Sequence of a sea urchin hsp70 gene and its 5' flanking region

We report the nucleotide sequence of a 4470-bp fragment derived from a sea urchin genomic clone containing part of a heat-shock protein 70 (Hsp70)-encoding gene. This fragment, named hsp70 gene II, contains 1271 bp of the flanking region and 3299 bp of structural gene sequence interrupted by five introns and encoding the N-terminal 371 amino acids (aa) of the protein. The 5' flanking region contains a putative TATA element, two CCAAT boxes, four heat-shock consensus sequence elements (hse) and one consensus sequence for binding of Sp1. Remarkable homologies were observed for deduced aa sequence and intron-exon organization between hsp70 gene II and rat hsc73 gene.     

Cloning and characterization of the Bacillus subtilis prkA gene encoding a novel serine protein kinase

We have cloned and sequenced a 3574-bp Bacillus subtilis (Bs) DNA fragment located between the nrdA and citB genes at about 169° on the chromosome. An Escherichia coli strain, LBG1605, carrying a mutated ptsH gene (encoding HPr (His-containing protein) of the bacterial phosphotransferase system (PTS)) and complemented for PTS activity with the ptsH of Staphylococcus carnosus, exhibited reduced mannitol fermentation activity when transformed with a plasmid bearing this 3574-bp Bs fragment. This fragment contained an incomplete and two complete open reading frames (ORFs). The product of the first complete ORF, a protein composed of 235 amino acids (aa) (25 038 Da), was found to be responsible for the observed reduced mannitol fermentation. The 3′ part of this 705-bp second ORF and the 428-bp incomplete first ORF encode aa sequences exhibiting almost 40% sequence identity. However, the function of these two proteins remains unknown. The third ORF, the 1893-bp prkA gene, encodes a protein (PrkA) of 72 889 Da. PrkA possesses the A-motif of nucleotide-binding proteins and exhibits distant homology to eukaryotic protein kinases. Several of the essential aa in the loops known to form the active site of cyclic adenosine 3′,5′-monophosphate (cAMP)-dependent protein kinase appeared to be conserved in PrkA. After expression of prkA and purification of PrkA, we could demonstrate that PrkA can indeed phosphorylate a Bs 60-kDa protein at a Ser residue.     

Cloning and nucleotide sequence of the murine hsp84 cDNA and chromosome assignment of related sequences

The nucleotide (nt) sequence of mouse 84-kDa heat shock protein (Hsp) cDNA has been determined using a combination of molecular cloning and oligodeoxynucleotide priming on poly(A) + RNA. The cDNA was 2.5 kb long, not including the poly(A) tail. It contained a 5' leader of about 94 nt that was G + C-rich, and a 243-nt 3'-untranslated region that was A + T-rich in the vicinity of the polyadenylation signal. Gene hsp84 codes for an acidic polypeptide of 724 amino acid (aa) residues. Mouse Hsp84 had 81% and 63% aa homology to Drosophila melanogaster Hsp82 and yeast Hsp90, respectively. The nucleotide sequence had 74% and 59% homology to Drosophila and yeast hsp sequences, respectively, in the coding regions of these genes. This homology did not extend to the 5' - and 3'-untranslated regions. Chromosomal analysis indicated that hsp84-related sequences are on at least three different chromosomes.     

Hsp 70 expression and function during gametogenesis

The dramatic transformations in nuclear content and cellular organization that occur during gametogenesis require unique regulation and execution of the mitotic and meiotic cell cycle, apoptotic cell death, DNA recombination and repair, and cellular differentiation. These processes are accompained by the constitutive and developmentally regulated expression of a number of hsp70 genes encoding 70 kDa heat shock proteins (Hsp70), including several hsp70s whose expression is unique to male germ cells. Examining the expression and function of Hsp70s in germ cells has provided significant insights into mechanisms of hsp70 gene regulation and Hsp70 protein function, as well as the developmental processes of gametogenesis.     

Isolation and sequence analysis of Clpgl,a gene coding for an endopolygalacturonase of the phytopathogenic fungus Colletotrichum lindemuthianum

Oligodeoxyribonucleotide primers designed from the N-terminal amino acid (aa) sequence of the endopolygalacturonase (EndoPG) of Colletotrichum lindemuthianum (Cl) race β and from an internal sequence conserved among different fungal EndoPG were used in a polymerase chain reaction (PCR) to amplify genomic related sequences of the fungus. A 542-bp fragment, designated pgA, was obtained and used as a probe to screen a partial genomic library of Cl. Among the positive clones, one was further analyzed. Nucleotide sequencing of this clone revealed an ORF encoding a 363-amino-acid (aa) polypeptide begining with a signal peptide of 26 aa interrupted by an intron of 70 bp, and showing a high degree of homology to ten fungal EndoPG sequences. Consensus sequences were identified in the 5′ non-coding region. This genomic clone was thereafter designated Clpgl. Southern analysis, performed with a Clpgl-specific probe, showed that this gene is present as a single copy in the Cl genome     

A Drosophila hsp70 gene contains long,antiparallel, coupled open reading frames (LAC ORFs) conserved in homologous loci

