SEQUENTIAL SILVER STAINING AND IN-SITU HYBRIDIZATION REVEAL A DIRECT ASSOCIATION BETWEEN RDNA LEVELS AND THE EXPRESSION OF HOMOLOGOUS NUCLEOLAR ORGANIZING REGIONS - A HYPOTHESIS FOR NOR STRUCTURE AND FUNCTION

We have developed a procedure for sequential silver staining and in si tu hybridization to analyze the relationship between the amount of rDN A present in nucleolar organizer regions, as estimated by in situ hybr idization, and their level of expression, as estimated by the silver s ignal, For simplicity we used cells from the insectivorous mole Talpa occidentalis, which have a single pair of nucleolar organizer regions in chromosome pair 3, The relative content of ribosomal cistrons was a lso related to the hierarchy of activation of the nucleolar organizer regions present in this chromosomal pair. Statistical analyses demonst rated that both the relative level of expression and the activation hi erarchy depended mainly on the number of ribosomal cistrons in nucleol ar organizer regions. We propose a functional two-step hypothesis, whi ch is consistent with most known data concerning interchromosomal, int ercellular and interindividual variation in a number of plant and anim al species, including Talpa occidentalis, In step one, the first avail able transcription factors bind randomly to the ribosomal promoters, s uch that larger nucleolar organizer regions are more likely to recruit them. In the second step the remaining transcription factors are recr uited in cooperative way, thus completing activation of one nucleolar organizer region,:before the next one becomes active.