Puffing activities and binding of ecdysteroid to polytene chromosomes of Drosophila melanogaster

Salivary glands of third instar Drosophila melanogaster larvae were incubated in vitro in the presence of 5 x 10(-6) M 20-hydroxy-ecdysone. Steroid hormone was localized on the polytene chromosomes of the salivary gland by a combination of photoaffinity-labeling and indirect immunofluorescence microscopy. Steroid hormone binding to chromosomal loci and their puffing activity was correlated for the larval/prepupal puffing cycle characterized by puff stages 1-10. In general, there was a good correlation between the sequential and temporal puffing activity induced by 20-hydroxy-ecdysone and the binding of ecdysteroid hormone to these puffs. Ecdysteroid hormone was detected at intermolt, and at early and late puffs with two notable exceptions. Ecdysteroid was not detected at the two well-studied puffs at 23E and at 25AC, the former being an early puff, which is activated in the presence of 20-hydroxy-ecdysone, and the latter being an intermolt puff, which regresses more rapidly in the presence of hormone. Ecdysteroid hormone was present at puffs as long as the respective puff was active. Also, it apparently accumulated at late puff sites after induction. Since ecdysteroid binding to chromosomal loci is temporal as well as sequential during the larval/prepupal puffing cycle, additional factors besides steroid hormone are necessary for sequentially regulating puffing and concomitant gene activity during development from larvae to prepupae.     

Patterns of puffing activity in the salivary gland chromosomes of Drosophila

The autosomal salivary gland chromosome puffing patterns of Drosophila simulans are described and compared with the puffing patterns of the sibling species D. melanogaster. During the late third larval instar and the prepupal period the patterns of puffing activity of these two species are similar — approximately 50% of the puffs common to both species showing identical activities. The remaining puffs differ in their timing of activity, or in their mean sizes, or in both of these parameters. A number of puffs (14) found in D. simulans have not been regularly observed in the Oregon stock of D. melanogaster but are active in other D. melanogaster strains. One puff (46 A) of D. melanogaster was absent from D. simulans and forms a heterozygous puff in hybrids, when the homologous chromosomes are synapsed. When the homologues are asynapsed a puff at 46 A is restricted to the melanogaster homologue. The puff at 63E on chromosome arm 3L is considerably smaller in D. simulans than in D. melanogaster and this size difference is autonomous in hybrids. Other puffs not common to both species behave non-autonomously in the species hybrid, even when the homologous chromosomes are asynapsed.     

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 effect of prolonged exposure to heat on the activity of salivary gland polytene chromosomes]

The influence of long-term heating on the puffing activity of polytene chromosomes in the early prepupa salivary glands was investigated. The activity of puffs was estimated by two criteria: size and frequency. The rearing of insects at a temperature of 29 degrees resulted in puff changes: the activity of some puffs increased or depressed, some puffs were inhibited, other puffs were induced newly. The differential response of each chromosome was observed. A possible mechanism of the effect of heating on the puff activity of polytene chromosomes is discussed.     

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.     

RNA Polymerase B (or II) in heat induced puffs of Drosophila polytene chromosomes

Using indirect immunofluorescence visualization techniques we investigated the distribution of RNA polymerase B (or II) and histone H1 at heat shock puff loci in Drosophila melanogaster polytene chromosomes at different times during and after heat shock. After heat treatments of from 5 to 45 min, the heat shock puff displayed intense fluorescence when stained for RNA polymerase B, but relatively little fluorescence when stained for histone H1. Returning heat shocked larvae to room temperature resulted in the appearance of a distinctive pattern of RNA polymerase-associated fluorescence in the heat shock puff at 87C, presumably reflecting events associated with the inactivation and regression of this puff. Large differences observed in the apparent RNA polymerase B content of puffs of similar size suggest that the interaction of RNA polymerase B with chromosomal loci does not depend on simply the state of condensation or decondensation of the chromatin.     

Distribution patterns of three subfractions of drosophila nonhistone chromosomal proteins: possible correlations with gene activity.

The distribution of three molecular weight subfractions of the Drosophila nonhistone chromosomal proteins (NHC proteins) has been studied using an immunofluorescent technique (Silver and Elgin, 1976). In all three cases, the fluorescence distribution patterns obtained are distinct and reproducible. The results imply that different NHC protein components have different distributions along the polytene chromosomes. A highly selective pattern is obtained using antiserum against subfraction ?; puffs (loci highly active in RNA synthesis) and many nonpuffed chromomeres which are known to puff at other times during the third larval instar or prepupal stages are brightly fluorescent. New RNA synthesis can be induced at 87A, 87B-C1 and other chromomeres by heat shock treatment; these loci, previously stained at low levels, are subsequently stained brightly using the ? serum. The staining of the heat shock puffs appears to be superimposed upon the prior ? pattern. The results suggest that a change in chromosomal structure, as indicated by staining using the ? serum, is associated with gene activity as indicated by puffing. This different chromosomal structure may be the consequence of either a redistribution of a ? antigenic determinant [a new association of specific protein(s) with the active sites] or a change in chromatin configuration [making the ? antigenic determinant(s) newly available to the antibody probe].     

Localization of nuclear proteins related to high mobility group protein 14 (HMG 14) in polytene chromosomes

An antibody was raised against high mobility group nuclear protein 14 (HMG 14) from calf thymus, known to be associated with actively transcribed chromatin. By means of indirect immunofluorescence, it was shown to react with the nuclei of mouse fibroblasts and of brain cells from Xenopus and Drosophila, but not of Xenopus erythrocytes. The antibody was used to detect immunologically related proteins in giant chromosomes of the midge, Chironomus pallidivittatus. Indirect immunofluorescence with anti-HMG 14 antibody in polytene nuclei was restricted to the active puffs. Giant puffs (Balbiani rings) exhibited especially intense fluorescence in their peripheral regions. An inducible puff site, the Balbiani ring 6 locus, showed no reaction with the antibody prior to induction. When puff formation began, the chromosome site assumed a very intense fluorescence, which disappeared again when the Balbiani ring was recondensed. — Protein extracts of salivary gland nuclei were found on immunoblots to contain one major protein fraction that reacted with the anti-HMG 14 antibody. The electrophoretic mobility of this fraction was similar to that of calf thymus HMG 17. — It is concluded that actively transcribed puffs in polytene chromosomes contain HMG 14-related protein(s) that are not present in potentially active gene loci prior to induction.This paper is dedicated to Prof. Hans Bauer on the occasion of his 80th birthday.