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

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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.

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Abbreviations

B-DNA:

Canonical (Watson–Crick) DNA structure

BSA:

Bovine serum albumin

CCD:

Charge-coupled device

DEAE:

Diethylaminoethyl

DAPI:

4′,6-Diamidino-2-phenylindole

EDTA:

Ethylenediaminetetraacetic acid

ELISA:

Enzyme-linked immunosorbent assay

IgG:

Immunoglobulin G

PBS:

Phosphate-buffered saline

Poly(dA):

Polydeoxyadenylic acid

Poly(dT):

Polydeoxythymidylic acid

Poly(rA):

Polyadenylic acid

Poly(rU):

Polyuridylic acid

rDNA:

Ribosomal DNA

RNase:

Ribonuclease

SSC:

Sodium saline citrate

TBS:

Tris-buffered saline

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Acknowledgements

The authors thank Dr. C. Vilela for maintenance of Drosophila stocks. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP).

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Authors and Affiliations

  1. 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

    Eduardo Gorab, José Mariano Amabis, Ann Jacob Stocker & Laura Drummond

  2. Centre for Environmental Stress and Adaptation Research, Department of Genetics, University of Melbourne, Parkville, Victoria, Australia

    Ann Jacob Stocker

  3. Department of Biochemistry, Tufts University School of Medicine, Boston, MA, USA

    Bernard David Stollar

Authors
  1. Eduardo Gorab
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  2. José Mariano Amabis
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  3. Ann Jacob Stocker
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  4. Laura Drummond
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  5. Bernard David Stollar
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Corresponding author

Correspondence to Eduardo Gorab.

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Responsible Editor: Dean A. Jackson.

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ESM S1

Mixing curves of polynucleotides obtained with pyrimidine percentage data after each addition step (X-axis) and the corresponding absorbance measured at 260 nm (Y-axis) (PPT 45.0 kb)

ESM S2

a Poly(rA) and poly(dT) solutions were mixed, adjusted to 2× SSC and incubated for 5 min at 37°C. After desalting the solution with Geneclean™, the sample was dissolved in 0.1× SSC and checked in agarose gels. Two (lanes 2, 3) of three tubes containing the same amount and volume (6 μl) of poly(rA).poly(dT) hybrid duplex were subjected to increasing volumes of poly(rU)/formamide solution varying from 1 to 2 μl per tube and corresponding to (2) 300 ng and (3) 600 ng of poly(rU). After incubation for 5 min at 37°C, the whole content of each tube was analysed in a 1.2% agarose gel. b Two (lanes 2, 3) of three tubes containing the same amount and volume of poly(rA).poly(dT) duplex were subjected to increasing volumes of formamide solution without poly(rU) varying from (lane 2) 1 μl to (lane 3) 2 μl per tube and subsequently analysed as described above. Size markers are in kilobase pairs. (PPT 107.0 kb)

ESM S3

a Localisation of a probe made of insect total RNA in polytene chromosome C of R. americana. b Results of localisation in polytene chromosome C when an excess of poly(rU) was added to total RNA and then used as a probe in R. americana chromosomes. Probe detection in a and b was done with anti-DNA.RNA duplex antibodies. (PPT 95.0 kb)

ESM S4

a Localisation of anti-poly(dA).poly(rU).poly(rU) antibodies in polytene chromosomes of wild-type Drosophila melanogaster (Canton S), previously treated with RNases, that were not subjected to in situ hybridisation with poly(rU) probes. b DAPI counterstaining and c the merged signals. (PPT 425 kb)

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Gorab, E., Amabis, J.M., Stocker, A.J. et al. Potential sites of triple-helical nucleic acid formation in chromosomes of Rhynchosciara (Diptera: Sciaridae) and Drosophila melanogaster . Chromosome Res 17, 821–832 (2009). https://doi.org/10.1007/s10577-009-9075-5

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