Potential sites of triple-helical nucleic acid formation in chromosomes of Rhynchosciara (Diptera: Sciaridae) and Drosophila melanogaster

Abstract

Antibodies to specific nucleic acid conformations are amongst the methods that have allowed the study of non-canonical (Watson-Crick) DNA structures in higher organisms. In this work, the structural limitations for the immunological detection of DNA.RNA hybrid duplexes were examined using specific RNA homopolymers as probes for homopolymer polydeoxyadenylic acid (poly(dA)).polydeoxythymidylic acid (poly(dT))-rich regions of Rhynchosciara americana (Diptera: Sciaridae) chromosomes. Anti-DNA.RNA duplexes did not react with the complex formed between chromosomal poly(dA) and exogenous polyuridylic acid (poly(rU)). Additionally, poly(rU) prevented the detection of polyadenylic acid.poly(dT) hybrid duplexes preformed in situ. These results raised the possibility that three-stranded structures rather than duplexes were formed in chromosomal sites. To test this hypothesis, the specificity of antibodies to triple-helical nucleic acids was reassessed employing distinct nucleic acid configurations. These antibodies were raised to the poly(dA).poly(rU).poly(rU) complex and have been used here for the first time in immunocytochemistry. Anti-triplex antibodies recognised the complex poly(dA).poly(rU).poly(rU) assembled with poly(rU) in poly(dA).poly(dT)-rich homopolymer regions of R. americana chromosomes. The antibodies could not detect short triplex stretches, suggesting the existence of constraints for triple-helix detection, probably related to triplex tract length. In addition, anti-poly(dA).poly(rU).poly(rU) antibodies reacted with the pericentric heterochromatin of RNase-treated polytene chromosomes of R. americana and Drosophila melanogaster. In apparent agreement with data obtained in cell types from other organisms, the results of this work suggest that significant triple-helix DNA extensions can be formed in pericentric regions of these species.

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

“…(dT) localisation at dipteran chromosome ends. Those results could be reproduced with the poly-(rA) probe followed by the immunological detection of DNA.RNA hybrids (Stocker et al 1993;Stocker and Gorab 2003;Gorab et al 2009), reinforcing the existence of local enrichment with homopolymeric (dA). (dT) DNA in the genome of this species.…”

“…(dT) homopolymers composed of 7 and 3 bp per repeat. While the poly-(rA) probe has fragments considerably longer than the T-14 length (Gorab et al 2009), it is able to hybridise to islands of homopolymeric chromosomal DNA forming DNA.RNA hybrids that are more stable than DNA.DNA duplexes produced by homopolymeric DNA probes. Although DNA.RNA hybrid duplexes are not continuous but rather interrupted in the T-14 array, the extension of the tandem repeat is likely to Rossato et al 2007).…”

“…Unusually enriched with (dA). (dT) homopolymeric DNA and localised at chromosome ends, pericentric regions and specific interstitial sites (Stocker et al 1993;Stocker and Gorab 2003), it also has the potential to form three-stranded structures with exogenous poly-(rU) (Gorab et al 2009). The repetitive sequence characterised in this work, 14 bp, represents the shortest tandem repeat that has been identified in the chromosome termini of dipterans up to this time.…”

Potential in vivo roles of nucleic acid triple-helices

“…The existence of triplex structures in the nucleus of insects, nematodes and mammals has been suggested by immunofluorescence using triple-helix specific monoclonal antibodies. [27][28][29][30][31][32][33][34] The antibodies recognize distinct triplex epitopes as indicated by different staining patterns on polytene chromosomes of Drosophila melanogaster. 30,34 Among the most compelling evidence for the existence of triplexes in vivo is immunodetection by triple-helix specific antibodies in human cell nuclei, 31 which detected an abundance of triplex signatures (Fig.…”

Triple helix-interacting proteins and cancer

“…Immunofluorescence experiments with triplex-specific monoclonal antibodies have shown distinct staining patterns on polytene chromosomes isolated from Drosophila melanogaster 18,19 . Similarly triplex-specific antibodies exhibit multiple positive foci disseminated throughout isolated human cell nuclei 20 .…”