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.     

Induction ofent-kaurene biosynthesis by low temperature in dwarf peas

Germinating pea (Pisum sativum L.) seeds of two dwarf cultivars, “Progress No. 9” and “Green Arrow”, and two tall cultivars, “Alaska” and “Alderman”, were treated with low temperature (3–5°C) for 14 days and then transferred to normal growing conditions (19–21°C for 16 h/14.5–16.5°C for 8 h) for an additional 10 days. Biosynthesis of [¹⁴C]ent-kaurene from [¹⁴C]2-mevalonic acid (2-MVA) was assayed in cell-free enzyme extracts prepared from shoot tips 10 days after cold treatment and was compared with activity in enzyme extracts prepared from noncold-treated, 10-day-old control plants. Shoot lengths of cold-treated plants were measured throughout a 35-day period and compared with shoot lengths of plants grown without cold treatment for 25–35 days. Low temperature induced a five-to 10-fold enhancement ofent-kaurene, hence potentially gibberellin (GA), biosynthesis in seedlings of the two dwarf cultivars but not in the tall cultivars. However, the lack of an increase in growth rate in the cold-treated dwarfs indicated that endogenous GA biosynthesis remained blocked at some point beyondent-kaurene in the biosynthetic pathway. Since the late-flowering “Alderman” cultivar did not exhibit enhanced biosynthesis ofent-kaurene, it appears that if vernalization in late-flowering cultivars of peas is correlated with enhanced GA biosynthesis, it is not the early part of the biosynthetic pathway which is affected.     

Characterization of a new high-temperature-induced 66-kDa heat-shock protein,antigenically related to heat-shock protein 72

M-14 human melanoma cells, following severe hyperthermic exposures, synthesized a heat-shock protein of 66 kDa (hsp 66), in addition to the major “classic” heat-shock proteins. This hsp 66 was not expressed following mild hyperthermic exposures sufficient to trigger the synthesis of the other heat-shock proteins. The induction of hsp 66 was observed also in Li human glioma cells treated at 45°C for 20 min. By contrast, hsp 66 was not induced in seven other human cell lines (both melanoma and nonmelanoma) when they were subjected to the same hyperthermic treatment. Immunological recognition experiments showed that hsp 66 cross-reacted with the inducible hsp 72, but not with the constitutive hsp 73. The possibility that hsp 66 is a breakdown product of hsp 72 was ruled out by the fact that Poly(A)⁺ RNA extracted from cells treated at 45°C for 20 min was able to direct the synthesis of hsp 66 (together with hsp 72) in a message-dependent rabbit reticulocyte lysate, as well as in microinjected Xenopus oocytes. By contrast, only the hsp 72 was expressed using Poly(A)⁺ RNA extracted from cells heated at 42°C for 1 h. Affinity chromatography experiments on ATP-agarose showed that hsp 66 did not bind ATP in vitro, hsp 66 was localized both in the cytoplasm (cytosol, mitochondria, and microsome fraction) and in the nuclei of cells recovered from a severe heat shock: this intracellular distribution closely corresponded to that of hsp 72. The nuclear-associated hsp 66 was found to be tightly bound to nuclear structures and could not be extracted by incubation in ATP-containing buffer. © 1996 Wiley-Liss, Inc.     

Veränderungen im Puffmuster und das Wachstum der Riesenchromosomen in Speicheldrüsen von Drosophila melanogaster aus spätlarvalen und embryonalen Spendern nach Kultur in vivo

Salivary glands from late third instar larvae of Drosophila melanogaster were transplanted into the abdomens of adult female and male flies and were kept in this medium from 6 to 120 h. Changes in the puffing pattern of chromosome arm III L were studied after the culture in vivo. Two noticeable puffs are induced. They are located in 68 B and 78 E. Neither of these loci show activity during normal development. — Front halves of embryos (6 to 9 h of age) were also transferred into adults. After 5 to 13 days in vivo they are able to develop and differentiate larval structures. Salivary glands, imaginal discs, fat body, Malpighian tubules and muscle fibers could be identified. Even 4 h old embryos can form polytene salivary gland chromosomes after a 13 day culture. These chromosomes can reach sizes comparable with the maximal size in normal development. In some nuclei an extensive growth leads to “supergiant” chromosomes. The puffs in 68B and 78E are formed in the polytenic chromosomes from embryonic implants as in cultured larval salivary gland chromosomes.     

Molecular cloning of the white locus region of Drosophila melanogaster using a large transposable element

We report the molecular cloning of a chromosome segment including the white locus of Drosophila melanogaster. This region was isolated using a deficiency extending from the previously cloned heat-shock puff sequences at 87A7 to a large transposable element containing the loci white and roughest.FB-NOF, a 7.5 kb element with partial homology to a family of inverted repeat sequences (Potter et al., 1980), is found very near the deficiency breakpoint, and is followed by DNA originating from the white locus region. Sequences totalling ˜60 kb surrounding this initial entry point were obtained by the cloning of successively overlapping fragments from a wild-type strain. Several rearrangement breakpoints have been mapped relative to the cloned DNA; these define the limits of the white locus and further differentiate the “white proximal region”, thought to function in gene regulation, from the remainder of the locus. Insertion of the dispersed repetitive element copia into the white locus is observed in strains carrying the white-apricot allele. Analysis of several white-apricot revertants suggests that copia insertion is responsible for the apricot eye color phenotype.     