A clone isolated from a Drosophila auraria heat-shock cDNA library presents two long, antiparallel, coupled (LAC) open reading frames (ORFs). One strand ORF is 1,929 nucleotides long and exhibits great identity (87.5% at the nucleotide level and 94% at the amino acid level) with the hsp70 gene copies of D. melanogaster, while the second strand ORF, in antiparallel in-frame register arrangement, is 1,839 nucleotides long and exhibits 32% identity with a putative, recently identified, NAD⁺-dependent glutamate dehydrogenase (NAD⁺-GDH). The overlap of the two ORFs is 1,824 nucleotides long. Computational analysis shows that this LAC ORF arrangement is conserved in other hsp70 loci in a wide range of organisms, raising questions about possible evolutionary benefits of such a peculiar genomic organization.Correspondence to: Z.G. Scouras     

Hsp70 Induction and hsp70 Gene polymorphisms as Indicators of acclimatization under hyperthermic conditions

The possibility of using Hsp70 and hsp70 gene polymorphisms as markers of acclimatization was investigated. Volunteers (22) were subjected to an acclimatization regimen and blood analysed for Hsp70 (Hsp72) and hsp70 polymorphisms before and after a heat tolerance test. Physiological parameters denoting acclimatization, or not, were correlated to levels of Hsp70 and combination of hsp70 genes. Only individuals that acclimatized had decreased basal Hsp70 levels and increased ability to induce Hsp70 together with a specific hsp70 genotype combination. We propose that Hsp70 levels (basal vs. induced) with the genotype combination have the potential to be used as markers of acclimatization.     

Expression of the HSP24 gene from Trichoderma harzianum in Saccharomyces cerevisiae

Hsp24 is a small heat-shock protein (sHSP). Such proteins are important endogenous cytoprotection factors involved in defense. A 1116-bp full-length cDNA of the Hsp24, with a 645-bp open reading frame nucleotide encoding a 24-kDa polypeptide consisting of 214 amino acid residues, was isolated from Trichoderma harzianum. Sequence analysis revealed that Hsp24 gene has more than 42–58% amino acid sequence homology with those of other fungi. The Hsp24 gene was integrated into pYES2 by inserting into a single site for recombination, yielding the recombinant of pYES2/Hsp24. Hsp24 expressed by pYES2/Hsp24 was induced by galactose. We tested whether Hsp24 could confer abiotic stress resistance when it was introduced into yeast cells. A transgenic yeast harboring T. harzianum Hsp24 was generated under the control of a constitutively expressed GAL promoter. The results indicated that Hsp24 yeast transformants had significantly higher resistance to salt, drought and heat stresses.     

Comparison of regulatory and structural regions of the Xenopus laevis small heat-shock protein-encoding gene family.

We have isolated several unique Xenopus laevis hsp30 (encoding heat-shock protein 30) genomic clones, one of which contains two complete hsp30 genes (hsp30C and hsp30D), as well as the promoter and N-terminal coding region of a third gene (hsp30E). Nucleotide sequence and restriction enzyme analysis revealed that this gene cluster is different from a cluster isolated previously. The hsp30C and hsp30D genes encode proteins of approx. 24 kDa. In all, the hsp30 gene family contains a minimum of seven genes. The strand exchange and breakage of the duplication events which generated this gene family appear to have occurred within tracts of DNA which potentially can assume a Z-DNA conformation. Comparing the amino acid (aa) sequences of each known Hsp30 protein with bovine alpha-crystallin revealed a high degree of shared conservation of aa that constitute the major structural feature(s) of alpha-crystallin.     

Cloning and sequencing of a cDNA encoding bovine intercellular adhesion molecule 3 (ICAM-3)

A cDNA encoding a putative bovine intercellular adhesion molecule (ICAM)-3, a ligand of the leukocyte integrin LFA-1 (CD11a/CD18), was sequenced and compared with human ICAM sequences. The 1635-bp bovine sequence codes for a protein of 544 amino acids (aa). This putative bovine ICAM-3 has five immunoglobulin (Ig)-like domains similar to human ICAM-1 and ICAM-3, and belongs to the Ig gene superfamily. The overall identities of the deduced aa sequence with those of human ICAM-3 and ICAM-1 are 61% and 58%, respectively. The predicted number and positions of Cys residues are all conserved between the bovine and human ICAM 3 aa sequences.     

Induction of hsp70, alterations in oxidative stress markers and apoptosis against dichlorvos exposure in transgenic Drosophila melanogaster: Modulation by reactive oxygen species

We examined a hypothesis that reactive oxygen species (ROS) generated by organophosphate compound dichlorvos modulates Hsp70 expression and anti-oxidant defense enzymes and acts as a signaling molecule for apoptosis in the exposed organism. Dichlorvos (0.015–15.0 ppb) without or with inhibitors of Hsp70, superoxide dismutase (SOD) and catalase (CAT) were fed to the third instar larvae of Drosophila melanogaster transgenic for hsp70 (hsp70-lacZ) Bg⁹ to examine Hsp70 expression, oxidative stress and apoptotic markers. A concentration- and time-dependent significant increase in ROS generation accompanied by a significant upregulation of Hsp70 preceded changes in antioxidant defense enzyme activities and contents of glutathione, malondialdehyde and protein carbonyl in the treated organisms. An inhibitory effect on SOD and CAT activities significantly upregulated ROS generation and Hsp70 expression in the exposed organism while inhibition of Hsp70 significantly affected oxidative stress markers induced by the test chemical. A comparison made among ROS generation, Hsp70 expression and apoptotic markers showed that ROS generation is positively correlated with Hsp70 expression and apoptotic cell death end points indicating involvement of ROS in the overall adversity caused by the test chemical to the organism. The study suggests that (a) Hsp70 and anti-oxidant enzymes work together for cellular defense against xenobiotic hazard in D. melanogaster and (b) free radicals may modulate Hsp70 expression and apoptosis in the exposed organism.