ULTRASTRUCTURAL IN-SITU HYBRIDIZATION TO NASCENT TRANSCRIPTS OF HIGHLY TRANSCRIBED RIBOSOMAL-RNA GENES IN CHROMATIN SPREADS

The amplified rRNA genes of amphibian oocytes were used as a model sys tem for the development of an in situ hybridization technique to label nascent transcripts in dispersed chromatin. A biotinylated complement ary RNA probe was hybridized to nascent transcripts from dispersed nuc leoli, and detected by a two step antibody technique utilizing colloid al gold as an electron dense marker. A specific sequence on the rRNA n ascent transcript was labeled in a pattern consistent with its locatio n; however, gene morphology was difficult to analyze following in situ hybridization owing to low sample contrast. Proteins associated with the transcripts were apparently lost during the procedure, leading to decreased electron density of the transcripts. The technique was syste matically modified in an attempt to identify conditions that preserved gene morphology adequately for ultrastructural analysis, while simult aneously maintaining sufficient levels of specific labeling.