Part I. Characterization of the RNA from the Mitochondrial Fraction of HeLa Cells. Part II. Properties of Membrane-Bound Ribosomes in HeLa Cells

Abstract

Part I of this thesis is concerned with the characterization of RNA components from the mitochondrial fraction of HeLa cells. Mitochondria prepared by differential centrifugation followed by buoyant density fractionation in sucrose gradient are contaminated by elements of the rough endoplasmic reticulum. In order to identify RNA components of mitochondrial origin in this fraction the following criteria were used: association of newly synthesized RNA with mitochondria (recognized by cytochrome oxidase as say), insensitivity in situ to ribonuclease digestion, linear kinetics of labeling after [ 3 H]-uridine pulses, sensitivity of their synthesis to ethidium bromide, and, especially, capacity to hybridize with purified closed-circular mitochondrial DNA. It has been found that the RNA synthesized on a mit-DNA template consists of both rapidly labeled heterogeneous RNA components, varying in sedimentation constant from 4 to 50 S or more, and discrete RNA species, migrating at 16 S, 12 S, and 4 S in sucrose gradient. Analysis of the sedimentation behavior of the 16 S and 12 S species under denaturing conditions has indicated that they are represented by continuous polynucleotide chains. From their migration in polyacrylamide gels relative to ribosomal RNA markers, the respective molecular weights of 16 S and 12 S have been estimated to be 0.7 x 10 6 and 0.4 x 10 6 daltons. The 16 S, 12 S, and 4 S RNA's appear to be methylated. Both the discrete and heterogeneous mit-DNA coded RNA components have a base composition clearly different from that of ribosomal RNA and cytoplasmic messenger RNA and complementary, as concerns the A and U content, to that of the heavy strand of mit-DNA. The results of an investigation concerning the properties of the ribosomes associated with the endoplasmic reticulum in HeLa cells are reported in Part II. From the distribution of ribosomal RNA among the subcellular fractions, it has been estimated that 10 -15% of the total ribosomes in HeLa cells are membrane-bound; 65-70% of these can be recovered in the form of polysomes after membrane lysis with sodium deoxycholate. The 18 S and 28 S RNA components of the membrane-bound ribosomes have similar kinetics of labeling and identical sedimentation properties and nucleotide composition to the homologous components of free ribosomes, these results pointing to a common nuclear origin. The ribosomal RNA of membrane-bound ribosomes is made acid-soluble to about the same extent as the ribosomal RNA of free polysomes under the conditions of ribonuclease digestion in situ enployed here. Treatment with EDTA releases about 85-90% of the small ribosomal subunits and 70% of the large ribosomal subunits, a situation which is similar to that which has been observed for rat liver microsomes. These results suggest that the great majority of the ribosomes in the mitochondrial fraction of HeLa cells are extramitochondrial, i.e. bound to the endoplasmic reticulum, but the existence of a small number of intramitochondrial ribosomes is not excluded.