Ecdysone-stimulated RNA synthesis in imaginal discs of Drosophila melanogaster

Cytoplasmic RNA from imaginal discs of Drosophila melanogaster, labeled by uridine incorporation in organ culture, has been assayed by hybridization to cytological preparations of polytene chromosomes. RNA labeled during the early stages (first four hours) of ecdysone stimulation was compared to RNA labeled in the absence of the hormone. For the poly(A)-containing fraction (oligo-dT bound), several loci hybridize only RNA labeled in the presence of ecdysone; one locus hybridizes only control RNA. The majority of hybridizing loci are unaffected by the hormone. Of the loci hybridizing RNA not bound to oligo-dT, several appear specific for the ecdysone-treated sample, though most are labeled more heavily with this RNA than with the control. None of the ecdysone-sensitive loci visualized by in situ hybridization are the sites of salivary gland puffs induced by ecdysone on the same time scale.     

Tritiated-histidine incorporation into Drosophila salivary chromosomes

An autoradiographic study of H³-histidine incorporation into nonhistone protein of explanted larval salivary gland chromosomes of D. virilis showed patterns of incorporation that were dependent upon the stage of larval development. The sequence of changes in the development of several puffs in a specific chromosomal region was followed using the appearance of pigment in the anterior spiracles as a means of larval staging. H³-histidine incorporation into these puffs in prepupae occurred as the puffs were regressing in size and protein staining. Acid extraction of histone and nucleic acid failed to alter the character of the autographs; presumably a non-histone protein is involved in the H³-histidine incorporation. Other puff sites in the same prepupal chromosomes showed various patterns of isotopic amino acid incorporation indicating that the pattern reported for a specific region may not be true for all puff sites.     

Chromosome location of the ribosomal RNA genes in Triturus vulgaris meridionalis (Amphibia,Urodela)

The mitotic chromosomes of six specimens from Triturus vulgaris meridionalis have been examined by both in situ hybridization with ³H 18S+28S rRNA and AS-SAT staining method. The results of these two sets of experiments can be summarized as follows: 1) in each specimen the NORs and the additional ribosomal sites, which react positively to in situ hybridization with ³H 18S + 28S rRNA, are also stained by silver; 2) other chromosomal regions, which do not hybridize in situ with ³H 18S+28S rRNA, are on the other hand stained by the AS-SAT method. These latter Ag-positive sites show a species-specific pattern of chromosomal distribution.     

Photoinduced bonding of endogenous ecdysterone to salivary gland chromosomes of Chironomus tentans

Endogenous ecdysterone has been bonded to chromosomal loci by irradiation of Ch. tentans salivary glands. The hormone has been localized on the polytene chromosomes by indirect immunofluorescence microscopy. Hormone binding to chromosomes is stage-specific. Seven chromosomal loci could be identified which specifically bound hormone in larval salivary glands, and 21 chromosomal loci which specifically bound hormone in prepupal salivary glands. All puffs that have been described by Clever (1961) as being inducible by ecdysterone have been found to contain irreversibly bound ecdysterone in prepupal salivary gland chromosomes. A small number of puff sites in larval salivary gland chromosomes exhibited varying amounts of bound ecdysterone, (as judged by fluorescence intensity) most notably 117B and Balbiani rings 1 and 3 on chromosome IV. In addition to stage specific binding sites, there were many others showing equal binding of the hormone in both, larval and prepupal, stages of development. — Fluorescence intensities (reflecting the amount of bonded hormone) at puff sites along the tip section of the prepupal salivary gland chromosome arm IR have been computed indicating that differences between fluorescence intensities of different puffs can be expressed as multiples of a basic fluorescence intensity. Thus, the amount of fluorescence intensity (bonded hormone) in the various puffs may be quantized. — The data indicate that in Ch. tentans salivary glands ecdysterone acts, at the chromosomal level. The development of larvae into prepupae generates more puff sites and more hormone binding. This is discussed in the light of current models of hormone-receptor function.     

