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.     

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.     

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.     

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.     

Factors Determining the Recruitment of Inositol Trisphosphate Receptor Channels During Calcium Puffs

Puffs are localized, transient elevations in cytosolic Ca²⁺ that serve both as the building blocks of global cellular Ca²⁺ signals and as local signals in their own right. They arise from clustered inositol 1,4,5-trisphosphate receptor/channels (IP₃Rs), whose openings are coordinated by Ca²⁺-induced Ca²⁺ release (CICR). We utilized total internal reflection fluorescence imaging of Ca²⁺ signals in neuroblastoma cells with single-channel resolution to elucidate the mechanisms determining the triggering, amplitudes, kinetics, and spatial spread of puffs. We find that any given channel in a cluster has a mean probability of ∼66% of opening following opening of an initial “trigger” channel, and the probability of puff triggering thus increases steeply with increasing number of channels in a cluster (cluster size). Mean puff amplitudes scale with cluster size, but individual amplitudes vary widely, even at sites of similar cluster size, displaying similar proportions of events involving any given number of the channels in the cluster. Stochastic variation in numbers of Ca²⁺-inhibited IP₃Rs likely contributes to the variability of amplitudes of repeated puffs at a site but the amplitudes of successive puffs were uncorrelated, even though we observed statistical correlations between interpuff intervals and puff amplitudes. Initial puffs evoked following photorelease of IP₃—which would not be subject to earlier Ca²⁺-inhibition—also showed wide variability, indicating that mechanisms such as stochastic variation in IP₃ binding and channel recruitment by CICR further determine puff amplitudes. The mean termination time of puffs lengthened with increasing puff amplitude size, consistent with independent closings of channels after a given mean open time, but we found no correlation of termination time with cluster size independent of puff amplitude. The spatial extent of puffs increased with their amplitude, and puffs of similar size were of similar width, independent of cluster size.     

Ecdysterone induced protein synthesis in salivary glands and in fat body of Drosophila melanogaster larvae

Salivary glands of 3rd instar larvae of Drosophila melanogaster were labeled with ³H-leucine in the presence and absence of ecdysterone. Twentysix ecdysterone inducible proteins were detected. Their induction was correlated with puff stage. Synthesis of fifteen proteins commenced during early puff stage (PS2); synthesis of seven others at late puff stages (PS8–10). Synthesis of four proteins was induced between puff stage 3/4 and 7/8. Thus, the hormonal induction of protein synthesis generally reflected the appearance of early and of late puffs as described by Ashburner (1972). Eleven ecdysterone inducible proteins were detected in larval fat body in vitro. Comparison of the fat body to the salivary gland proteins revealed that one of the ecdysterone induced fat body proteins was identical in molecular weight and charge to one of the proteins induced by ecdysterone in salivary glands.     

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.     

A STUDY OF THE NUCLEOLAR MATERIAL IN SCIARA COPROPHILA

In the polytene chromosomes of Sciara coprophila, in addition to a nucleolus, large numbers of nucleolarlike structures or micronucleoli are formed. A detailed mapping localized the nucleolar organizer at one end of the X chromosome and revealed that approximately 18% of the bands of each chromosome are potentially capable of producing micronucleoli. Most of these sites are in regions known from a previous study to show asynchronous DNA replication: DNA puffs and certain heterochromatic regions. Micronucleoli are rarely found in association with bulbs. The RNA metabolism of the polytene chromosomes during late fourth instar was studied using radioautographic techniques. Isolated glands were incubated in tritiated uridine for 10 to 30 min, and radioautographs were made of squash preparations. Despite the wide range of variation found among different larval cultures, the following pattern was observed. Just prior to and at the beginning of DNA puff formation, a period of intense extrachromosomal nucleolar and micronucleolar RNA synthesis occurs. After maximal development of the DNA puffs, the synthesis of extrachromosomal RNA is at a low point, while incorporation into bulbs and DNA puffs remains high. With the onset of the prepupal stage, all nuclear RNA synthesis ceases.     

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.     

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).     

Changes of enzyme activity in larval salivary glands following the induction of respiration dependent puffs in giant chromosomes of Drosophila melanogaster.

Changes in the activity of 3 enzymes--lactate dehydrogenase (LDH), soluble malate dehydrogenase (sMDH), and pyruvate kinase (PK)--in homogenates of larval salivary glands of Drosophila melanogaster were studied before and after the induction of heat sensitive puffs with trinactin. The activities of 2 enzymes (LDH and sMDH) were enhanced after puff induction, an effect that was abolished in the presence of cycloheximide. Pyruvate kinase activity did not change after puff induction. The results indicate a relation between the increase of LDH and sMDH activity due to de novo synthesis and the induction of heat sensitive puffs.     

