Purification of major abundance class of poly(A+)-RNA from rat ventral prostate.

Abstract Poly(A + )-RNA from rat ventral prostate was isolated using oligo(dT)-cellulose chromatography. 45% of the total poly(A + )-RNA was a single peak at 10S as demonstrated by centrifugation in a 5–20% sucrose gradient containing 1% SDS. By using complementary DNA probes, it was shown that the 10S RNA contained the major abundance class of poly(A + )-RNA. Denaturing agarose-gel analysis revealed 2 major bands in the 10S poly(A + )-RNA preparation approx. 600 NT and 500 NT (NT = nucleotides) long, resp. Double-stranded 32 P-DNAs complementary to light side and heavy side of the 10S poly(A + )-RNA peak were synthesized and isolated using reverse transcriptase and hydroxyapatite (HAP) chromatography. Approx. 40% of the first strand of the cDNAs were converted to double-stranded structures with a T m of 88°C. HAP purified double-stranded material was 92% resistant to S 1 nuclease. The DNA-DNA reannealing profile of double stranded 32 P-cDNA enriched for the 500 NT band gave a Cot 1 2 of ~7 × 10 −4 moles × sec × 1 −1 indicating a complexity for this enriched synthetic gene of 500–600 nucleotide pairs (NTP).