Structural paradox of polytene chromosomes

The observation of thick chromatin fibers in interbands of Dipteran polytene chromosomes suggests that there should be 5 to 10 times more mass and DNA in interbands than is commonly thought to be present. To resolve this paradox, the chromatin content of interbands was estimated, using whole-mounted polytene chromosomes from Drosophila melanogster. Densitometry of high voltage electron microscopic negatives provides an estimate of less than 4:1 for the average ratio of cross-sectional dry mass (or mass per unit chromosome length) of bands relative to interbands. This ratio, combined with an estimate of the length of chromosome composed of interbands, indicates that at least 26% of chromosome mass is contributed by interband chromatin. Since DNA comprises a similar proportion of chromatin mass in bands and interbands (Laird et al., 1980b), these data imply that DNA sequences in interbands represent at least 26% of the euchromatic genome of D. melanogaster. This result calls for reinterpretation of some of the genetic and molecular data from Diptera. The discrepancy between this higher estimate of interband mass and DNA, and previous estimates of 3-5%, is discussed. One possibility is that previous measurements were made on prominent interbands, which are proposed here to be in regions that are delayed in DNA replication. Such interbands would be reduced in polyteny and DNA content compared with the average interband region. The concept of local variations in polyteny is also used here to explain major differences in the cross-sectional mass of bands. This leads to a revised model of polytene chromosomes in which at least three levels of polyteny, rather than one or two levels, can be present within one euchromatic region.     

Identical functional organization of nonpolytene and polytene chromosomes in Drosophila melanogaster

Salivary gland polytene chromosomes demonstrate banding pattern, genetic meaning of which is an enigma for decades. Till now it is not known how to mark the band/interband borders on physical map of DNA and structures of polytene chromosomes are not characterized in molecular and genetic terms. It is not known either similar banding pattern exists in chromosomes of regular diploid mitotically dividing nonpolytene cells. Using the newly developed approach permitting to identify the interband material and localization data of interband-specific proteins from modENCODE and other genome-wide projects, we identify physical limits of bands and interbands in small cytological region 9F13-10B3 of the X chromosome in D. melanogaster, as well as characterize their general molecular features. Our results suggests that the polytene and interphase cell line chromosomes have practically the same patterns of bands and interbands reflecting, probably, the basic principle of interphase chromosome organization. Two types of bands have been described in chromosomes, early and late-replicating, which differ in many aspects of their protein and genetic content. As appeared, origin recognition complexes are located almost totally in the interbands of chromosomes.     

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.     

The distribution of DNA and RNA in salivary gland chromosomes of Chironomus tentans as revealed by fluorescence microscopy

Summary In acridine orange stained chromosomes of Chironomus tentans, ribonuclease-sensitive reddish-orange fluorescence is found in all bands and in all interband regions as well as in nucleoli and Balbiani rings.Following ribonuclease digestion, deoxyribonuclease-sensitive yellowish-green fluorescence is found in all bands and in all interband regions. Banded fibres, apparent in Balbiani rings and in nucleoli, and formed by the splitting of the chromosome axis, also show no evidence of discontinuities in their yellowish-green fluorescence. From these results it is concluded that DNA is present in interbands and (at least at the level of the light microscope), is continuous through these regions of polytene chromosomes.     

<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.     

Analysis of DNA interband regions 3A5/A6, 3C5-6/c7 and 60E8-9/E10 of Drosophila melanogaster polytene chromosomes]

Using electron microscopic (EM) data on the formation of a novel band from the P-element material after its insertion in the interband and the procedure of P-target rescue, DNA interband regions 3A5/A6, and 60E8-9/E10 of Drosophila melanogaster polytene 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 sequences removed by deletion Df(1)faswb. 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 Drosophila genome and heterogeneity of their molecular organization. Interband 60E8-9/E10 contains gene rpl19 transcribed 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 Drosophila interbands may contain both housekeeping genes and regulatory sequences of currently inactive genes from adjacent bands.     

Banding patterns in Drosophila melanogaster polytene chromosomes correlate with DNA-binding protein occupancy

The most enigmatic feature of polytene chromosomes is their banding pattern, the genetic organization of which has been a very attractive puzzle for many years. Recent genome-wide protein mapping efforts have produced a wealth of data for the chromosome proteins of Drosophila cells. Based on their specific protein composition, the chromosomes comprise two types of bands, as well as interbands. These differ in terms of time of replication and specific types of proteins. The interbands are characterized by their association with "active" chromatin proteins, nucleosome remodeling, and origin recognition complexes, and so they have three functions: acting as binding sites for RNA pol II, initiation of replication and nucleosome remodeling of short fragments of DNA. The borders and organization of the same band and interband regions are largely identical, irrespective of the cell type studied. This demonstrates that the banding pattern is a universal principle of the organization of interphase polytene and non-polytene chromosomes.     

Polytene chromosome interband DNA is organized into nucleosomes

The molecular basis that underlies the maintenance of polytene chromosome banding pattern remains unclear. To test the possibility that the decondensed state of interbands is provoked by the absence of nucleosomes, we have subjected chromatin from the previously defined 61C7/C8 interband to digestion with micrococcal nuclease. We have demonstrated that interband DNA forms nucleosomes both in salivary glands and in the bulk of larval tissues. This finding strongly suggests that the difference in compaction between DNA in polytene chromosome bands and interbands results from differences that appear at the higher levels of chromatin organization.     

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.