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.     

Formation and morphology of dark puffs in Drosophila melanogaster polytene chromosomes

The formation of unusual dark puffs in Drosophila melanogaster polytene chromosomes has been studied by electron microscopic (EM) analysis. Fly stocks transformed by the P[ry; Prat:bw] and P[hs-BRC-z1] constructs were used. In the former the bw gene is under the promoter of a housekeeping gene, Prat; in the latter the Br-C locus, mapping to the dark puff 2B, is under the promoter of a heat-shock gene, hsp70. Inserted into region 65A of the 3L chromosome, the Prat:bw copies give rise to structures which are morphologically reminiscent of the so-called "dark" puffs. In contrast, insertion of P[hs-BRC-z1] into region 99B of the 3R chromosome causes a regular "light" puff of form. Comparative analysis of the dark puffs--both transgenic and natural--suggests that there might be at least two mechanisms underlying their formation. One is a local incomplete decondensation of activated bands, characteristic of the so-called small puffs. The other is the formation of ectopic-looking contacts between the bands adjacent to the puffing zone. Transposition of the DNA, from which such a puff develops, causes a regular light puff to form at the new location. Heterochromatic regions do not appear to be directly involved in puffing.     

The 82F late puff contains the L82 gene, an essential member of a novel gene family.

Metamorphosis in Drosophila results from a hierarchy of ecdysone-induced gene expression initiated at the end of the third larval instar. A now classical model of this hierarchy was proposed based on observations of the activity of polytene chromosome "puffs" which distinguished "early" puffs as those directly induced by ecdysone and "late" puffs as those which become active as a secondary response to the hormone. We report here the isolation and characterization of the L82 gene corresponding to the extensively characterized late puff at 82F. L82 is a complex gene that spans at least 50 kb of genomic DNA, produces at least seven different nested mRNAs, and has homology to a novel gene family. In contrast to most previously characterized puff genes, the broad developmental expression pattern of L82 suggests that it is controlled by both ecdysone-dependent and ecdysone-independent regulatory mechanisms. L82 mutations were identified by transgene rescue of developmental delay and eclosion lethal phenotypes.     

Electron microscopical analysis of Drosophila polytene chromosomes

Mapping of 16 regions of polytene chromosomes in which 18 one-band puffs develop was carried out with the use of electron microscopy (EM). In most cases a uniform decondensation of the whole band was observed. However, there were examples in which only a part of the band was activated (three puffs) or its right and left parts decondensed simultaneously (three puffs). Splitting of the band into two parts with their further decondensation was also found (one puff). This suggests structural and functional complexity of the bands. On the basis of the data obtained here and those published earlier, a classification of 52 puffs by the number of bands participating in their formation is given. Four classes numbering 22, 21, 7, 2 puffs, developing from 1, 2, 3 and 4 bands, respectively, are revealed. The data show that active chromosome regions are rather diverse in both the pattern of decondensation and expansion of the decondensed region, thus providing evidence of the informational complexity of the majority of active regions.     

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.     

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.     

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.     

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     

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.     

The effect of prolonged exposure to heat on the activity of salivary gland polytene chromosomes]

The influence of long-term heating on the puffing activity of polytene chromosomes in the early prepupa salivary glands was investigated. The activity of puffs was estimated by two criteria: size and frequency. The rearing of insects at a temperature of 29 degrees resulted in puff changes: the activity of some puffs increased or depressed, some puffs were inhibited, other puffs were induced newly. The differential response of each chromosome was observed. A possible mechanism of the effect of heating on the puff activity of polytene chromosomes is discussed.     

Cytological and autoradiographic studies in Sciara coprophila salivary gland chromosomes

Summary Morphological and metabolic changes on the salivary chromosomes of Sciara coprophila were followed during the later half of the fourth larval instar.Cytological maps were prepared for five successive stages from mid-fourth instar to the prepupal stage. These maps, which constitute a revision of those published earlier by Crouse, summarized our cytological findings and were the basis for studies on DNA replication of these chromosomes.Similar to earlier studies in Chironomidae, differences in the puffing pattern were noted between the anterior and the posterior portions of the salivary gland. The most striking difference was noted in region 2B on chromosome III which produces a large puff only in nuclei from the anterior part of the gland. Other autosomal puffs, although present in both parts of the gland, showed constant differences in size.An increase in the number of bands from mid-fourth to late fourth instar was observed. The new bands are all of the light-staining kind.In Sciara the puffed area may include a large number of bands in addition to the bands which originated the puff. The maximal extent of puffs was determined in terms of chromosomal map regions and the number of bands subject to obliteration.In the autoradiographic experiments use was made of H³-thymidine as DNA precursor. The aim of these studies was to detect any asynchronies in the replication time of bands. In fact, marked differences in the relative rates of uptake of H³-thymidine of a number of bands in a certain proportion of chromosomes have been observed, while others showed uniform incorporation. Since these latter were found with higher frequency the period of uniform labeling must comprise a larger part of the replication cycle then the periods of localized labeling. To assess the validity and constancy of the observed patterns of unequal incorporation, a semiquantitative analysis was carried out. It showed that the bands showing localized uptake may be separated into two broad groups. In one of these groups are the centromere regions and certain chromosomal ends, which are presumably heterochromatic. The other group comprises most of the puff sites and bulbs. Since late replication is characteristic of heterochromatin, we assumed that bands of the former group (C) replicate late in the cycle, while puffs and bulbs start replication early, and the period of equal labeling is intermediate. Other intermediate labeling patterns were observed and are described.It is known that in the fourth instar from two to three DNA replications occur in the salivary gland nuclei, the last of which coincides with puffing. Several stages may be distinguished in the puffing process based on morphology and rates of isotope uptake of the puffs. The first sign of puffing is a very high rate of incorporation at puffs. It is maintained throughout this last DNA synthesis period and only declines when all other chromosomal regions have ceased to replicate. A pattern of high and exclusive uptake at the heterochromatic sites (pattern C) was never observed in this replication; instead puffs are the last regions to terminate DNA synthesis.These results are discussed in relation to several current problems, such as, asynchronous DNA replication, the problem of metabolic DNA, and the concept of the heterochromatic state.Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, in the Faculty of Pure Science, Department of Zoology, Columbia University, New York. This work has been supported by U.S. Public Health Training Grant No. 2Tl-GM-216-05; partial support has been received also from Grants GB 42 and G-14043 from the National Science Foundation to Dr. H. V. Crouse.