Genetic and molecular analysis of the 87A7 and 87C1 heat-inducible loci of D. melanogaster.

Two different types of heat-inducible sequences are found at the cytogenetic loci 87A7 and 87C1 of D. melanogaster. One of these codes for the 70,000 dalton heat shock protein (hsp 70) and is found at both loci. The other type of sequence (alpha beta) codes for an RNA of unknown function and is found only at 87C1. We have completed a study of the organization of the two loci, using deficiencies that delete one or other locus, and have estimated the number of the hsp 70 genes at each locus. Thus in at least three strains of files there are a total of five coding sequences, three at 87C1 and two at 87A7. Restriction mapping of the coding regions at the two loci reveals that each of the two cytogenetic loci has its own characteristic coding sequence. The overall organization of the two loci appears to differ considerably. The alpha beta and hsp 70 heat-induced sequences at 87C1 are closely linked and are contained within two Eco RI restriction fragments.     

Two hybrid plasmids with D. melanogaster DNA sequences complementary to mRNA coding for the major heat shock protein.

The isolation and partial characterization of two cloned segments of Drosophila melanogaster DNA containing "heat shock" gene sequences is described. We have inserted sheared embryonic D. melanogaster DNA by the poly(dA-dt) connector method (Lobban and Kaiser, 1973) into the R1 restriction site of the ampicillin-resistant plasmid pSF2124 (So, Gill and Falkow, 1975). A collection of independent hybrid plasmids was screened by colony hybridization (Grunstein and Hogness, 1975) for sequences complementary to in vitro labeled polysomal poly(A)+ heat shock RNA. Two clones were identified which contain sequences complementary to a heat shock mRNA species that directs the in vitro synthesis of the 70,000 dalton heat-induced polypeptide. Both cloned segments hybridize in situ to the heat-induced puff sites located at 87A and 87C of the salivary gland polytene chromosomes.     

Deletion mapping of two D. melanogaster loci that code for the 70,000 dalton heat-induced protein.

Using deficiencies in D. melanogaster that lack either the 87A or 87C heat-induced puffs, we have shown that the 70,000 dalton heat-induced protein (hsp 70) is encoded at both these loci. Embryos deleted for one of the two loci retain the ability to make hsp 70 after heat shock, but deleting both loci eliminates synthesis of hsp 70. Thus both loci encode hsp 70 and can be active following heat shock. We have analyzed the proteins made by embryos lacking either 87A or 87C, and have compared the 87A- and 87C-coded hsp 70 by isoelectric focusing and tryptic peptide fingerprinting. The hsp 70 made by the two loci is very similar, although a variant tryptic peptide appears to be encoded only at 87C. Using deficiencies with slightly different breakpoints, we have mapped the 87A locus to band 87A7, the site of the 87A heat-induced puff. The 87C locus maps within 87C1.     

Physical map of two D. melanogaster DNA segments containing sequences coding for the 70,000 dalton heat shock protein.

The isolation of the two hybrid plasmids 56H8 and 132E3, which contain D. melanogaster (Dm) DNA sequences complementary to the mRNA coding for the 70,000 dalton heat shock protein, has been reported (Schedl et al., 1978). Here we compare the sequence arrangement in the two cloned Dm DNA segments by restriction, cross-hybridization and heteroduplex analysis. The results show that the two cloned DNA segments derive from nonoverlapping regions of the Dm genome; that they contain homologous regions present once in 56H8 and twice in 132E3; and that each homologous region is composed of three distinct contiguous sequence elements, x, y and z, which together define a 3 kb common unit. While the 2.5 kb z elements show a high degree of sequence homology in all three common units, the three x and y elements display an intriguing relationship. The localization of the mRNA coding sequences within each of these common units is presented in the accompanying paper (Artavanis-Tsakonas et al., 1979).     

Evolutionary implications of a complex pattern of DNA sequence homology extending far upstream of the hsp70 genes at loci 87A7 and 87C1 in Drosophila melanogaster

Genes coding for the major 70,000 M_r heat shock protein (hsp70) are found at two loci, 87A7 and 87C1, in Drosophila melanogaster. At 87A7 they are present as two genes in diverging orientation, whilst at 87C1 two tandemly repeated distal copies are separated from a single copy in divergent orientation by about 40,000 bases of DNA. Within this 40,000 bases are found the αβ heat-induced genes, interspersed with γ elements. In this paper we report the isolation and characterization of the proximal hsp70 gene from locus 87C1. The DNA sequence upstream from this gene shows greater than 98% homology with that of αγ, suggesting that the γ element interspersed with αβ sequences originated from this position. In addition, we present the DNA sequence between the two genes in a cloned DNA segment from 87A7, and compare the sequence with those from 87C1. We find a complex pattern of nucleotide sequence homology extending far upstream of the hsp70 genes at the two loci. The evolution of the present arrangement at these two loci is discussed.     

