A human gene family with sequence homology to Drosophila melanogaster Hsp70 heat shock genes

A growing literature describes the structure and regulation of prokaryotic and eukaryotic heat shock genes. We here report the isolation of several members of a human heat shock protein 70 (hsp 70) multigene family which contains at least 10 different genes and/or pseudogenes exhibiting sequence homology to the hsp70 gene of Drosophila melanogaster. Eight nonoverlapping recombinant lambda phages from a lambda-Charon4A human genomic library were studied by restriction mapping. One lambda clone was sequenced and characterized as a hsp70 pseudogene inserted into a rearranged human HindIII 1.9-kilobase repeated DNA sequence. This pseudogene is probably located on the X chromosome. Its predicted amino acid sequence shows extensive homology to those of Drosophila hsp70, trout hsp70, Xenopus hsp70, yeast hsp70, and some homology to the heat-inducible dnaK gene product of Escherichia coli. Amino acid homology is clustered, suggesting evolutionary conservation of domains critical to the function of this protein in both prokaryotic and eukaryotic cells.     

Assignment of the functional gene for human adrenodoxin to chromosome 11q13----qter and of adrenodoxin pseudogenes to chromosome 20cen----q13.1.

Adrenodoxin is a small iron/sulfur protein serving as an electron-transport intermediate for all mitochondrial forms of cytochrome P450. Southern blots of normal genomic DNA cleaved with six restriction endonucleases probed with full-length human adrenodoxin cDNA revealed complex patterns indicating the presence of multiple adrenodoxin genes. Southern blots of DNA from a panel of mouse/human somatic cell hybrids identified cross-hybridizing adrenodoxin DNA in two loci, chromosome 11q13----qter and chromosome 20cen----q13.1. Examination of adrenodoxin clones from a genomic DNA library in phage lambda revealed some clones bearing gene fragments interrupted by introns and other clones bearing processed pseudogenes. By probing the mouse/human hybrids with unique intronic DNA and by correlating restriction maps of the phage clones with that of uncloned genomic DNA, we show that the authentic transcribed adrenodoxin gene lies on chromosome 11, while pseudogenes lie on chromosome 20.     

Evolution of human cytoplasmic actin gene sequences: chromosome mapping and structural characterizations of three cytoplasmic actin-like pseudogenes including one on the Y chromosome

Eight recombinant phage clones containing cytoplasmic actin-like gene sequences have been isolated from a human genomic library for structural characterization. Kpn I family repeat sequences flank six of these actin genes isolated, and Alu family repeats are scattered throughout the DNA inserts of all eight phage clones. Three of these genes are γ actin-like, and the other five are β actin-like. The complete nucleotide sequence analysis of one β and one γ actin-like genes and their flanking regions demonstrates that they both are processed pseudogenes. Using unique DNA sequences flanking these two pseudogenes as hybridization probes for human-mouse somatic cell hybrid DNAs, we have mapped the two actin pseudogenes on human chromosomes 8 and 3, respectively. We have also determined the DNA sequence of a human Y chromosome-linked, processed actin pseudogene. The different values of sequence divergence of these processed pseudogenes and their functional counterparts allow us to estimate the time of generation of the pseudogenes. The results suggest that the cDNA insertion events generating the human cytoplasmic actin-like pseudogenes have occurred at significantly different times during the evolution of primates, after their separation from other mammalian species.     

DNA sequences complementary to human 7 SK RNA show structural similarities to the short mobile elements of the mammalian genome

A complementary DNA clone of 7 SK RNA from HeLa cells was used to study the genomic organization of 7 SK sequences in the human genome. Genomic hybridizations and genomic clones show that 7 SK is homologous to a family of disperse repeated sequences most of which lack the 3' end of the 7 SK RNA sequence. Only few of the genomic K sequences are homologous to both 3' and 5' 7 SK probes and presumably include the gene(s) for 7 SK RNA. The sequence of four genomic 7 SK clones confirms that they are in most cases pseudogenes. Although Alu sequences are frequently found near the 3' and 5' end of K DNA, the sequences immediately flanking the pseudogenes are different in all clones studied. However, direct repeats were found flanking directly the K DNA or the K-Alu unit, suggesting that the K sequences alone or in conjunction with Alu DNA might constitute a mobile element.     

