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 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     

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     

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.     

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.     

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.     

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.     

Dosage compensation of X-linked heat-shock puffs in Drophila pseudoobscura

Four major puffs are inducible by heat shock in the larval salivary gland chromosomes of D. pseudoobscura. Two of these puffs are present at 23 and 39–40 on the right arm of the X chromosome and two are present at 53 and 58 on chromosome 2. By means of in situ hybridization, residual homologies were demonstrated between the puffs at 23 in D. pseudoobscura and at 63C in D. melanogaster, and between the two chromosome 2 puffs of D. pseudoobscura and 87A and 87C of D. melanogaster. RNA synthesis was monitored as a function of ³H-uridine incorporation in the major heat-induced puffs of D. pseudoobscura and was found to be equivalent in males and females indicating dosage compensation of the two X-linked loci. The evolution of the regulatory controls of these genes is discussed.     

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).     

A comparison of banding patterns in salivary gland chromosomes of two species ofDrosophila

An attempt was made to compare the details of chromosome structure between two distantly related species ofDrosophila, D. melanogaster andD. hydei, which belong to different subgenera. Several short stretches of salivary gland chromosomes were selected on the basis of presumed homologous markers, either mutants associated with chromosome breaks, or experimental puffs. Banding patterns were compared in map diagrams, compiled from photographs. It proved possible to correlate with fair accuracy at least a short sequence of bands in each of the examples studied. The map regions were however very different otherwise. It was not possible to judge in how far this is a consequence of major or very small rearrangements, or other types of change. The chromosomal location of one permanent puff and of several others some of which are formed normally during pupation, and some of which appear after a period of oxygen deprivation, was found to be in complete agreement with genetical data which indicate homology of chromosomes 2 (hydei) with3R (melanogaster), and of 4 with3L. On the other hand, relative position and sequence of these puffs within the chromosome are different in the two species. Supported by the Netherlands Organization for the Advancement of Pure Research, Grant 913-28.     

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.     

The role of the hormone ecdysterone in the control of the activity of the Balbiani rings and the other puffs of Drosophila auraria

The activity of the Balbiani rings and the other puffs of Drosophila auraria salivary gland chromosomes from various stages of development was studied in vitro in the presence or the absence of various concentrations of the hormone ecdysterone. It was found that of 81 sites affected by these conditions, 69 (including the BRs) exhibit changes during normal development as well, while the remaining 12 change only under culture conditions. The results indicate that the normal profiles of certain puffs (and the BRs) are approximated more closely by the lower concentrations of the hormone and it is suggested that such low concentrations are necessary to induce the normal course of events in vivo. Various hypotheses concerning the influence of ecdysterone on the puffing patterns are discussed in view of the data presented in this report.     

Chromosomal control of foot pad development in Sarcophaga bullata

The puffing schedule of 71 puffs of chromosome B in Sarcophaga bullata polytene foot pad cells were analyzed during an 8 day period of pupal development. The number of puffs fluctuates, with maxima at about day 7 and day 10. The nucleolar volume reaches two maxima which precede the maxima in the number of puffs by approximately one day. With respect to qualitative changes in the puffing pattern, the present study shows that all puffs appear and disappear in a sequential fashion which is related to development. The pattern of potentially active loci and the sequence in which they form puffs are identical in all foot pad cells. Nevertheless, two neighboring cells may differ characteristically in the timing of the activity of a few individual loci relative to the rest of the puffing sequence. Furthermore, pulvilli cells from different pairs of legs may differ in the timing of the entire puffing sequence. It is concluded that puffing, at least in part, is under the control of intracellular processes rather than of external factors such as hormones.     

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.     

Immunoelectron microscopic localization of RNA polymerase B on isolated polytene chromosomes of Chironomus tentans

RNA polymerase B (or II) was localized by immunoelectron microscopy in ultrathin sections of polytene chromosomes isolated from larval salivary glands of Chironomus tentans. The enzyme was found at decondensed sites (puffs and interbands), whereas no detectable RNA polymerase B was present in condensed loci (bands). Within each of the large puffs the highest enzyme concentration was observed wherever the chromatin was in the most decondensed state. Otherwise the enzyme appeared homogeneously distributed within puffs and interbands. This immunoelectron microscopic study, along with the recently published immunofluorescent and autoradiographic analysis of isolated Chironomus chromosomes (Sass, 1982) unequivocally demonstrates that RNA polymerase B is present in most, if not all interbands.     

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.     

