Heat-shock proteins, Hsp84 and Hsp86, of mice and men: two related genes encode formerly identified tumour-specific transplantation antigens

Mouse cDNA clones have been isolated with the help of Drosophila melanogaster 82-kDa heat-shock protein (Hsp82)-coding sequences as hybridization probe. Sequencing of the overlapping mouse clones reveals a long open reading frame (ORF) that encodes a polypeptide of 83.3 kDa which shows about 80% similarity to the respective Drosophila Hsp82 amino acid sequence. The N-terminal half of this cDNA cross-hybridizes to a different class of mouse cDNA clones indicating a related gene. Northern blot hybridization experiments reveal a 2.6-kb poly(A)+RNA when probed with the hsp84 clone and a 2.85-kb signal with the hsp84-related cDNA. The amino acid sequences deduced from the contiguous ORF of the hsp84 and the hsp84-related cDNA coincide with the N-terminal sequence of formerly identified 84-kDa and 86-kDa tumour-specific transplantation antigens (Ullrich et al., 1986). In addition, the amino acid composition of the putative 84-kDa mouse Hsp described here is very similar to that of the 84-kDa tumour antigen described by Ullrich et al. (1986). Both observations corroborate the assumption that these Hsps are identical to the described 84-kDa and 86-kDa tumour-specific transplantation antigens. Using these mouse hsp gene clones as hybridization probes we also isolated the corresponding pair of human cDNA clones. Comparison of the respective sequences reveals a strong evolutionary constraint on these two genes in mouse and man.     

The genome of Trypanosoma cruzi contains a constitutively expressed, tandemly arranged multicopy gene homologous to a major heat shock protein.

cDNA libraries have been constructed in the plasmid vector pUC18 with mRNA isolated from both epimastigotes and trypomastigotes of the Peru strain of Trypanosoma cruzi. Pools of randomly selected clones were analyzed by hybridization-selection-translation. Translation products were immunoprecipitated either with normal human sera or with sera from patients with Chagas' disease (chagasic sera), and the immunoprecipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With this approach, a cDNA clone (pEC5) was identified which encodes a portion of an 85,000-Mr polypeptide. A genomic clone was subsequently isolated (FG1) by using oligonucleotide probes derived from the DNA sequence of this cDNA clone. A portion of this clone was isolated and sequenced, and the coding region for the protein was identified. Computer analysis of the predicted protein sequence indicates that this protein is closely related to the 83,000-Mr heat shock protein (hsp83) of Drosophila melanogaster, the hsp90 of Saccharomyces cerevisiae, and the hsp90 of chicken. This gene is tandemly organized in the T. cruzi genome as a cluster of 6 to 10 copies.     

Characterization of a novel 23-kilodalton protein of unactive progesterone receptor complexes.

Immunoprecipitation of unactivated avian progesterone receptor results in the copurification of hsp90, hsp70, and three additional proteins, p54, p50, and p23. p23 is also present in immunoaffinity-purified hsp90 complexes along with hsp70 and another protein, p60. Antibody and cDNA probes for p23 were prepared in an effort to elucidate the significance and function of this protein. Antibodies to p23 detect similar levels of p23 in all tissues tested and cross-react with a protein of the same size in mice, rabbits, guinea pigs, humans, and Saccharomyces cerevisiae, indicating that p23 is a conserved protein of broad tissue distribution. These antibodies were used to screen a chicken brain cDNA library, resulting in the isolation of a 468-bp partial cDNA clone encoding a sequence containing four sequences corresponding to peptide fragments isolated from chicken p23. This partial clone was subsequently used to isolate a full-length human cDNA clone. The human cDNA encodes a protein of 160 amino acids that does not show homology to previously identified proteins. The chicken and human cDNAs are 88% identical at the DNA level and 96.3% identical at the protein level. p23 is a highly acidic phosphoprotein with an aspartic acid-rich carboxy-terminal domain. Bacterially overexpressed human p23 was used to raise several monoclonal antibodies to p23. These antibodies specifically immunoprecipitate p23 in complex with hsp90 in all tissues tested and can be used to immunoaffinity isolate progesterone receptor complexes from chicken oviduct cytosol.     