Isolation and mapping of 62 new RFLP markers on human chromosome 11.

To obtain new RFLP markers on human chromosome 11 for a high-resolution map, we constructed a cosmid library from a Chinese hamster x human somatic hybrid cell line that retains only human chromosome 11 in a Chinese hamster genomic background. A total of 3,500 cosmids were isolated by colony hybridization with labeled human genomic DNA. DNA was prepared from 130 of these cosmid clones and examined for RFLP. In 62 of them, polymorphism was detected with one or more enzymes; four RFLPs were VNTR systems. All polymorphic clones were assigned to one of 22 intervals obtained by mapping on a deletion panel of 15 somatic hybrid cell lines containing parts of chromosome 11; 11 clones were finely mapped by in situ hybridization. Although RFLP markers were scattered on the whole chromosome, they were found predominantly in the regions of R-banding. These DNA markers will contribute to fine mapping of genes causing inherited disorders and tumor-suppressor genes that reside on chromosome 11. Furthermore, as one-third of the cosmid clones revealed a band or bands in Chinese hamster DNA, indicating sequence conservation, this subset of clones may be useful for isolating biologically important genes on chromosome 11.     

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.     

Identification and analysis of a third mouse Polycomb gene, MPc3

Betaine-homocysteine S-methyltransferase (BHMT) is one of the enzymes involved in the branch point metabolism of homocysteine. Elevated levels of plasma homocysteine may be a risk factor for the development of vascular disease; however, whether BHMT has a significant role in the regulation of plasma levels of homocysteine remains to be determined. As a prelude to creating a mouse strain deficient in BHMT activity, we screened a lambda library containing mouse SvJ 129 genomic DNA for the mouse BHMT gene using random probes made from the human cDNA. One genomic isolate was completely sequenced and found to encode an intronless BHMT pseudogene (mBHMT-ps). mBHMT-ps was then used as a template for the generation of random probes that were used to screen a BAC library containing mouse 129 Sv/Ev genomic DNA. In order to discriminate between pseudogenes and the authentic BHMT gene, a secondary PCR-based screen was employed which used primers designed from the pseudogene sequence that would predictably amplify across introns. Using this strategy, we isolated six mouse genomic clones that tested positive for the presence of all seven introns characteristic of the human gene, and the BHMT gene of one clone was completely sequenced. Like the human BHMT gene, the mouse gene spans 21 kb and is encoded by eight exons interrupted by seven introns. The structure of the mouse BHMT gene is described herein as well as the 5′-flanking region of the gene adjacent to exon 1, which we demonstrate is capable of conferring basal promoter activity in Chinese Hamster Ovary cells.     

Mapping of the gene encoding the multifunctional protein carrying out the first three steps of pyrimidine biosynthesis to human chromosome 2

Summary The CAD gene encodes a trifunctional protein that carries the activities of the first three enzymes (carbamyl phosphate synthetase II, aspartate transcarbamylase, and dihydroorotase) of de novo pyrimidine biosynthesis. Genomic fragments of the human CAD gene have been obtained by screening a human genomic library in bacteriophage lambda using a Syrian hamster cDNA clone as a probe. These human genomic clones have been used to assign the CAD gene to human chromosome 2 using in situ hybridization to human metaphase chromosomes and Southern blot hybridization analysis of DNA isolated from a panel of Chinese hamster/human hybrid cells. In situ hybridization analysis has allowed further localization of this gene to the chromosomal region 2p21-p22.     

Human ERCC5 cDNA-cosmid complementation for excision repair and bipartite amino acid domains conserved with RAD proteins of Saccharomyces cerevisiae and Schizosaccharomyces pombe.

