Kinetic complexity of RNA molecules

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A procedure has been developed for calculating the base sequence complexity of polyribonucleotides using RNA-DNA hybridization on membrane filters. Optimal rate temperature ( T opt) in hybridization media containing 6 × SSC (SSC is 0.15 m-NaCl, 0.015 m-sodium citrate) (pH 7.2), 50% formamide or 1.15 m-sodium acetate (pH 5), 60% formamide were determined for a variety of RNA species. The T opt in formamide were found to be unexpectedly high for all RNA species investigated. It is suggested that the high T opt are caused by increased stability of RNA-DNA hybrids in these incubation mixtures. It is demonstrated that RNA-DNA reactions with homogeneous RNA species present in excess are most easily characterized by C r t 1 2 where C r is the molar concentration of RNA nucleotides in solution and t 1 2 is the time taken to reach the 50% saturation value. Within certain limits C r t 1 2 is independent of the amount of DNA on filter disks and the degree of fragmentation of the RNA. C r t 1 2 increases linearly with the base sequence complexity of RNA molecules. Using complementary RNA derived from a balanced synthetic RNA-ΦX DNA hybrid we show that the slope of the straight linear standard curve which links C r t 1 2 to base sequence complexity is 9.3 × 10 −9 moles.sec/l. dalton for 6 × SSC, 50% formamide. In 1.15 m-sodium acetate (pH 5), 60% formamide, the slope is 2.3 × 10 −8 moles.sec/l. daltons. Applications of the kinetic standard curve for evaluation of purity and concentration of isolated messenger RNA's are discussed. We show that transfer RNA of both Escherichia coli and Xenopus laevis is made up from 30 to 40 basic nucleotide sequences distinguishable by RNA-DNA hybridization. In Xenopus there are some 6500 cistrons for tRNA and so each tRNA sequence is, on average, 160-fold reiterated. 5 s RNA of Xenopus, coded by some 9000 cistrons, and ribosomal RNA of Bacillus subtilis, rabbit and Xenopus coded by 8, 250 and 610 cistrons, respectively, each contain a single family of nucleotide sequences and are therefore highly conserved in these species.