In situ hybridization to plant telomeres using synthetic oligomers

Abstract

The distribution of telomeric repeats in Hordeum vulgare (barley) and Secale cereale (rye) was studied by DNA–DNA in situ hybridization to root-tip chromosome preparations. Biotinylated synthetic oligomers, (TTTAGGG)6 and (CCCTAAA)6, homologous to the consensus sequence of the Arabidopsis thaliana telomeric repeat, were used as probes, and hybridization sites were detected by Texas red fluorescence or horseradish peroxidase catalyzed precipitation of diaminobenzidine. After examination in the light microscope, the same preparations were transferred to the electron microscope for high-resolution analysis. Sites of hybridization were visualized as single or double dots at the end of most chromosome arms. The sizes of the signal dots varied widely, indicating that individual telomeres may contain different numbers of repeats of the telomeric sequence. In contrast to many mammals, no telomere repeats were detected other than at the ends of the chromosomes. At interphase, signals were concentrated in one region of the nucleus, as would be expected from the Rabl orientation typical for dividing cells from cereal root tips.Key words: barley, rye, nuclear architecture, DNA–DNA in situ hybridization, telomeres.