Patterns of puffing activity in the salivary gland chromosomes of Drosophila

The patterns of puffing activity have been studied during the late larval and prepupal stages of Drosophila melanogaster. On the major salivary gland autosomes (chromosomes 2 and 3) 108 loci form puffs at some time during these developmental stages. The timing and pattern of activity of 83 of these puffs is found to be strictly dependent upon the age of the animals. Two major peaks in puffing activity occur. The first of these is at the time of puparium formation and the second in 8 hr. old prepupae. Both of these puffing peaks precede a moult by 4 hrs. 30 puffs are active before or at the time of both of these two moults. However, the sequence of appearance and regression of many of this group of puffs is different at the prepupal moult than at the pupal moult. 12 puffs occur only before or at the time of the prepupal moult and 13 puffs only before or at the time of the pupal moult. The functional significance of these periods of puffing activity is discussed and it is concluded that one function of this genetic activity in the salivary glands of metamorphosing Drosophila is the production of substances to be utilised during the histogenesis of the adult tissues.     

Autonomous puffing patterns in thoracic and abdominal polytene bristle cell chromosomes of the flesh fly Sarcophaga barbata

The puffing patterns of the thoracic and abdominal polytene bristle cell chromosomes were investigated in Sarcophaga barbata during a 10-day period of pupal development. The autonomous differentiation of imaginal disk descendants is visualized microscopically at the chromosomal level by the cell autonomous puff activities of the polytene bristle cell chromosomes. The sequence of chromosomal activities is strictly stage specific in both cell types. The changes in the puffing pattern are closely corelated with development. The puffing pattern changes synchronously in all bristle cells of a certain body region, e.g., the scutellum or the fifth abdominal tergit. However, there is no synchrony between the puffing pattern changes of the thoracic and abdominal bristle cells. The loci of the abdominal bristle cells are activated one day later than those of the thoracic cells. Each particular puffing pattern truly represents a particular developmental state of the bristle, regardless of body location. That is, the bristle cell chromosomes of various body segments control the timing of their puffing activities autonomously and puff formation and puff regression are not hormonally synchronized.     

Puff induction in larval salivary gland chromosomes ofDrosophila hydei sturtevant

The puffing pattern in salivary chromosomes of third instar larvae ofDrosophila hydei was studied following treatment with various gases, potassium cyanide, or vacuum. It was found that a number of specific puffs appear when anaerobiosis is followed by exposure to air or oxygen. These puffs seem to be independent of larval age, and are identical with some of those puffs which can be induced by raised temperature. It is suggested that the chromosomal loci involved, are connected with respiration.     

Die experimentelle Beeinflussung des Puffmusters von Riesenchromosomen

By treating larvae and prepupae of Ch. thummi with 2 mg/ml oxytetracycline (OTC) about 30 puffs not present in normal development are induced in the salivary gland chromosomes. Already existing puffs become enlarged (cf. Fig. 4). A considerable number of induced puffs appeared in heterozygous condition (cf. Fig. 1a-c). The species Ch. strenzkei does not react in any way to the same treatment. Other inhibitors of protein synthesis such as cycloheximide and chloramphenicole do not influence the puffing pattern in both species. — Animals which had been treated with OTC for 2 hrs show the first signs of puffing. Fully developed OTC-induced puffs are detectable 20 hrs after treatment. At this time the Balbiani rings and the nucleolus are mostly regressed. — Both the induced puffing pattern and the number of heterozygous puffs depend on the genetic constitution of the animals. Animals derived from different locations can be shown to possess different specific spectra of induced puffs. The induced puffing pattern of animals bred from single egg masses is reduced, and heterozygous puffs are rare or absent. — OTC-induced puffs show a strong uptake of tritiated uridine (cf. Fig. 2). Heterozygous puffs are labelled only in the puffed half of the band (cf. Fig. 3).     

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.     

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.     

