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.     

Characteristics of structures of Drosophila polytene chromosomes formed by transposable DNA fragments

An electron microscopic analysis of regions of Drosophila melanogaster polytene chromosomes into which DNA fragments of different genetic composition were inserted by the P element-mediated transformation was performed. In 4 of 5 regions studied with integrated DNA sequences of the hsp28-ry, hsp70-Adh, ry-hsp70-beta-gal genes new bands appeared. Apparently their generation is mainly caused by integration of the DNA fragments in interbands. Absence of a new band in transformed region in one of the stocks can be explained by fusion of the insertion with a band existed in the initial untransformed stock. Among the transformants studied, the minimum length of DNA fragment revealed as a new band is about 5 kb. DNA packing ratio of such the bands varies from 30 to 50. The activation of the inserted genes by heat shock allows to trace peculiarities of the new bands puffing. The puff sizes correlate with the length of the activated genes. If the DNA of the fragment consists of the sequence of one gene, its activation will lead to decondensation of the whole band. In the case when DNA fragment consists of 2 genes and the promoter of activated gene is situated inside the sequence, the band is splitted after gene activation at the beginning and then separated portion of the band is decondensed and puffed. The data obtained evidence that a band of polytene chromosome is not a unit of decondensation. DNA packing ratio in puffs is equal to 1.5-3.5.     

Use of immunogold labelling technique for immunoelectron microscope localization of proteins in Drosophila polytene chromosomes

Using gold labeled antibodies, we developed and tested an immunoelectron microscope (IEM) method for detection of protein localization in Drosophila melanogaster polytene chromosomes. This method is based on procedures widely used for indirect immunofluorescent (IF) staining of salivary gland polytene chromosome squashes. The application of IEM was evaluated by using specific antibodies against proteins earlier localized in both decondensed (interbands and puffs) and compact (bands) regions of polytene chromosomes. In all the experiments, IEM and IF images for homologous chromosome regions were compared. When applied to regions of loose structures, IEM enabled us to localize, with high precision, signals in fine bands, interbands and puffs. There was a good correspondence between immunogold EM and IF data. However, there was no correspondence for dense bands: gold particles were distributed at their boundaries, while the entire bands showed bright fluorescence. This discrepancy probably resulted from a poor penetration of antibodies conjugated to gold particles in the tightly packaged structures. From the results obtained it may by concluded that the IEM method is advantageous for studying the fine protein topography of loose decompacted regions of polytene chromosomes. And this must be taken into consideration when protein localization in polytene chromosomes is performed.     

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.     

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.     

Characterization of Drosophila heterochromatin

A number of preliminary experiments have shown that the fluorescence pattern of Hoechst 33258, as opposed to that of quinacrine, varies with the concentration of dye. The metaphase chromosomes of D. melanogaster, D. simulans, D. virilis, D. texana, D. hydei and D. ezoana have therefore been stained with two concentrations of H 33258 (0.05 and 0.5 mug/ml in phosphate buffer at pH 7) and with a single concentration of quinacrine (0.5% in absolute alcohol). The three fluorescence patterns so obtained were shown to be somewhat different in some of the species and the coincide in others. All three stainings gave an excellent longitudinal differentiation of heterochromatin while euchromatin fluoresced homogeneously. Living ganglion cells of the six species mentioned above were treated with quinacrine and H 33258. Quinacrine induced a generalized lengthening and swelling of the chromosomes and H 33258 the decondensation of specific heterochromatic regions. A correlation of the base composition of the satellite DNAs contained in the heterochromatin of the species studied with the relative fluorescence and decondensation patterns showed that: 1) the extremely fluorochrome bright areas and those decondensed are present only in species containing AT rich satellite DNA; 2) the opposite is not true since some AT-rich satellite DNAs are neither fluorochrome bright nor decondensed; 3) there is no good correspondence between Hoechst bright areas and the decondensed ones. AT richness therefore appears to be a necessary but not sufficient condition both for bright fluorescence and decondensation. Some cytological evidence suggests that similarly AT rich satellite DNAs respond differently in fluorescence and decondensation because they are bound to different chromosomal proteins. A combination of the results of fluorescence and decondensation revealed at least 14 types of heterochromatin; 4-7 of which are simultaneously present in the same species. Since closely related species (i.e. D. melanogaster and D. simulans; D. virilis and D. texana) show marked differences in the heterochromatic types they contain, it can be suggested that within the genus Drosophila qualitative variations of heterochromatin have played an important role in speciation.     

Electron microscope autoradiographic study on transcriptional activity of Drosophila melanogaster polytene chromosomes

Incorporation of ³H-uridine into three chromosome regions 21D, 100AB, 7EF showing no puffs was studied by means of EM autoradiography. These regions show rather good coincidence between EM and Bridges' revised maps. The reduction of band number observed in the EM map was mainly at the expense of “doublet” bands. — Theoretical silver grain distributions were calculated on the basis of “universal curves” (Salpeter et al., 1969, J. Cell Biol. v. 41, 1–20) on condition that either bands or interbands are linear sources of radioactivity. From these curves the resolution of EM autoradiography was deduced to be sufficient with regard to the investigated region. — The results show that in addition to the puffs peaks of silver grains occur over the interbands and diffuse bands. The lowest incorporation level is observed over the dense bands. The possibility of utilizing the data obtained for the location of RNA-synthesising regions is discussed.     

