The Enigma of Y Chromosome Degeneration: Tram, a Novel Retrotransposon Is Preferentially Located on the Neo-Y Chromosome of Drosophila Miranda

We have cloned a novel transposable element from the neo-Y 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 tRNA(Trp). 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 X2 chromosome is devoid of TRAM sites. 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.     

Accumulation of Spock and Worf,two novel non-LTR retrotransposons,on the neo-Y chromosome of Drosophila miranda

Transposable elements constitute a major fraction of eukaryotic genomes. Here, I characterize two novel non-LTR retrotransposons, cloned from the neo-Y chromosome of Drosophila miranda. Worf is 4.1 kb in size and shows homology to the T1-2 non-LTR transposon characterized in Anopheles. Spock is 4.9 kb in size and shows similarity to the Doc element of D. melanogaster. Southern blot analysis of both elements yielded stronger signals for male DNA. In situ hybridization to polytene chromosomes revealed that both elements are accumulating on the neo-Y chromosome of D. miranda. PCR analysis was conducted to investigate the frequency of spock and worf and of the previously identified transposons, TRIM and TRAM, at individual chromosomal sites among 12 strains of D. miranda. Contrary to the observation that element frequencies are usually kept low at individual sites in Drosophila, the four transposons investigated are fixed at their genomic locations on the neo-Y chromosome. These results support the hypothesis that transposons accumulate in nonrecombining regions and may be one cause of the heteromorphism of sex chromosomes.     

Identification of a new hobo element in the cabbage moth, Mamestra brassicae (Lepidoptera)

A complete hobo-like element, called Mbhobo, was identified in the cabbage moth, Mamestra brassicae. This element has a high sequence similarity to the HFL1 hobo element of Drosophila melanogaster. Amplification of Mbhobo termini indicated that transposition occurred into a 5'-GTGGGTAC-3' target sequence that was duplicated upon insertion. This target site conforms to the consensus sequence established for the insertion sites of insect hAT elements. Mbhobo has a single 1935 bp long ORF with significant homology to the D. melanogaster HFL1 hobo transposase. FISH experiments evidenced Mbhobo clusters located in heterochromatic regions of Z and W sex chromosomes and in heterochromatic areas of chromosome pair 10.     

GEM, a cluster of repetitive sequences in the Drosophila subobscura genome

GEM is a new family of repetitive sequences detected in the D. subobscura genome. Two of the four described GEM elements encompass a heterogeneous central module, with no detectable ORF, flanked by two long inverted repeats. These elements are composed of a set of repetitive modules, which are inverted repeat (IR), direct repeat (DR), palindromic sequence (PS), long sequence (LS) and short sequence (SS). These five modules can be found either clustered or dispersed as single modules in the D. subobscura genome, in euchromatic and heterochromatic regions. In addition to the 3' region of Adh retrosequences, single IR and LS blocks were found associated with the promoter region of different genes, in particular, LS-like blocks have also been found associated with functional genes in D. melanogaster and D. virilis. Conversely, the DR block is highly similar to satellite DNAs from some other species of the obscura group. In addition, GEM elements share some structural features with IS elements described in different Drosophila species. It is likely that both GEM and IS sequences would be vestiges of an ancestral transposable element.     

Molecular characterization of the Drosophila melanogaster urate oxidase gene, an ecdysone-repressible gene expressed only in the malpighian tubules.

The urate oxidase (UO) gene of Drosophila melanogaster is expressed during the third-instar larval and adult stages, exclusively within a subset of cells of the Malpighian tubules. The UO gene contains a 69-base-pair intron and encodes mature mRNAs of 1,224, 1,227, and 1,244 nucleotides, depending on the site of 3' endonucleolytic cleavage prior to polyadenylation. A direct repeat, 5'-AAGTGAGAGTGAT-3', is the proposed cis-regulatory element involved in 20-hydroxyecdysone repression of the UO gene. The deduced amino acid sequences of UO of D. melanogaster, rat, mouse, and pig and uricase II of soybean show 32 to 38% identity, with 22% of amino acid residues identical in all species. With use of P-element-mediated germ line transformation, 826 base pairs 5' and approximately 1,200 base pairs 3' of the D. melanogaster UO transcribed region contain all of the cis elements allowing for appropriate temporal regulation and Malpighian tubule-specific expression of the UO gene.     

