Cytogenetic analysis of region 2B3-4-2B11 of the X-chromosome of Drosophila melanogaster

About 160 kb of DNA were cloned from the 2B region of the X chromosome, where the early ecdysone puff develops and the ecs locus is located. On the physical map of this sequence the positions of 13 chromosome rearrangement breakpoints interfering with both puff development and the ecs locus proximally and distally, were plotted by means of in situ hybridization. The maximal size of the ecs locus is about 100 kb (between the breakpoint of In(1)Hw ^49c and the proximal end of Df(1)St472) The DNA sequences essential for normal puffing are located within the ecs locus between the In(1)br ^lt103 and Df(1)St472 breakpoints and comprise about 65 kb. Thus the puff develops as a result of ecs activation. Since Df(1)P154, which reduces the puff size and removes the proximal part of the ecs locus, does not prevent puff induction by ecdysone, while removing the distal part of the locus by Df(1)St469 completely stops development of the puff, we conclude that the regulatory zone of the locus, which reacts to hormone is located in the distal parts of both the puff and the locus, proximal to the breakpoint of In(1)br ^lt103 .Since In(1)br ^lt103 , Df(1)pn7b and Df(1)br ^R1 damage ecs but do not prevent puffing it is proposed that there is a second regulatory zone for this locus with a minimal size of 15–20 kb (between the breakpoints of Df(1)br ^R1 and In(1)br ^lt103). After cytogenetic and electron microscopic analysis of 2B puff formation it seems very likely that the site of puff formation is situated in the proximal part of 2B3-4 and after enhancement of ecs expression by hormone it spreads proximally to the 2B6 band which does not puff. When the puff regresses at puff stages (PS)10-11 its material does not condense completely and a zone of residual puffing joins the condensed material located distal to it. This material can give the impression of a separate band, designated 2B5 in Bridges' map. For convenience we propose to call the site giving rise to the puff as 2B3-5.     

Cytogenetic analysis of the 2B3-4-2B11 Region of the X chromosome of Drosophila melanogaster

Larvae homozygous or hemizygous for the l(l) t435 mutation located within the early ecdysteroid puff 2B5, or carrying a deletion of the 2B5 band, die at the end of the third larval instar. In the salivary gland chromosomes of these larvae only intermoult puffs are detected. If these salivary glands are incubated in vitro with 20-OH ecdysone for 6 h the intermoult puff 68 C remains large, some early puffs (74EF and 75B) are induced to 30–40% of their normal size, other early (63F) and all late puffs (62E, 78D, 82F and 63E) are not induced at all. Puff 2B5 reaches its normal size but does not regress after 6h incubation with 20-OH ecdysone, as it does in normal stocks. The data obtained in this study show the existence of a locus (or loci) in the band (puff) 2B5 which is necessary for the normal response of the salivary gland chromosomes to the hormone 20-OH ecdysone.     

The hormone ecdysone as effector of specific changes in the pattern of gene activities of Drosophila hydei

The hormone ecdysone induces a large number of changes in the puffing pattern of mid third instar larvae of Drosophila hydei. The pattern of changes occurring after experimental administration of the hormone are identical with those observed in normal development during a 6 hour period before puparium formation. After administration of the hormone a considerable number of puffs react with a change in activity within 15–20 min. During this period 3 puffs arise newly, 12 puffs show a strong increase in activity, 6 puffs show a less pronounced increase in activity and 12 puffs show a decrease in activity. At a period of 4–6 hours after administration of the hormone another 5 puffs arise newly. The effect of the hormone was identical in both in vivo and in vitro experiments. — Diameter measurements on several puffs reacting within 30 min with an increase in diameter showed that these puffs reacted simultaneously. Most of the puffs that showed a decrease in activity reacted with some delay. — A study of the effect of different hormone concentrations revealed that the kinetics of 4 puffs with respect to the relationship between concentration and puff size was identical over a range of concentrations from 33·10^–5 to 33CU/l. Three of these puffs showed a reaction with even lower concentrations. Maximum puff size is attained by all puffs at a concentration of 33·10^–4CU/l. Among the puffs studied no difference in their reaction threshold was found. — A study of the behavior of 5 puffs of the group reacting within 15–20 min and one of the group reacting after 4–6 hours in midintestine and Malpighian tubules revealed that these puffs showed the same reaction after injection of the hormone as observed in the salivary glands. — All puffs activated by administration of the hormone showed particularly strong uptake of tritiated uridine and accumulation of acidic protein. — It is concluded that the hormone ecdysone induces a pattern of changes in gene activity that is far more complex in Drosophila hydei than in Chironomus tentans.     

