Investigations on (i) Chromosomal Ribonucleic Acid of Ascites Tumour, (ii) RNA Polymerase of E. Coli

Citation

McConnell, David John (1971) Investigations on (i) Chromosomal Ribonucleic Acid of Ascites Tumour, (ii) RNA Polymerase of E. Coli. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/D6DK-F035. https://resolver.caltech.edu/CaltechTHESIS:05252018-095226235

Abstract

PART I: When chromatin isolated from rat ascites cells is dissociated in the presence of high salt and the chromosomal proteins separated from the DNA by buoyant density centrifugation, a portion of the RNA contained in the chromatin remains associated with the chromosomal proteins. This RNA (chromosomal RNA) is characterized by its small size, s_20,w = 3.3S, its high content of dihydroribothymidine and its ability to form hybrid with about 4% of the nuclear DNA. It appears to have no sequences in common with ascites transfer, ribosomal, or messenger RNA.

A class of RNA (cytoplasmic 3S RNA) with similar proper­ties but associated with the cytoplasmic proteins has also been isolated. This RNA hybridizes to about 2% of the nuclear DNA and contains very few, if any, sequences not also contained in chromosomal RNA. This fraction of RNA is however unable to compete with about 50% of the sequence present in chromosomal RNA indicating that a large portion of chromoso­mal RNA is confined to the chromatin. A further class of RNA associated with the nuclear sap proteins appears to be identical to the RNA associated with cytoplasmic proteins.

A further class of RNA (nuclear 3S RNA) with hybridiza­tion properties similar to the cytoplasmic 3S RNA has been isolated from the nuclear sap. It hybridises to a lower extent than chromosomal RNA and is strongly competed by cyto­plasmic 3S RNA.

PART II. RNA polymerase, more than 95% pure, has been prepared from E. coli D-10 and B. It is free from ribonu­clease and phosphatase. It carries out poly A synthesis on E. coli or ascites tumour DNA but not on T7 DNA. Phosphate incorporation (TCA precipitable) from the γ phosphate of ATP in the absence of primer may be caused by a slight contami­nation with polyphosphate kinase. This can be controlled. Initiation of synthesis on T7, E. coli and ascites tumour DNA differ in the response to the σ sub-unit. RNA synthe­sized on T7 DNA can initiate with A or G. A salt- or rifam­- picin-stable complex between T7 DNA can be formed when a single nucleoside triphosphate (commercial reagent) is present, but requires a small amount of propagation. After purification of the nucleotides the efficiency of complex foration is greatly reduced.

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