Dmitry E. Koryakov

Polytene chromosomes: 70 years of genetic research.

Polytene chromosomes were described in 1881 and since 1934 they have served as an outstanding model for a variety of genetic experiments. Using the polytene chromosomes, numerous biological phenomena were discovered. First the polytene chromosomes served as a model of the interphase chromosomes in general. In polytene chromosomes, condensed (bands), decondensed (interbands), genetically active (puffs), and silent (pericentric and intercalary heterochromatin as well as regions subject to position effect variegation) regions were found and their features were described in detail. Analysis of the general organization of replication and transcription at the cytological level has become possible using polytene chromosomes. In studies of sequential puff formation it was found for the first time that the steroid hormone (ecdysone) exerts its action through gene activation, and that the process of gene activation upon ecdysone proceeds as a cascade. Namely on the polytene chromosomes a new phenomenon of cellular stress response (heat shock) was discovered. Subsequently chromatin boundaries (insulators) were discovered to flank the heat shock puffs. Major progress in solving the problems of dosage compensation and position effect variegation phenomena was mainly related to studies on polytene chromosomes. This review summarizes the current status of studies of polytene chromosomes and of various phenomena described using this successful model.

Vital genes in the heterochromatin of chromosomes 2 and 3 of Drosophila melanogaster

Heterochromatin has been traditionally regarded as a genomic wasteland, but in the last three decades extensive genetic and molecular studies have shown that this ubiquitous component of eukaryotic chromosomes may perform important biological functions. In D. melanogaster, about 30 genes that are essential for viability and/or fertility have been mapped to the heterochromatin of the major autosomes. Thus far, the known essential genes exhibit a peculiar molecular organization. They consist of single-copy exons, while their introns are comprised mainly of degenerate transposons. Moreover, about one hundred predicted genes that escaped previous genetic analyses have been associated with the proximal regions of chromosome arms but it remains to be determined how many of these genes are actually located within the heterochromatin. In this overview, we present available data on the mapping, molecular organization and function of known vital genes embedded in the heterochromatin of chromosomes 2 and 3. Repetitive loci, such as Responder and the ABO elements, which are also located in the heterochromatin of chromosome 2, are not discussed here because they have been reviewed in detail elsewhere.

Intercalary heterochromatin in Drosophila melanogaster polytene chromosomes and the problem of genetic silencing

The morphological characteristics of intercalary heterochromatin (IH) are compared with those of other types of silenced chromatin in the Drosophila melanogaster genome: pericentric heterochromatin (PH) and regions subject to position effect variegation (PEV). We conclude that IH regions in polytene chromosomes are binding sites of silencing complexes such as PcG complexes and of SuUR protein. Binding of these proteins results in the appearance of condensed chromatin and late replication of DNA, which in turn may result in DNA underreplication. IH and PH as well as regions subject to PEV have in common the condensed chromatin appearance, the localization of specific proteins, late replication, underreplication in polytene chromosomes, and ectopic pairing.

Dynamic organization of the beta-heterochromatin in the Drosophila melanogaster polytene X chromosome.

Abstract Region 20 of the polytene X chromosome of Drosophila melanogaster was studied in salivary glands (SG) and pseudonurse cells (PNC) of otu mutants. In SG chromosomes the morphology of the region strongly depends on two modifiers of position effect variegation: temperature and amount of heterochromatin. It is banded in XYY males at 25° C and β-heterochromatic in X0 males at 14° C, i.e. it shows dynamic transitions. In PNC chromosomes region 20 is not heterochromatic, but demonstrates a clear banding pattern. Some molecular markers of mitotic heterochromatin were localized by means of in situ hybridization on PNC chromosomes: DNA of the gene su(f) in section 20C, the nucleolar organizer and 359-bp satellite in 20F. The 359-bp satellite, which has been considered to be specific for heterochromatin of the mitotic X chromosome, was found at two additional sites on chromosome 3L, proximally to 80C. The right arm of the X chromosome in SG chromosomes was localized in the inversion In(1LR)pn2b: the telomeric HeT-A DNA and AAGAG satellite from the right arm are polytenized, having been relocated from heterochromatin to euchromatin.