A Duplication Including the Y Allele of Lcp2 and the Trim Retrotransposon at the Lcp Locus on the Degenerating Neo-Y Chromosome of Drosophila Miranda: Molecular Structure and Mechanisms by Which It May Have Arisen

Evolutionary changes during the process of sex chromosome differentiation in Drosophila miranda are associated with massive DNA rearrangements. Comparing the DNA structure of the larval cuticle protein (Lcp) region from the X2 and neo-Y chromosome pair, we observed insertions, deletions and a large duplication at the neo-Y chromosomal locus. The duplication encompasses a complete copy of the neo-Y allele of Lcp2, and the ISY3 and the ISY4 insertion sequences. The latter was identified as a retrotransposon, termed TRIM. ISY3 shows DNA sequence similarity to P element homologs identified in the Drosophila obscura species group. We were interested in mechanistic aspects generating the duplication. We cannot exclude unequivocally that unequal sister-chromatid exchange could give rise to the observed duplication; however, recombination is a rare event in Drosophila males. Location and sequence of the retrotransposon TRIM served as molecular markers allowing us to reconstruct two intrachromosomal transposition events that could lead to the observed duplication.     

The Drosophila mobile element jockey is a LINE element and contains coding sequences homologous to retroviral proteins

A detailed investigation of Drosophila melanogaster mobile dispersed repetitive element jockey is performed. Its structural features resemble those of LINE elements. Sequencing of the complete jockey 5020 bp in length revealed two long open reading frames ORF1 and ORF2 overlapping with a frameshift-1. Judging by amino acid homologies, ORF1 encodes a nucleic acid binding protein, characteristic of replication competent retroviruses; the 3' part of ORF2 encodes an RNA-dependent DNA polymerase which has an amino acid sequence, similar to recently published sequences of LINE elements of Drosophila, Trypanosoma and mammals. This fact demonstrates their evolutionary relationship. Sequencing of several deleted copies of jockey revealed the absence of the major part of ORF2, though the rest of the element, including the ends, is highly conservative.     

A selective sweep associated with a recent gene transposition in Drosophila miranda

In Drosophila miranda, a chromosome fusion between the Y chromosome and the autosome corresponding to Muller's element C has created a new sex chromosome system. The chromosome attached to the ancestral Y chromosome is transmitted paternally and hence is not exposed to crossing over. This chromosome, conventionally called the neo-Y, and the homologous neo-X chromosome display many properties of evolving sex chromosomes. We report here the transposition of the exuperantia1 (exu1) locus from a neo-sex chromosome to the ancestral X chromosome of D. miranda. Exu1 is known to have several critical developmental functions, including a male-specific role in spermatogenesis. The ancestral location of exu1 is conserved in the sibling species of D. miranda, as well as in a more distantly related species. The transposition of exu1 can be interpreted as an adaptive fixation, driven by a selective advantage conferred by its effect on dosage compensation. This explanation is supported by the pattern of within-species sequence variation at exu1 and the nearby exu2 locus. The implications of this phenomenon for genome evolution are discussed.     

Rates and patterns of chromosomal evolution in Drosophila pseudoobscura and D. miranda

Comparisons of gene orders between species permit estimation of the rate of chromosomal evolution since their divergence from a common ancestor. We have compared gene orders on three chromosomes of Drosophila pseudoobscura with its close relative, D. miranda, and the distant outgroup species, D. melanogaster, by using the public genome sequences of D. pseudoobscura and D. melanogaster and approximately 50 in situ hybridizations of gene probes in D. miranda. We find no evidence for extensive transfer of genes among chromosomes in D. miranda. The rates of chromosomal rearrangements between D. miranda and D. pseudoobscura are far higher than those found before in Drosophila and approach those for nematodes, the fastest rates among higher eukaryotes. In addition, we find that the D. pseudoobscura chromosome with the highest level of inversion polymorphism (Muller's element C) does not show an unusually fast rate of evolution with respect to chromosome structure, suggesting that this classic case of inversion polymorphism reflects selection rather than mutational processes. On the basis of our results, we propose possible ancestral arrangements for the D. pseudoobscura C chromosome, which are different from those in the current literature. We also describe a new method for correcting for rearrangements that are not detected with a limited set of markers.     

