Molecular Analysis of Human Ribosomal-Dna: Electron Microscopic Analysis of Repeat Length Heterogeneity and Sequence Analysis of the Transcription Termination Region.

Abstract

In higher eukaryotes, the DNA template for 18S, 5.8S, and 28S ribosomal RNA (rRNA) is transcribed as a single RNA molecule, which is subsequently processed. The transcription unit plus a length of nontranscribed spacer DNA make up the repeating unit of ribosomal DNA (rDNA). Approximately 300 units are distributed in t and emly repeated clusters which are located at the nucleolus organizers of the five pairs of D and G group chromosomes in the human. The observation that rDNA undergoes concerted evolution, in which repeat units on separate chromosomes have maintained homology, suggests a mechanism for genetic exchange among nonhomologous chromosomes. Analysis of nucleotide sequence variation and length heterogeneity have been used to examine the relationship between concerted evolution and the chromosomal organization of rDNA. This thesis presents the analysis of two areas of rDNA organization. In the first, electron microscopic R-loop mapping of human genomic DNA, enriched for ribosomal sequences, is used to analyze the distribution of length variants of rDNA repeat units from a single individual. 86% of repeat units fall into the single length class of 43.85 (+OR-) 1.59 kilobase pairs (kb). The remaining repeat units fall into a second size class of 49.81 (+OR-) 0.72 kb. Two examples of molecules containing two complete repeat units were measured. In one molecule, the repeat unit lengths were found to be in the major size class, while in the other molecule, both repeat unit lengths were in the minor size class. The second area of rDNA organization analyzed was the transcription termination region. The 3' end of a human 28S rRNA gene was localized by R-loop mapping of cloned rDNA and is within 50 base pairs on either side of a Taq I restriction enzyme site. The sequence of 296 nucleotides surrounding the rRNA transcription termination site has been determined. Comparison of the human 3' end sequence with those of other species reveals strong conservation of certain blocks of nucleotides as well as conservation of potential secondary structure.