DNA sequence comparison of micropia transposable elements fromDrosophila hydei andDrosophila melanogaster

Members of the retrotransposon family micropia were discovered as constituents of wild-typeY chromosomal fertility genes fromDrosophila hydei. Several members of the micropia family have subsequently been recovered fromDrosophila melanogaster and four micropia elements, micropia-DhMiF2, -DhMiF8, -Dm11 and-Dm2, two each fromD. hydei andD. melanogaster, have been totally sequenced (17 kb of micropia sequences and 6.8 kb from insertions)¹. Comparative analysis of micropia sequences revealed a complex pattern of divergence within a singleDrosophila genome. The divergence includes deletions, possibly by a slipped mispairing mechanism, insertions of a retroposon, and of another retrotransposon (copia) and positional nucleotide shuffling within the tandem repeats of the 3 non-protein-coding region of micropia elements. A 10 bp long sequence of each repeat unit of the 3 tandem repeats of micropia elements is highly conserved and is therefore a candidate of functional importance either in transposition events or in regulatory activity on flanking DNA sequences.Abbreviations LTR long terminal repeat - PBS primer binding site - PolII RNA polymerase II - bp base pairs - kb kilobases (pairs) - LINE long interspersed sequence - MHC major histocompatibility complex This paper is dedicated to the 90^th birthday of Prof. Dr. Bernhard Rensch.     

Regulation of the segmentation gene fushi tarazu has been functionally conserved in Drosophila.

An evolutionary approach was applied to identify elements involved in the regulation of the segmentation gene fushi tarazu (ftz) by comparing the Drosophila melanogaster ftz gene with its Drosophila hydei homologue. The overall organization of the ftz gene is very similar in both species. Surprisingly, ftz proved to be inverted in the ANT-C of D. hydei with respect to D. melanogaster. Strong homologies extend over the entire 6 kb of the ftz upstream region with the best match in the 'upstream element'. We identified several highly conserved boxes embedded in unrelated sequences that correspond extremely well to two germ layer specific enhancers in the upstream element. Transformation experiments revealed that D. hydei ftz gene products can restore D. melanogaster ftz function and, furthermore, that trans-acting factors from D. melanogaster recognize and control D. hydei ftz regulatory elements. These findings indicate a conservation of the entire regulatory network among segmentation genes for several millions of years during the evolution of Drosophila.     

Evolution of gypsy endogenous retrovirus in the Drosophila obscura species group

The Ty3/gypsy family of retroelements is closely related to retroviruses, and some of their members have an open reading frame resembling the retroviral gene env. Sequences homologous to the gypsy element from Drosophila melanogaster are widely distributed among Drosophila species. In this work, we report a phylogenetic study based mainly on the analysis of the 5' region of the env gene from several species of the obscura group, and also from sequences already reported of D. melanogaster, Drosophila virilis, and Drosophila hydei. Our results indicate that the gypsy elements from species of the obscura group constitute a monophyletic group which has strongly diverged from the prototypic D. melanogaster gypsy element. Phylogenetic relationships between gypsy sequences from the obscura group are consistent with those of their hosts, indicating vertical transmission. However, D. hydei and D. virilis gypsy sequences are closely related to those of the affinis subgroup, which could be indicative of horizontal transmission.     

Localization of ecs, dor and swl genes in eight Drosophila species]

The DNA sequences from Drosophila melanogaster early ecdysterone-inducible puff 2B have been located in 8 Drosophila species by in situ hybridization. The location site of the ecs, dor and swi genes in D. funebris, D. virilis, D. hydei, D. repleta, D. mercatorum, D. paranaensis is a puff on the telomeric and of X chromosome; in D. kanekoi it is the puff in distal part of X chromosome; and in D. pseudoobscura it is the puff in proximal portion of X chromosome. So, conservative organization of DNA sequences located in D. melanogaster 2B puff could be suggested. Dispersed distribution of some DNA segments from the region studied in D. hydei chromosomes was revealed.     

Identification of Two Subfamilies of micropia Transposable Element in Species of the repleta Group of Drosophila

The occurrence, number of insertion sites and antisense RNA expression of micropia transposable element were studied in 26 species that belong to three subgroups (mercatorum, mulleri and hydei) of repleta group of Drosophila. Under high specific PCR, micropia sequences were detected in 11 species, but under less stringent condition, this retrotransposon was detected in all species. The widespread distribution of micropia suggests that this element was already present at the common ancestor of the repleta group of Drosophila. Southern blot analysis showed a variation from 0 to 17 different insertion sites and the occurrence of male-specific sequences. We found that the expression of the 1.0 kb micropia antisense RNA is variable among the species and tissues (soma and testis), which suggests that more than one mechanism regulates transposition in these species. Variation of amplification by PCR and of antisense RNA expression, as well as divergence of nucleotide sequences among the species allow us to suggest that at least two subfamilies of micropia transposable element are harbored by the genome of this species group.     

The endogenous Drosophila melanogaster retrovirus gypsy can propagate in Drosophila hydei cells

The endogenous Drosophila melanogaster retrovirus gypsy (mdg4) forms virus-like particles (VLPs) which are found as extracellular particles in the medium used to culture D. melanogaster cells. The D. hydei somatic cell line DH14, which does not harbour gypsy sequences, was exposed to D. melanogaster VLPs. Subsequent PCR and Southern analysis revealed that gypsy elements had penetrated into the D. hydei cells, suggesting interspecific transmission of the retrovirus. A D. hydei cell line containing gypsy sequences was established and grown in a mixed culture together with the G418-resistant D. hydei cell line DH33, and gypsy was shown to be transmitted from cell to cell. The proportion of cells carrying gypsy increased with time. The rate of gypsy invasion of the lines DH14 and DH33 was 10(-3) and 10(-2) per cell per generation, respectively. The results demonstrate the possibility of interspecific horizontal transfer of gypsy in the form of its VLPs.     

