[Insulator Protein CP190 Regulates Expression of Spermatocyte Differentiation Genes in Drosophila melanogaster Male Germline].

CP190 protein is one of the key components of Drosophila insulator complexes, and its study is important for understanding the mechanisms of gene regulation during cell differentiation. However, Cp190 mutants die before reaching adulthood, which significantly complicates the study of its functions in imago. To overcome this problem and to investigate the regulatory effects of CP190 in adult tissues development, we have designed a conditional rescue system for Cp190 mutants. Using Cre/loxP-mediated recombination, the rescue construct containing Cp190 coding sequence is effectively eliminated specifically in spermatocytes, allowing us to study the effect of the mutation in male germ cells. Using high-throughput transcriptome analysis we determined the function of CP190 on gene expression in germline cells. Cp190 mutation was found to have opposite effects on tissue-specific genes, which expression is repressed by CP190, and housekeeping genes, that require CP190 for activation. Mutation of Cp190 also promoted expression of a set of spermatocyte differentiation genes that are regulated by tMAC transcriptional complex. Our results indicate that the main function of CP190 in the process of spermatogenesis is the coordination of interactions between differentiation genes and their specific transcriptional activators.

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.

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

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 degrees C and beta-heterochromatic in X0 males at 14 degrees 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(ILR)pn2b: the telomeric HeT-A DNA and AAGAG satellite from the right arm are polytenized, having been relocated from heterochromatin to euchromatin.

Su(UR)ES: a gene suppressing DNA underreplication in intercalary and pericentric heterochromatin of Drosophila melanogaster polytene chromosomes.

A genetic locus suppressing DNA underreplication in intercalary heterochromatin (IH) and pericentric heterochromatin (PH) of the polytene chromosomes of Drosophila melanogaster salivary glands, has been described. Found in the In(1)scV2 strain, the mutation, designated as Su(UR)ES, was located on chromosome 3L at position 34. 8 and cytologically mapped to region 68A3-B4. A cytological phenotype was observed in the salivary gland chromosomes of larvae homozygous and hemizygous for Su(UR)ES: (i) in the IH regions, that normally are incompletely polytenized and so they often break to form "weak points," underreplication is suppressed, breaks and ectopic contacts disappear; (ii) the degree of polytenization in PH grows higher. That is why the regions in chromosome arm basements, normally beta-heterochromatic, acquire a distinct banding pattern, i. e., become euchromatic by morphological criteria; (iii) an additional bulk of polytenized material arises between the arms of chromosome 3 to form a fragment with a typical banding pattern. Chromosome 2 PH reveals additional alpha-heterochromatin. Su(UR)ES does not affect the viability, fertility, or morphological characters of the imago, and has semidominant expression in the heterozygote and distinct maternal effect. The results obtained provide evidence that the processes leading to DNA underreplication in IH and PH are affected by the same genetic mechanism.

Cytological study of the brown dominant position effect.

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 degrees 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.

Alpha and beta heterochromatin in polytene chromosome 2 of Drosophila melanogaster.

The formation of alpha and beta heterochromatin in chromosomes of Drosophila melanogaster was studied in salivary glands (SGs) and pseudonurse cells (PNCs). In SGs of X0, XY, XYY, XX and XXY individuals the amounts of alpha heterochromatin were similar, suggesting that the Y 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 with Rsp 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.