Abstract
The aim of the present study is to make available a replicating molecule of known sequence. Accordingly, we sought a molecule that has the following properties: (a) replicates in vitro in a manner similar to phage Qβ RNA; (b) produces antiparallel complementary strands that can be separated from one another; and (c) is small enough to yield its sequence with reasonable effort.
We report here the isolation of a replicating RNA molecule that contains 218 nucleotides and possesses the other features desired for a definitive analysis of the replicating mechanism. Despite its small size, this molecule can mutate to previously determined phenotypes. It will, therefore, permit the precise identification of the base changes required to mutate from one phenotype to another in the course of extracellular Darwinian selection experiments.
Keywords: phage Qβ RNA, antiparallel complementary strands, Darwinian selection
Full text
PDFSelected References
These references are in PubMed. This may not be the complete list of references from this article.
- Banerjee A. K., Rensing U., August J. T. Replication of RNA viruses. X. Replication of a natural 6 s RNA by the Q-beta RNA polymerase. J Mol Biol. 1969 Oct 28;45(2):181–193. doi: 10.1016/0022-2836(69)90098-9. [DOI] [PubMed] [Google Scholar]
- Billeter M. A., Dahlberg J. E., Goodman H. M., Hindley J., Weissmann C. Sequence of the first 175 nucleotides from the 5' terminus of Qbeta RNA synthesized in vitro. Nature. 1969 Dec 13;224(5224):1083–1086. doi: 10.1038/2241083a0. [DOI] [PubMed] [Google Scholar]
- Eikhom T. S., Spiegelman S. The dissociation of Q-beta-replicase and the relation of one of the components to a poly-C-dependent poly-G-polymerase. Proc Natl Acad Sci U S A. 1967 Jun;57(6):1833–1840. doi: 10.1073/pnas.57.6.1833. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Glynn I. M., Chappell J. B. A simple method for the preparation of 32-P-labelled adenosine triphosphate of high specific activity. Biochem J. 1964 Jan;90(1):147–149. doi: 10.1042/bj0900147. [DOI] [PMC free article] [PubMed] [Google Scholar]
- HARUNA I., NOZU K., OHTAKA Y., SPIEGELMAN S. AN RNA "REPLICASE" INDUCED BY AND SELECTIVE FOR A VIRAL RNA: ISOLATION AND PROPERTIES. Proc Natl Acad Sci U S A. 1963 Nov;50:905–911. doi: 10.1073/pnas.50.5.905. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Haruna I., Spiegelman S. Autocatalytic synthesis of a viral RNA in vitro. Science. 1965 Nov 12;150(3698):884–886. doi: 10.1126/science.150.3698.884. [DOI] [PubMed] [Google Scholar]
- Haruna I., Spiegelman S. Recognition of size and sequence by an RNA replicase. Proc Natl Acad Sci U S A. 1965 Oct;54(4):1189–1193. doi: 10.1073/pnas.54.4.1189. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Haruna I., Spiegelman S. Specific template requirments of RNA replicases. Proc Natl Acad Sci U S A. 1965 Aug;54(2):579–587. doi: 10.1073/pnas.54.2.579. [DOI] [PMC free article] [PubMed] [Google Scholar]
- JOSSE J., KAISER A. D., KORNBERG A. Enzymatic synthesis of deoxyribonucleic acid. VIII. Frequencies of nearest neighbor base sequences in deoxyribonucleic acid. J Biol Chem. 1961 Mar;236:864–875. [PubMed] [Google Scholar]
- Kacian D. L., Mills D. R., Spiegelman S. The mechanism of Q replication: sequence at the 5' terminus of a 6-S RNA template. Biochim Biophys Acta. 1971 May 13;238(2):212–223. [PubMed] [Google Scholar]
- Kamen R. Characterization of the subunits of Q-beta replicase. Nature. 1970 Nov 7;228(5271):527–533. doi: 10.1038/228527a0. [DOI] [PubMed] [Google Scholar]