The activity of two heat shock loci of Drosophila hydei in tissue culture cells and salivary gland cells as analyzed by in situ hybridization of complementary DNA

Complementary DNA was made to poly A⁺ nuclear or polysomal RNA isolated from heat shock tissue culture cells of Drosophila hydei. A number of loci other than the four major heat shock loci are labelled after in situ hybridization of these cDNA preparations, while solution hybridization indicated that only about 10% of the cDNA was specific for heat shocked cells. Removal of the fraction of cDNA which could react with 25° C RNA and subsequent in situ hybridization of heat shock specific cDNA indicated that locus 4–81 B, a major salivary gland heat shock locus, is also active at 25° C in tissue culture cells, while locus 4–85 B is specifically activated by heat shock in tissue culture cells. This latter locus is not seen to be clearly puffed in salivary glands, but was shown to be active in that tissue both by direct autoradiography of salivary gland chromosomes after 3H-uridine labeling and by hybridization of cDNA to chromosomal RNA.     

The effect of ecdysterone on nonhistone proteins in salivary gland chromosomes of Sciara coprophila.

Synthesis and transport of proteins to the cell nucleus during puff induction was studied in S. coprophila. Changes in grain distribution along chromosomes (L-methionine [35S] incorporation into protein) were correlated with puffs induced by ecdysterone in vitro; A pattern of specific labelling at the sites of incipient puffs was noted within 2 h after the addition of the hormone, i.e. grains on the chromosomes were in clusters, characteristic for this time point and not seen in the controls (where only non-specific labeling was noted 0-4 h). Characteristic chromosomal puffs appeared between 3-4 h after the addition of ecdysterone. It was concluded that during ecdysterone-induced puff formation in salivary gland chromosomes, proteins which had been previously synthesized were selectively transported from the cytoplasm to specific sites on the chromosomes.     

Digitonin treatment activates specific genes including the heat-shock genes in salivary glands of Drosophila melanogaster

A large number of chromosomal sites were found to form puffs in Drosophila salivary glands after treatment with the mild detergent digitonin and incubation in a defined medium for 2 hr. The cytological locations of these puffs were determined, and the puff size was measured at 43 loci in both digitonin-treated salivary glands and intact glands. On the basis of comparisons of puffing between digitonin-treated and intact salivary glands, the puffs were classified into three categories: (1) digitonin-unaffected preexisting puffs (8 sites), (2) digitonin-activated preexisting puffs (6 sites), and (3) digitonin-induced new puffs ("digitonin puffs", 29 sites). The digitonin puffs included some of the developmentally regulated puffs and all the heat-shock puffs known in Drosophila melanogaster. The activation of the specific loci by digitonin treatment suggests that gene expression at these loci is suppressed in salivary glands by a mechanism(s) sensitive to digitonin.     

Induction of chromosome puffs in Drosophila hydei salivary glands after inhibition of RNA synthesis by α-amanitin

The effect of injection of various concentrations (4 ng to 0.5 g/larva) of -amanitin on chromosomal RNA synthesis in larval salivary glands of D. hydei was investigated at subsequent time intervals (90 min to 20 hrs) after injection. — As revealed by autoradiography, ³H-uridine incorporation into the polytene chromosomes was greatly reduced as compared with that in control larvae. In -amanitin injected larvae, new chromosome puffs could be induced by a temperature treatment. These puffs never attained a size comparable to that in the controls. The newly induced puffs did not show specific incorporation of ³H-uridine. — Puffs which were active before the toxin was applied undergo a reduction in size and incorporation of ³H-uridine.     

Cytological and autoradiographic studies in Sciara coprophila salivary gland chromosomes

