Cytogenetic analysis of the 2B3-4-2B11 Region of the X chromosome of Drosophila melanogaster

Larvae homozygous or hemizygous for the l(l) t435 mutation located within the early ecdysteroid puff 2B5, or carrying a deletion of the 2B5 band, die at the end of the third larval instar. In the salivary gland chromosomes of these larvae only intermoult puffs are detected. If these salivary glands are incubated in vitro with 20-OH ecdysone for 6 h the intermoult puff 68 C remains large, some early puffs (74EF and 75B) are induced to 30–40% of their normal size, other early (63F) and all late puffs (62E, 78D, 82F and 63E) are not induced at all. Puff 2B5 reaches its normal size but does not regress after 6h incubation with 20-OH ecdysone, as it does in normal stocks. The data obtained in this study show the existence of a locus (or loci) in the band (puff) 2B5 which is necessary for the normal response of the salivary gland chromosomes to the hormone 20-OH ecdysone.     

Comparison of the molecular and genetic map of the 2B6-2B7-8 of Drosophila melanogaster X-chromosome]

Molecular and genetic data were compared for the 2B6-2B7-8 region of the Drosophila melanogaster X chromosome. This region contains the dor (deep orange) and swi (single wing) genes influencing ecdysterone-dependent gene expression. Genes which had not been identified previously by genetic methods were shown to be present in this region. Two novel loci, designated a6 and b6, were characterized in detail. Both genes are expressed throughout Drosophila embryogenesis. The product of b6 has a homology with mammalian pentraxins. This is the first Drosophila gene found to contain the pentraxin motif.     

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.     

Localization of ecs, dor and swl genes in eight Drosophila species]

The DNA sequences from Drosophila melanogaster early ecdysterone-inducible puff 2B have been located in 8 Drosophila species by in situ hybridization. The location site of the ecs, dor and swi genes in D. funebris, D. virilis, D. hydei, D. repleta, D. mercatorum, D. paranaensis is a puff on the telomeric and of X chromosome; in D. kanekoi it is the puff in distal part of X chromosome; and in D. pseudoobscura it is the puff in proximal portion of X chromosome. So, conservative organization of DNA sequences located in D. melanogaster 2B puff could be suggested. Dispersed distribution of some DNA segments from the region studied in D. hydei chromosomes was revealed.     

Modeling dark puffs using P-transposons in Drosophila melanogaster polytene chromosomes

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 puffs 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.     

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.     

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.     

Cytogenetical analysis of the 2B3-4-2B11 region of the X chromosome of Drosophila melanogaster

Summary Of 13 ecs mutations, which affect female fertility, as revealed by complementation analysis, 7 are chromosome rearrangements involving the br complementation group. The other six show no cytologically detectable rearrangements and behave as completely or partially noncomplementing ecs alleles. All viable combinations of these 13 mutations were characterized by partial or complete female sterility. Viable heterozygotes carrying any of these mutations and the rearrangements Df(1)sta, T(1,3)sta, Df(1)St490, previously localized distal to the ecs locus, were also sterile. Using deletions and an electrophoretic mobility variant from the Staket strain, a minor chorion gene S70 has been mapped. It had been thought this gene was located in the 2B3-5 region, and corresponded to the ecs locus. However, in the present study, this gene was shown to map in the region removed by Df(1)sta (1E1-2-2B3-4) but outside that removed by Df(1)At127 (1E1-2-2A1-2), i.e. within the 2A1-2-2B3-4 region which is distal to the ecs locus. Rearrangements and point mutations at the ecs locus that result in female sterility had no effect on synthesis of the chorion protein s70. It may therefore be suggested that the chorion protein gene is not functionally associated with the ecs locus and that sterility is caused not by disruptions of the chorion protein gene but by lesions in the ecs gene itself. Thus, an ecs product, which controlls cell sensitivity to ecdysterone is also necessary for female fertility. Data on the locations of lesions affecting female fertility indicate that at least two elements at the ecs locus are essential for this function: a cis-acting distal zone with no effect on viability and a sequence within the essential part of the ecs locus. A defect in either of these zones or their separation by chromosomal rearrangement leads to female sterility.     

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.     

