Enzymic polyadenylation of 5S ribosomal ribonucleic acid and synthesis of a complementary deoxyribonucleic acid

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Abstract
The 5S ribosomal RNA was isolated, pure and intact, from rat liver (5 mg of 5S RNA from 150 g of liver). The 5S RNA serves as a primer for calf thymus poly(A) polymerase with 20% of the efficiency of (Ap)3A. Bacterial [Escherichia coli] 5S RNA and transfer RNA also serve as primers; rat liver 18S and 28S ribosomal RNAs support poly(A) synthesis poorly. Neither the 5S RNA primer nor the appended poly(A) tract is nicked or degraded by poly(A) polymerase, and initiation of poly(A) tracts on 5S RNA primers continues throughout the reaction period. The rate of initiation is dependent on the enzyme concentration; the ATP concentration affects the rate of elongation. The polyadenylated material increases in size over time, with the largest material reaching a size of 6.8 S in 5 h, corresponding to an appended poly(A) tract of 140 nucleotides. Using polyadenylated 5S RNA, oligo(dT) as primer, and avian myeloblastosis virus reverse transcriptase, DNA complementary to 5S RNA was synthesized. The complementary DNA has an apparent MW (in alkaline sucrose gradients) of 4.3 .times. 104. Base composition analysis and nearest-neighbor analysis of the DNA are as expected for a complement of 5S RNA, indicating that the entire 5S sequence is copied. The complementary DNA hybridizes to 5S RNA with a Rot1/2 [half renaturation time] of 8.9 .times. 10-4 mol .cntdot. l-1. No hybrid is formed with E. coli 16S and 23S ribosomal RNA, E. coli 5S ribosomal RNA, yeast transfer RNA, rat liver transfer RNA, or rat liver 18S and 28S ribosomal RNA. The Tm [melting temperature] of the 5S RNA:5S DNA hybrid in 15 mM NaCl containing 1.5 mM sodium citrate is 74.degree. C, 2.5.degree. C below the theoretical melting temperature of a DNA duplex of 60% G + C. Analysis of the hybrid in buoyant density gradients also indicates that hybridization is both specific and precise. The complementary [c] DNA anneals to calf thymus, rat liver and salmon sperm DNA but not to E. coli DNA. Annealing of 5S cDNA to calf thymus DNA with a Cot1/2 of 2.1 suggests that there are several thousand 5S RNA genes in the calf thymus genome (haploid). At least that number of 5S RNA genes is present in the salmon sperm genome.