Developmental changes in genome activity in Drosophila lebanonensis casteeli pipkin

Puparium formation in Drosophila lebanonensis casteeli is obviously restricted to a certain phase in circadian oscillation. The question whether or not the release of molting hormone is the actual process which is controlled by the circadian oscillation could be approached by using molting hormone-specific changes in genome activity as indication for changes in hormone titer. The identification of hormone specific changes in the puffing pattern of polytene chromosomes should provide a basis for this study.—To this end, a chromosome map of the 7 polytene chromosome arms (1 acrocentric and 3 metacentric chromosomes) of the species was made. Changes in the puffing pattern associated with puparium formation are described and compared with those occurring in response to experimental administration of -ecdysone.—89 puffs were regularly observed in midthird instar larvae. Prior to puparium formation 5 new puffs arise, one at an early stage and 4 attaining their maximum size immediately before puparium formation. Concomitantly, 5 puffs increase considerably in size. These changes in the puffing pattern can be reproduced by injection of ecdysone.—Upon injection of the hormone a clear differentiation between fast reacting loci (within 30–60 min) and slow reacting loci (after 3–4 hours) can be found. As in other Drosophila species the immediate response (within 30–60 min) comprises more than one (5) locus.In memory of Professor Dr. J. Schultz.     

Conservation of the 93D puff of Drosophila melanogaster in different species of Drosophila

Temperature shock (TS) results in activation of a specific set of puffs in polytene nuclei of D. melanogaster. Earlier studies in this species from several laboratories revealed certain unique features of the major TS puff at 93D locus, which is also specifically induced by benzamide (BM) and by incubation of glands in heat shocked glands' homogenate (HSGH). We have now extended studies on TS response to several other species of Drosophila to ascertain whether loci homologous to 93D puff of D. melanogaster are present in other species. In polytene nuclei of two closely related (D. ananassae, D. kikkawai) and in two distantly related species (D. hydei, D. nasuta), six to nine puffs are induced by TS. Interestingly, in each species one of the major TS puffs, viz., 2L-2C in D. ananassae, E-11BC in D. kikkawai, 2R-48A in D. nasuta and 2-48C in D. hydei, is also specifically induced by BM, autologous species' HSGH and vitamine-B₆ (vit-B₆) treatment. HSGH of a different species fails to induce these puffs. These puffs thus resemble the 93D locus of D. melanogaster, although the 93D puff does not respond to vit-B₆. These observations are discussed in relation to the conservation of 93D puff locus in different species of Drosophila.     

Comparative characteristics of the puffing pattern and the endocrine system in an oregon laboratory stock and in l(2)gl Drosophila melanogaster mutants differing in the time of their death