CYTOLOGICAL STUDY OF THE BROWN DOMINANT POSITION EFFECT

Abstract.Classic recessive position effect variegation is related to inactivation of genes juxtaposed to heterochromatin and accompanied by cytologically visible heterochromatization (compaction) of the chromosome region containing these genes. Compaction and gene inactivation occur only in the rearranged homologue. In contrast to this, dominant variegation of the bw gene is known to involve transcriptional silencing in both the cis and trans copy, if they are paired. Our paper describes a cyto- logical approach to understanding this phenomenon. Analysis of salivary gland chromosomes carrying In(2R)bwVDe1 and In(2R)bwVDe2, evoking strong dominant bw variegation, has shown that in the rearranged homologues typical heterochromatization of the bw region and proximal neighbouring bands occurs. Heterochromatization was never observed on a normal homologue paired with a rearranged one. The insertion into the chromosome region 59E in the bwD strain is similar to pericentric heterochromatin. The insertion seems to induce heterochromatization of the neighbouring chromosome region and as a result the material of the insert and the 59E1–2 band join into a single block. When variegation is suppressed, the 59E1–2 band can be seen as a separate structure located proximal to the insert. This occurs in salivary gland polytene chromosomes of XYY males at 29°C and in pseudonurse cell polytene chromosomes of otu11/otu11 females. All bands in the region of the non-rearranged homologue show normal morphology. Thus, although in all strains studied we observed heterochromatization in cis, the homologous regions in trans are not visibly affected.

Abnormal tissue-dependent polytenization of a block of chromosome 3 pericentric heterochromatin in Drosophila melanogaster

Heterochromatic DNA sequences in the polytene chromosomes of Drosophila melanogaster salivary glands are under-replicated in wild-type strains. In salivary glands of SuUR and in the nurse cells of otu mutants, under-replication is partly suppressed and a banded structure appears within the centric heterochromatin of chromosome 3. This novel banded structure in salivary gland chromosomes was called Plato Atlantis. In order to characterize the heterochromatic component of Plato Atlantis, we constructed a fine-scale cytogenetic map of deletions with break points within centric heterochromatin (Df(3L)1-16, Df(3L)2-66, Df(3R)10-65, Df(3R)4-75 and Df(3L)6B-29 + Df(3R)6B-29). Salivary gland chromosomes show that Df(3L)1-16 removes the complete Plato Atlantis, while Df(3L)2-66 deletes the most proximal 3L regions. These deletions therefore show a substantial cytological overlap. However, in the chromosomes of nurse cells, the same deficiencies remove distinct heterochromatic blocks, with the region of overlap being almost invisible. Satellite (AATAACATAG, AAGAG) and dodecasatellite DNAs mapped in a narrow interval in salivary glands but were found in three clearly distinguishable blocks in nurse cells. The 1.688 satellite was found at a single site in salivary glands but at two sites in nurse cells. We show that newly polytenized heterochromatic structures include blocks h47-h50d of mitotic heterochromatin in salivary glands, but the additional blocks h50p, h53 and h57 are also included in nurse cell chromosomes. Tissue specificity of the patterns of abnormal heterochromatic polytenization implies differential control of DNA replication in somatic and germline cells.

ALPHA AND BETA HETEROCHROMATIN IN POLYTENE CHROMOSOME 2 OF DROSOPHILA MELANOGASTER

The formation of alpha and beta heterochromatin in chromosomes ofDrosophila melanogaster was studied in salivary glands (SGs) and pseudonurse cells (PNCs). In SGs ofX0, XY, XYY, XX andXXY individuals the amounts of alpha heterochromatin were similar, suggesting that theY chromosome does not substantially contribute to alpha heterochromatin formation. Pericentric heterochromatin developed a linear sequence of blocks in PNCs, showing morphology of both alpha and beta heterochromatin. In situ hybridization withRsp sequences (Ho clone) revealed that the most proximal heterochromatic segment of the mitotic map (region h39) formed a polytenized block in PNCs. Dot analysis showed that the clone had a hybridization rate with PNC-DNA very close to that with DNA from mainly diploid head cells, whereas the homologous SG-DNA was dramatically underrepresented. A similar increase of DNA representation in PNC was found for AAGAC satellite DNA. The mitotic region h44 was found not to polytenize in the SG chromosome, whereas in PNC chromosome 2 this region was partly polytenized and presented as an array of several blocks of alpha and beta heterochromatin. The mapping of deficiencies with proximal breakpoints in the most distal heterochromatin segments h35 in arm 2L and h46 in 2R showed that the mitotic eu-heterochromatin transitions were located in SG chromosomes distally to the polytene 40E and 41C regions, respectively. Thus, the transition zones between mitotic hetero- and euchromatin are located in banded polytene euchromatin. A scheme for dynamic organization of pericentric heterochromatin in nuclei with polytene chromosomes is proposed.