Sequence of three copies of the gene for the major Drosophila heat shock induced protein and their flanking regions

The primary sequence of the major heat shock gene of D. melanogaster, that for the 70,000 protein, has been determined. One of the reading frames is devoid of stop codons for over 2000 bp. The region between the first ATG and the first stop codon encodes a protein of molecular weight 70,270. The 5' end of the messenger RNA was localized in the DNA sequence by two independent methods. The 5' flanking sequences of three distinct 70K genes were also determined. Extensive homology in the primary sequences extends about 500 bp upstream from the ATG, which is the presumptive initiation of protein synthesis. Each 70K gene has the putative promoter sequence TATAAATA about 325 bp upstream from this ATG. A heptanucleotide sequence identified as the capping site for other messengers is found 24-30 bp downstream from the ends of the A-T-rich sequence. A 12 bp sequence with dyad symmetry begins 23 bp upstream from the beginning of the above A-T-rich sequence.     

Sequence organization of two recombinant plasmids containing genes for the major heat shock-induced protein of D. melanogaster.

We have isolated recombinant DNA clones which include cDNA and chromosomal DNA sequences of the major heat shock-inducible gene of Drosophila. With the cDNA fragments used as specific hybridization probes, DNA:DNA reassociation and in situ hybridization analysis demonstrated that the DNA sequences are repeated approximately 7 times in the haploid Drosophila genome, and that gene sequences are present at both the 87A and 87C loci on the cytological map. The cloned cDNA and homologous cloned chromosomal DNA hybridized to mRNA which translated in vitro into the major 70K heat shock-specific protein. Here we summarize a study of the organization of genes coding for the 70K heat shock-specific protein contained in the two recombinant chromosomal DNA plasmids pG3 and pG5. On the basis of R loop hybridization experiments and restriction enzyme analysis, we conclude that a 14 kb fragment, G3, contains three copies of the gene coding for the 70K protein. A second 9.2 kb fragment, G5, contains one copy of the gene coding for the 70K protein. Hybridization of labeled poly(A)-containing RNA to restriction endonuclease-cleaved DNA indicates that the mRNA coding regions in G3 and G5 are each approximately 2100 bp long. The three tandemly repeated genes of G3 are separated by approximately 1400 bp of spacer DNA. The two internal spacer regions in G3 appear to be identical, whereas differences in restriction enzyme sites indicate that the sequences adjacent to the cluster differ from the internal spacer and from each other.     

Heat-shock DNA homology in distantly related species of Drosophila

Polytene chromosomes of D. melanogaster and D. virilis were hybridized in situ with ¹²⁵I labeled mRNA isolated from polysomes of D. melanogaster tissue culture cells incubated at 37° C. ¹²⁵I mRNA hybridized preferentially with subdivisions 87A and 87Cl of the D. melanogaster 3R chromosome; grains were also observed at regions 93D, 95D and over the chromocenter. A considerable cross hybridization of this mRNA with D. virilis polytene chromosomes was observed. The 29C region of the D. virilis second chromosome was the main site of hybridization. Significant grain numbers also appeared in region 20F of the same chromosome. The two regions mentioned belong to heat shock loci in the latter species. Based on label intensity we conclude that region 29C of D. virilis contains DNA sequences retaining molecular homology with those at subdivisions 87A and 87Cl of D. melanogaster. SDS-polyacrylamide gel electrophoresis revealed similar distributions of heat shock proteins in the two species studied.     

Divergence of the Two Albumins of X. laevis

The mRNAs coding for the 68,000 and 74,000 dalton serum albumins of Xenopus laevis were purified by hybridisation to their corresponding cloned cDNA and translated using the reticulocyte lysate. The primary translational product of the 68,000 dalton albumin has a molecular weight of 70,000 daltons suggesting that it is synthesised with a signal peptide which is cleaved during secretion. In contrast, the primary translational product of the 74,000 dalton albumin has a molecular weight of 72,000 daltons suggesting that it must be posttranslationally modified to account for the increased molecular weight of the mature protein. X. laevis oocytes injected with albumin mRNA secrete proteins of the same molecular weights as the mature albumins. When these translational products were chromatographed on concanavalin A Sepharose, the 74,000 dalton albumin was bound suggesting that it is glycosylated. Comparison of X. laevis and X. tropicalis albumins suggests that the 68,000 dalton albumin is similar to the primitive Xenopus albumin and that since the genome duplication which occurred in X. laevis , differences have arisen in both the length and processing of the primary translational product to account for the current difference in the molecular weights of the two X. laevis albumins.     

Divergence of the two albumins of X. laevis. Evidence for the glycosylation of the major 74K albumin

The mRNAs coding for the 68,000 and 74,000 dalton serum albumins of Xenopus laevis were purified by hybridisation to their corresponding cloned cDNA and translated using the reticulocyte lysate. The primary translational product of the 68,000 dalton albumin has a molecular weight of 70,000 daltons suggesting that it is synthesised with a signal peptide which is cleaved during secretion. In contrast, the primary translational product of the 74,000 dalton albumin has a molecular weight of 72,000 daltons suggesting that it must be posttranslationally modified to account for the increased molecular weight of the mature protein. X. laevis oocytes injected with albumin mRNA secrete proteins of the same molecular weights as the mature albumins. When these translational products were chromatographed on concanavalin A Sepharose, the 74,000 dalton albumin was bound suggesting that it is glycosylated. Comparison of X. laevis and X. tropicalis albumins suggests that the 68,000 dalton albumin is similar to the primitive Xenopus albumin and that since the genome duplication which occurred in X. laevis, differences have arisen in both the length and processing of the primary translational product to account for the current difference in the molecular weights of the two X. laevis albumins.     

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.