This study shows that homozygotes for different alleles of the lethal mutant, l(2)gl, differing in the time of death also vary in the state of their endocrine system and the puffing patterns of their salivary gland chromosomes. Homozygotes which die at the larval stage have underdeveloped prothoracic glands and normal corpora allata (CA); in those dying at the prepupal stage both the prothoracic glands and the CA are equally underdeveloped. — All the early third instar larval puffs (96–110 h., PS 1–2) develop in homozygotes; however, the reduction of some early larval puffs, normally occurring before pupariation or at puparium formation, is delayed. Some puffs are more developed than normal. — The differences in puffing patterns chiefly concerned puffs which normally appear 4–5 h before puparium formation and at puparium formation. In homozygotes lethal as larvae some of the puffs normally active at this time did not develop. However, along with some of the late larval puffs, there appeared many puffs characteristic of prepupae. — In homozygotes lethal as prepupae only the time and sequence of puff appearance was altered. Many late larval puffs were active in prepupae rather than in larvae, whereas some of the puffs, normally appearing in prepupae, were active in the larval stage.Accordingly, we propose to distinguish two groups of puff loci. 1) Hormone dependent puffs: These do not develop in larval lethals and are active only after puparium formation in pupariated lethals. 2) Autonomous puffs: Their appearance depends more on the time of development, than on hormonal background. It is suggested that the induction of hormone dependent puffs and of puparium formation is possible at low ecdysone levels, provided that the juvenile hormone level is also low.     

Cytogenetic analysis of the 2B3-4 — 2B11 region of the X chromosome of Drosophila melanogaster

Puffing patterns have been studied both in homozygotes t10/t10, a gene located in the area of the early ecdysone puff 2B5, and in a yellow (y) control stock, at the end of the third instar and during prepupal development. In mutants t10 at the end of the third instar puffing develops normally in general, however, 21 puffs (5 early and 16 late ones) underdevelop or do not develop at all, some larval intermoult puffs regressing slower. The next cycle of puffs (mid prepupal) in mutants t10 proceeds normally, but in the late prepupal cycle 21 puffs underdevelop again or are not formed at all. A model for the induction of early ecdysone puffs is proposed, assigning a key role to the 2B5 puff product in stimulating other early puffs. It is suggested that defects in the activity of early puffs in the mutant t10 may cause underdevelopment of late puffs.Dedicated to Professor W. Beermann on the occasion of his 60th birthday     

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.     

Comparative study of the function of polytene chromosomes in laboratory stocks of Drosophila melanogaster and the l(3)tl mutant (lethal tumorous larvae)

A study of the puffing pattern of the salivary gland autosomes of D. melanogaster was performed through the last 24 hours of larval development and 0-hour prepupae. Since both prominent and small puffs were taken into account, the total puff number amounted to 275. Of these, 116 are almost constant in size during the 24 hours observation period, 106 increase in size or appear before pupation. 37 puffs are active in 96 hour larvae and disappear or decrease sharply in size by 115–118 hours. 12 biphasic puffs have been found with higher activity in 96 hour larvae and 0-hour prepupae and lower activity by 115–118 hours. Three extremely irregular puffs have been detected in chromosome 4. The data obtained evidence that a larger number of D. melanogaster polytene chromosome loci are active during larval development than it has been thought earlier. It has also been shown that only 38% of autosomal puffs change before the beginning of metamorphosis. The functional significance of small puffs and strain specificity of puffs are discussed.     

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.     

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.     

Cytogenetic analysis of the X-chromosome region 2B1-2-2B9-10 of Drosophila melanogaster

In salivary glands of yellow control stock the puffing pattern in the ecdysone-added artificial C46P medium was on the whole similar to that observed during larval development in vivo. However, underdevelopment of a series of late puffs and a delay in the regression of early puffs were observed. In addition a set of medium puffs not visible in vivo appeared. Late puffs differed from those developing in Grace medium.When salivary glands of homozygotes for the lethal dor ^lt187, a mutation that causes death in the third instar with no signs of ecdysone induction were incubated with ecdysterone, the development of puffs was restored, i.e., the puffing pattern of mutant cells in vitro practically did not differ from that in cells of the control stock. This implies that the dor ^lt187 lethal allele belongs to the class of ecdysone-deficient mutations.     