Developmental Studies in Drosophila

The salivary gland chromosomes of 3rd instar Drosophila pseudoobscura larvae were observed for puffing changes after injection of larvae with ecdysterone solution. Chromosomes from the salivary glands of 3rd instar larvae and prepupae were similarly examined after incubation in ecdysterone-containing medium. The larvae, after treatment, showed advancement of the puffing process with the occurrence of a pattern similar to that observed during the pre-spiracle eversion period of normal development. At least 92 puffs showed changes in size. For the prepupae, the puffing changes resembled those occurring normally during the late prepupal period. A group of puffs were selected for detailed study. Among these were four puffs on the XR chromosome which exhibited large increases before spiracle eversion and pupation in normal development. As in normal development, two of these became the most prominent puffs observed within h after hormone treatment. In chromosomes from larval glands, the other two XR chromosome puffs were among the largest puffs to appear later in the sequence. However, in chromosomes from prepupal glands one of these later puffs failed to appear. The significance of this large number of hormone-inducible puffing changes at two different periods in development is discussed.     

Patterns of puffing activity in the salivary gland chromosomes of Drosophila

Exposure of Drosophila melanogaster larvae to high temperature for short periods of time results in marked changes in the puffing patterns of salivary gland chromosomes. Temperature shock induces puffing at 9 specific loci; this pattern of induced puffs shows little developmental specificity and is similar in three strains of D. melanogaster (including the mutant lethal giant-larvae) and in D. simulans. Temperature shock also (i) retards the regression of some developmentally specific puffs and (ii) results in the regression of all other puffs normal to development. The effect of temperature treatment is similar in vivo and after in vitro treatment of salivary glands. The in vitro response is not sensitive to cycloheximide. A similar pattern of induced puffs to that found after temperature treatment is found during recovery of larvae from anoxia, but additional puffs are induced after anoxia. The size and duration of activity of the induced puffs is dependent upon the magnitude of the treatment.     

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.     

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.     

Different structure of polytene chromosomes ofPhaseolus coccineus suspensors during early embryogenesis

Summary The pattern of DNA and RNA puffs in pair VII of polytene chromosomes has been investigated in the suspensor ofPhaseolus coccineus during early embryo development. The pattern of³H-TdR and³H-U incorporation has been also detected. Collected data indicate that: 1. both heterochromatic regions, p₁₁ and q_(111+112), of chromosome pair VII, organize large DNA puffs; 2. DNA puffs of both regions are specific of different embryo differentiation steps; 3. a seasonal influence on the DNA puffing seems also to be present, as demonstrated by the comparison of the results collected in two different crops; 4. the incorporation experiment by³H-TdR evidences that not all DNA puffs show clustered labeling; 5. the RNA puffing of the two regions seems also to be specific of determined embryo stages.     

Developmental puffing activity in the salivary gland and Malpighian tubule chromosomes ofAcricotopus lucidus (Diptera,Chironomidae)

A detailed map of the salivary gland chromosomes ofAcricotopus lucidus is presented. Differences in puffing and developmental Puffing sequences of the three salivary gland lobes were investigated from mid fourth larval instar to pupation and compared with the puffing pattern of the Malpighian tubules. The intraglandular differentiation is quite extensive; the differences in the pattern of gene activity between the anterior lobe and the main and side lobes are as great as between the salivary gland and the Malpighian tubules. In the main and side lobes all developmental puffing changes proceed synchronously whereas in the anterior lobe both asynchronous and synchronous changes occur. In the anterior lobe the asynchronous regression of BR 3 and BR 4 is followed by a characteristic sequence of activation and inactivation of puffs.     