Structure and expression of a human gene coding for a 71 kd heat shock ''cognate'' protein.

In all eukaryotes examined so far, hsp70 gene families include cognate genes (hsc70) encoding proteins of about 70 Kd which are expressed constitutively during normal growth and development. We have investigated the structural relationship of heat-inducible and cognate members of the human hsp70 gene family. Among several human genomic clones isolated using Drosophila hsp/hsc70 probes, one contained an hsc70 gene. Its complete sequence is reported here. It is split by eight introns and encodes a predicted protein of 70899 d that would be 81% homologous to hsp70. Structural comparisons with corresponding genes from other species provide one of the most striking examples of gene conservation. Isolation of a corresponding cDNA clone, RNA-mapping and in vitro translation data demonstrate that the gene is expressed constitutively and directs the synthesis of a 71 kd protein. The latter is very likely to be identical to a clathrin uncoating ATPase recently identified as a member of the hsp70-like protein family.     

Structure and Expression of a Heat-Shock Protein 83 Gene of Pharbitis nil

Four cDNA clones representing mRNAs whose levels were affected by a photoperiod that induces flowering in Pharbitis nil were isolated by a differential hybridization screening procedure. The level of mRNAs represented by three clones (12L, 15L, and 17L) increased following a photoperiod that induces flowering and that represented by the fourth clone (clone 27) increased under conditions in which flowering was inhibited. DNA sequence analysis showed that one cDNA, clone 17L, is homologous to members of the 83- to 90-kD heat-shock protein (hsp) gene family. The corresponding gene, hsp83A, was isolated and its DNA sequence was determined. hsp83A encodes a protein that exhibits 70% amino acid identity with Drosophila melanogaster HSP83. The P. nil hsp83A gene contains two introns within the coding region. hsp83A mRNA was not detectable in cotyledons of plants grown in continuous light, but its level increased transiently following a 14-h dark period and reached a maximum 2 h after the lights were turned on. A dramatic increase in the level of hsp83A mRNA was also found 2 h after an end-of-day dark treatment. Genomic Southern blot analysis demonstrated that the P. nil hsp83-90 gene family consists of at least six members, one of which appears to be constitutively expressed in the light.     

Beta-internexin is a microtubule-associated protein identical to the 70-kDa heat-shock cognate protein and the clathrin uncoating ATPase

We have examined the relationship of the ubiquitous 68-70-kDa cytoskeletal-associated protein beta-internexin (Napolitano, E. W., Pachter, J. S., Chin, S. S. M., and Liem, R. K. H. (1985) J. Cell Biol. 101, 1323-1331) to heat-shock cognate 70 (hsc70), the major constitutive member of the mammalian heat-shock protein 70 (hsp70) family of stress proteins. We purify beta-internexin from rat brain microtubules and confirm its identity with hsc70 and the clathrin-uncoating ATPase by the following criteria: 1) The partial sequence of a cyanogen bromide-derived peptide from beta-internexin matches the inferred amino acid sequence of the cDNA clone pRC62 encoding hsc70 from rat brain (O'Malley, K., Mauron, A., Barchas, J. D., and Kedes, L. (1985) Mol. Cell. Biol. 5, 3476-3483). 2) Mixing experiments followed by two-dimensional gel analyses reveal the precise co-migration of beta-internexin, the clathrin-uncoating ATPase, and the in vitro translation product of cDNA clone pHSP-4 encoding rat brain hsc70. 3) beta-Internexin is recognized by a monoclonal antibody reactive against the class of hsp70 proteins. 4) beta-Internexin purified from a microtubule-associated protein-enriched fraction of rat brain by virtue of high affinity binding to ATP-agarose possesses clathrin cage-specific ATPase activity.     

