Potential sites of triple-helical nucleic acid formation in chromosomes of Rhynchosciara (Diptera: Sciaridae) and Drosophila melanogaster.
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.
Gorab
Eduardo
E
Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, Rua do Matão 277, Cidade Universitária, CEP 05508-090 São Paulo, São Paulo, Brazil. egorab@usp.br
Amabis
José Mariano
JM
Stocker
Ann Jacob
AJ
Drummond
Laura
L
Stollar
Bernard David
BD
D000818:Animals; D000911:Antibodies, Monoclonal; D033481:Chromosomes, Mammalian / Q000235:genetics; D004247:DNA / Q000737:chemistry*; D004175:Diptera / Q000235:genetics*; D004331:Drosophila melanogaster / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D007124:Immunoenzyme Techniques; D017403:In Situ Hybridization; D009690:Nucleic Acid Conformation; D011061:Poly A / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D011072:Poly U / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D012313:RNA / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D011061:Poly A; D011072:Poly U; D012313:RNA; D004247:DNA