On Bacterial and ØX-174 Messenger-RNA
<p>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 o...
| Summary: | <p>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.</p>
<p>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.</p>
<p>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 10<sup>4</sup>
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.</p>
<p>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.</p>
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