Puff expression in relation to genotype]

The studies of genotype influence on puff size in salivary gland chromosomes of Drosophila virilis (stocks 9, 101, 142, 151) and D. texana (the stock 123) reveal significant differences between the species concerning the structure of puff in the 3-C-6 region at the stage of puparium formation. In reciprocal F1 hybrids the size of the puff was intermediate in comparison with parental forms having a slight maternal effect. The differences in puff size in the 5th chromosome between interspecific hybrids and the special stock of D. virilis carrying a region of D. texana 5th chromosome in heterozygous condition (inserted into D. virilis 5the chromosome by double crossing-over) were observed. The transfer of the region of the third chromosome to near centrimetric heterochromatic of the 5th chromosome by translocation resulted in the increase in the 3-B-2 puff size. However, the transposition of the 3-B1 region in the proximal direction with respect to chromocenter did not affect the puff size.     

The probability of triggering calcium puffs is linearly related to the number of inositol trisphosphate receptors in a cluster

Puffs are local Ca(2+) signals that arise by Ca(2+) liberation from the endoplasmic reticulum through concerted opening of tightly clustered inositol trisphosphate receptor/channels (IP(3)R). They serve both local signaling functions and trigger global Ca(2+) waves. The numbers of functional IP(3)R within clusters differ appreciably between different puff sites, and we investigated how the probability of puff occurrence varies with cluster size. We imaged puffs in SH-SY5Y cells using total internal fluorescence microscopy, and estimated cluster sizes from the magnitude of the largest puff observed at each site relative to the signal from a single channel. We find that the initial triggering rate of puffs following photorelease of IP(3), and the average frequency of subsequent repetitive puffs, vary about linearly with cluster size. These data accord well with stochastic simulations in which opening of any individual IP(3)R channel within a cluster triggers a puff via Ca(2+)-induced Ca(2+) release. An important consequence is that the signaling power of a puff site (average amount of Ca(2+) released per puff × puff frequency) varies about the square of cluster size, implying that large clusters contribute disproportionately to cellular signaling and, because of their higher puff frequency, preferentially act as pacemakers to initiate Ca(2+) waves.     

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.     

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.     

Patterns of puffing activity in the salivary gland chromosomes of Drosophila

Patterns of puffing activity during the third larval instar and the prepupal period of two different strains of D. melanogaster (Oregon and vg6) are compared. The variation in puffing activity observed is both quantitative (involving the mean size or timing of activity of individual puffs) and qualitative. The pattern of activity of 64% of the puffs is the same in the two strains, 12% show strain differences in puff size and 19% in the time of their activity. One puff (64C) is active only in one of the strains (vg6). In genetic experiments this puff segregates normally and the puff locus has been mapped genetically to a site coincident with, or at least very close to, the cytogenetic position of the puff. In heterozygotes the puff is homozygous only when the maternal and paternal homologues are synapsed. When the homologues are asynapsed only the homologue from the vg6 parent is puffed at 64C. With the exeption of some strains closely related to vg6 no other strain of D. melanogaster has been found to possess puffing activity at 64C. In vg6/In(3LR)C165 heterozygotes 64C forms a heterozygous puff even when the homologues are synapsed. In the discussion consideration is given to the various factors that control puff size.     

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.     

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.     

Respiratory functions involved in the induction of puffs in Drosophila salivary glands

Salivary glands from third instar larvae of Drosophila melanogaster were incubated in vitro with various substances affecting oxidative phosphorylation. After an incubation time of 1–3 h changes in puff size and in cellular ATP level were registered. 10^?6 M trinactin, 10^?5 or 10^?4 M oligomycin both induce puff 63BC together with some other puffs and reduce the cellular ATP level by about 80–90%. The trinactin-dependent puff induction can be inhibited, if the medium is supplemented with 10^?3 M ATP or 10^?3 M ITP or 10^?6 M antimycin or 10^?2 M KCN. The effect of exogenous ATP is prevented by adding 10^?6 M oligomycin to the incubation mixture; 10^?6 M oligomycin alone, however, has no inductive effect on 63BC. The presence of exogenous ITP, furthermore, prevents the ATP level from being reduced by trinactin. 10^?4 M atractyloside lowers the ATP level by about 75 %, whereas a puff induction cannot be observed. The same is true for various concentrations of KCN. It is concluded that ATP itself is not involved directly in the regulation of puff activity but that it acts on a phosphorylating reaction that can be inhibited by oligomycin.     

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.     

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.     

Experimental induction of gene activity in the salivary gland chromosomes of Trichosia pubescens (Diptera: Sciaridae)

During the course of experiments with larvae of Trichosia pubescens, we have unexpectedly found that diethyl ether or chloroform anesthesia induces a large puff in a specific band in the polytene chromosomes of the salivary glands. This puff develops a few minutes after the treatment, attaining its maximum size after 60-100 min, and regresses completely 200 min after its activation. Through autoradiography, an intense incorporation of RNA precursors into that puff was observed. A few other smaller puffs are also induced by the treatment. The treatment with diethyl ether or chloroform does not induce puffing in the polytene cells of malpighian tubules and of midgut.     

Puffs and salivary gland function: The fine structure of the larval and prepupal salivary glands ofDrosophila melanogaster

Summary The salivary glands ofDrosophila melanogaster have been examined by electron microscopy for fine structural alterations occurring during larval and prepupal stages. The changes observed in the glands have been correlated with the puffing patterns of the polytene chromosomes at corresponding stages. In early third instar larvae, the lumen of the salivary gland appears empty, and no signs of secretory activity are visible in the glandular cytoplasm. From puff stages 1 to 6 the endoplasmic reticulum becomes reorganized and increases in volume. Electron dense material appears within its cisternae and subsequently within the Golgi saccules. Dense secretory granules then appear to be elaborated from the Golgi by terminal budding; these granules represent the glue for adhering the pupa to its substrate, and gradually increase in size and complexity. By puff stage 6 their contents have been liberated into the glandular lumen. Following puparium formation, those granules which are not extruded coalesce to form larger granules. Other dense bodies and autophagic vacuoles, considered to be lysosomes, appear, and the surplus secretory granules begin to display myelination at their peripheries; ultimately they are reduced to dense residual bodies. At puparium formation, the lumen is depleted of the glue and contains flocculent material. Histolysis commences after puff stage 11, and the cytoplasm becomes vacuolated and opaque; the nucleus becomes reduced in volume and crenated in outline. Nuclear blebbing occurs after puff stage 12, and material seemingly moves from the nucleus into the cytoplasm; the glandular lumen now becomes empty. An attempt has been made to ascertain how the chromosomal puffing activity relates to these cytoplasmic developments.     

Effect of pilocarpine on the puffing of polytene chromosomes in the salivary glands of Chironomus thummi]

Under the influence of pylocarpine on polytene chromosomes of the salivary glands of Chironomus thummi at the end of the 4th age, the puff size increased in the region 3-A5cd, 3-C3ghi, 4-KB1 and 4-KB2, in parallel with the increase of the amount of secrete in the central reservoire. In this connection, the above indicated puff may be considered as closely correated in their function with the secretory process of salivary gland cells.