Date of Award
Doctor of Philosophy (PhD)
The ribosomal RNA genes of Physarum polycephalum are encoded on multiple copies of linear, palindromic extrachromosomal DNA molecules (rDNA), which have been isolated both as a satellite DNA molecule, and as a "minichromosome" with associated chromatin proteins. The genes coding for the 19 S, 26 S, and 5.8 S rRNAs are transcribed as a single, large precursor molecule, beginning nearer the center of the palindrome and transcribing outward. The genes are in the order 19 - 5.8 - 26, and the 26 S gene contains two intervening sequences. The intervening sequences are transcribed, and later excised out. The region of the rDNA previously identified as the transcription initiation site was cloned in Charon 28 bacteriophage. The initial large clone was subcloned in M13mp9, and a small clone containing the initiation site was isolated. The transcription initiation site was mapped within this clone using SI nuclease protection experiments, and in vitro transcription run-off synthesis. The DNA sequence of over 700 nucleotides of this clone has been determined, including the putative transcription initiation site. Comparison of this sequence to other rRNA transcription initiation sites finds no consistent region of homology with any other set of initiator sequences. A consistent structural feature, an inverted repeat at approximately -30 to -50 nucleotides, was identified in this and other rRNA transcription initiation sites examined. The transcribed regions of this rDNA minichromosome were found to be packaged into an altered, extended nucleosomal arrangement during active stages of the Physarum life cycle. This altered conformation is specific to transcribed areas, is more sensitive to nuclease digestion, and is separable as a slowly sedimenting monomer peak on sucrose gradients. This altered monomer nucleosome or "lexosome" contains, after staphylococcal nuclease digestion, a monomeric length of DNA (1M4 bp), and has been shown subsequently to contain a full complement of core histones, along with two specific associated proteins (Prior et. al., 1983 and Prior, 1982). A model for the activation of the transcription unit, based on a combination of the conserved inverted repeat and the chromatin structural studies, is proposed. The ends of the linear molecule have been investigated, and shown to contain inverted repeats, length and sequence heterogeneities, single-strand one-nucleotide gaps in one or both strands at variable intervals, and a covalently bound protein. The rDNA terminal Eco RI fragment was cloned in Charon 13, and a large, representative clone was restriction mapped, subcloned into M13 vectors, and partially sequenced. The mapping and sequence data confirm the presence of inverted repeats, and identify several components of these repeats. A model is proposed to explain how these features may function to allow the replication of the 5' end of the linear rDNA molecule.
Campbell, Gerald Ray, "Sequence and Functional Structures of the Extrachromosomal rDNA Minichromosome of Physrum Polycephalum" (1983). Student Theses and Dissertations. 442.