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

Microdissection and sequence analysis of pericentric heterochromatin from the Drosophila melanogaster mutant Suppressor of Underreplication

In the Suppressor of Underreplication (SuUR) mutant strain of Drosophila melanogaster, the heterochromatin of polytene chromosomes is not underreplicated and, as a consequence, a number of P-heterochromatic regions acquire a banded structure. The chromocenter does not form in these polytene chromosomes, and heterochromatic regions, normally part of the chromocenter, become accessible to cytological analysis. We generated four genomic DNA libraries from specific heterochromatic regions by microdissection of polytene chromosomes. In situ hybridization of individual libraries onto SuUR polytene chromosomes shows that repetitive DNA sequences spread into the neighboring euchromatic regions. This observation allows the localization of eu-heterochromatin transition zones on polytene chromosomes. We find that genomic scaffolds from the eu-heterochromatin transition zones are enriched in repetitive DNA sequences homologous to those flanking the suppressor of forked gene [su(f) repeat]. We isolated and sequenced about 300 clones from the heterochromatic DNA libraries obtained. Most of the clones contain repetitive DNA sequences; however, some of the clones have unique DNA sequences shared with parts of unmapped genomic scaffolds. Hybridization of these clones onto SuUR polytene chromosomes allowed us to assign the cytological localizations of the corresponding genomic scaffolds within heterochromatin. Our results demonstrate that the SuUR mutant renders possible the mapping of heterochromatic scaffolds on polytene chromosomes

Accession numbers of chromosome proteins.

<p>*modENCODE data used (<a href="http://www.modencode.org/Genomes.shtml" target="_blank">http://www.modencode.org/Genomes.shtml</a>).</p><p>**GEO data used (<a href="http://www.ncbi.nlm.nih.gov/gds/" target="_blank">http://www.ncbi.nlm.nih.gov/gds/</a>).</p

Comparison of extents of band/interbands in polytene chromosomes according to Flybase r5.25 (A, B) and to the data of this study (C).

<p>Physical DNA map is situated between 10792800 and 11220400 positions of the map of Flybase.</p

Colocalization of DNA probes limiting the band 10B1–2, and Chriz/CHRO protein.

<p>Banding pattern in the region 10A–B (DAPI) (<b>A</b>), immunostaining of Chriz/CHRO and FISH of the DNA probe (<b>A</b>), immunostaining of Chriz/CHRO, FISH of the DNA probe and DAPI (<b>C</b>). Bar represents 5 µm.</p

Relation of genetic map, and band/interband pattern in the region 9F13 – 10B3.

<p><b>A</b> - predicted bands <b>B</b> - FlyBase genes <b>C</b> – 30 chromatin states in BG3 and S2 cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025960#pone.0025960-Kharchenko1" target="_blank">[48] </a><b>D</b> - DNase I hypersensitivity sites (DHS) in S2, BG3 and Kc cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025960#pone.0025960-Kharchenko1" target="_blank">[48] </a><b>E</b> - ORC-binding sites in S2, BG3 and Kc cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025960#pone.0025960-Eaton1" target="_blank">[59] </a><b>F</b> – Nucleosome Density (modENCODE, Henikoff group) <b>G</b> - active chromatin specific <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025960#pone.0025960-Kharchenko1" target="_blank">[48]</a> and - interbands specific proteins. Predicted interbands (dotted vertical lines are according to peaks in distribution of corresponding elements, solid lines reflect the edges of distributions of different characteristics).</p

DNA compaction ratio of the bands in the 9F13 -10B3 region.

a<p>– estimated on electron microscope sections of 50 polytene chromosomes.</p><p>Notes: IB-interband; B-band.</p><p>Data of release FB2011_03 were used.</p

Comparison of several Electron Microscope sections of the region 9F11-12 – 10B (A, B) with revised Bridges map

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025960#pone.0025960-Bridges1" target="_blank">[<b>56</b>] </a><b>(C).</b> Vertical lines connect homologous bands. Scale represents 1 µm.</p

Coordinates and sizes of bands and interbands on physical map of the 9F13 – 10B3 region.

<p>Notes: IB-interband; B-band.</p><p>Data of release FB2011_03 were used.</p