On Bacterial and ΦX-174 Messenger-RNA

Citation

Sedat, John W. (1971) On Bacterial and ΦX-174 Messenger-RNA. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/5Z4D-EC83. https://resolver.caltech.edu/CaltechTHESIS:04172018-094635838

Abstract

General methods for the chromatography of nucleic acids on benzoylated (naphthoylated) DEAE cellulose are described. This procedure results in well-resolved peaks with good recovery and seems to separate nucleic acids on the basis of their secondary structure almost independently of their molecular weight. These methods have proved to be useful in the analysis of the replicative intermediates of MS-2 RNA, ØX RFDNA, and ØX single stranded DNA, for example.

Specific methods for the purification of E. coli. pulse-labeled RNA based on the chromatography on benzoylated DEAE cellulose at pH 7.5 and pH 3.5 were developed. Greater than 60% of the pulse label with less than 4% of the mass label is recovered, and the RNA size distribution in a denaturing solvent (99% dimethyl sulfoxide) after chroma­tography shows that the mass label closely parallels the pulse label as a function of size; there appears to be no selection or degradation. Pulse-chase experiments indicate that a large fraction of both the pulse and mass labels are chased out, suggesting reasonable purity of the mRNA. These data imply that there is a difference, structural or chemical, between mRNA and other known RNAs that allows all E. Coli. mRNA to act as a group regardless of size during the purification.

The in vivo ØX mRNA has been studied using the above procedure for purification and analysis. The results show that the ØX mRNA size distribution at early and late times after infection is very broad ranging from a distinct maximum size of 1.7 megadaltons (one genome length of poly- cistronic mRNA), to a peak in the distribution at 0.2-0.3 megadaltons, and with significant mRNA as small as 10⁴ daltons. The only difference observed between the early and late times after infection appears to be in the amount of RNA present: approximately 100 molecules of mRNA per cell are present at 4 min. after infection compared to 1000 molecules per cell at 25 min. after infection. Little if any difference could be found in the size distribution of mRNA made by the strongly polar OP6 ØX mutant or upon infection of the ØX replication-restrict. A variety of experimental approaches showed an absence of significant methylation and 5'triphosphate termini in the mRNA.

Attempts were made to ask which RF, parental or progeny, was the template for the transcription of the ØX mRNA. One type of experiment indicated that there was a small amount of pulse labeled RNase-, phenol-, and detergent- resistant RNA specifically attached to the density labeled parental RF. However, other types of direct experiments (such as analysis of non-RNase treated RF in CsCl density equilibrium gradients) demonstrated that neither type of RF was shifted to higher densities due to attached nascent mRNA; nor could significant pulse labeled RNA be detected in the RF region of the CsCl density gradients. Thus, no unambiguous answer can be given to the template question.

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