Digitonin Activates Different Sets of Puff Loci Depending on Developmental Stages in Drosophila melanogaster Salivary Glands

We showed previously that treatment of Drosophila melanogaster salivary glands with a mild detergent, digitonin, induces heat shock puffs and many developmentally regulated puffs. To find if the mechanism underlying the puff induction by digitonin is related to the temporal control of gene expression in salivary glands, we examined effects of digitonin on salivary glands at various puff stages from late third instar larva to white prepupa. The results indicate that (a) all the heat shock puffs are induced by digitonin irrespective of the developmental stage of the treated glands, (b) intermolt and early puff loci are always irresponsive to digitonin, and (c) late puff loci respond to digitonin to form puffs only before the stage of their developmentally programmed puffing. Based on the stage at which the locus becomes digitonin responsive, the digitonin-responsive late puff loci were divided into two groups: group A loci, responsive to digitonin continuously from PS1 until programmed puffing begins, and group B loci, responsive to digitonin only in a short period of time immediately before the programmed puffing. The results suggest that a digitonin-sensitive suppression mechanism(s) is involved in the temporal control of gene expression in Drosophila salivary glands.     

The Drosophila Eip78c Gene Is Not Vital but Has a Role in Regulating Chromosome Puffs

We have generated a number of chromosomal aberrations that disrupt the early-late ecdysone-induced 78C puff gene (Eip78C, ecdysone-induced protein, FlyBase name for the E78 gene of STONE and THUMMEL 1993), which encodes the two members of the nuclear hormone receptor superfamily Eip78C-A and Eip78C-B. The aberrations include deletions of the ligand-binding/dimerization domain of both, inversions that split Eip78C-A but retain residual Eip78C-B expression, and a small deletion specific for Eip78C-B. We find that wild-type Eip78C functions are completely dispensable for normal development under laboratory conditions. However, we show that Eip78C-B is required for the maximal puffing activity of a subset of late puffs (63E and 82F) since these puffs are reduced in size in Eip78C-B mutant backgrounds. Paradoxically the same late puffs are reduced, as well as at least one other, when the Eip78C-B cDNA is overexpressed from a heat shock promoter. These data indicate either that Eip78C function is redundant or that it plays a subtle modulating role in the regulation of chromosome puffing.     

Formation and morphology of dark puffs in Drosophila melanogaster polytene chromosomes

The formation of unusual dark puffs in Drosophila melanogaster polytene chromosomes has been studied by electron microscopic (EM) analysis. Fly stocks transformed by the P[ry; Prat:bw] and P[hs-BRC-z1] constructs were used. In the former the bw gene is under the promoter of a housekeeping gene, Prat; in the latter the Br-C locus, mapping to the dark puff 2B, is under the promoter of a heat-shock gene, hsp70. Inserted into region 65A of the 3L chromosome, the Prat:bw copies give rise to structures which are morphologically reminiscent of the so-called "dark" puffs. In contrast, insertion of P[hs-BRC-z1] into region 99B of the 3R chromosome causes a regular "light" puff of form. Comparative analysis of the dark puffs--both transgenic and natural--suggests that there might be at least two mechanisms underlying their formation. One is a local incomplete decondensation of activated bands, characteristic of the so-called small puffs. The other is the formation of ectopic-looking contacts between the bands adjacent to the puffing zone. Transposition of the DNA, from which such a puff develops, causes a regular light puff to form at the new location. Heterochromatic regions do not appear to be directly involved in puffing.     

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.     

A change in the puff spectrum during development of Drosophila virilis]

The puff spectrum has been studied in the salivary gland chromosomes of D. virilis from 48 hrs of the 3rd larval until the gland lysis. 142 puffs were observed in the D. virilis genome. Among them, 43% were observed at all developmental stages and other puffs were unstable: 47.1% appear at a certain stage and degrade or remain until the gland lysis; 9.9% are characterized by the "pulse" activity, i.e. they appear at a certain stage, degrade and reappear. The number of newly appeared puffs exceeds that of fully degraded ones, i.e. the number of puffs during the gland development until its lysis increases constantly. At the stage of puparium formation, sexual differences in the puff length were found: puffs were longer in males than in females.     

Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. I. Dependence upon ecdysone concentration

The response of the three major classes of puff in salivary gland chromosomes of larval Drosophila melanogaster to varying β-ecdysone concentrations has been studied in in vitro cultured glands. Two (25AC and 68C) of the intermolt puffs regress at a rate dependent upon the hormone concentration. Three rapidly reacting puffs (23E, 74EF and 75B) respond in a graded way to β-ecdysone concentrations over a range of at least 600 ×. In contrast, five late-reacting puffs (62E, 78D, 22C, 63E, and 82F) do not respond below 5 × 10^?8M and at 2.5 × 10^?7M react maximally. The 50% response of the early puff sites 74EF and 75B and of the late puff sites occurs at 1 × 10^?7M. Two points are discussed in detail: whether ecdysone is necessary as a sustained stimulus or only as a trigger for the sequential puffing response and an evaluation of the absolute ecdysone concentration necessary for induction.     

Modeling dark puffs using P-transposons in Drosophila melanogaster polytene chromosomes

Modeling of morphologically unusual "dark" puffs was conducted using Drosophila melanogaster strains transformed by construct P[ry; Prat:bw], in which gene brown is controlled by the promoter of the housekeeping gene Prat. In polytene chromosomes, insertions of this type were shown to form structures that are morphologically similar to small puffs. By contrast, the Broad-Complex (Br-C) locus, which normally produce a dark puff in the 2B region of the X chromosome, forms a typical light-colored puffs when transferred to the 99B region of chromosome 3R using P[hs-BRC-z1]. A comparison of transposon-induced puffs with those appearing during normal development indicates that these puff types are formed via two different mechanisms. One mechanism involves decompaction of weakly transcribed bands and is characteristic of small puffs. The other mechanism is associated with contacts between bands adjacent to the puffing zone, which leads to mixing of inactive condensed and actively transcribed decondensed material and forming of large dark puffs.     

Developmental changes in midgut chromosomes of Drosophila gibberosa

In Drosophila gibberosa, differences between midgut and salivary gland chromosomes fall into two categories: tissue-specific band modulations which persist throughout the 90 h developmental period that we studied and tissue-specific puffs. Puffs that are common to both tissues tend to appear earlier in the midgut. Some major early ecdysteroid-induced puffs appear simultaneously in both tissues at the end of the third larval instar; however, the many late puffs that follow in the salivary glands are absent from the midgut. Intense puff activity in the early third larval instar midgut declines at the time of the hormonal pulse that initiates intense gene and secretory activity in salivary glands; the sloughing of midgut cells ensues.     

Ecdysone-regulated puff genes 2000.

The Ashburner model for the hormonal control of polytene chromosome puffing has provided a strong foundation for understanding the basic mechanisms of steroid-regulated gene expression (Cold Spring Harbor Symp. Quant. Biol. 38 (1974) 655). According to this model, the steroid hormone 20-hydroxyecdysone (referred here as ecdysone) directly induces the expression of a small set of early regulatory genes. These genes, in turn, induce a much larger set of late target genes that play a more direct role in controlling the biological responses to the hormone. The recent characterization of two early puff genes, E63-1 and E23, and three late puff genes, D-spinophilin, L63, and L82, provide further confirmation of the Ashburner model. In addition, these studies provide exciting new directions for our understanding of ecdysone signaling. Overexpression studies of E63-1 implicate this gene in directing calcium-dependent salivary gland glue secretion. In contrast, overexpression of E23 indicates that this ABC transporter family member may negatively regulate ecdysone signaling by actively transporting the hormone out of target cells. Finally, genetic studies of the L63 and L82 late genes reveal unexpected possible functions for ecdysone in controlling developmental timing and growth. This review surveys the recent characterization of these ecdysone-inducible genes and provides an overview of how they expand our understanding of ecdysone functions during development.     