Several human genes related to DNA excision repair (ER) have been isolated via ER cross-species complementation (ERCC) of UV-sensitive CHO cells. We have now isolated and characterized cDNAs for the human ERCC5 gene that complement CHO UV135 cells. The ERCC5 mRNA size is about 4.6 kb. Our available cDNA clones are partial length, and no single clone was active for UV135 complementation. When cDNAs were mixed pairwise with a cosmid clone containing an overlapping 5'-end segment of the ERCC5 gene, DNA transfer produced UV-resistant colonies with 60 to 95% correction of UV resistance relative to either a genomic ERCC5 DNA transformant or the CHO AA8 progenitor cells. cDNA-cosmid transformants regained intermediate levels (20 to 45%) of ER-dependent reactivation of a UV-damaged pSVCATgpt reporter plasmid. Our evidence strongly implicates an in situ recombination mechanism in cDNA-cosmid complementation for ER. The complete deduced amino acid sequence of ERCC5 was reconstructed from several cDNA clones encoding a predicted protein of 1,186 amino acids. The ERCC5 protein has extensive sequence similarities, in bipartite domains A and B, to products of RAD repair genes of two yeasts, Saccharomyces cerevisiae RAD2 and Schizosaccharomyces pombe rad13. Sequence, structural, and functional data taken together indicate that ERCC5 and its relatives are probable functional homologs. A second locus represented by S. cerevisiae YKL510 and S. pombe rad2 genes is structurally distinct from the ERCC5 locus but retains vestigial A and B domain similarities. Our analyses suggest that ERCC5 is a nuclear-localized protein with one or more highly conserved helix-loop-helix segments within domains A and B.     

Structure and Light-Induced Expression of a Small Heat-Shock Protein Gene of Pharbitis nil

To isolate genes that are regulated by a photoperiod that promotes flowering in Pharbitis nil, a cDNA library representing mRNA of induced cotyledons was screened by differential hybridization. The DNA sequence of one cDNA clone isolated by this approach, clone 12L, showed homology to plant small heat-shock protein (hsp) genes. P. nil genomic clones hybridizing to clone 12L were isolated, and the DNA sequences of two P. nil small hsp (shsp) genes, shsp-1 and shsp-2, were determined. The derived amino acid sequences of shsp-1 and shsp-2 showed maximum homology to the 17.9-kD soybean hsp, a member of the class II cytoplasmic hsps found in plants. A study of the expression of shsp-1 and shsp-2 genes by RNase protection assay indicated that shsp-1 is induced by photoperiod, by light treatment of dark-grown P. nil seedlings, and by heat shock, and that shsp-2 is induced only by heat shock. Analysis of the sequences of the nontranscribed region indicates that both genes contain multiple heat-shock elements. The shsp-1 gene, in addition, contains sequences homologous to the GT-1-binding site, which may play a role in its light-regulated expression.     

Nucleotide sequence of mouse HSP60 (chaperonin, GroEL homolog) cDNA

The cDNA sequence of the 60 kDa heat-shock protein from mouse 3T3 cells has been determined. The deduced amino acid sequence of mouse hsp60 protein differs from the corresponding proteins from Chinese hamster and human cells in 7 and 13 residues, respectively, most of which are conservative replacements.     

ERCC2: cDNA cloning and molecular characterization of a human nucleotide excision repair gene with high homology to yeast RAD3

Human ERCC2 genomic clones give efficient, stable correction of the nucleotide excision repair defect in UV5 Chinese hamster ovary cells. One clone having a breakpoint just 5' of classical promoter elements corrects only transiently, implicating further flanking sequences in stable gene expression. The nucleotide sequences of a cDNA clone and genomic flanking regions were determined. The ERCC2 translated amino acid sequence has 52% identity (73% homology) with the yeast nucleotide excision repair protein RAD3. RAD3 is essential for cell viability and encodes a protein that is a single-stranded DNA dependent ATPase and an ATP dependent helicase. The similarity of ERCC2 and RAD3 suggests a role for ERCC2 in both cell viability and DNA repair and provides the first insight into the biochemical function of a mammalian nucleotide excision repair gene.