The characterization of chromosome end sequences of Rhynchosciara americana was initiated with the screening of a plasmid microlibrary made from a microdissected polytene chromosome tip. A 268 bp insert chosen for analysis hybridized specifically to non-telocentric chromosome ends in which reverse transcriptase had been previously identified. Southern-blot hybridization of R. americana genomic DNA cut with XbaI, the only restriction site identified in the 268 bp probe, showed a ladder composed of multimers of a band in the range of 400 bp indicating a tandem array of the repeat partially represented in the cloned fragment. The complete repeat unit obtained by inverse PCR is 414 bp long, 67% AT-rich, characterized by the restriction sites XbaI and SalI and displays features typical of a nematoceran telomeric satellite. The telomere-like repeat is apparently absent from the chromosomes of two other Rhynchosciara species, R. baschanti and R. milleri. Double staining for satellite hybridization and reverse transcriptase in R. americana suggests that the arrays composed of the telomeric-like satellite do not reach the chromosome ends. This may indicate that telomeric-like features of some nematoceran terminal satellites do not warrant their telomeric position.

The characterisation of chromosome end (terminal and sub-terminal) sequences of Rhynchosciara americana chromosomes was continued with the screening of a plasmid library made of amplified DNA fragments from a microdissected chromosome tip. An insert chosen for analysis hybridised to two chromosome ends and contains two microsatellite arrays in close vicinity to a sequence (named M-47), part of which is significantly similar to minisatellites of Salmonidae that are frequently present in the vicinity of microsatellite arrays. PCR results using a single primer representative of M-47 elements suggest that they are also repetitive in Rhynchosciara genomes. In addition, total single primer PCR products hybridised to the non-telocentric end subset of R. americana chromosomes. Another plasmid microlibrary made of chromosome tips amplified by a single M-47 primer was screened for repeats of Rhynchosciara chromosomes. Selected inserts that hybridised strongly to non-telocentric ends of R. americana and R. hollaenderi have a tandem array of 22 bp repeats (M-22). There is sequence divergence among M-22 repeats but their mean similarity is significantly high in relation to the M-22 consensus sequence derived from the cloned tandem array. M-22 elements lie distal to the 414 bp sub-telomeric satellite array characterised previously as suggested by double labelling for M-22 hybridisation and reverse transcriptase. M-47 elements, formerly identified in Salmonidae, thus contribute to specify unusually short repeats composing the sub-telomeric structure of two Rhynchosciara species.

The characterisation of sequences at chromosome ends of Rhynchosciara americana was continued with the screening of a genomic library using as a probe a short repeat identified in a previous report (M-22, 22 bp) which was found to be specific for noncentromeric termini of this species. Simple repeats, complex tandem and apparently dispersed repeats were present in the genomic clones analysed. Repetitive sequences do not define individual chromosome tips as they were found in all noncentromeric ends. A novel and unusually short tandem repeat type for dipteran chromosome ends (named M-16) composed of 16 nucleotides and frequently associated with M-22 arrays was characterised in this work. Islands of M-16 and M-22 tandem repeats were found in all the genomic clones analysed. Individual probes representative of each repetitive element hybridised not only to all noncentromeric ends of R. americana chromosomes but also to inter-telomeric bridges. This contrasted with the other repeat types which displayed sub-telomeric localisation as seen by double detection of hybridised probe and telomeric reverse transcriptase. Some stretches composed of M-16 and M-22 tandem repeats localised in different regions of the analysed genomic clones were either identical or showed sequence similarity that was unexpectedly higher than the mean sequence similarity observed among repeats within each of their tandem arrays. The occurrence of segmental duplications, as deduced by sequence analyses involving the two repeats that appeared to reach chromosome ends, might indicate the involvement of this type of duplication process in the chromosome end maintenance in this species.

The chromosomal localization of ribosomal DNA (rDNA) was studied in polytene and diploid tissues of four sciarid species, Trichosia pubescens, Rhynchosciara americana, R. milleri and Schwenkfeldina sp. While hybridization to mitotic chromosomes showed the existence of a single rDNA locus, ribosomal probes hybridized to more than one polytene chromosome region in all the species analyzed as a result of micronucleolar attachment to specific chromosome sites. Micronucleoli are small, round bodies containing transcriptionally active, probably extrachromosomal rDNA. In T. pubescens the rDNA is predominantly localized in chromosome sections X-10 and X-8. In R. americana the rDNA is frequently found associated with centromeric heterochromatin of the chromosomes X, C, B and A, and also with sections X-1 and B-13. Ribosomal probes in R. milleri hybridized with high frequency to pericentric and telomeric regions of its polytene complement. Schwfenkfeldina sp. displays a remarkably unusual distribution of rDNA in polytene nuclei, characterized by the attachment of micronucleoli to many chromosome regions. The results showed that micronucleoli preferentially associate with intercalary or terminal heterochromatin of all sciarid flies analyzed and, depending on the species, are attached to a few (Trichosia), moderate (Rhynchosciara) or a large (Schwenkfeldina sp.) number of polytene chromosome sites.

In Drosophila, telomere retrotransposons counterbalance the loss of telomeric DNA. The exceptional mechanism of telomere recovery characterized in Drosophila has not been found in lower dipterans (Nematocera). However, a retroelement resembling a telomere transposon and termed "RaTART" has been described in the nematoceran Rhynchosciara americana. In this work, DNA and protein sequence analyses, DNA cloning, and chromosomal localization of probes obtained either by PCR or by screening a genomic library were carried out in order to examine additional features of this retroelement. The analyses performed raise the possibility that RaTART represents a genomic clone composed of distinct repetitive elements, one of which is likely to be responsible for its apparent enrichment at chromosome ends. RaTART sequence in addition allowed to assess a novel subtelomeric region of R. americana chromosomes that was analyzed in this work after subcloning a DNA fragment from a phage insert. It contains a complex repeat that is located in the vicinity of simple and complex tandem repeats characterized previously. Quantification data suggest that the copy number of the repeat is significantly lower than that observed for the ribosomal DNA in the salivary gland of R. americana. A short insertion of the RaTART was identified in the cloned segment, which hybridized preferentially to subtelomeres. Like RaTART, it displays truncated sequences related to distinct retrotransposons, one of which has a conceptual translation product with significant identity with an endonuclease from a lepidopteran retrotransposon. The composite structure of this DNA stretch probably reflects mobile element activity in the subtelomeric region analyzed in this work.