C7, a novel nucleolar protein, is the mouse homologue of the Drosophila late puff product L82 and an isoform of human OXR1

The C7 gene was identified in a project aimed to characterize differential gene expression upon attachment of cells to extracellular matrix proteins in vitro. C7 is the homologue of Drosophila L82, a late puff gene (Stowers et al. (1999) Dev. Biol. 213, 116-130) and human OXR1, a gene, which protects cells against oxidation (Volkert et al. (2000) Proc. Natl. Acad. Sci. USA 97, 14530-14535). All are transcribed into multiple splice forms with a common 3' domain. Additional members of this novel gene family are found in a number of eukaryotic species. In the mouse, the C7 gene is highly and broadly expressed during development in at least 4 splice forms, 3 of which were sequenced. In the adult, the C7 gene is most highly expressed in brain and testis. Antibodies to recombinant C7 protein localized to nucleoli in a variety of cell types, suggesting that C7 may be involved in the formation or function of this important organelle.     

Electron microscopical analysis of Drosophila polytene chromosomes

Data are presented of electron microscopic (EM) analysis of consecutive developmental stages of Drosophila melanogaster complex puffs, formed as a result of simultaneous decondensation of several bands. EM mapping principles proposed by us permitted more exact determination of the banding patterns of 19 regions in which 31 puffs develop. It is shown that 20 of them develop as a result of synchronous decondensation of two bands, 7 of three and 4 of one band. Three cases of two-band puff formation when one or both bands undergo partial decondensation are described. In the 50CF, 62CE, 63F and 71CF regions puffing zones are located closely adjacent to each other but the decondensation of separate band groups occurs at different puff stages (PS). These data are interpreted as activation of independently regulated DNA sequences. The decondensation of two or three adjacent bands during formation of the majority of the puffs occurs simultaneously in the very first stages of their development. It demonstrates synchronous activation of the material of several bands presumably affected by a common inductor. Bands adjacent to puffing centres also lose their clarity as the puff develops, probably due to "passive" decondensation connected with puff growth. The morphological data obtained suggest a complex genetic organisation of many puffs.     

Studies on the fine structure of puffs in Sciara coprophila

Fine structure of RNA and DNA puffs of Sciara coprophila was studied during late developmental stages of the fourth larval instar. In RNA puffs the predominant structure seen seems to be a diffuse, lampbrush-like thread or threads sectioned in a variety of planes. The thread is composed of filamentous and granular material. Three types of RNA puffs, each with a slightly different morphology, are found. In their development DNA puffs pass through a precise sequence of stages, each with its distinct morphologic and metabolic characteristics. At the initial and final stages, when much of the puff chromatin is in the compacted state, DNA puffs resemble condensed chromosomal bands. In contrast, at stages when most chromatin is diffuse, DNA puffs share many structural characteristics of RNA puffs. Most of the expanded puff area is permeated by lampbrush-like threads composed of fibrils and granules. RNA and DNA puffs were compared with respect to granule size and distribution by means of electron micrographs of known magnification. The results of the statistical analysis show that: 1) The coefficient of variation (C.V.) of the method of measurement falls between 5 and 7%. 2) There is a fluctuation in granule sizes within each puff with a C.V. of 24–26%. 3) The average granule diameter is 238 Å for DNA puffs and 310 Å for RNA puffs; the difference is statistically significant. 4) The variation in mean granule size in a sample of DNA puffs is rather small (C.V. 12%), while the variation in granule size between different RNA puffs is somewhat larger (C.V. 20%). 5) The relative spread of granule sizes in DNA puffs is more restricted than that in RNA puffs. It is evident then that, on the average, DNA puff granules are smaller and more uniform than granules found in RNA puffs.     

Modeling of Dark Puffs Using P Transposons in Polytene Chromosomes of Drosophila melanogaster

Modeling of morphologically unusual dark puffs was conducted using Drosophila melanogaster strains transformed by construct P[ry; Prat:bw], in which gene brown is controlled by the promoter of the housekeeping gene Prat. In polytene chromosomes, insertions of this type were shown to form structures that are morphologically similar to small puffs. By contrast, the Broad-Complex (Br-C) locus, which normally produce a dark puff in the 2B region of the X chromosome, forms a typical light-colored puff when transferred to the 99B region of chromosome 3R using P[hs-BRC-z1]. A comparison of transposon-induced puffs with those appearing during normal development indicates that these puff types are formed via two different mechanisms. One mechanism involves decompaction of weakly transcribed bands and is characteristic of small puffs. The other mechanism is associated with contacts between bands adjacent to the puffing zone, which leads to mixing of inactive condensed and actively transcribed decondensed material and forming of large dark puffs.     

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.