JBC, Vol. 251, Issue 9, 2592-2599, May, 1976
Appearance of rapidly labeled, high molecular weight RNA in nuclear ribonucleoprotein. Release from chromatin and association with protein
L. H. Augenlicht and M. Lipkin
Chromatin and nuclear ribonucleoprotein (nRNP) have been prepared from a
human carcinoma cell line. Following a 1-hour (3H)uridine pulse, 60 to 70%
of the nuclear radioactivity, after removal of nucleoli, was found in the
chromatin, the balance in nRNP. This was true whether the chromatin and
nRNP were separated by velocity centrifugation or by isopycnic
centrifugation on Metrizamide gradients. Radioactivity in chromatin and
nRNP was found in high molecular weight RNA, with mean sedimentation
coefficients of 20 S and 15 S, respectively, as determined on sodium
dodecyl sulfate-sucrose gradients. Experiments on the kinetics of
appearance of radioactivity in the RNA of the two fractions suggest that
some of the chromatin-associated RNA is precursor to nRNP-RNA. The proteins
of nRNP are complex as revealed by sodium dodecyl sulfate gel
electrophoresis. The contamination by chromatin protein was estimated to be
5%. Experiments involving short pulses of (3H)tryptophan, and pulse-chase,
suggested that the rapidly turning over proteins of nRNP were not complexed
with RNA while still associated with chromatin. However, it was also shown
that the radioactivity in nRNP following short pulses of (3H)tryptophan did
not correspond to the major bands seen on stained sodium dodecyl sulfate
gels. It is therefore concluded that the protein of nRNP consists of two
classes: species present in large amounts, possibly common to all RNA in
nRNP, which are relatively stable and may be complexed to RNA still
associated with chromatin; and a large number of rapidly turning over
species, each present in small amounts and associated with nRNP only after
its release from chromatin.
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