E. S. Belyaeva

EcR isoforms in Drosophila: testing tissue-specific requirements by targeted blockade and rescue

The three Drosophila EcR isoforms differ only at their N termini; thus, they share the conserved ligand-binding domain transcriptional activation function (AF2) and only differ in the unconserved A/B region, which contains a second, isoform-specific, activation function (AF1). We have developed a dominant-negative mutant EcR (EcR-DN), expressed it in flies with the GAL4/UAS system, and used it to block ecdysone signaling in eight tissues or groups of tissues. Localized EcR-DN arrests ecdysone-dependent development in the target cells and often — because of a molting checkpoint — arrests development globally. Simultaneously expressing individual wild-type EcR isoforms in the same target tissues suppresses the EcR-DN phenotype and identifies the rescuing isoform as sufficient to support the development of the target. Every isoform, and even an N-terminal truncated EcR that lacks any AF1, supports development in the fat body, eye discs, salivary glands, EH-secreting neurosecretory cells and in the dpp expression domain, implying that AF1 is dispensable in these tissues. By contrast, only EcR-A is able to support development in the margins of the wing discs, and only EcR-B2 can do so in the larval epidermis and the border cells of the developing egg chamber. In light of our results, the simplest explanations for the widespread spatial and temporal variations in EcR isoform titers appear untenable.

Influence of the SuUR gene on intercalary heterochromatin in Drosophila melanogaster polytene chromosomes

Abstract. Salivary gland polytene chromosomes of Drosophila melanogaster have a reproducible set of intercalary heterochromatin (IH) sites, characterized by late DNA replication, underreplicated DNA, breaks and frequent ectopic contacts. The SuUR mutation has been shown to suppress underreplication, and wild-type SuUR protein is found at late-replicating IH sites and in pericentric heterochromatin. Here we show that the SuUR gene influences all four IH features. The SuUR mutation leads to earlier completion of DNA replication. Using transgenic strains with two, four or six additional SuUR+ doses (4–8×SuUR+) we show that wild-type SuUR is an enhancer of DNA underreplication, causing many late-replicating sites to become underreplicated. We map the underreplication sites and show that their number increases from 58 in normal strains (2×SuUR+) to 161 in 4–8×SuUR+ strains. In one of these new sites (1AB) DNA polytenization decreases from 100% in the wild type to 51%–85% in the 4×SuUR+ strain. In the 4×SuUR+ strain, 60% of the weak points coincide with the localization of Polycomb group (PcG) proteins. At the IH region 89E1–4 (the Bithorax complex), a typical underreplication site, the degree of underreplication increases with four doses of SuUR+ but the extent of the underreplicated region is the same as in wild type and corresponds to the region containing PcG binding sites. We conclude that the polytene chromosome regions known as IH are binding sites for SuUR protein and in many cases PcG silencing proteins. We propose that these stable silenced regions are late replicated and, in the presence of SuUR protein, become underreplicated.

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.

MOLECULAR CHARACTERISATION OF THE DEEP ORANGE (DOR) GENE OF DROSOPHILA MELANOGASTER

Abstract Mutations of the dor gene of Drosophila melanogaster cause defects in different stages of development. Heterozygotes for lethal or viable dor alleles and the rearrangement T(1;2)dorvar7, which causes position effect variegation of dor, exhibit traits such as rough eyes, reduction of bristles on the thorax and scutellum and wavy wings. The dor gene was mapped to the proximal part of the 2B3-5 band or in the interband between 2B3-5 and 2B6 and localised within an interval of 5 kb on the physical map of the cloned 2B region. The 3.0–3.1 kb dor transcript was detected by Northern hybridization at all stages of development and is expressed in salivary glands of third instar larve. This RNA was not expressed in the dor mutants with insertions in the 5′ part of the gene. The sequence of the 3180 bp dor cDNA predicts a 115.3 kDa protein that contains a cysteine- and histidine-rich zinc finger-like motif CX2CX13CXHX2HX2CX2H at the C-terminus. The protein sequence reveals 23% identity to the Saccharomyces cerevisiae PEP3 protein. The most significant homology (57% similarity and 32% identity) between the DOR and PEP3 proteins is observed at the C-termini of the proteins.

Three distinct chromatin domains in telomere ends of polytene chromosomes in Drosophila melanogaster Tel mutants

Drosophila melanogaster telomeric DNA is known to comprise two domains: the terminal tract of retrotransposons (HeT-A, TART and TAHRE) and telomere-associated sequences (TAS). Chromosome tips are capped by a protein complex, which is assembled on the chromosome ends independently of the underlying terminal DNA sequences. To investigate the properties of these domains in salivary gland polytene chromosomes, we made use of Tel mutants. Telomeres in this background are elongated owing to the amplification of a block of terminal retroelements. Supercompact heterochromatin is absent from the telomeres of polytene chromosomes: electron microscopy analysis identifies the telomeric cap and the tract of retroelements as a reticular material, having no discernible banding pattern, whereas TAS repeats appear as faint bands. According to the pattern of bound proteins, the cap, tract of retroelements and TAS constitute distinct and non-overlapping domains in telomeres. SUUR, HP2, SU(VAR)3-7 and H3Me3K27 localize to the cap region, as has been demonstrated for HP1. All these proteins are also found in pericentric heterochromatin. The tract of retroelements is associated with proteins characteristic for both heterochromatin (H3Me3K9) and euchromatin (H3Me3K4, JIL-1, Z4). The TAS region is enriched for H3Me3K27. PC and E(Z) are detected both in TAS and many intercalary heterochromatin regions. Telomeres complete replication earlier than heterochromatic regions. The frequency of telomeric associations in salivary gland polytene chromosomes does not depend on the SuUR gene dosage, rather it appears to be defined by the telomere length.

