A three-dimensional structural dissection of Drosophila polytene chromosomes.

Abstract

Abstract. We have analyzed the three-dimensional structural details of Drosophila melanogaster polytene chromosome bands and interbands using three-dimensional light microscopy and a novel method of sample preparation that does not involve flattening or stretching the chromosomes. Bands have been visualized in unfixed chromosomes stained with the DNA specific dye 4,6-Diamidino-2-phenylindole (DAPI). Interbands have been visualized using fixed chromosomes that have been immunostained with an antibody to RNA polymerase II. Additionally, these structures have been analyzed using in situ hybridization with probes from specific genetic loci (Notch and white).Bands are seen to be composed of ~36 substructural features that measure 0.2-0.4 txm in diameter. We suggest that these substructural features are in fact longitudinal fibers made up of bundles of chromatids. Band shape can be a reproducible characteristic of a particular band and is dependent on the spatial relationship of these bundles, varying from bands with a uniform distribution of bundles to bands with a peripheral concentration of chromatin. Interbands are composed of bundles of chromatids of a similar size and number as those seen in the bands. The distribution of bundles is similar between a band and the neighboring interband, implying that there is a long range organization to the DNA that includes both the coding and the noncoding portions of genes. Finally, we note that the polytene chromosome has a circular shape when viewed in cross section, whether there are one or two homologs present. fundamental unsolved problem in biology is the organization of the eukaryotic genome in the interphase nucleus. During interphase, chromatin must be maintained in structures that allow site-specific regulated transcription and temporally controlled DNA replication. Normally, it is not possible to resolve individual chromosomes in the interphase nucleus by light microscopy. However, due to the process of polytenization, the giant salivary gland chromosomes of Drosophila melanogaster are large enough to visualize, thus providing an excellent model system for studying interphase processes. Polytenization occurs in nearly all larval tissues and is a result of a modified cell cycle, which alternates between S phase and G phase (Smith and Orr-Weaver, 1991). The polytene nuclei in salivary gland tissue can undergo up to 10 cycles of replication without intervening divisions. The unsegregated sister chromatids and homologs remain Y. Urata and S. J. Parmelee both contributed equally to the work presented herein.

A Quantitative Study of the Quality of Deconvolved Wide-field Microscopy Images as Function of Empirical Three-dimensional Point Spread Functions

“…Therefore, a locally defined PSF is unlikely to remove the effects of spherical aberration using a global deconvolution. However, there is evidence suggesting that high-quality deconvolution of images of thick objects can be achieved without introducing this complexity into the deconvolution algorithm [11,26]. In our lab, we have recently obtained similar images, using setups that are up to 25 μm below the cover slip [17].…”

Linker histone H1 is essential for Drosophila development, the establishment of pericentric heterochromatin, and a normal polytene chromosome structure

“…Previous studies suggested that interchromatid cohesion is generated and maintained in the banded regions (Ananiev and Barsky 1985;Urata et al 1995). H1 is widely distributed in euchromatic arms of polytene chromosomes; however, it localizes predominantly to bands of compacted chromatin (Fig.…”

c(3)G encodes a Drosophila synaptonemal complex protein

“…Prior to viewing, immunostained tissue was embedded in polyacrylamide gel using a procedure based on methods published previously (Urata et al 1995;Bass et al 1997). Briefly, the immunostained ovarioles in PBST were placed on a coverslip within an area enclosed by dried nail polish or Scotch invisible tape, and as much PBST as possible was aspirated.…”