Nucleotide sequences and novel structural features of human and Chinese hamster hsp60 (chaperonin) gene families

A number of clones that specifically hybridize to the human hsp60 cDNA (chaperonin protein; GroEL homolog) were isolated from human and Chinese hamster ovary cell genomic libraries. DNA sequence analysis shows that one of these clones, pGem-10, is completely homologous to the human hsp60 cDNA (in both coding and noncoding regions) with no intervening sequences. The other human clones analyzed were all nonfunctional pseudogenes containing numerous small additions, deletions, and base substitutions, but no introns. On the basis of sequence data, six different hsp60 pseudogenes were identified in human cells. In addition, we also cloned and completely sequenced a genomic clone from CHO cells. This clone, which was also a pseudogene, contained a small 87-nucleotide intron near the 3' end. Southern blot analysis of human, mouse, and Chinese hamster DNA, digested with unique restriction enzymes (no sites in cDNA), indicates the presence of about 8-12 genes for hsp60 in the vertebrate genomes. The sequence data, however, suggest that most of these genes, except one (per haploid genome), are likely to be nonfunctional pseudogenes.     

Isolation and sequence analysis of a complementary DNA encoding rat liver L-gulono-gamma-lactone oxidase, a key enzyme for L-ascorbic acid biosynthesis

L-Gulono-gamma-lactone oxidase, one of the microsomal flavin enzymes, catalyzes the last step of L-ascorbic acid biosynthesis in many animals; however, it is missing in scurvy-prone animals such as humans, primates, and guinea pigs. A cDNA clone for this enzyme was isolated by screening a rat liver cDNA expression library in lambda gt11 using antibody directed against the enzyme. The cDNA clone contained 2120 nucleotides and an open reading frame of 1320 nucleotides encoding 440 amino acids of the protein with a molecular weight of 50,605. The amino-terminal sequence (residues 1-33) of the enzyme isolated from rat liver completely coincided with the corresponding part of the deduced amino acid sequence. The identity of the cDNA clone was further confirmed by the agreement of the composition of the deduced amino acids with that determined by amino acid analysis of the enzyme. Hydropathy analysis of the deduced amino acid sequence revealed several hydrophobic regions, suggesting that they anchor the protein into the microsomal membrane. The deduced amino acid sequence showed no obvious homology with the flavin-binding regions of other eight flavoenzymes.     

Molecular cloning and primary structure of rat alpha 1-antitrypsin

A cDNA clone encoding rat alpha 1-antitrypsin has been isolated from a lambda gt-11 rat liver cDNA library using an antigen-overlay immunoscreening method. The nucleotide sequence of this cDNA clone is 1306 base pairs in length and has a coding region of 1224 base pairs which can be translated into an alpha 1-antitrypsin precursor protein consisting of 408 amino acid residues. The cDNA sequence contains a termination codon, TAA, at position 1162 and a polyadenylation signal sequence, AATAAT, at position 1212. The calculated molecular weight of the translated mature protein is 43,700 with 387 amino acid residues; this differs from purified rat alpha 1-antitrypsin's apparent molecular weight of 54,000 because of glycosylation. Five potential glycosylation sites were identified on the basis of the cDNA sequence. The translated mature protein sequence from the cDNA clone matches completely with the N-terminal 33 amino acids of purified rat alpha 1-antitrypsin, which has an N-terminal Glu. The cDNA encoding rat alpha 1-antitrypsin shares 70% and 80% sequence identity with its human and mouse counterparts, respectively. The reactive center sequence of rat alpha 1-antitrypsin is highly conserved with respect to human alpha 1-antitrypsin, both having Met-Ser at the P1 and P1' residues. Genomic Southern blot analysis yielded a simple banding pattern, suggesting that the rat alpha 1-antitrypsin gene is single-copy. Northern blot analysis using the cDNA probe showed that rat alpha 1-antitrypsin is expressed at high levels in the liver and at low levels in the submandibular gland and the lung.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of a cDNA clone encoding the major hsp70 of the pea chloroplastic stroma

