| Visualization of the genetic activity of ribosomal
RNA genes in wheat by in situ hybridization using biotinylated probe
N. Metsugi1, M.Yamamoto2 and Y. Mukai1 1 Department of Biology, Osaka Kyoiku University, Ikeda, Osaka, Japan 2 Department of Biotechnology, Kansai Women's Junior College, Kashiwara, Osaka, Japan Ribosomal RNA genes (rDNA) are fundamental components of the protein synthetic machinery and are tandemly repeated in higher plants. They are mostly present in the secondary constrictions of satellited chromosomes. In situ hybridization studies established the location of the rDNA on chromosomes 1B, 6B and 5D of Triticum aestivum cv. Chinese Spring. In the present study, behavior of the rDNA during the wheat cell cycle was visualized using in situ hybridization with a biotinylated rDNA probe of wheat. The in situ hybridization experiment showed that the rDNA sites were seen as brown color on the blue chromosomes at early prophase to metaphase stages (Fig. 1). Nucleoli were stained purple. In prophase, rDNA of chromosome 6B separated into two condensed parts, while that of 1B localized near its short arm only. Further, rDNA on chromosomes 1B and 6B was always accompanied with nucleolus. Ribosomal RNA genes being active were recognized as dispersed part within the nucleolus. We considered the condensed parts to be genetically inactive. These parts were probably methylated. The rDNA on chromosome 5D was seldom accompanied with nucleolus. Stretched chromosomes in early metaphase showed that most rDNA on chromosomes 1B and 6B were condenced on both the ends of secondary constriction. In both interphase and early prophase nuclei of the F1 hybrid between Chinese Spring wheat and Prolific rye, wheat rDNA was always accompanied with nucleolus, whereas rye rDNA was not, showing no activity of rye ribosomal gene (Fig. 2). Wheat rDNA on chromosomes 1B and 6B at metaphase was partially stretched. None of the rye rDNA was found stretched but remained condensed. Thus, in the presence of wheat NOR chromosomes 1B and 6B, the obvious suppression of rDNA on the chromosome 1R is evident, as the rye rDNA is no longer dispersed. In all cases of other derivatives of wheat-rye hybrids, such as triticale, addition lines, substitution and translocation lines (Fig. 3), we found that rDNA on the chromosome 1R of rye was suppressed by the presence of wheat chromosomes. In conclusion, as also reported by Gustafson et al (1988), in situ hybridization using biotin-labeled probe can be effectively used to visualize genetic activity or gene expression at the rDNA sites with the help of light microscope. |