- Levisohn R., Spiegelman S. Further extracellular Darwinian experiments with replicating RNA molecules: diverse variants isolated under different selective conditions. Proc Natl Acad Sci U S A. 1969 Jul;63(3):805–811. doi: 10.1073/pnas.63.3.805. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Levisohn R., Spiegelman S. The cloning of a self-replicating RNA molecule. Proc Natl Acad Sci U S A. 1968 Jul;60(3):866–872. doi: 10.1073/pnas.60.3.866. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mills D. R., Bishop H. L., Spiegelman S. The mechanism and direction of RNA synthesis templated by free minus strands of a "little" variant of Q-beta RNA. Proc Natl Acad Sci U S A. 1968 Jun;60(2):713–720. doi: 10.1073/pnas.60.2.713. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mills D. R., Pace N. R., Spiegelman S. The in vitro synthesis of a noninfectious complex containing biologically active viral RNA. Proc Natl Acad Sci U S A. 1966 Dec;56(6):1778–1785. doi: 10.1073/pnas.56.6.1778. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mills D. R., Peterson R. L., Spiegelman S. An extracellular Darwinian experiment with a self-duplicating nucleic acid molecule. Proc Natl Acad Sci U S A. 1967 Jul;58(1):217–224. doi: 10.1073/pnas.58.1.217. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pace N. R., Bishop D. H., Spiegelman S. The immediate precursor of viral RNA in the Q-beta-repicase reaction. Proc Natl Acad Sci U S A. 1968 Jan;59(1):139–144. doi: 10.1073/pnas.59.1.139. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pace N. R., Bishop D. H., Spiegelman S. The kinetics of product appearance and template involvement in the in vitro replication of viral RNA. Proc Natl Acad Sci U S A. 1967 Aug;58(2):711–718. doi: 10.1073/pnas.58.2.711. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pace N. R., Spiegelman S. In vitro synthesis of an infectious mutant RNA with a normal RNA replicase. Science. 1966 Jul 1;153(3731):64–67. doi: 10.1126/science.153.3731.64. [DOI] [PubMed] [Google Scholar]
- Saffhill R., Schneider-Bernloehr H., Orgel L. E., Spiegelman S. In vitro selection of bacteriophage Q-beta ribonucleic acid variants resistant to ethidium bromide. J Mol Biol. 1970 Aug;51(3):531–539. doi: 10.1016/0022-2836(70)90006-9. [DOI] [PubMed] [Google Scholar]
- Sanger F., Brownlee G. G., Barrell B. G. A two-dimensional fractionation procedure for radioactive nucleotides. J Mol Biol. 1965 Sep;13(2):373–398. doi: 10.1016/s0022-2836(65)80104-8. [DOI] [PubMed] [Google Scholar]
- Spiegelman S. An approach to the experimental analysis of precellular evolution. Q Rev Biophys. 1971 Aug;4(2):213–253. doi: 10.1017/s0033583500000639. [DOI] [PubMed] [Google Scholar]
- Spiegelman S., Haruna I., Holland I. B., Beaudreau G., Mills D. The synthesis of a self-propagating and infectious nucleic acid with a purified enzyme. Proc Natl Acad Sci U S A. 1965 Sep;54(3):919–927. doi: 10.1073/pnas.54.3.919. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Spiegelman S., Pace N. R., Mills D. R., Levisohn R., Eikhom T. S., Taylor M. M., Peterson R. L., Bishop D. H. The mechanism of RNA replication. Cold Spring Harb Symp Quant Biol. 1968;33:101–124. doi: 10.1101/sqb.1968.033.01.015. [DOI] [PubMed] [Google Scholar]
- Weissmann C., Feix G., Slor H. In vitro synthesis of phage RNA: the nature of the intermediates. Cold Spring Harb Symp Quant Biol. 1968;33:83–100. doi: 10.1101/sqb.1968.033.01.014. [DOI] [PubMed] [Google Scholar]