridized to a 2.6-kilobase polyadenylated mRNA from HeLa cells exposed to 43 degrees C for 2 h. The 2.6-kilobase mRNA was shown to direct the translation in vitro of a 70,000-dalton protein similar in electrophoretic mobility to the HSP70 synthesized in vivo. From the analysis of S1 nuclease-resistant mRNA-DNA hybrids, the HSP70 gene appears to be transcribed as an uninterrupted mRNA of 2.3 kilobases. We show that the cloned HSP70 gene contains the sequences necessary for heat shock-induced expression by two criteria. First, hamster cells transfected with a subclone containing the HSP70 gene and flanking sequences synthesized a HSP70-like protein upon heat shock. Second, human cells transfected with a chimeric gene containing the 5' flanking sequences of the HSP70 gene and the coding sequences of the bacterial chloramphenicol acetyltransferase gene transcribed the chimeric gene upon heat shock. We show that the HSP70 mRNA transcribed in an adenovirus 5 transformed human cell line (293 cells) is identical to the HSP70 mRNA induced by heat shock.

Structure and expression of the human gene encoding major heat shock protein HSP70.

We have cloned a human gene encoding the 70,000-dalton heat shock protein (HSP70) from a human genomic library, using the Drosophila HSP70 gene as a heterologous hybridization probe. The human recombinant clone hybridized to a 2.6-kilobase polyadenylated mRNA from HeLa cells exposed to 43 degrees C for 2 h. The 2.6-kilobase mRNA was shown to direct the translation in vitro of a 70,000-dalton protein similar in electrophoretic mobility to the HSP70 synthesized in vivo. From the analysis of S1 nuclease-resistant mRNA-DNA hybrids, the HSP70 gene appears to be transcribed as an uninterrupted mRNA of 2.3 kilobases. We show that the cloned HSP70 gene contains the sequences necessary for heat shock-induced expression by two criteria. First, hamster cells transfected with a subclone containing the HSP70 gene and flanking sequences synthesized a HSP70-like protein upon heat shock. Second, human cells transfected with a chimeric gene containing the 5' flanking sequences of the HSP70 gene and the coding sequences of the bacterial chloramphenicol acetyltransferase gene transcribed the chimeric gene upon heat shock. We show that the HSP70 mRNA transcribed in an adenovirus 5 transformed human cell line (293 cells) is identical to the HSP70 mRNA induced by heat shock.