Sequence of puff formation inRhynchosciara polytene chromosomes

The chief characteristics of the life cycle ofRhynchosciara sp. are: egg stage (12 days); three larval instars of approximately 6 days each, followed by a 4th instar of approximately 40 days duration; pupation (6 days); and adult form (5–6 days). Maps of the 4 polytene chromosomes ofRhynchosciara sp. have been prepared, and the temporal sequence of puff formation on the chromosomes described. The cocoon is synthesized during the prepupal period, and at this time major puffs are seen on all chromosomes. The largest and most numerous puffs occur on the salivary gland chromosomes during the 24 hours prior to the last or prepupal molt. Three of the puffs that occur at this time are DNA-puffs (Summary see p. 249). Research sponsored by the U.S. Atomic Energy Commission under contract with the Union Carbide Corporation.     

Localization of nuclear proteins related to high mobility group protein 14 (HMG 14) in polytene chromosomes

An antibody was raised against high mobility group nuclear protein 14 (HMG 14) from calf thymus, known to be associated with actively transcribed chromatin. By means of indirect immunofluorescence, it was shown to react with the nuclei of mouse fibroblasts and of brain cells from Xenopus and Drosophila, but not of Xenopus erythrocytes. The antibody was used to detect immunologically related proteins in giant chromosomes of the midge, Chironomus pallidivittatus. Indirect immunofluorescence with anti-HMG 14 antibody in polytene nuclei was restricted to the active puffs. Giant puffs (Balbiani rings) exhibited especially intense fluorescence in their peripheral regions. An inducible puff site, the Balbiani ring 6 locus, showed no reaction with the antibody prior to induction. When puff formation began, the chromosome site assumed a very intense fluorescence, which disappeared again when the Balbiani ring was recondensed. — Protein extracts of salivary gland nuclei were found on immunoblots to contain one major protein fraction that reacted with the anti-HMG 14 antibody. The electrophoretic mobility of this fraction was similar to that of calf thymus HMG 17. — It is concluded that actively transcribed puffs in polytene chromosomes contain HMG 14-related protein(s) that are not present in potentially active gene loci prior to induction.This paper is dedicated to Prof. Hans Bauer on the occasion of his 80th birthday.     

An overview of gene activity in the fat body of Drosophila gibberosa

In the larval fat body of Drosophila gibberosa, polytene chromosome structure and activity exhibit cytological differences from chromosomes of midgut and salivary glands. These differences include long-persisting puffs, transient puffs and long-persisting band modulations. Some early ecdysteroid-induced puffs are present in all three organs but few late puffs are present in the fat body. Comparative studies reveal, therefore, that late larval-early pupal puffing is enhanced in salivary glands relative to gut, fat body and Malpighian tubules. After the fat body breaks up in the prepupa, the rate of programmed cell death and the corresponding slow decline of chromosomal activity also differ from cell to cell and from other organs.by M.L. Pardue     

Replication in Drosophila chromosomes

Prolongation of larval life in Drosophila melanogaster, by growing wild type larvae at lower temperature, or in animals carrying the X-linked mutation giant is known to result in a greater proportion of nuclei in salivary glands showing the highest level of polyteny. We have examined by autoradiography the patterns of ³H-thymidine incorporation during 10 min or 1 min pulses in salivary gland polytene chromosomes of older giant larvae and of wild type late third instar larvae of D. melanogaster grown since hatching either at 24 ° C or at 10 ° C. The various patterns of labelling and their relative frequencies are generally similar in glands from the warm-(24 ° C) or cold (10 ° C)-reared wild type larvae, except the interband (IB) labelling patterns which are very frequent in the later group but rare in the former. The IB type labelled nuclei in cold-reared wild type larvae show labelling ranging from only a few puffs/interbands labelled to nearly all puffs/interbands labelled. In warm-reared wild type larvae, very low labelled IB patterns are not seen. In older giant larvae, the ³H-thymidine labelling patterns are in most respects similar to those seen in cold-reared wild type larvae. In 1 min pulsed preparations from all larvae, the IB patterns are relatively more frequent than in corresponding 10 min pulsed preparations. No nuclei with the continuous (2C or 3C) type of labelling pattern, with all bands and interbands/puffs labelled, were seen in 1 min pulsed preparations from cold-reared wild type or in giant larvae, and only a few nuclei in 1 min pulsed preparations from warm-reared wild type larvae exhibited the 2C labelling pattern. Analysis of silver grain density on specific late replicating sites in late discontinuous (1D) type labelled nuclei suggests that the rate of DNA synthesis per chromosomal site is not different at the two developmental temperatures. It is suggested that correlated with the prolongation of larval life under cold-rearing conditions or in giant larvae, the polytene replication cycles are also prolonged. It is further suggested that the polytene S-period in these larvae is longer due to a considerable asynchrony in the initiation and termination of replication of different sites during a replication cycle.