Polytene Chromosomes From Ovarian Nurse Cells of Drosophila melanogaster otu Mutants

Preface ..............................................................................................................v Contributors ..................................................................................................... ix Color Plates ................................................................................................... xiii 1. The Chromosomes of Drosophila melanogaster Daryl S. Henderson ............................................................................... 1 2. Spermatogenesis: Analysis of Meiosis and Morphogenesis Helen White-Cooper ........................................................................... 45 3. Time-Lapse Imaging of Male Meiosis by Phase-Contrast and Fluorescence Microscopy Elena Rebollo and Cayetano González ............................................... 77 4. Immunocytological Analysis of Oogenesis Endre Máthé ........................................................................................ 89 5. Cytological Analysis of Oogenesis Seppo Nokkala and Christina Nokkala ............................................. 129 6. Polytene Chromosomes From Ovarian Nurse Cells of Drosophila melanogaster otu Mutants Dmitry E. Koryakov, Natalia I. Mal’ceva, Robert C. King, and Igor F. Zhimulev .................................................................... 139 7. Feulgen–DNA Cytophotometry for Estimating C Values Ellen M. Rasch ................................................................................... 163 8. Fluorescent BrdU Labeling and Nuclear Flow Sorting of the Drosophila Ovary Brian R. Calvi and Mary A. Lilly ....................................................... 203 9. Studying Nuclear Organization in Embryos Using Antibody Tools Kristen M. Johansen and Jørgen Johansen ........................................ 215 10. Whole-Mount Fluorescence In Situ Hybridization to Chromosomes of Embryos Daryl S. Henderson ........................................................................... 235 11. Orcein Staining and the Identification of Polytene Chromosomes John Tonzetich .................................................................................. 249 12. Salivary Chromosome Analysis of Aberrations Adelaide T. C. Carpenter .................................................................. 257 13. In Situ Hybridization to Polytene Chromosomes Robert D. C. Saunders ...................................................................... 279

The effects of SUUR protein suggest its role in repressive chromatin renewal during replication in Drosophila

Replication of chromosomes is central to heredity. To become available for replication machinery, DNA invariably needs to dissociate from chromatin proteins. Yet, chromatin landscape must be promptly re-established during or soon after replication. Although this process underlies the epigenetic inheritance, little is known about its molecular mechanisms. This mini-review is focused on Drosophila melanogaster SUppressor of UnderReplication (SUUR) protein, which is involved both in replication and chromatin maintenance in polytene tissues. Existing data suggest that it is involved in the regulation of chromatin renewal during replication. According to this model, SUUR protein moves along the chromosomes together with the replication complex. When the replication fork enters the repressed, H3K27me3- or H3K9me3-enriched, chromatin, SUUR-containing complex slows down the replisome until these histone modifications are properly placed on the newly-synthesized DNA strands. Suggested model provides an insight into the mechanism of epigenetic information inheritance. This hypothesis could be tested by further analysis of the interplay between local enrichment of repressive histone modifications and the replication fork progression rate.

Tethering of SUUR and HP1 proteins results in delayed replication of euchromatic regions in Drosophila melanogaster polytene chromosomes

We analyze how artificial targeting of Suppressor of Under-Replication (SUUR) and HP1 proteins affects DNA replication in the “open,” euchromatic regions. Normally these regions replicate early in the S phase and display no binding of either SUUR or HP1. These proteins were expressed as fusions with DNA-binding domain of GAL4 and recruited to multimerized UAS integrated in three euchromatic sites of the polytene X chromosome: 3B, 8D, and 18B. Using PCNA staining as a marker of ongoing replication, we showed that targeting of SUURGAL4DBD and HP1GAL4DBD results in delayed replication of appropriate euchromatic regions. Specifically, replication at these regions starts early, much like in the absence of the fusion proteins; however, replication completion is significantly delayed. Notably, delayed replication was insufficient to induce underreplication. Recruitment of SUURGAL4DBD and HP1GAL4DBD had distinct effects on expression of a mini-white reporter, found near UAS. Whereas SUURGAL4DBD had no measurable influence on mini-white expression, HP1GAL4DBD targeting silenced mini-white, even in the absence of functional SU(VAR)3-9. Furthermore, recruitment of SUURGAL4DBD and HP1GAL4DBD had distinct effects on the protein composition of target regions. HP1GAL4DBD but not SUURGAL4DBD could displace an open chromatin marker, CHRIZ, from the tethering sites.