Cloning and characterization of a dispersed, multicopy, X chromosome sequence in Drosophila melanogaster.

Abstract

We have isolated and characterized a dispersed middle repetitive DNA sequence from Drosophila melanogaster that is concentrated on the euchromatic portion of the X chromosome. In situ hybridization of the repeat unit to salivary gland chromosomes shows the sequence is distributed among approximately 10 major and 20 minor X chromosomal sites. Based on DNA sequence analysis of homologous sequences from three different cytogenetic regions, the 372-base-pair repeat unit appears to be (A+T)-rich and noncoding and shows strong sequence conservation among units from different chromosomal regions. The nature and distribution of this sequence are suggestive of the hypothetical X chromosome DNA sequences thought to be involved in the primary establishment of sex determination and dosage compensation in Drosophila.The Drosophila genome consists of three kinetic components, highly repetitive, middle repetitive, and unique DNA (1). The middle repetitive fraction constitutes -417% of the total genomic DNA and includes the tandemly repeated rRNA, 5S RNA, and histone genes. Most of the middle repetitive DNA in Drosophila melanogaster, however, is not tandemly repeated but rather is dispersed over the entire genome (2). It is composed of at least 50-100 different sequence families, each of which generally consists of 10-100 closely related sequences (2). The number of sequences within a family seems to be conserved in different strains of the species, but their chromosomal positions are highly variable. In marked contrast, in this paper we describe a dispersed middle repetitive family with repeats at chromosomal positions that are conserved between strains. More importantly, the repeat units are concentrated in the euchromatic portion of the X chromosome. A functional link between family members is suggested by the extensive sequence homology found between repeats from different chromosomal regions. The intriguing possibility that these sequences may play a role in the primary establishment of sex determination and dosage compensation in Drosophila is considered. MATERIALS AND METHODSPreparation of Nucleic Acids. The Maniatis X bacteriophage library of D. melanogaster genomic DNA (3) was screened by in situ plaque hybridization (4) under low stringency hybridization [4x SET (0.6 M NaCl/8 mM EDTA/0.12 M Tris HCl, pH 8), 43% formamide, 37°C] and wash conditions (0.3 M NaCl/30 mM sodium citrate, pH 7, 37°C) with the 32P-labeled single-stranded RNA probe described in the text.Phage from single plaques were purified from liquid cultures by centrifugation in CsCl block gradients (5); phage DNA was purified with NaDodSO4, proteinase K, and phenol. For smaller quantities, DNA was prepared from phage lysates with RNase, NaDodSO4, and phenol.Subclones were generated by inserting gel-purified restriction fragments into the appropriate sites of the pGem-1 and pGem-2 transcription vectors (Promega Biotec, Madison, WI). Recombinant plasmids were used to transfect Escherichia coli HB101. To confirm their identity, alkaline minipreparat...

A model of segmental duplication formation in Drosophila melanogaster

“…2). Some of these duplications with >5 copies correspond to clusters of minisatellites that have already been described (Waring and Pollack 1987) as SARDM and X_DMR in Repbase Update (Supplemental Table S2; Jurka 2000). A repeat with 13 copies contained the "euchromatic Stellate gene cluster."…”

siRNAs from an X-linked satellite repeat promote X-chromosome recognition in Drosophila melanogaster

“…This suggests that siRNAproducing sequences on the X chromosome might participate in the identification of X chromatin. The X-limited distribution of 1.688 g/cm 3 satellite-related repeats (1.688 X repeats) has prompted speculation that they function in dosage compensation, an idea supported by the remarkable enrichment of rapidly evolving repeats on the X chromosomes of related species (10)(11)(12)(13). The D. melanogaster 1.688 X repeats are arranged in short tandem arrays and, unlike most satellite repeats, are associated with transcriptionally active regions (14).…”

Transcription of the 1.688 Satellite DNA Family Is Under the Control of RNA Interference Machinery inDrosophila melanogasterOvaries

“…Short tandem arrays (two to four copies) of sequences homologous to the 1.688 satellite are dispersed throughout the euchromatin of the X chromosome. These 1.688 satellite-related sequences are often situated in the vicinity of genes and their requirement for some sexchromosome-specific functions has been discussed (Waring and Pollack 1987;DiBartolomeis et al 1992;Losada and Villasante 1996).…”