Isolation and Purification of Ribosomal Rnas of Euglena Gracilis; Their Evolutionary Significance as Determined by Oligonucleotide Catalogue Analysis
Kissil, Martin Samuel
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https://hdl.handle.net/2142/77578
Description
Title
Isolation and Purification of Ribosomal Rnas of Euglena Gracilis; Their Evolutionary Significance as Determined by Oligonucleotide Catalogue Analysis
Author(s)
Kissil, Martin Samuel
Issue Date
1981
Department of Study
Biology
Discipline
Biology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, General
Language
eng
Abstract
The Euglena chloroplast DNA shares a relatively high degree of homology with the ribosomal RNAs of various cyanobacteria. This level of homology may indicate a phylogenetic relationship between this chloroplast and various prokaryotic organisms. Therefore, this study, involving the technique of oligonucleotide 'fingerprinting', was undertaken to study the phylogenetic relatedness of the Euglena chloroplast in greater detail. In order to accomplish this project, methodology had to be developed for the in vivo radioactive labelling of the cellular ribosomal RNAs to high specific activities, and the isolation and purification of these molecules.
A method for the isolation and purification of Euglena gracilis chloroplasts by sucrose flotation is described. This method yields high levels of purified chloroplasts as compared to previous methods. The isolated chloroplasts are characterized biochemically, show incorporation of radio-actively-labeled amino acids into acid-precipitable material, and yield polyribosomes, DNA and RNA free of significant contamination from cytoplasmic nucleic acids.
Three bleached mutants, in addition to the wild type Euglena gracilis, are employed to optimize growth conditions in a phosphorus limiting medium in order to facilitate the isolation and purification of radioactively labeled (('32)P) ribosomal RNAs.
Various media, both simple and complex, are employed with varying concentrations of phosphorus and ('32)P(,i). Chloroplast rRNAs are labeled during maturation of the chloroplasts in response to light. Although this process occurs in the presence of high levels of radioactive phosphorus (250 (mu)Ci/(mu)g), the normal growth of these strains exhibits various radioactive sensitivities; the least sensitive being W(,1)ZHL (a heat bleached mutant of wild type, strain Z).
Isolation and purification of chloroplast ribosomal RNAs, radioactively labeled to high specific activity (5-7.5 x 10('5) cpm/(mu)g RNA) is demonstrated, whereas, cytoplasmic ribosomal RNAs are found to incorporate ('32)P(,i) to a much lower level, 1.25 x 10('5) cpm/(mu)g RNA. A discussion of phosphorus metabolism is included.
Chloroplast 16S and 5S rRNAs have been analysed with two-dimensional ionophoresis of T(,1)-ribonuclease generated oligonucleotides and subsequent comparison with oligonucleotide catalogues of other organisms (prokaryotes and eukaryotes).
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