Induction of ecdysterone-stimulated chromosomal puffs in permeabilized Drosophila salivary glands: A new method for assaying the gene-regulating activity of cytoplasm

A simple assay system for gene regulation using chromosomal puffing as an index of gene activity was established. Salivary glands of Drosophila melanogaster treated with a mild detergent, digitonin, were permeable to high molecular substances, including β-galactosidase (MW 465,000). The permeabilized salivary glands retained the ability to form puffs at the ecdysterone-stimulated loci (74EF and 75B) in response to the hormone. Incubation of the permeabilized salivary glands at puff stage 1 (PS1) for 2 hr in a medium containing both ecdysterone and a homogenate of intact salivary glands at puff stage 8–9 (PS8–9) induced a puff at 78C, where puffing occurs only at puff stages 6–11 in vivo. The puff at 78C was not induced when the permeabilized PS1 glands were incubated with the combination of ecdysterone and a homogenate of the PS1 salivary glands. Likewise, the 78C puff was not induced in intact PS1 salivary glands by a 2-hr incubation with ecdysterone and PS8–9 gland homogenate. These results indicate that a factor(s) required for 78C puff formation is present in PS8–9 but not in PS1 salivary glands and that factor(s) can permeate digitonin-treated salivary glands but not intact glands. The effectiveness of the permeabilized salivary glands as an assay system for gene-regulating factors is discussed.     

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

Relative values for the dry mass in puff-forming regions of Drosophila hydei salivary gland chromosomes were established with a Leitz double beam interference microscope. All measurements were made after RNA digestion.Optical path differences per unit area of the induced puffs 2-48C and 4-81B (temperature induced) and a cytoplasm-free background were recorded. In each of the nuclei used for these measurements, the same procedure was applied to two reference regions in the vicinity of the puff, region 2, 47A-48B and region 4, 81C-82C respectively. For comparison of the dry mass values of a puff region at various time intervals after the onset of puff induction, ratios of the optical path differences of the puff region over that of the reference region were calculated.These ratios were established at 5, 10, 30, 60 and 120 min after the onset of a temperature treatment and compared with the ratio in non-treated animals. From the data it can be concluded that the dry mass in the induced puffs increases gradually up to 30 min. At this time the dry mass ratio for puff 2-48C has reached a value of 150% and that of puff 4-81B, 210%. It is concluded that this increase in dry mass is due almost entirely to a local accumulation of non-histone protein.     

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.     

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.     

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.     

A Cytogenetic Analysis of the Punch-Tudor Region of Chromosome 2r in Drosophila Melanogaster

Eleven chromosomal deficiencies and several rearrangements in the Pu-tud region of chromosome 2R have been generated and examined cytologically. The Pu locus has been localized to chromosome bands 57C5-6 and tud to 57C7-8. Mutagenesis within the region defined by the deletion intervals has resulted in the isolation of 92 new lethal mutations. Seventy-six of these mutations have been separated into 16 complementation groups that have been ordered and placed cytologically by deletion mapping. All new alleles fully complement tud for both lethal and grandchildless phenotypes. The largest number of new mutations, a total of 25, are Pu alleles.     

Fine cytogenetical analysis of the band 10a1-2 and the adjoining regions in the Drosophila melanogaster X chromosome

The region 9E1-2 - 10B1-2 of the Drosophila melanogaster X chromosome was analysed under the light (LM) and the electron (EM) microscope using different fixatives and an EM map of the region was constructed. EM analysis revealed 21 bands in the region 9E1-2 - 10B1-2 instead of 36 bands in Bridges' map. This discrepancy mainly results from the fact that 14 bands indicated as "doublets" by Bridges appear as a single bands. No doublets were found in the whole 9B1-2 - 10C1-2 region after fixation of salivary glands in 3% glutaraldehyde, 3% formaldehyde and 3 : 1 ethanol-acetic acid mixture. 45% acetic acid is the only fixative which results in strongly vacuolated appearance of the bands. - The break points of 30 chromosome rearrangements in the region 9E1-2 - 10B1-2 were located under EM or LM within the limits of the EM map of this region.     

Genetic and Phenotypic Analysis of Thirteen Essential Genes in Cytological Interval 22f1-2; 23b1-2 Reveals Novel Genes Required for Neural Development in Drosophila

In an attempt to identify mutations in the Drosophila synaptotagmin gene we have isolated many new rearrangements, point mutations and P element insertions in the 22F1-2; 23B1-2 cytological interval on chromosome arm 2L. This interval encompasses 13 cytological bands and is shown to contain 13 essential complementation groups, including decapentaplegic, synaptotagmin and Curly. Through chemical and P element mutagenesis we have isolated seven new deletions, which combined with previously isolated rearrangements, have allowed us to order most genes in the interval. A genomic walk covering approximately 100 kb within this interval spans at least five essential genes as identified by chromosomal aberrations. Preliminary phenotypic characterizations of the mutant phenotype and lethal phase is presented for many mutations. Three loci within this interval are shown to be required for proper neural development. Given that the average number of alleles per complementation group is greater than seven, it is very likely that all essential genes within this cytological interval have been identified.