Chemical investigation of different extracts and essential oil from the tubers of (Tunisian)Cyperus rotundus. Correlation with their antiradical and antimutagenic properties

The mutagenic potential of aqueous, Total Oligomers Flavonoids (TOF), ethyl acetate, and methanol extracts as well as essential oil (EO) obtained from tubers ofCyperus rotundus L. was assessed by “Ames assay”, usingSalmonella tester strains TA98 and TA100, and “SOS chromotest” usingEscherichia coli PQ37 strain with and without an exogenous metabolic activation system (S9). None of the different extracts showed a mutagenic effect. Likewise, the antimutagenicity of the same extracts was tested using the “Ames test” and the “SOS chromotest”. Our results showed thatC. rotundus extracts have antimutagenic effects withSalmonella typhimurium TA98 and TA100 strains towards the mutagen Aflatoxin B1 (AFB1), as well as withE. coli PQ37 strain against AFB1 and nifuroxazide mutagens. A free radical scavenging test was used in order to explore the antioxidant capacity of the extracts obtained from the tubers ofC. rotundus. TOF, ethyl acetate and methanol extracts showed an important free radical scavenging activity towards the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical. These extracts showed IC50 values of respectively 5, 20 and 65 μg/ml. The beneficial effects of TOF, ethyl acetate, methanol and essential oil extracts ofC. rotundus have been assessed by antioxidant and antimutagenic activities.     

Ultrastructure of salivary glands of Drosophila lebanonensis during normal development and after in vivo ecdysterone administration

Changes in the morphology of the salivary glands of Drosophila lebanonensis have been followed at both the light and electronmicroscopic level during a period of 30 hr before puparium formation and during puparium formation itself. Three striking differences were observed in comparison to other Drosophila species studied: (1) the secretion product of Drosophila lebanonensis has a different stainability to PAS reagent and uranyl acetate and no internal structures or “caps” can be observed; (2) the release of this secretion product is not restricted to a time period shortly before puparium formation but is a continuous process starting about 24 hr before puparium formation; and (3) the histolysis of these glands starts immediately after puparium formation, whereas in other Drosophila species this event starts 5 hr later.Puparium formation of Drosophila lebanonensis is controlled by the circadian oscillation. Injection of ecdysterone before the “gate” period results in changes in the cuticle as observed during normal development, but is not followed by the histolysis of the glands. Injection of ecdysterone after the “gate” is not followed by changes in the cuticle but histolysis is induced.     

Genetic Characterization of the 87c Region of the Third Chromosome of DROSOPHILA MELANOGASTER

Ethyl methanesulphonate (EMS) was used to induce 39 lethal and 13 karmoisin mutations within Df(3R)kar^3J, a nine-band deficiency extending from 87C1 to 87C9 (inclusive). Five complementation groups (four lethal and one visible) were identified and cytologically mapped between 87C4–5 and 87C9, one complementation group per band, with the exception of complementation group A, which is localized to 87C4–5. These positions were determined using a set of overlapping deficiencies, each having at least one breakpoint in the 87C1–9 region. Mutations within a single complementation group have similar lethal phases or subvital phenotypes, consistent with the notion that each complementation group represents a single functional locus. No mutations localized to 87C1–C3. The inability to induce mutations in the 87C1 heat-shock puff locus is consistent with the current interpretation of a duplication of coding sequences at the 87A7 and 87C1 heat-shock puffs.     

Kinetic basis of spontaneous mutation. Misinsertion frequencies, proofreading specificities and cost of proofreading by DNA polymerases of Escherichia coli

Repeating members of multiple-copy sequence families display high levels of sequence homogeneity. In order to examine the rates at which this is achieved, and to compare the rates with those assessed for the ribosomal DNA and histone gene families (Coen et al., 1982, accompanying paper), we have examined the patterns of variation in the Drosophila melanogaster species subgroup for the “complex” noncoding families of high copy-number. Our analysis reveals that the evolution of some of the families has involved the gradual replacement of ancestral repeats by variant repeats, independently within each species. Hybridizations between genomes at different levels of stringency indicate the presence of two basic ancestral families (the “500” and “360” families) within the subgroup. The majority of repeats representative of these families can be characterized by restriction sites and patterns of organization that are uniquely diagnostic for each species, excepting the two most closely related species. Drosophila mauritiana and Drosophila simulans. Another family (the “180” family) is confined to the one species. Drosophila orena, with features suggestive of a more rapid origin. The wide karyotypic distribution of some members of the 500 and 180 families, revealed by hybridization in situ, shows that chromosomes are evolving in concert with respect to gradual and rapidly evolving families. The distribution of sequence and pattern variation within the subgroup shows that the time required for gradual fixation (concerted evolution) of variants within large families, distributed throughout the karyotype, is longer than that required for the smaller and chromosomally restricted families of rDNA and histone genes (Coen et al., 1982). We discuss the forces that might either accelerate or retard the fixation of variants in karyotypically dispersed families.