Evolutionary Conservation of the Structure and Expression of Alternatively Spliced Ultrabithorax Isoforms from Drosophila

In Drosophila melanogaster, alternatively spliced mRNAs from the homeotic gene Ultrabithorax (Ubx) encode a family of structurally distinct homeoprotein isoforms. The developmentally regulated expression patterns of these isoforms suggest that they have specialized stage- and tissue-specific functions. To evaluate the functional importance of UBX isoform diversity and gain clues to the mechanism that regulates processing of Ubx RNAs, we have investigated whether the Ubx RNAs of other insects undergo similar alternative splicing. We have isolated and characterized Ubx cDNA fragments from D. melanogaster, Drosophila pseudoobscura, Drosophila hydei and Drosophila virilis, species separated by as much as 60 million years of evolution, and have found that three aspects of Ubx RNA processing have been conserved. (1) These four species exhibit identical patterns of optional exon use in a region adjacent to the homeodomain. (2) These four species produce the same family of UBX protein isoforms with identical amino acid sequences in the optional exons, even though the common amino-proximal region has undergone substantial divergence. The nucleotide sequences of the optional exons, including third positions of rare codons, have also been conserved strongly, suggesting functional constraints that are not limited to coding potential. (3) The tissue- and stage-specific patterns of expression of different UBX isoforms are identical among these Drosophila species, indicating that the developmental regulation of the alternative splicing events has also been conserved. These findings argue for an important role of alternative splicing in Ubx function. We discuss the implications of these results for models of UBX protein function and the mechanism of alternative splicing.     

Intra- and interspecies variation among Bari-1 elements of the melanogaster species group.

We have investigated the distribution of sequences homologous to Bari-1, a Tc1-like transposable element first identified in Drosophila melanogaster, in 87 species of the Drosophila genus. We have also isolated and sequenced Bari-1 homologues from D. simulans, D. mauritiana, and D. sechellia, the species constituting with D. melanogaster the melanogaster complex, and from D. diplacantha and D. erecta, two phylogenetically more distant species of the melanogaster group. Within the melanogaster complex the Bari-1 elements are extremely similar to each other, showing nucleotide identity values of at least 99.3%. In contrast, Bari-1-like elements from D. diplacantha and D. erecta are on average only 70% similar to D. melanogaster Bari-1 and are usually defective due to nucleotide deletions and/or insertions in the ORFs encoding their transposases. In D. erecta the defective copies are all located in the chromocenter and on chromosome 4. Surprisingly, while D. melanogaster Bari-1 elements possess 26-bp inverted terminal repeats, their D. diplacantha and D. erecta homologues possess long inverted terminal repeats similar to the terminal structures observed in the S elements of D. melanogaster and in several other Tc1-like elements of different organisms. This finding, together with the nucleotide and amino acid identity level between D. diplacantha and D. erecta elements and Bari-1 of D. melanogaster, suggests a common evolutionary origin and a rapid diversification of the termini of these Drosophila Tc1-like elements.     

Isolation of Y-chromosomal repetitive DNA sequences of Drosophila hydei via enrichment of chromosome-specific sequences by heterogeneous hybridization between female and male DNA

Male or female DNA of Drosophila hydei was sheared by sonication, denatured, reannealed to different C0t-values and fractionated by hydroxyapatite. The highly repetitive, moderately repetitive and unique fractions of female DNA were denatured again and coupled via diazotization or cyanogen bromide to macroporous Sephacryl S-500. Enrichment of Y-chromosomal sequences was achieved by cycling each of the different fractions of male DNA under optimized hybridization conditions over a column with a manifold excess of immobilized female DNA of the corresponding complexity. Thereby, Y-chromosomal sequences of D. hydei could be enriched about 100-fold for highly and moderately repetitive DNA and about 10-fold for unique DNA. When a library of male D. hydei DNA was screened with Y-enriched highly repetitive DNA, more than 98% of all hybridizing phages contained inserts of repetitive Y-chromosomal DNA of at least four different sequence families.     

Evolution of Polypyrimidines in Drosophila

We surveyed 101 different Drosophila species for the presence of a particular highly repetitive DNA sequence containing long tracts of polypyrimidine/polypurine DNA, first found in D. melanogaster. Out of 55 tested species in the melanogaster group, only the sibling species D. simulans and D. mauritiana, as well as one distant relative in the ananassae subgroup, D. varians, contained the same sequence. All four of these species have long pyrimidine tracts as shown by acid hydrolysis of labelled DNA. All four species have the same sequence, bu the amount of this polypyrimidine/polypurine DNA varies greatly. Four other species in the hydei subgroup were found to contain a polypyrimidine/polpurine sequence, with an oligonucleotide composition different from that of D. melanogaster. This polypyrimidine DNA varies from as much as 10% of the total DNA in D. nigrohydei, to as little as 0.4% in D. neohydei. The long pyrimidine tracts in the hydei subgroup are often more than a thousand nucleotides in length, representing exceedingly homogeneous repetitious sequences.--These results show a rapid but discontinuous pattern of evolution for polypyrimidine/polypurine DNA . These sequences are not species specific, yet closely related species have greatly different amounts of polypyrimidines. Drastic changes occur in the amounts of these satellite type DNA sequences, as if the sequence had no continuous selective advantage in evolution. The implications of these results with regard to the general function and evolution of satellite DNA are discussed.