Immunofluorescent localization of transcriptional activity on lampbrush chromosomes

Abstract

Immunofluorescent reactions specific for distinct chromosomal structures are obtained by using antiserum to oocyte proteins. Antisera diluted more than 1000-fold are used to localize proteins associated with RNA transcripts in unfixed lampbrush chromosomes of Triturus cristatus carnifex. Of the several protein components of nuclear RNP, at least some are bound to all chromosome transcripts and may form the basis of the regular 20 nm beads characteristic of the RNP chain. On the other hand some proteins are peculiar to a restricted number of loci (loops). For example, a nuclear RNP protein in the size range 30–35,000 daltons is bound to the transcripts of only several of the many thousands of loops. Also, the two highly basic proteins of a cytoplasmic 40S particle have a special chromosomal location, one with the 5S locus, the other with putative tRNA loops. In these instances it is likely that there is recognition by proteins of particular RNA nucleotide sequences, or their resulting secondary structure, as they are generated. The specific immunostaining of distinct loops can be used not only to identify the genetic sequences being transcribed but also to give information on the pattern of transcription and processing. In contrast to the RNA-binding proteins, most of the chromosomal histone is seen, by immunofluorescence, to be localized in chromatin which is condensed in chromomeric structures and not active in transcription.