Action of hydroxyurea on chromosome physiology in Rhynchosciara angelae

Experiments have been performed to investigate the action of hydroxyurea (H.U.) on the polytene chromosomes of the salivary gland of Rhynchosciara angelae. After different times of H.U. treatment, larvae were injected with ³H-thymidine for a pulse of 10 min. DNA puffs were analysed especially in those regions where differential incorporation of thymidine occurs. H.U. progressively inhibited thymidine incorporation all over the chromosome. The maximum of inhibition occurs 9 hours after the treatment. However, after 227 hours the chromosome label was similar to that in the controls and puff 2B recovered its original size. The puff 3C showed a delay in its appearance. Our results show that H.U. inhibits temporarily the opening rate of the puff, as well as DNA synthesis. There is no reaction on RNA puffs.This work was supported by a grant from the National Institutes of Health (GM 17590-03), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) of which one of us (G.M.M.S.) was a fellow during this research, and the Conselho Nacional de Pesquisas (CNPq).     

Cytogenetic analysis of the X chromosome region 2B3-4 — 2B11 of Drosophila melanogaster

Mutation t467, belonging to the swi complementation group, and causing death in late prepupa, is located in the interval from 2B6 to the left part of 2B7-8. In this region puffing is absent in salivary gland chromosomes. In t467/t467 homozygotes intermoult early and early-late larval 20-OH ecdysone puffs do not differ from the controls. Mid-prepupal puffs are normal too with a few exceptions. However, all late larval and prepupal puffs are reduced or absent in the mutant. Both, hormone incubation of t467 glands in vitro and hormone injection have shown: i) 20-OH ecdysone in vitro does not restore the normal larval puffing pattern. ii) Withdrawal of the hormone from glands at PS6 causes premature appearance of late larval puffs, which, however, do not reach control sizes. It is concluded that the swi gene product is necessary for induction of late puffs. Thus in the 2B3-4—2B7-8 region three genes, affecting 20-OH ecdysone induction processes, have become known.     

Temperature-induced Balbiani rings in Chironomus thummi

The formation of a new telomeric Balbiani ring in the right arm of chromosome III (T-BR III) has been induced in Chironomus thummi larvae by applying a wide range of temperature treatments (33 °–39 ° C). In this paper we present some kinetic and functional characteristics of this structure. T-BR III incorporates tritiated uridine, and during its formation accumulation of acidic proteins takes place. However, induction and maintenance of this puff structure appear to be insensitive to Actinomycin treatment. An additional T-BR can be induced in chromosome I by employing the most drastic temperature treatments (37 °–39 ° C). We also report the existence of a group of puffs active after heat treatments in Chironomus polytene chromosomes which could be homologous with the T-puffs of Drosophila.     

Cytogenetic and molecular aspects of position effect variegation in Drosophila

Variations in compaction of chromosomal material of the rearrangements Dp(1;f) 1337, Dp(1;f) R, Dp(1;1)pn2b, and T(1;4)w ^m258-21, which display an extended position effect, were characterized. Morphological changes found in these rearangements were assigned to two major types: (i) continuous compaction, in which bands and interbands located distal to the eu/heterochromatin junction fuse into one compacted block of chromatin. The extent of compaction is increased by enhancers of position effect (low temperature, removal of the Y or 2R chromosome heterochromatin). In extreme cases compaction extends over dozens of bands. (ii) Discontinuous compaction, in which at least two zones of compaction separated by morphologically normal zones can readily be identified. As a result, some regions located at a greater distance from heterochromatin may be compacted more frequently than others than map nearer to it. A few regions (1D, 2B1-12, 2D) were shown to be most frequently compacted in all rearrangements investigated. The 2B13-18, 2C1-2, 2E, and 2F regions exhibited the lowest frequencies of compaction. Compaction of the zone containing the 2B1-12 bands is always accompanied by inactivation of the ecs locus, which maps in the 2B3-5 puff. At the same time the 2C1-2 and 2E bands located nearer to the breakpoint can retain normal morphology and puffing in response to ecdysterone. The results are interpreted as morphological manifestations of the discontinuity of the spreading effect.by W. Beermann     

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.