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.     

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.     

Similarities in chromosomal puffing induced by temperature shocks and dinitrophenol in Drosophila

A comparison between chromosomal puffs induced by temperature shocks and dinitrophenol in regions 87 A and 87 B of salivary gland chromosome 3 R of Drosophila melanogaster is presented. The size of both puffs can be regulated by either the temperature of a heat-shock or by the concentration of dinitrophenol. The two agents are additive with respect to their effects upon puff size, supporting the idea that both result in a change in ATP synthesis within the cell. A possible relationship between these agents and puff formation and RNA metabolism is discussed.This investigation was supported by research grant No. 119-70 from the Cancer Association of Greater New Orleans.     

Dosage compensation of X-linked heat-shock puffs in Drophila pseudoobscura

Four major puffs are inducible by heat shock in the larval salivary gland chromosomes of D. pseudoobscura. Two of these puffs are present at 23 and 39–40 on the right arm of the X chromosome and two are present at 53 and 58 on chromosome 2. By means of in situ hybridization, residual homologies were demonstrated between the puffs at 23 in D. pseudoobscura and at 63C in D. melanogaster, and between the two chromosome 2 puffs of D. pseudoobscura and 87A and 87C of D. melanogaster. RNA synthesis was monitored as a function of ³H-uridine incorporation in the major heat-induced puffs of D. pseudoobscura and was found to be equivalent in males and females indicating dosage compensation of the two X-linked loci. The evolution of the regulatory controls of these genes is discussed.     

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.     

Studies of cloned sequences from four Drosophila heat shock loci.

DNA cloned from the D. melanogaster (Oregon R) heat shock loci at 63BC and 95D codes for the 83,000 and the 68,000 dalton heat shock proteins, respectively. Both coding sequences occur once per haploid genome. Sequences complementary to messenger RNA for the 70,000 dalton heat shock protein are represented five times, twice at 87A and three times at 87 C. The copies at 87A differ characteristically from those at 87C in an interval of a few hundred bp near the 5' end of the messenger sequence, and the corresponding two classes of hsp 70 messenger RNA are found on polysomes after heat shock. Within this differential region, there is about 15% divergence between messenger sequences cloned from the two loci, while in the rest of the messenger region examined the homology is much closer although still imperfect. Unexpectedly, considerable homology is found between the sequence for the 68,000 dalton heat shock protein at 95D and the sequences for the 70,000 dalton protein at 87A and 87C, and between these sequences and a site in 87D. Messenger RNA molecules of 2.4, 2.55 and 3.05 kb code for the 68,000, 70,000 and 83,000 dalton heat shock proteins and hybridize to apparently uninterrupted DNA sequences of 2.1, 2.25 and 2.6 kb, respectively.     

Factors involved in the expression of gene activity in polytene chromosomes

In order to separate some of the factors involved in the formation of puffs the antibiotic actinomycin D was applied at different stages of puff activity. Puffs were induced by temperature shocks or eodysone.Inhibition of RNA synthesis with actinomycin D before application of a puff inducing stimulus prevents neither the appearance of the stimulus specific puffs nor the accumulation of acidic proteins in the puff regions. The puffs attained under these conditions approximately 1/3 of the size normally produced by the stimulus.Indications were obtained that during puff formation acidic protein accumulation precedes the onset of RNA synthesis.Synthesis and storage of newly synthesized RNA within the puff region was studied on the basis of grain distribution in uridine-H³ autoradiographs after various incubation periods. RNA synthesis appears to be restricted to a particular area of the puff region. After a 3 min temperature shock following injection of uridine-H³ silver grains are located only over a particular area of the newly formed puff. The same area becomes labeled during a 1 min pulse of uridine-H³ applied at a stage of maximum puff development. Longer periods of incubation result in a random distribution of the grains over the whole puff region. Grain counts on different areas of experimentally induced puffs and on the same areas at a stage of puff regression indicate that the newly synthesized RNA becomes transferred from the area where it was synthesized and is stored for a certain period within the puff region. Complete release of newly synthesized RNA from puffs in which RNA synthesis was inhibited by actinomycin D at a stage of maximal activity is accomplished within 30 to 35 min.     

Two-step regulation of ecdysone-inducible late puffs in salivary glands of Drosophila melanogaster

Our previous study showed that some ecdysone-inducible late puffs could also be induced by a mild detergent (digitonin) in Drosophila salivary glands. However, they could only be induced at the stage immediately prior to when developmentally programmed puffing occurred, suggesting that these late puff loci were under two-step regulation. Using an in vitro culture of salivary glands, we have examined whether ecdysone or the protein products of early puff genes participate in either of the two steps of late puff regulation. This study has revealed that (i) the acquisition of digitonin-responsiveness (the first step) could be induced in vitro by incubating salivary glands with ecdysone; (ii) the first step could also be induced by protein synthesis inhibition even in the absence of ecdysone; (iii) the second step required both ecdysone and protein synthesis unless treated with digitonin; and (iv) the first step, rather than the second step, determines the timing of normal puff formation in the loci. These results suggest that, during normal development, ecdysone controls both steps by activating two types of early genes; the first type, whose function can be mimicked by cycloheximide, renders the loci responsive to digitonin and the second type, whose function can be mimicked by digitonin, activates the loci to form puffs.     

