Novel simple sequence repeats (SSRs) detected by ND-FISH in heterochromatin of Drosophila melanogaster

<p>Abstract</p> <p>Background</p> <p>In recent years, substantial progress has been made in understanding the organization of sequences in heterochromatin regions containing single-copy genes and transposable elements. However, the sequence and organization of tandem repeat DNA sequences, which are by far the majority fraction of <it>D. melanogaster </it>heterochromatin, are little understood.</p> <p>Results</p> <p>This paper reports that the heterochromatin, as well as containing long tandem arrays of pentanucleotide satellites (AAGAG, AAGAC, AATAT, AATAC and AACAC), is also enriched in other simple sequence repeats (SSRs) such as A, AC, AG, AAG, ACT, GATA and GACA. Non-denaturing FISH (ND-FISH) showed these SSRs to localize to the chromocentre of polytene chromosomes, and was used to map them on mitotic chromosomes. Different distributions were detected ranging from single heterochromatic clusters to complex combinations on different chromosomes. ND-FISH performed on extended DNA fibres, along with Southern blotting, showed the complex organization of these heterochromatin sequences in long tracts, and revealed subclusters of SSRs (several kilobase in length) flanked by other DNA sequences. The chromosomal characterization of C, AAC, AGG, AAT, CCG, ACG, AGC, ATC and ACC provided further detailed information on the SSR content of <it>D. melanogaster </it>at the whole genome level.</p> <p>Conclusion</p> <p>These data clearly show the variation in the abundance of different SSR motifs and reveal their non-random distribution within and between chromosomes. The greater representation of certain SSRs in <it>D. melanogaster </it>heterochromatin suggests that its complexity may be greater than previously thought.</p

Both piRNA and siRNA Pathways Are Silencing Transcripts of the Suffix Element in the Drosophila melanogaster Germline and Somatic Cells

In the Drosophila melanogaster germline, the piRNA pathway silences retrotransposons as well as other transcribed repetitive elements. Suffix is an unusual short retroelement that was identified both as an actively transcribed repetitive element and also as an element at the 3′ ends of the Drosophila non-LTR F element. The copies of suffix that are F element-independent are far more actively transcribed than their counterparts on the F element. We studied the patterns of small RNAs targeting both strands of suffix in Drosophila ovaries using an RNase protection assay and the analysis of the corresponding RNA sequences from the libraries of total small RNAs. Our results indicate that suffix sense and antisense transcripts are targeted mainly by 23–29 nucleotides in length piRNAs and also by 21 nucleotides in length siRNAs. Suffix sense transcripts actively form longer RNA species, corresponding either to partial digestion products of the RNAi and Piwi pathways or to another RNA silencing mechanism. Both sense and antisense suffix transcripts accumulated in the ovaries of homozygous spn-E, piwi and aub mutants. These results provide evidence that suffix sense and antisense transcripts in the germ line and soma are targeted by both RNAi and Piwi pathways and that a Dicer-independent pathway of biogenesis of siRNAs could exist in Drosophila cells

Late Replication Domains in Polytene and Non-Polytene Cells of Drosophila melanogaster

In D. melanogaster polytene chromosomes, intercalary heterochromatin (IH) appears as large dense bands scattered in euchromatin and comprises clusters of repressed genes. IH displays distinctly low gene density, indicative of their particular regulation. Genes embedded in IH replicate late in the S phase and become underreplicated. We asked whether localization and organization of these late-replicating domains is conserved in a distinct cell type. Using published comprehensive genome-wide chromatin annotation datasets (modENCODE and others), we compared IH organization in salivary gland cells and in a Kc cell line. We first established the borders of 60 IH regions on a molecular map, these regions containing underreplicated material and encompassing ∼12% of Drosophila genome. We showed that in Kc cells repressed chromatin constituted 97% of the sequences that corresponded to IH bands. This chromatin is depleted for ORC-2 binding and largely replicates late. Differences in replication timing between the cell types analyzed are local and affect only sub-regions but never whole IH bands. As a rule such differentially replicating sub-regions display open chromatin organization, which apparently results from cell-type specific gene expression of underlying genes. We conclude that repressed chromatin organization of IH is generally conserved in polytene and non-polytene cells. Yet, IH domains do not function as transcription- and replication-regulatory units, because differences in transcription and replication between cell types are not domain-wide, rather they are restricted to small “islands” embedded in these domains. IH regions can thus be defined as a special class of domains with low gene density, which have narrow temporal expression patterns, and so displaying relatively conserved organization

The Effect of Central Loops in miRNA:MRE Duplexes on the Efficiency of miRNA-Mediated Gene Regulation

