Abstract
We report a new technique—nondenaturing FISH (ND-FISH)—for the rapid detection of plant telomeres without the need for prior denaturation of the chromosomes. In its development, two modified, synthetic oligonucleotides, 21 nt in length, fluorescently labelled at their 5′ and 3′ ends and complementary to either the cytidine-rich (C3TA3) or guanosine-rich (T3AG3) telomeric DNA strands, were used as probes. The high binding affinity of these probes and the short hybridization time required allows the visualization of plant telomeres in less than an hour. In tests, both probes gave strong signals visualized as double spots at both chromosome ends; this was true of both the mitotic and meiotic chromosomes of barley, wheat, rye, maize, Brachypodium distachyon and Rhoeo spathacea. They were also able to detect telomere motifs at certain intercalary sites in the chromosomes of R. spathacea. To investigate the nature of the target structures detected, the chromosomes were treated with RNase A and single strand-specific nuclease S1 before ND-FISH experiments. Signal formation was resistant to standard enzymatic treatment, but sensitive when much higher enzyme concentrations were used. The results are discussed in relation to current knowledge of telomere structure.
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Abbreviations
- dsDNA:
-
double stranded DNA
- ND-FISH:
-
nondenaturing FISH
- ssDNA:
-
single-stranded DNA
References
Adams SP, Hartman TP, Lim KY et al (2001) Loss and recovery of Arabidopsis-type telomere repeat sequences 5′-(TTTAGGG)(n)-3′ in the evolution of a major radiation of flowering plants. Proc Biol Sci 268:1541–1546
Azzalin CM, Reichenbach P, Khoriauli L, Giulotto E, Linger J (2007) Telomeric repeat-containing RNA and RNA surveillance factors at mammalian chromosome ends. Science 318:798–801
Breslauer KJ, Frank R, Blocker H, Marky LA (1986) Predicting DNA duplex stability from the base sequence. Proc Natl Acad Sci U S A 83:3746–3750
Burr B, Burr FA, Matz EC, Romero-Severson J (1992) Pinning down loose ends: mapping telomeres and factors affecting their length. Plant Cell 8:953–960
Cesare AJ, Quinney N, Willcox S, Subramanian D, Griffith JD (2003) Telomere looping in P. sativum (common garden pea). Plant J 36:271–279
Cuadrado A, Jouve N (2007a) The non-random distribution of long clusters of all possible classes of trinucleotide repeats in barley chromosomes. Chromosome Res 15:711–720
Cuadrado A, Jouve N (2007b) Similarities in the chromosomal distribution of AG and AC repeats within and between Drosophila, human and barley chromosomes. Cytogenet Genome Res 119:91–99
De la Herrán R, Cuñado N, Navajas-Pérez R et al (2005) The controversial telomeres of lily plants. Cytogenet Genome Res 109:144–147
Fajkus J, Sýkorová E, Leitch AR (2005) Techniques in plant telomere biology. Biotechniques 38:233–243
Fuchs J, Brandes A, Schubert I (1995) Telomere sequence localization and karyotype evolution in higher plants. Pl Syst Evol 195:227–241
Golczyk H, Hasterok R, Joachimiak AJ (2005) FISH-aimed karyotyping and characterization of Renner complexes in permanent heterozygote Rhoeo spathacea. Genome 48:145–153
Gonzalo S, Garcoa-Cao M, Fraga MF et al (2005) Role of the R1 family in stabilizing histone methylation at constitutive heterochromatin. Nat Cell Biol 7:420–428
Griffith JD, Comeau L, Rosenfield S et al (1999) Mammalian telomeres end in a large duplex loop. Cell 97:419–422
Nabetani A, Ishikawa F (2008) Unusual telomeric DNAs in human telomerase-negative immortalized cells. Mol Cell Biol 29:703–713
Ohno M, Fukagawa T, Lee JS, Ikemura T (2002) Triplex-forming DNAs in the human interphase nucleus visualized in situ by polypurine/polypyrimidine DNA probes and antitriplex antibodies. Chromosoma 111:201–213
Raices M, Verdun RE, Compton SA et al (2008) C. elegans telomeres contain G-strand and C-strand overhangs that are bound by distinct proteins. Cell 132:745–757
Richards EJ, Ausubel FM (1988) Isolation of a higher eukaryotic telomere form Arabidopsis thaliana. Cell 53:127–136
Schoeftner S, Blasco MA (2008) Developmentally regulated transcription of mammalian telomeres y DNA-dependent RNA polymerase II. Nat Cell Biol 10:228–236
Schwarzacher T, Leitch AR, Bennett MD, Heslop-Harrison JS (1989) In situ localization of parental genomes in a wide hybrid. Ann Bot 64:315–324
Sugimura K, Takebayashi S, Ogata S, Taguchi H, Okumura K (2007) Non-denaturing fluorescence in situ hybridization to find replication origins in a specific genome region on the DNA fiber. Biosci Bioechnol Biochem 71:627–632
Vershinin AV, Heslop-Harrison JS (1998) Comparative analysis of the nucleosomal structure of rye, wheat and their relatives. Plant Mol Biol 36:149–161
Zellinger B, Riha K (2007) Composition of plant telomeres. Biochim Biophys Acta 1769:299–409
Acknowledgements
This study was supported by grants from the Spanish Ministry of Education and Science (AGL2006-09018-C02) and the Polish State Committee for Scientific Research (N301 116 32/4008). The authors thank Adrian Burton for linguistic assistance.
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Cuadrado, Á., Golczyk, H. & Jouve, N. A novel, simple and rapid nondenaturing FISH (ND-FISH) technique for the detection of plant telomeres. Potential used and possible target structures detected. Chromosome Res 17, 755–762 (2009). https://doi.org/10.1007/s10577-009-9060-z
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DOI: https://doi.org/10.1007/s10577-009-9060-z