Transposition of elements of the 412, copia and 297 dispersed repeated gene families in Drosophila.

The stability of elements of three different dispersed repeated gene families in the genome of Drosophila tissue culture cells has been examined. Different amounts of sequences homologous to elements of 412, copia and 297 dispersed repeated gene families are found in the genomes of D. melanogaster embryonic and tissue culture cells. In general the amount of these sequences is increased in the cell lines. The additional sequences homologous to 412, copia and 297 occur as intact elements and are dispersed to new sites in the cell culture genome. It appears that these elements can insert at many alternative sites. We also describe a DNA sequence arrangement found in the D. melanogaster embryo genome which appears to result from a transposition of an element of the copia dispersed repeated gene family into a new chromosomal site. The mechanism of insertion of this copia element is precise to within 90 bp and may involve a region of weak sequence homology between the site of insertion and the direct terminal repeats of the copia element.     

Complete foldback transposable elements encode a novel protein found in Drosophila melanogaster.

An apparently complete foldback (FB) transposable element homologous to FB white-crimson (FBwc) was analyzed. A complete FB element could encode one or more proteins required for regulation of FB transposition. The central DNA region (the loop) and the junctions between the loop and the inverted terminal repeats were sequenced. Three open reading frames (ORFs) are present in the loop, and a novel 308 bp inverted repeat is present at the junctions. No significant homologies were found when the DNA sequences of the loop region and the novel inverted repeat were screened against the Gene data bank. Antibodies were prepared in guinea-pigs against a peptide present near the amino terminus of ORF1, the longest ORF. A 71,000 dalton protein was isolated from an extract of Drosophila melanogaster early-stage embryos on an anti-ORF1 peptide-affinity column. Immunohistochemical studies of adult flies demonstrate localization of this protein in egg chambers.     

Site-specific ribosomal DNA insertion elements in Anopheles gambiae and A. arabiensis: nucleotide sequence of gene-element boundaries.

The nucleotide sequence of the junctions between the 28S ribosomal gene and site-specific insertion elements from two sibling mosquito species, Anopheles gambiae and A. arabiensis, is reported. In both species, elements insert at the same point within the 28S gene, but this site is 634 basepairs (bp) 3' of the R1 (Type I) insertion site in Drosophila melanogaster. The two mosquito elements each have poly A tails and a polyadenylation signal, but the extreme 3' and 5' ends show no other similarity to each other or to any other insertion element. In both mosquito species, identical target site duplications of 17 bp are generated. The sequence TNTCCCTNT found in this duplication is also found in the 14 bp target site duplications that flank R1 elements in D. melanogaster. Another sequence in this duplication, GGGATAACT, is very similar to the sequence GGGAGTAACT found in the 24 base sequence required by the Bombyx mori R2 endonuclease.     

Differentiation of Muller''s Chromosomal Elements D and E in the Obscura Group of Drosophila

Twenty-two markers located on Muller's elements D or E have been mapped by in situ hybridization in six species of the obscura group of Drosophila and in D. melanogaster. The obscura species can be grouped into a Palearctic cluster (D. subobscura, D. madeirensis and D. guanche) and a Nearctic one (D. pseudoobscura, D. persimilis and D. miranda). Eleven of the probes contain known genes: E74, Acp70A, Est5, hsp28/23, hsp83, emc, hsp70, Xdh, Acph-1, Cec and rp49. The remaining probes are recombinant phages isolated from a D. subobscura genomic library. All these markers hybridize to the putative homologous chromosome or chromosomal arm of elements D and E. Thus, these elements have conserved their genic content during species divergence. Chromosomal homologies proposed previously for each element among the species of the same cluster have been compared with the present results. The distribution of markers within each element has changed considerably as inferred from pairwise comparisons of obscura species included in the two different clusters. Only chromosomal segments defined by closely linked markers have been conserved: one such segment has been detected in element D and three in element E between D. subobscura and D. pseudoobscura.