NeeY Chromosome of Drosophila miranda

We have cloned a novel transposable element from the nepY chromosome of Drosophila miranda. The size of the element, designated as TRAM, is 3.452 bp, including on both sides long terminal direct repeats (LTRs) of 372 bp, respectively. The element is flanked by a 5-bp target site duplication, ATATG. The putative primer binding site (PBS) for minus-strand priming is complementary to 18 nucleotides of the 3"end of tRNATv. Data base screens for DNA sequence identities were negative, apart from the sequence motif of the PBS. The deduced amino acid sequence from the large ORF does not' reveal identities described for other transposons. In situ hybridizations with TRAM subclones show a biased distribution in the genome, with a massive accumulation of TRAM in the neo-Y chromosome, while the former homologue, the XZchromosome is devoid of TRAMsites. The enriched occurrence of the TRAM element at the evolving neo-Y chromosome of D.miranda adds compelling evidence in favor of the view that Y chromosome degeneration is driven by the accumulation of transposable elements. Y CHROMOSOME degeneration (MULLER 1918, 1932) is a process that involves structural changes in chromosome architecture and expansion of genetic inertness along the Y chromosome (6 CHARLESWORTH 1978,1991,1996; LUCCHESI 1978,1994; CHARLESWORTH and CHARLESWORTH 1979; BULL 1983; RICE 1987,1994; STEINEMANN and STEINEMANN 1992). It is generally assumed that the heteromorphic sex chromosome pair has developed from a pair of homologues. Causes of the evolution of the structurally and functionally different X and Y chromosomes have been the object of speculation since the discovery of sex chromosomes. To analyze the molecular basis of this evolutionary process we chose a model system, Drosophila miranda. D. miranda belongs to the American representatives of the obscura group. The separation of D. mirandu from its next relatives D. pseudoobscura and D. persimilis occurred relatively recently, about 2 mya ago according to mtDNA restriction analysis (BARRIO et al. 1992). Due to the fusion of one of the autosomes to the Y chromosome, a neo-Y chromosome and a neo-X chromosome, designated as X2, were formed (DOBZHANSKY 1935; MACKNIGHT 1939; STEINEMANN 1982). Thus formerly autosomal genes are transmitted in association with the sex chromosomes. We have cloned the larval cuticle protein (Lcp) genes from the X 2 and neo-Y chromosome (STEINEMANN and STEINEMANN 1990). Analyzing the DNA sequences from the X 2 and neo-Y region, we observed a massive accumulation of DNA insertions on the neo-Y chromosome. In Corresponding author; Manfred Steinemann, Institut fur Genetik, Heinrich Heine Univenitat Dusseldorf, Univeniatsstr. 1, D40225 Dtisseldorf, FR Germany. E-mail: [email protected] Genetics 145 261-266 (February, 1997) polytene chromosome squashes, the male X chromosome in Drosophila can be distinguished by the presence of an isoform of histone H4 acetylated at lysine 16, H4.Ac16. Staining D. miranda with H4.Ac16 antibodies showed that dosage compensation extends across part of the X 2 (STEINEMANN et al. 1996). The X 2 shows a much brighter staining over -90% chromosome length, reflecting the predominant association of H4.Ac16. A terminal region constituting -10% of the X 2 chromosome is not labeled. This finding fits nicely with quantitative autoradiographic studies, showing that RNA synthesis in this region is not upregulated (STROBEL et al. 1978). Here we describe a novel LTR-retrotransposon cloned from the neo-Y-chromosomal Lcpl-4 locus. While the neo-Y shows an enrichment of TRAM sites (-50-60), we find the former homologue, the X 2 chromosome, to be devoid of sites. Together with the distribution of the non-LTR TRIM retrotransposon (STEINEMANN and STEINEMANN 1991) and the ISY3 insertion elements (STEINEMANN and STEINEMANN 1992, 1993), we present compelling evidence that the first step in Y chromosome degeneration is driven by the accumulation of transposable elements, especially retrotransposons. MATERIALS AND METHODS Cloning of the TRAM element from the neo-Y chromosome and the homologous region on the X 2 chromosome: High molecular weight DNA from D. miranda was isolated according to STEINEMANN 1982. Genomic EMBL4 lambda libraries from partial Sau3A (Boehringer Mannheim) digests were described in STEINEMANN and STEINEMANN (1990). Using a polymorphic restriction site, overlapping clones with X2 or neo-Y chromosomal origin were isolated, covering -30 kb 262 Steinemann and Steinemann