Summary Morphological and metabolic changes on the salivary chromosomes of Sciara coprophila were followed during the later half of the fourth larval instar.Cytological maps were prepared for five successive stages from mid-fourth instar to the prepupal stage. These maps, which constitute a revision of those published earlier by Crouse, summarized our cytological findings and were the basis for studies on DNA replication of these chromosomes.Similar to earlier studies in Chironomidae, differences in the puffing pattern were noted between the anterior and the posterior portions of the salivary gland. The most striking difference was noted in region 2B on chromosome III which produces a large puff only in nuclei from the anterior part of the gland. Other autosomal puffs, although present in both parts of the gland, showed constant differences in size.An increase in the number of bands from mid-fourth to late fourth instar was observed. The new bands are all of the light-staining kind.In Sciara the puffed area may include a large number of bands in addition to the bands which originated the puff. The maximal extent of puffs was determined in terms of chromosomal map regions and the number of bands subject to obliteration.In the autoradiographic experiments use was made of H³-thymidine as DNA precursor. The aim of these studies was to detect any asynchronies in the replication time of bands. In fact, marked differences in the relative rates of uptake of H³-thymidine of a number of bands in a certain proportion of chromosomes have been observed, while others showed uniform incorporation. Since these latter were found with higher frequency the period of uniform labeling must comprise a larger part of the replication cycle then the periods of localized labeling. To assess the validity and constancy of the observed patterns of unequal incorporation, a semiquantitative analysis was carried out. It showed that the bands showing localized uptake may be separated into two broad groups. In one of these groups are the centromere regions and certain chromosomal ends, which are presumably heterochromatic. The other group comprises most of the puff sites and bulbs. Since late replication is characteristic of heterochromatin, we assumed that bands of the former group (C) replicate late in the cycle, while puffs and bulbs start replication early, and the period of equal labeling is intermediate. Other intermediate labeling patterns were observed and are described.It is known that in the fourth instar from two to three DNA replications occur in the salivary gland nuclei, the last of which coincides with puffing. Several stages may be distinguished in the puffing process based on morphology and rates of isotope uptake of the puffs. The first sign of puffing is a very high rate of incorporation at puffs. It is maintained throughout this last DNA synthesis period and only declines when all other chromosomal regions have ceased to replicate. A pattern of high and exclusive uptake at the heterochromatic sites (pattern C) was never observed in this replication; instead puffs are the last regions to terminate DNA synthesis.These results are discussed in relation to several current problems, such as, asynchronous DNA replication, the problem of metabolic DNA, and the concept of the heterochromatic state.Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, in the Faculty of Pure Science, Department of Zoology, Columbia University, New York. This work has been supported by U.S. Public Health Training Grant No. 2Tl-GM-216-05; partial support has been received also from Grants GB 42 and G-14043 from the National Science Foundation to Dr. H. V. Crouse.     

Segments of chromosomal DNA from Rhynchosciara americana that undergo additional rounds of DNA replication in the salivary gland DNA puffs have only weak ARS activity in yeast

We have constructed a library of recombinant phage containing DNA from salivary gland chromosomes of Rhynchosciara americana. We have isolated phage from this library that carry sequences homologous to cDNA clones that hybridize in situ to the DNA puffs at the polytene chromosome regions C3 and C8. This has enabled us to demonstrate a 16-fold amplification of the genomic DNA sequences at these regions during DNA-puffing. At the C8 site there is a sequence element that has characteristics of 'scrambled' moderately repetitive DNA. This is located within 3 kb from the gene encoding a 1.95-kb mRNA. We have assayed restriction fragments from the two DNA puffs for Ars activity in yeast. The only strong Ars activity is associated with a part of the moderately repetitive DNA element from the C8 puff which is not present at this site in all animals.     

Replication in Drosophila chromosomes

Prolongation of larval life in Drosophila melanogaster, by growing wild type larvae at lower temperature, or in animals carrying the X-linked mutation giant is known to result in a greater proportion of nuclei in salivary glands showing the highest level of polyteny. We have examined by autoradiography the patterns of ³H-thymidine incorporation during 10 min or 1 min pulses in salivary gland polytene chromosomes of older giant larvae and of wild type late third instar larvae of D. melanogaster grown since hatching either at 24 ° C or at 10 ° C. The various patterns of labelling and their relative frequencies are generally similar in glands from the warm-(24 ° C) or cold (10 ° C)-reared wild type larvae, except the interband (IB) labelling patterns which are very frequent in the later group but rare in the former. The IB type labelled nuclei in cold-reared wild type larvae show labelling ranging from only a few puffs/interbands labelled to nearly all puffs/interbands labelled. In warm-reared wild type larvae, very low labelled IB patterns are not seen. In older giant larvae, the ³H-thymidine labelling patterns are in most respects similar to those seen in cold-reared wild type larvae. In 1 min pulsed preparations from all larvae, the IB patterns are relatively more frequent than in corresponding 10 min pulsed preparations. No nuclei with the continuous (2C or 3C) type of labelling pattern, with all bands and interbands/puffs labelled, were seen in 1 min pulsed preparations from cold-reared wild type or in giant larvae, and only a few nuclei in 1 min pulsed preparations from warm-reared wild type larvae exhibited the 2C labelling pattern. Analysis of silver grain density on specific late replicating sites in late discontinuous (1D) type labelled nuclei suggests that the rate of DNA synthesis per chromosomal site is not different at the two developmental temperatures. It is suggested that correlated with the prolongation of larval life under cold-rearing conditions or in giant larvae, the polytene replication cycles are also prolonged. It is further suggested that the polytene S-period in these larvae is longer due to a considerable asynchrony in the initiation and termination of replication of different sites during a replication cycle.