Cloning and characterisation of a novel chromosome end repeat enriched with homopolymeric (dA)/(dT) DNA in Rhynchosciara americana (Diptera: Sciaridae)

Abstract

Short tandem DNA repeats and telomerase compose the telomere structure in the vast majority of eukaryotic organisms. However, such a conserved organisation has not been found in dipterans. While telomeric DNA in Drosophila is composed of specific retrotransposons, complex terminal tandem repeats are present in chromosomes of Anopheles and chironomid species. In the sciarid Rhynchosciara americana, short repeats (16 and 22 bp long) tandemly arrayed seem to reach chromosome ends. Moreover, in situ hybridisation data using homopolymeric RNA probes suggested in this species the existence of a third putative chromosome end repeat enriched with (dA).(dT) homopolymers. In this work, chromosome microdissection and PCR primed by homopolymeric primers were employed to clone these repeats. Named T-14 and 93 % AT-rich, the repetitive unit is 14 bp long and appears organised in tandem arrays. It is localised in five non-centromeric ends and in four interstitial bands of R. americana chromosomes. To date, T-14 is the shortest repeat that has been characterised in chromosome ends of dipterans. As observed for short tandem repeats identified previously in chromosome ends of R. americana, the T-14 probe hybridised to bridges connecting non-homologous polytene chromosome ends, indicative of close association of T-14 repeats with the very end of the chromosomes. The results of this work suggest that R. americana represents an additional example of organism provided with more than one DNA sequence that is able to reach chromosome termini. Abbreviationsbp base pairs DAPI 4′,6-diamidino-2-phenylindole DOP-PCR degenerate oligonucleotide primed PCR dNTP deoxy-ribonucleotide 5′-triphosphate dUTP 2′-deoxy-uridine 5′-triphosphate FITC fluorescein isothiocyanate kbp kilobase pairs PCR polymerase chain reaction SDS Sodium dodecyl sulfate SSC sodium saline citrate TBS Tris-buffered saline TBST Tris-buffered saline 0.1 % Triton X-100 Chromosome Res (2012) 20:435-445

Telomere structure in insects: A review

“…The telomeric retrotransposons are reverse transcribed directly onto the end of the chromosome, extending the end by successive transposition (Pardue & DeBaryshe, ). Moreover, the telomerase gene was not detected in D. melanogaster genome, which also can be explained by the deep modification of the process of telomere replication in this species (Osanai et al, ).Interestingly, short telomeric repeats (14, 16, and 22 bp) and RaTART, which is apparently related to Drosophila's TART, were also detected in a particular member of Nematocera, Rhynchosciara americana (Wiedemann, 1821) that belongs to the family Sciaridae (dark‐winged fungus gnats) (Fernandes, Madalena, & Gorab, ; Madalena, Amabis, & Gorab, ; Madalena, Fernandes, Villasante, & Gorab, ). In the same species, reverse transcriptase‐related proteins were also found at non‐centromeric chromosome ends (Gorab, ), which likely represents a unique character state within Nematocera.…”

“…Interestingly, short telomeric repeats (14, 16, and 22 bp) and RaTART, which is apparently related to Drosophila's TART, were also detected in a particular member of Nematocera, Rhynchosciara americana (Wiedemann, 1821) that belongs to the family Sciaridae (dark‐winged fungus gnats) (Fernandes, Madalena, & Gorab, ; Madalena, Amabis, & Gorab, ; Madalena, Fernandes, Villasante, & Gorab, ). In the same species, reverse transcriptase‐related proteins were also found at non‐centromeric chromosome ends (Gorab, ), which likely represents a unique character state within Nematocera.…”

Telomeric transcriptome from Chironomus riparius (Diptera), a species with noncanonical telomeres

“…The localization of reverse transcriptase in Chironomid telomeres (López et al ., ; Gorab, ; Díez et al ., ) suggests that retrotranscription processes are also involved in the maintenance mechanism of these atypical telomeres. Curiously, composite structures of a retrotransposon and a complex repeat associated with chromosome ends have been found in Rhynchosciara [Diptera: Sciaridae (Madalena & Gorab, ; Madalena et al ., ; Fernandes et al ., )].…”

Aspectos da estrutura da cromatina e da atividade de transcrição induzida na glândula salivar de Rhynchosciara americana (Diptera: Sciaridae)

“…Dados de caracterização de sequências repetitivas de R. americana não mostraram que as sequências clonadas se encontram enriquecidas nesta região (Madalena & Gorab, 2005;Rossato et al, 2007;Madalena et al, 2010a;Madalena et al, 2010b;Fernandes et al, 2012). Dada a inexistência de sondas capazes de acessar a região, o passo seguinte foi o da microdissecção cromossômica para amplificação do DNA e posterior clonagem dos produtos de PCR.…”

“…15). Este método tem sido corriqueiramente empregado no laboratório para tal finalidade (Madalena & Gorab, 2005;Rossato et al, 2007;Madalena et al, 2010a;Madalena et al, 2010b;Fernandes et al, 2012) e A ponta de seta assinala a região A-9. Notar nestas imagens a localização da sonda em regiões terminais dos cromossomos.…”