Messenger-like RNA synthesis and DNA chromosomal puffs in the salivary glands of Rhynchosciara americana

RNA synthesis was studied mainly in the proximal sections of Rhynchosciara salivary glands in late fourth instar at two typical periods of development. These are characterized either by the absence or presence of the so-called “DNA puffs” in the salivary gland chromosomes. It was found that simultaneously with the appearance of the DNA puffs there is a great increase in the synthesis of all RNA species. The greatest increase was found to take place in the rate of synthesis of messenger-like RNA. Four main classes of messenger-like RNA were detected, having mobilities corresponding to 33, 23, 16, and 14 S RNA. There is a correlation between the abundance of the 16 S messenger-like RNA and the degree of opening of the B-2 DNA puff. This species might therefore be transcribed from this puff.     

Puff induction in permeabilized Drosophila salivary glands in a chemically defined medium

We showed previously that digitonin-permeabilized salivary glands form prominent puffs in response to ecdysterone only when the incubation medium is supplemented with a homogenate of intact glands. To develop a chemically defined medium that supports puff formation in permeabilized salivary glands, we examined the requirement of ribonucleoside triphosphates (NTPs), precursors of RNA synthesis, for puff formation in permeabilized salivary glands. We found that prominent ecdysone puffs were induced in permeabilized salivary glands when the concentration of each NTP in the medium was higher than 0.5 mM. The puff size was significantly reduced if the volume of the medium were more than 2.0 microliter per gland. This suggests the existence of a factor(s), in addition to NTPs, which is required for puff formation and is diffusible from permeabilized glands.     

Similarities in chromosomal puffing induced by temperature shocks and dinitrophenol in Drosophila

A comparison between chromosomal puffs induced by temperature shocks and dinitrophenol in regions 87 A and 87 B of salivary gland chromosome 3 R of Drosophila melanogaster is presented. The size of both puffs can be regulated by either the temperature of a heat-shock or by the concentration of dinitrophenol. The two agents are additive with respect to their effects upon puff size, supporting the idea that both result in a change in ATP synthesis within the cell. A possible relationship between these agents and puff formation and RNA metabolism is discussed.This investigation was supported by research grant No. 119-70 from the Cancer Association of Greater New Orleans.     

Deletions of two heat-activated loci in Drosophila melanogaster and their effects on heat-induced protein synthesis.

Deficiencies that delete two heat-induced puffs in Drosophila melanogaster have been isolated. Two deficiencies delete the puff arising from 87C1, and one deletes the two puffs at 87A and 87C1. Homozygotes for these deletions can be recognized by their abnormal, transparent malpighian tubules. The influence on heat-induced protein synthesis of deleting one or both of these puffs has been examined. Unexpectedly, deleting only the 87C1 puff has no apparent effect. Deleting both the 87A and 87C1 puffs eliminates synthesis of a 71,000 dalton protein. We map a coding locus for the 71,000 dalton heat-induced protein to 87A7-B3, a seven band region which also contains a heat-induced puff. The roles of the 87A and 87C1 puffs in coding for heat-induced proteins are discussed in the light of these results.     

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.     

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.     

Cytogenetic and molecular aspects of position effect variegation in Drosophila melanogaster

A new genetic model system for studying position effect variegation in Drosophila melanogaster was found. It allows the analysis of genetic inactivation and changes in chromosome morphology in the same cells. In T(1;2)dor ^var7 strains the 2B5 early ecdysone puff, and the ecs locus which maps in this puff are translocated into the vicinity of centromeric heterochromatin. The ecs locus plays a key role in the system of ecdysone puffs: genetic damage to this locus results in loss of sensitivity of cells to the hormone and, as a consequence, ecdysone-induced puffs do not develop. In the T(1;2)dor ^var7 chromosome the ecs and at least five adjoining loci are inactivated in a variegated fashion. In the salivary gland cells of T(1;2)dor ^var7/ ecs^lt435 0 h prepupae which do not show the ecdysone puffs, the morphology of the 2B region was analysed. In all cases where the ecs locus was inactivated, a dense block of chromatin reminiscent of a solid band was found in the 2B region instead of the four bands 2B1–2, 3–4, 5 and 6. Sometimes compaction of the chromatin reached the 2A1–2 or even 1E1–4 bands. Formation of the compact block of chromatin coincided with late replication in this region. In situ hybridization of polytene chromosomes with a DNA clone from the ecs locus showed that when the dense chromatin block was present, no DNA was accessible for hybridization in 2B5. Hybridization of DNA of another clone located in the region of the translocation breakpoint (2B7–8) was found only in polytene chromosomes of larvae grown at 25° C, and never in those grown at 18° C, independently of the morphology of the 2B5 puff. The possibility that in the case of block formation both late replication and, as a consequence, underreplication of chromosome DNA take place, is discussed.Dedicated to Professor W. Beermann on the occasion of his 65th birthday