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.     

Bands,interbands and puffs in native Drosophila polytene chromosomes are recognized by a monoclonal antibody to an epitope in the carboxy-terminal tail of histone H1

A monoclonal antibody was raised against Drosophila melanogaster histone H1. Immunoscreening of proteolytic cleavage fragments of H1 and of a set of all possible overlapping synthetic octapeptides corresponding to the amino acid sequence of H1, revealed that the antibody recognizes an epitope within the sequence ²⁰⁷VTAAKPKA²¹⁴ near the centre of the carboxy-terminal tail. This antibody gives positive immunofluorescence over the entire length of native D. melanogaster polytene chromosomes isolated from salivary glands by microdissection at physiological pH and ionic strength. Bands, interbands and puffs are all seen to contain H1. The immunofluorescence over puffs, albeit lower than that over bands and interbands, indicates that chromatin decondensation can occur without complete loss of H1 in these structures. The reaction of the antibody with bands suggests that the segment of the C-terminal tail containing the epitope may be exposed in the condensed 30 nm chromatin filament.     

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.     

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.     

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.     

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.     

<Emphasis Type="Italic">Drosophila</Emphasis> polytene chromosome bands formed by gene introns

Genetic organization of bands and interbands in polytene chromosomes has long remained a puzzle for geneticists. It has been recently demonstrated that interbands typically correspond to the 5’-ends of house-keeping genes, whereas adjacent loose bands tend to be composed of coding sequences of the genes. In the present work, we made one important step further and mapped two large introns of ubiquitously active genes on the polytene chromosome map. We show that alternative promoter regions of these genes map to interbands, whereas introns and coding sequences found between those promoters correspond to loose grey bands. Thus, a gene having its long intron “sandwiched” between to alternative promoters and a common coding sequence may occupy two interbands and one band in the context of polytene chromosomes. Loose, partially decompacted bands appear to host large introns.     

Molecular and genetic organization of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>polytene chromosomes: evidence for two types of interband regions

The 3A and 60E regions of Drosophila melanogaster polytene chromosomes containing inserted copies of the P{lArB} transposon have been subjected to an electron microscopic (EM) analysis. We show that both inserts led to formation of new bands within the interband regions 3A4/A6 and 60E8-9/E10. This allowed us to clone DNA of these interbands. Their sequences, as well as those of DNA from other four interbands described earlier, have been analyzed. We have found that, with the exception of 60E8-9/E10 interband, all other five regions under study corresponded to 5' or 3' ends of genes. We have further obtained the evidence for 60E8-9/E10 interband to harbor the 'housekeeping' RpL19 gene, which is transcribed in many tissues, including salivary glands. Based upon the genetic heterogeneity of the interbands observed a revised model of polytene chromosome organization is discussed.     

Mapping of the pattern of DNA replication in polytene chromosome from Chironomus thummi using monoclonal anti-bromodeoxyuridine antibodies

We present results from a nonautoradiographic study of DNA replication in polytene chromosomes from dipteran larvae. Monoclonal antibodies with specificity for 5-bromodeoxyuridine (BrdUrd) were used to localize by indirect immunofluorescence the sites of BrdUrd incorporation and to follow the dynamics of DNA synthesis in salivary gland cells of 4th instar Chironomus thummi larvae. This technique presents numerous advantages over autoradiographic procedures and allows mapping of DNA synthesis patterns at the level of resolution of one chromosomal band. Several replication patterns were observed, classified according to characteristic features, and tentatively assigned to specific periods of the S-phase. In early S-phase, DNA synthesis is first detectable in puffs and interbands, later in bands. Most chromosomal bands appear to initiate DNA synthesis synchronously; however, in bands within centromeric and heterochromatic regions the start of synthesis is delayed. At mid S-phase, all the bands show uniform staining. Subsequent staining patterns are increasingly differential with the bands displaying characteristic fluorescence intensities. As replication progresses through the late S-phase period, the chromosomes show a decreasing number of fluorescent bands. The last bands to terminate replication are located in centromeric and heterochromatic DNA-rich regions and a few bands of low DNA content in region IIAa-c.     

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.     

Early replicating DNA in heterochromatin ofPlagiochila ovalifolia (Liverworts)

The timing of DNA replication of heterochromatin in malePlagiochila ovalifolia was investigated by the use of³H-thymidine autoradiography. The estimated duration of the mitotic cycle was as follows: S period, 19 hr: G₂+prophase, 10 hr; G₁+meta-, ana-, telophase, 5 hr; total mitotic cycle, 34 hr. The first appearance of silver grains over the chromosomes was observed at 8 hr after the beginning of pulse labelling at which time the silver grains were only over the euchromatic regions, not over the heterochromatic regions. This labelling pattern was also observed at 10 to 15 hr. The heterochromatic regions having more grains than the euchromatic regions were observed at 20 to 25 hr. These results show that the DNA of the heterochromatin of this species is replicated earlier than the euchromatin.     

Analysis of DNA Interband Regions 3A5/A6, 3C5-6/C7, and 60E8-9/E10 in Polytene Chromosomes of Drosophila melanogaster

Using electron microscopic (EM) data on the formation of a novel band from theP-element material after its insertion in the interband and the procedure of P-target rescue, DNA interband regions 3A5/A6, 3C5-6/C7, and 60E8-9/E10 of Drosophila melanogasterpolytene chromosomes were cloned and sequenced. EM analysis of the 3C region have shown that the formation of the full-size 3C5-6/C7 interband requires a 880-bp DNA sequence removed by deletion Df(1)fa ^swb. A comparison of DNA sequences of six bands, two of which were obtained in the present work and four were described earlier, demonstrated the uniqueness of each of them in the Drosophilagenome and heterogeneity of their molecular organization. Interband 60E8-9/E10 contains gene rpl19transcribed throughout the development, in particular in salivary glands. In the other interbands examined 5" and 3" nontranslated gene regions are located. These results suggest that Drosophilainterbands may contain both housekeeping genes and regulatory sequences of currently inactive genes from adjacent bands.     

The replicative organization of DNA in polytene chromosomes ofDrosophila hydei

Salivary-gland nuclei ofDrosophila hydei were pulse-labeledin vitro with³H-thymidine and studied autoradiographically in squash preparations. The distribution of radioactive label over the length of the polytene chromosomes was discontinuous in most of the labeled nuclei; in some nuclei the pattern of incorporation was continuous. Comparison of the various labeling patterns of homologous chromosome regions in different nuclei showed that specific replicating units are replicated in a specific order. By combining autoradiography with cytophotometry of Feulgen-stained chromosomes, it was possible to correlate thymidine labeling of specific bands with their DNA content. The resulting data indicate that during the S-period many or perhaps all of the replicating units in a salivary-gland nucleus start DNA synthesis simultaneously but complete it at different times. Furthermore, the data support the hypothesis that the chromomere is a unit of replication or replicon. The DNA content of haploid chromomeres was found to be about 5×10^-4 pg for the largest bands inDrosophila hydei. From the results of H³-thymidine autoradiography and Feulgen-cytophotometry on neuroblast and anlage nuclei it was concluded that during growth of the polytenic nucleus heterochromatin is for the most part excluded from duplication. The results of DNA measurements in interbands of polytene chromosomes do not agree with a multistrand structure for the haploid chromatid. A chromosome model is proposed which is in accordance with the reported results and with current views concerning the replicative organization of chromosomes.