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

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, s20,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.