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.     

Reversible differential decondensation of unfixed Chinese hamster chromosomes induced by change in calcium ion concentration of the medium

Differential decondensation of isolated unfixed Chinese hamster metaphase chromosomes was obtained by decreasing the calcium ion concentration in the surrounding medium. A banded appearance of the swollen chromosomes could be observed either directly by phase contrast microscopy or after glutaraldehyde fixation and staining. There was a gradual transition from homogeneously dense to banded and finally to extensively decondensed chromosomes. The patterns induced at different stages were similar to those observed on fixed chromosomes after standard banding procedures (i.e., G-, C-, C_d–, Ag-NOR-staining). Chromosome decondensation could be reversed by the addition of calcium ions to the medium. Ca⁺⁺-dependent reversible differential chromosome decondensation was not observed if the chromosomes were previously treated with 0.35 M NaCl. Chromosome regions which had incorporated BrdU into their DNA were more resistant to a decrease in calcium ion concentration than BrdU non-substituted regions.     

Differences in the structural organization of the G- and R-bands detectable during the differential decondensation of chromosomes

The ultrastructure of G- and R-bands in differentially decondensed chromosomes of Chinese hamster was studied with a gradual decrease in CaCl2 concentration in the medium. The gradual reduction of CaCl2 concentration leads to the decondensation of compact G-bands into chromonemes, chromomeres and further into DNP-fibrils. In the complete local decondensation zones (R-bands), the DNP-fibril orientation is parallel to the chromosome longitudinal axis. These zones have no lateral loops or chromomeres. Thus, different chromosome regions corresponding to G- and R-bands possess different sensibility to the decondensing action. Following the complete decondensation in the calcium-free medium chromosomes can be "reconstructed" by adding Ca2+. The data obtained permit to suggest a "fastener" model of the mitotic chromosome organization in which the chromosome represents an hierarchy of discrete structures--G-bands, chromomeres, nucleomeres (superbeads) and nucleosomes. The structural integrity of these levels is supported by specific protein "fasteners".     

Ultrastructural organization of polytene chromosomes of Chironomus thummi salivary glands]

An electronmicroscopical mapping of a number of regions of the polytene chromosomes of Ch. thummi salivary glands (3rd chromosome, right arm of the 1st chromosome, centromere regions, puffs 1-A2e, 1-A3ij, III-A5c and others) was done by the method of oriented ultrastructural sections of the unsquashed polytene chromosomes. The banding pattern on the electron micrograph was similar to the observed with the light microscope. The difference was that some doublets appeared as single cavity-containing bands with the double structure only in short regions under the electron microscope. It was also difficult to distinguish single bands in those regions where heavy adjacent bands were connected by dens, protrusions and anastomoses. These connections were most pronounced in the regions of the centromerers which had "spongy" appearance on the electron micrographs. These pictures may be connected with small interbands between heavy bands. Thin bands and some broad bands were frequently dotted. The puffs examined contained mainly RNP granules 200-400 A in diameter and RNP fibrils; BR-1 and BR-2 contained granules 500 A, RNP fibrils and smaller granules (200-400 A). BR and puffs were characterized by loop-like structures composed of granules arranged along the central DNP fibril. Only fibrils were presented in small interbands (0.05 mk), while larger interbands could include a small number of granules similar to those observed in puffs. It was found that centromere, telomeres and some heavy bands formed characteristic contacts with the nuclear membrane.     

Sites of gene activity and of inactive genes in polytene chromosomes of diptera

DNA has been found between bands, in puffs, and in RNA-rich lamellae of bands as dispersed DNA double helices coiled to form secondary helices with a diameter and period of about 400Å. In the bands and heterochromatic regions the secondary helix is further coiled or folded into compact masses. RNA-containing granules are present in interbands and RNA-rich lamellae as well as in puffs. The dispersed DNA of puffs, interbands and the RNA-rich lamellae of bands may all represent the part of the genome that is active in the salivary gland nucleus, while the compact DNA of bands as well as heterochromatic parts may comprise inactive genes.     

A model of chromatin-dependent DNA replication sequences based on the decondensation units hypothesis

A model of chromatin-dependent DNA replication sequences was developed on the previously reported "decondensation units" hypothesis and its kinetic properties were examined by way of calculating various numerical indices using a Monte Carlo procedure. The model has much in common with the previous one but a fundamental difference is that the unit is assumed to consist of linearly arranged H-, D-, A- and S-zones each containing genes of different functional categories which are called H-, D-, A- and S-genes, respectively. The units are decondensed by the action of D-factors, i.e. decondensation factors, from H-zone to the end of S-zone and the genes in decondensed regions release signals to produce housekeeping enzymes, D-factors, A-factors and S-factors. These products are stored and at the same time degraded. A-factors activate replication origins in the decondensed regions and S-factors induce DNA synthesis at the activated origins. Replicated DNA is recondensed and gene activities are shut down in the recondensed chromatin. The factors are produced under the control of chromosome cycle and in turn affect chromosomes. Thus, dual control mechanism operates as Mazia and Prescott have argued. Biochemical and cytogenetic basis of this model was reviewed briefly and some results of simulation presented which include DNA synthesis rate vs. DNA content relationships. An outstanding characteristic of the model is the constancy of cellular state in A-subphase located in the late G1.     

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.