The synthesis of polyadenylic acid-containing ribonucleic acid by isolated mitochondria from Ehrlich ascites cells

Abstract

Isolated mitochondria from mouse Ehrlich ascites cells incorporate [3H]AMP or [3H]UTP into an RNA species that absorbs on oligo(dT)-cellulose columns or Millipore filters. Hydrolysis of the poly(A)-containing RNA with pancreatic and T1 RNase released a poly(A) sequence that had an electrophoretic mobility slightly faster than 4SE. Ascites-cell cytosolic poly(A)-containing RNA had a poly(A) tail that had an electrophoretic mobility of about 7SE. Sensitivity of the incorporation of [3H]AMP into poly(A)-containing RNA to ethidium bromide and to atractyloside and lack of sensitivity to immobilized RNase added to the mitochondria after incubation indicated that the site of incorporation was mitochondrial. The poly(A)-containing RNA sedimented with a peak of about 18 S, with much material of higher S value. After denaturation at 70.degree. C for 5 min the poly(A)-containing RNA separated into 2 components of 12S and 16S on a 5-20% (wt/vol) sucrose gradient at 4.degree. C, or at 4.degree. and 25.degree. C in the presence of formaldehyde. Poly(A)-containing RNA synthesized in the presence of ethidium bromide sedimented at 5-10 S in a 15-33% (wt/vol) sucrose density gradient at 24.degree. C. The poly(A) tail of this RNA was smaller than that synthesized in the absence of ethidium bromide. The size of the poly(A)-containing RNA (approximately 1300 nucleotides) is about the length necessary for that of mRNA species for the products of mitochondrial protein synthesis.