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.     

Genetic Characterization of the Region between 86f1,2 and 87b15 on Chromosome 3 of DROSOPHILA MELANOGASTER

The region between 86F1,2 and 87B15 on chromosome 3 of Drosophila melanogaster, which contains about 27 polytene chromosome bands including the 87A7 heat-shock locus, has been screened for EMS-induced visible and lethal mutations. We have recovered 268 lethal mutations that fall into 25 complementation groups. Cytogenetic localization of the complementation groups by deficiency mapping is consistent with the notion that each band encodes a single genetic function. We have also screened for mutations at the 87A7 heat shock locus, using a chromosome that has only one copy of the gene encoding the 70,000 dalton heat-shock protein (hsp70). No lethal or visible mutations at 87A7 were identified from 10,719 mutagenized chromosomes, and no female-sterile mutations at 87A7 were recovered from the 1,520 chromosomes whose progeny were tested for female fertility. We found no evidence that a functional hsp70 gene is required for development under laboratory conditions.     

Extensive regions of homology in front of the two hsp70 heat shock variant genes in Drosophila melanogaster

The major heat shock protein of 70,000 M_r in Drosophila melanogaster is encoded by two variant gene types located, respectively, at the chromosomal sites 87A7 and 87C1. We present the DNA sequence of a complete hsp70² gene of the 87A7 type and of the adjacent regions from both variants, extending to 1·2 × 10³ bases upstream from the start of the messenger coding region. We find an untranslated region of 250 nucleotides at the 5′ end of the messenger coding sequence in both variants. There is only one open reading frame which allows coding of a 70,000 M_r protein within the 87A7 variant, as found for an 87C1 variant (Ingolia et al., 1980). We observe 4·2% nucleotide divergence between these two variants with complete conservation of the reading frame. There is a conserved sequence of 355 nucleotides in front of each hsp70 gene, which is 85% homologous between the two variants. The presence of the same sequence element in γ, in front of the αβ heat shock genes (R. W. Hackett & J. T. Lis, personal communication) suggests that this element contains the regulatory signals for the coordinate expression of both the hsp70 and the αβ heat shock genes. Finally we find a very A + T-rich sequence of 150 basepairs which is highly conserved (91·8%) 0·6 × 10³ bases upstream from two hps70 gene variants.     

Genetic and physical analysis of the mitochondrial gene for subunit II of yeast cytochrome c oxidase.

The mitochondrial genetic locus oxi 1 contains the structural gene for subunit II of Cytochrome c oxidase. In this study, the oxi 1 locus, or at least a major portion of it, has been localized to a 2·4 kb2 HpaII fragment of mitochondrial DNA, by examining the mtDNA of oxi 1 mutants, and rho^? yeast strains that selectively retained in amplified form, this region of the mitochondria) genome. The 2·4 kb fragment is missing from the mtDNA of an oxi 1 locus deletion mutant, but is present in the mtDNAs retained by two rho^? strains that genetically recombine with all 16 oxi 1 mutants tested, to produce respiring progeny. Two other rho^? strains, that retained different but overlapping portions of the oxi 1 locus as determined genetically, contained mtDNAs consisting of “cloned” segments derived from within the 2·4 kb fragment: these rho^? mtDNAs hybridized only to the 2·4 kb HpaII fragment of wild-type mtDNA and could not be cleaved with HpaII. Furthermore, these two rho^? mtDNAs were found to correspond to sequences from opposite sides of the 2·4 kb fragment that overlap for 100 to 300 base-pairs near the middle of the fragment. Thus, five oxi 1 mutations that recombine with both of these rho^? strains could be further localized to this relatively short region of overlap. One such mutation, of particular interest because it produces an altered form of subunit II, was shown to lie on a 75-base-pair fragment that maps in this region of the overlap. The 75-base-pair fragment from the mutant migrates slightly faster during electrophoresis than the corresponding wild-type fragment. In contrast, the mobility of the fragment from a spontaneous revertant was indistinguishable from wild type.     

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.     

Position Effects and Variegation Enhancers in an Autosomal Region of DROSOPHILA MELANOGASTER

A dominant eye color mutation was found associated with a third chromosome inversion broken distally at or near the karmoisin (kar) locus in 87C and proximally within centric heterochromatin. Suppressibility of the mutant phenotype by an extra Y chromosome indicated that this was an example of dominant position-effect variegation. When heterozygous with deficiencies uncovering the kar locus, this inversion chromosome was found to be lethal unless a region in 87EF was also deleted. Extra Y chromosomes rescued inversion/deletion heterozygotes, while removal of the Y chromosome from heterozygous males deficient for the region in 87EF was lethal. Thus, a variegating lethal lies near the breakpoint in 87C, and a wild-type gene that enhances its variegation lies in 87EF. Furthermore, deletion of the region in 87EF was found to strongly suppress white-mottled-4 (w^m4) variegation, while deletion of another region in 87BC suppressed less strongly. These results indicate that essential genes on autosomes are sensitive to position effects, and loci that enhance variegation, as defined by deficiency mapping, are very common.