MicroRNAs (miRNAs) guide posttranscriptional repression of mRNAs. Hundreds of miRNAs have been identified but the target identification of mammalian mRNAs is still a difficult task due to a poor understanding of the interaction between miRNAs and the miRNA recognizing element (MRE). In recent research, the importance of the 5′ end of the miRNA:MRE duplex has been emphasized and the effect of the tail region addressed, but the role of the central loop has largely remained unexplored. Here we examined the effect of the loop region in miRNA:MRE duplexes and found that the location of the central loop is one of the important factors affecting the efficiency of gene regulation mediated by miRNAs. It was further determined that the addition of a loop score combining both location and size as a new criterion for predicting MREs and their cognate miRNAs significantly decreased the false positive rates and increased the specificity of MRE prediction

MicroRNA miR-328 Regulates Zonation Morphogenesis by Targeting CD44 Expression

Morphogenesis is crucial to initiate physiological development and tumor invasion. Here we show that a microRNA controls zonation morphogenesis by targeting hyaluronan receptor CD44. We have developed a novel system to study microRNA functions by generating constructs expressing pre-miRNAs and mature miRNAs. Using this system, we have demonstrated that expression of miR-328 reduced cell adhesion, aggregation, and migration, and regulated formation of capillary structure. Protein analysis indicated that miR-328 repressed CD44 expression. Activities of luciferase constructs harboring the target site in CD44, but not the one containing mutation, were repressed by miR-328. Zonation morphogenesis appeared in cells transfected by miR-328: miR-328-transfected cells were present on the surface of zonating structures while the control cells stayed in the middle. MiR-328-mediated CD44 actions was validated by anti-CD44 antibody, hyaluronidase, CD44 siRNA, and CD44 expression constructs. In vivo experiments showed that CD44-silencing cells appeared as layers on the surfaces of nodules or zonating structures. Immuno-histochemistry also exhibited CD44-negative cells on the surface layers of normal rat livers and the internal zones of Portal veins. Our results demonstrate that miR-328 targets CD44, which is essential in regulating zonation morphogenesis: silencing of CD44 expression is essential in sealing the zonation structures to facilitate their extension and to inhibit complex expansion

Model of continuous and discontinuous chromatin compaction, resulting from position effect variegation

A position effect variegation model based on statistical distribution of molecules of compactisator-proteins (position effect variegation modifier genes products) around compactization initiation centers is suggested. The model is founded on two theses: 1) The centers, consisting of DNA motifs, are present in both euchromatin and heterochromatin; 2) the proteins bind to the DNA and to each other acting in synergy. When a definite level of the DNA-protein binding is exceeded heterochromatic domains are formed. The model explains continuous and discontinuous compaction as a consequence of the distribution of irregular initiation centers along the chromosome

Position Effect Variegation Model of Chromatin Compaction

A position effect variegation (PEV) model of chromatin compaction is proposed, based on the concept of a statistical distribution of compaction protein (CP) molecules around compaction initiation centers (CICs). The principles of the model are as follows: (1) CICs are present in both hetero- and euchromatin, and (2) different CP molecules interact not only with DNA but also with one another, forming a multimeric complex. When a certain level of DNA-protein binding is exceeded, heterochromatic domains are formed. The model suggests that continuous and discontinuous chromatin compaction resulting from PEV is due to an irregular CIC distribution along the chromosome

Observations On the Induction of Position Effect Variegation of Euchromatic Genes in Drosophila-Melanogaster

In the T(1;2)dor(var7) translocation, the IA-2B7-8 segment of the X chromosome is brought to the vicinity of 2R-chromosome heterochromatin resulting in position effect variegation of dor, BR-C and more distal genes, as well as compaction of chromatin in this segment. By irradiation of T(1;2)dor(var7), nine reversions (rev) to a normal phenotype were recovered. In two cases (rev27, rev226), the 1A-2B7-8 section is relocated to the 19A region of the X chromosome, forming free duplications (1A-2B7--8/19A-20F-X-het). Modifiers of position effect do not change the normal expression of the BR-C and dor genes in these duplications. In five reversions (rev3, rev40, rev60, rev167, rev175), free duplications have formed from the 1A-2B7-8 fragment and X chromosome heterochromatin. In these rearrangements, modifiers of position effect (low temperature, removal of Y and 2R-chromosome heterochromatin and a genetic enhancer (E-var(3)201) induce position-effect again. Two reversions (rev45 and rev110) are associated with additional inversions in the original dor(var7) chromosomes. The inversions relocate part of the heterochromatin adjacent to the IA-2B7-8 section into new positions. In T(1;2)dor(rev45), position-effect is seen in the 2B7-8-7A element as compaction spreading from 2B7-8 proximally in some cases as far as the 5D region. Thus, in rev45 the pattern of euchromatin compaction is reciprocal to that of the initial dor(var7) strain. Apparently, it is due to the same variegation-evoking center near the 2R centromere in both cases. In all nine revertants, weakening or complete disappearance of the position-effect is observed despite retention of the 20- kb heterochromatic segment adjacent to the IA-2B7-8 region. Thus, a 20-kb heterochromatic sequence does not inactivate euchromatin joined to it

Molecular characterisation of the deep orange (dor) gene of Drosophila melanogaster

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)dor(var7), 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 for 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 CX(2)CX(13)CXHX(2)HX(2)CX(2)H 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