Cytogenetic and molecular aspects of position effect variegation in Drosophila melanogaster

The peculiarities of compact blocks appearing as a consequence of position effect variegation were studied in male polytene chromosomes. In T(1;2)dorvar7/Y males the frequency of nuclei with a block in the 2B region was lower at all temperature and the chromosome regioninvolved in compaction was shorter than in T(1;2)dorvar7/FM6 females. The fraction of nuclei with blocks was considerably increased indorvar7/0 males, especially at 18° C when the viability of these males is sharply reduced. The following features distinguish theblocks in males from those in females: (i) compaction of the 2B region in the males results in genetic inactivation only to a very small extent; (ii) the structure of the blocks in males is diffuse; and(iii) the male blocks still maintain some transcriptional activity as indicated by 3H-uridine incorporation. The temperaturesensitive period of both block formation and geneticinactivation was found to be during the first 3 h of embryonic development.

SUUR joins separate subsets of PcG, HP1 and B-type lamin targets in Drosophila.

Drosophila melanogaster Suppressor of Under-Replication (SuUR) gene encodes a protein that modulates replicative properties of heterochromatin in endocycles of polytene cells. The SuUR mutation abolishes underreplication of intercalary heterochromatin and results in partial underreplication of pericentric heterochromatin. We performed a genome-wide mapping of SUUR target genes in non-polytenic Drosophila Kc cells by using the DamID approach. We show that SUUR preferentially binds genes that are transcriptionally silent and late-replicated. Distinct subsets of SUUR targets are associated with PcG proteins (Pc and Esc; Polycomb and Extra sexcombs), heterochromatic proteins [HP1 and SU(VAR)3-9] and B-type lamin. The SUUR binding profile negatively correlates with the DNA polytenization levels of salivary gland polytene chromosomes. Finally, SUUR target genes are repressed in Drosophila embryos and gradually activated later in development. Together these results suggest that SUUR is a ubiquitous marker of heterochromatin in different cell types.

Electron microscopical analysis of Drosophila polytene chromosomes

An electron microscopical (EM) analysis was performed on regions of polytene chromosomes which contained DNA segments of different genetic composition, inserted by P element-mediated transformation into the Drosophila melanogaster genome. In seven of ten regions examined, containing insertions of the hsp28-ry, hsp70-Adh, ryhsp 70-β-gal genes and of the ry gene tetramer, new bands appeared. Lack of new bands in three other strains is apparently connected with the fusion of the inserted material to preexisting bands. The new bands do not differ morphologically from the usual bands of polytene chromosomes, and their formation is likely due to predominant insertion of DNA segments into interbands. Among the constructs examined, the minimal length of a DNA segment which appears as a new band is about 5 kb; the DNA packing ratio in the new bands varies from 30 to 50. Activation of the inserted genes by heat shock has enabled us to observe the puffing characteristics of new bands. A sequence of some one kb forms a large interband, or micropuff; the puff size is correlated with the length of the genes being activated. If a DNA segment contains a single gene, then its activation causes the decompaction of the whole band; however, when a DNA segment consists of two genes and the promoter element of the activated gene is positioned in the middle of the sequence, the band splits and only part is decompacted and puffed. The DNA packing ratio in the puffs is 1.4–3.5. The subsequent deletion of the hsp70 promoter but retention of 23, 59, and 73 bp from the transcription start points leads to failure of puff formation. In all the transformed sites an increase in the total length of the interbands adjacent to the insert as compared with the initial interband was observed. This increase appears to be due to decompaction of the P element DNA flanking the inserted segments. It is shown that a DNA segment, consisting of four tandemly repeated ry gene copies and interspersed by material which includes P DNA, forms a complex of loose chromatin in which, however, four bands can be resolved. We also observed a lengthening of interband regions containing only the P element sequence itself. Insertion of the complete 2.9 kb P element into the large single 10A1-2 band of the X chromosome (an insertion in the region approximately 10 kb to the right of the v gene) causes splitting of the band into two parts and formation of a new interband. However, insertion of the 412 mobile genetic element from the copia family into the same region results in no such effect. These facts together with data on puffing initiation in the centre of the band, when the hsp70 promoter is inside the insert, necessitate a reappraisal of the putative unit character of polytene chromosome bands as regards decompaction.

Constitutive heterochromatin in early embryogenesis of Drosophila melanogaster

SummaryThe formation of constitutive heterochromatin was studied during the embryonic development of Drosophila melanogaster, using the C-banding technique. During embryonic cleavage, C-banded material is not seen in mitotic chromosomes; the differentiation between euchromatin and heterochromatin only occurs at blastoderm. This event correlates with the establishment of position-effect variegation.

The introduction of a transpositionally active copy of retrotransposon GYPSY into the Stable Strain of Drosophila melanogaster causes genetic instability

A previously described genetic system comprising a Mutator Strain (MS) and the Stable Strain (SS) from which it originated is characterized by genetic instability caused by transpositions of the retrotransposon gypsy. A series of genetic crosses was used to obtain three MS derivatives, each containing one MS chromosome (X, 2 or 3) in the environment of SS chromosomes. All derivatives are characterized by elevated frequencies of spontaneous mutations in both sexes. Mutations appear at the premeiotic stage and are unstable. Transformed derivatives of SS and another stable strain 208 were obtained by microinjection of plasmid DNA containing transpositionally active gypsy inserted into the Casper vector. In situ hybridization experiments revealed amplification and active transposition of gypsy in SS derivatives, while the integration of a single copy of gypsy into the genome of 208 does not change the genetic properties of this strain. We propose that genetic instability in the MS system is caused by the combination of two factors: mutation(s) in gene(s) regulating gypsy transposition in SS and its MS derivatives, and the presence of transpositionally active gypsy copies in MS but not SS.