Puffing activity in the salivary gland chromosomes of Rhynchosciara under experimental conditions

By using the techniques of ligation of the larvae (brain and endocrine glands extirpation) and salivary gland implantation, the hormonal dependence of the activity of certain puffs of Rhynchosciara was investigated. Our results have shown that the puffing behaviour — activation and deactivation — varies according to the developmental stage in which the larvae were ligated. When the larvae were ligated just before the drastic changes in the puffing pattern, which occur prior to pupation, these changes fail to occur. When the larvae were ligated after the onset of these changes we have observed: a) some of the puffs active at the time of the ligature regress promptly, earlier than their normal timing observed in controls; b) others remain active indefinitely and c) there are still some which regress accordingly to the normal timing.The puff B2 which behaves as those in b was double checked by means of implantation experiments. Salivary glands which had puff B2 at its maximum expansion were implanted into younger larvae and that puff also remained active in the body cavity of these larvae. Hypotheses to explain the results obtained are discussed.     

Developmental changes in fat body and midgut chromosomes of Drosophila auraria

Changes in puffing activity of fat body (FB) and midgut (MG) chromosomes of Drosophila auraria during late larval and white prepupal development as well as after in vitro culture with or without ecdysterone were studied and compared with those of the salivary gland (SG). The Balbiani Rings characteristic of the SG chromosomes of D. auraria, are not formed in FB and MG. Most of the inverted tandem chromosomal duplications that have been found to be common to all three tissues showed differentiation of puffing activity of the bands considered to be homologous. The major early ecdysone puffs 73A and 73B (considered to be homologues of D. melanogaster puffs 74EF and 75B, respectively), together with other early ecdysone puffs were present in all three tissues. Clear intermoult and postintermoult puffs were not evident in FB and MG chromosomes. However, a small set of late ecdysone puffs could be scored in FB, while no late ecdysone puffs were abserved in MG. Other tissue-specific puffs were identified, but a very small number of them were limited to MG.by W. Beermann     

Das Puffmuster der Borstenapparat-Chromosomen vonSarcophaga barbata

The chromosome complement ofSarcophaga barbata polytene scutellar trichogen and tormogen cells is described and the puffing sequences of chromosome IV were analysed from day 5 to day 17 of pupariation, i. e. from the beginning up to the end of bristle and socket formation at 21°C. There are 5 normal polytene chromosomes and a complex of fibrillar and granular heterochromatin in the giant cell nuclei. It is supposed that the heterochromatic masses represent the underreplicaetd sex-chromosomes. During a 13 day period of development 105 puffs appear in the trichogen cell chromosome IV respective 102 puffs in the tormogen cell chromosome IV. The puffing patterns of these two sister cells show many similarities. However, according to the differences in development, morphology and function of bristle and socket, there are also specific differences in the puffing patterns of the trichogen and tormogen cell. Preliminary observation suggest that the hormone bursicon induces some new puffs in the tormogen cell chromosomes of freshly emerged adults.     

Patterns of puffing activity in the salivary gland chromosomes of Drosophila

Salivary gland X chromosome puffing patterns are described for the Oregon stock of Drosophila melanogaster and for the Berkeley stock of D. simulans. In D. melanogaster regular phase specific puffing was recorded at 21 loci in the third larval instar and subsequent prepupal stage. A comparison of the X chromosome puffing patterns of male and female larvae failed to show any qualitative differences although in the males a group of puffs were active for a longer time during development than in females. The X chromosome puffing patterns of D. simulans are similar to those described for D. melanogaster although two puffs (4F 1–4 and 7B 1–3) were active in D. simulans but not in D. melanogaster. The sex differences in puffing observed in D. melanogaster were also observed in D. simulans.     

Gene activity of polytene chromosomes in Drosophila species of the obscura group

The polytene chromosome puffing patterns of Drosophila guanche were established and compared with those of Drosophila subobscura. A total of 150 loci, active in some of the 17 developmental stages studied, were described and 23 of them were found to form the characteristic puffing pattern of D. guanche. Taking into account the number of puffs as well as the gene activity of each chromosome and the total gene activity, D. guanche seems to be less active than D. subobscura. Although both species show a degree of homology in their puffing patterns lower than that found for sibling species, the degree of homology is stronger than that between species belonging to the same group but to different subgroups. Thus, D. guanche and D. subobscura must be considered as phylogenetically closely related species, belonging to the same subgroup.     

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.     

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.     

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.