The 70-kD heat shock proteins (hsp70s) are a group of ubiquitous, highly conserved molecular chaperones that have been implicated in a variety of processes, ranging from DNA replication to protein folding and transport. To learn more about the evolution and possible functions of higher plant chloroplastic hsp70s, we isolated a cDNA clone encoding the major stromal hsp70 of pea chloroplasts, which we term CSS1 (Chloroplastic Stress Seventy). This cDNA clone encodes a 75,490-D protein that is very closely related to an hsp70 from the cyanobacterium, Synechocystis. CSS1 is nuclear encoded and synthesized as a higher molecular mass precursor with a chloroplastic transit peptide approximately 65 amino acids long. CSS1 mRNA was detected in RNA samples from leaves and roots of pea (Pisum sativum) plants grown at 18°C but increased 9- and 6-fold, respectively, after brief exposure of the plants to elevated temperature. We discuss the possible role(s) of CSS1 in chloroplastic protein transport and other processes.     

Immunological evidence for the association of p53 with a heat shock protein, hsc70, in p53-plus-ras-transformed cell lines.

A rabbit antiserum was prepared against the C-terminal peptide of 21 amino acids from the human heat shock protein hsp70. These antibodies were shown to be specific for this highly inducible heat shock protein (72 kilodaltons [kDa] in rat cells), and for a moderately inducible, constitutively expressed heat shock protein, hsc70 (74 kDa). In six independently derived rat cell lines transformed by a murine cDNA-genomic hybrid clone of p53 plus an activated Ha-ras gene, elevated levels of p53 were detected by immunoprecipitation by using murine-specific anti-p53 monoclonal antibodies. In all cases, the hsc70, but not the hsp70, protein was coimmunoprecipitated with the murine p53 protein. Similarly, antiserum to heat shock protein coimmunoprecipitated p53. Western blot (immunoblot) analysis demonstrated that the hsc70 and p53 proteins did not share detectable antigenic epitopes. The results provide clear immunological evidence for the specific association of a single heat shock protein, hsc70, with p53 in p53-plus-ras-transformed cell lines. A p53 cDNA clone, p11-4, failed to produce clonable cell lines from foci of primary rat cells transfected with p11-4 plus Ha-ras. A mutant p53 cDNA clone derived from p11-4, SVKH215, yielded a 2- to 35-fold increase in the number of foci produced after transfection of rat cells with SVKH215 plus Ha-ras. When cloned, 87.5% of these foci produced transformed cell lines. SVKH215 encodes a mutant p53 protein that binds preferentially to the heat shock proteins of 70 kDa compared with binding by the parental p11-4 p53 gene product. These data suggest that the p53-hsc70 protein complex could have functional significance in these transformed cells.     

Cloning and regulation by glucocorticoid receptor ligands of a rat hsp90

We have isolated a full length cDNA that encodes a heat shock protein, hsp90, from a rat brain library and present the nucleotide sequence and deduced amino acid sequence. Comparison of the entire nucleotide sequence with mouse hsp84 and human hsp90β cDNAs reveal sequence similarities of 92 and 87%, respectively. The coding region of 2172 nucleotides corresponds to a polypeptide chain of 724 amino acids. Comparison with mouse hsp84 and human hsp90β amino acid sequences indicates a similarity of 97%, respectively. Characterization of the constitutive expression of this cDNA both by RNA blot hybridization and immunoblotting, reveals that it is expressed in all rat tissues examined. Hsp90 has been shown to form a transient complex with steroid hormone receptors. In order to further elucidate the role of hsp90 in the endocrine response of cells, we have examined the effects of dexamethasone and RU38486 on the level of hsp90 mRNA in a system in which glucocorticoids down-regulate glucocorticoid receptor mRNA levels. In this system, a subtle but reproducible approx. 2-fold decrease in hsp90 mRNA levels is observed after 48 h treatment with dexamethasone.