Date of Award

1988

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Elizabeth A. Zimmer

Abstract

The restriction endonuclease HpaII was utilized to examine ribosomal RNA gene (rDNA) methylation in maize and teosinte leaf DNA. Much of the rDNA was inaccessible to HpaII cleavage indicating that these repeat units were completely methylated. In all of the DNAs examined, a significant fraction (10-25%) of the rDNA was cleaved at least once by HpaII into repeat unit length (9.1 kbp) fragments. The undermethylated HpaII sites mapped to the intergenic spacer (IGS) region of the rDNA. The major site of undermethylation was located in a region near the transcriptional start site. An EcoRI polymorphism, present in the 26S gene of certain maize inbred lines, was utilized to correlate rDNA undermethylation, DNaseI sensitivity and expression. In double digest experiments with EcoRI and HpaII, the fragments originating from repeat units with two EcoRI sites (8.0 kbp) are sensitive to HpaII digestion, but the fragments originating from repeat units with a single EcoRI site (9.1 kbp) in polymorphic lines are resistant to HpaII digestion. To examine rDNA chromatin structure, intact nuclei were purified and digested briefly with increasing amounts of DNaseI. Analysis of this DNA with EcoRI showed that the 8.0 kbp fragments were extremely sensitive to DNaseI digestion, but the 9.1 kbp fragments were relatively resistant even at high levels of DNaseI. Specific sites hypersensitive to DNaseI cleavage were located in the IGS of the rDNA in a region near the major undermethylated site. Experiments utilizing the polymerase chain reaction and direct rRNA sequencing indicated that the EcoRI polymorphism is due to sequence change in the rDNA. Oligonucleotide probes specific for the region surrounding and including the EcoRI polymorphic site were used to examine rRNA transcripts in inbred lines and hybrids created by crossing EcoRI polymorphic and nonpolymorphic inbred lines. Results from these experiments indicate that the majority of the rRNA transcripts in a hybrid originate from the EcoRI polymorphic arrays when the maternal parent is EcoRI polymorphic. In the reciprocal cross, the EcoRI nonpolymorphic rRNA transcripts seem to be predominate. This suggests that a transcription factor in the maternal genome may determine which rDNA arrays are transcribed.

Pages

115

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