Local protein accumulation during gene activation

Measurements of the integrated absorbancy of naphthol yellow S binding to protein (430 nm) and Feulgen-stained DNA (550 nm) of two puff regions in Drosophila hydei polytene chromosomes revealed a significant increase in the naphthol yellow S binding capacity during the first 5 min of puff induction. The ratio of integrated absorption values at 430 and 550 nm of two chromosome regions, 2-48 C and 4-81 B were determined relative to the ratio of absorption values at 430 and 550 nm of a reference band. These determinations were carried out in a non-puffed state and at 5, 10, 30, 60 and 120 min after onset of a temperature treatment inducing puffs in these regions. The quotient of the absorption ratio of the puff region and the ratio of the reference band provides a relative measure for naphthol yellow S binding to protein. The staining reaction was absent after pronase treatment.—The relative increase in naphthol yellow S binding was most obvious during the first 5 min after onset of puff induction. The binding of naphthol yellow S was increased by a factor 1.7 for puff 2-48 C, and a factor 1.9 for puff 4-81 B. The maximum value, indicating a relative increase by a factor 1.8 in puff 2-48 C and a factor 2.2 in puff 4-81 B was attained in both puffs at 30 min after onset of puff induction.—Among staining procedures performed on sulphydryl groups, free -amino acids and indole groups of tryptophane, only a positive result with the staining reaction on the indole groups was obtained for induced puffs.—Injection of tritiated sodium acetate, methionine-H³-methyl, ethionine-H³-ethyl, C¹⁴-sodium bicarbonate, a mixture of 15 H³-labelled L-amino acids and H³-tryptophane at various time intervals prior to puff induction failed to result in a specific incorporation of any of these radioactive substances into newly induced puffs.     

Comparative study of protein synthesis and heat-shock puffing activity in Drosophila salivary glands treated with chloramphenicol

The effects of chloramphenicol (CAP) on puffing activity and incorporation of tritiated amino acids in proteins synthesized by cultured larval salivary glands of Drosophila melanogaster were examined. CAP concentrations exceeding 1 mM were found to inhibit cellular protein synthesis and to induce the special group of heat-shock puffs in the polytene chromosomes. Recovery from a transient treatment with 5 mM CAP for 120 min led to rapid regression of the puffs and resumption of protein synthesis giving a pattern of labelled polypeptides similar to that produced by cells submitted to a temperature shift from 25 to 37 degrees C. Only slight inhibition of protein synthesis was found with thiamphenicol, the methylsulphonyl analogue of CAP, which induced a single puff in the 93D region, but did not alter the pattern of polypeptides. In contrast to the results obtained with CAP, recovery from a transient inhibition of protein synthesis by cycloheximide led to the synthesis of normal proteins as produced by control cells at 25 degrees C. Different effects of CAP which may interfere with protein synthesis and puffing activity are discussed.     

Monoclonal antibody directed against RNA polymerase II of Drosophila melanogaster

Summary Monoclonal antibodies were raised against purified RNA polymerase II (or B) from Drosophila melanogaster. The antibody produced by one hybridoma cell clone was found to be directed against the two large subunits of the enzyme. The absence of antibodies directed against proteins possibly contaminating the antigens used for immunization allowed us to identify RNA polymerase unequivocally in interbands and puffs of polytene chromosomes. Within a single heat shock puff (87C1) RNA polymerase was found to be clustered in two separate areas suggesting two distint regions of RNA polymerase activity in this puff.Abbreviations FITC fluorescein isothiocyanate - PAGE polyacrylamide gel electrophoresis - PBS phosphate buffered saline - SDS sodium dodecyl sulfate - Enzyme DNA-dependent RNA polymerase or nucleotide-triphosphate - RNA nucleotidyltransferase (EC 2.7.7.6)     

Ecdysone-regulated chromosome puffing in the salivary glands of the Mediterranean fruit fly, Ceratitis capitata.

The effect of ecdysone on the puffing activity of the polytene chromosomes of Ceratitis capitata has been studied in organ cultures of late-larval salivary glands. Culture of glands from 120-h-old larvae (puff stage 1) in the presence of ecdysone resulted in the initiation of the late-larval puffing cycle that is normally observed in 145-h-old larvae (puff stage 4). During a 7-h period in the presence of ecdysone, the puffing patterns of most loci resembled the in vivo patterns observed in the period between puff stages 4 and 10, indicating that the first puffing cycle can be initiated by the hormone and proceed almost to completion, in vitro. Culture of salivary glands in the presence of ecdysone and a protein-synthesis inhibitor, as well as ecdysone withdrawal and readdition experiments, indicated that most of the ecdysone-regulated puffs could be categorized into three classes: (i) the puffs that were suppressed immediately by ecdysone, even in the absence of protein synthesis; (ii) the puffs that were induced directly by ecdysone; and (iii) the puffs that were induced indirectly by ecdysone, that is, they were induced after a lag period of a few hours and required protein synthesis for their induction.