Abstract
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.
Highlights
Chromatin and nucleai ribonucleoprotein have been prepared from a human carcinoma cell line
It has been reported that all of the hnRNA can be isolated free of chromatin in the form of nRNP [5, 7], we have found that in cultured colonic carcinoma cells a considerable proportion of uridine pulse-labeled nuclear RNA is associated with chromatin, and some of this appears to be precursor to species isolated as nRNP
When the isolated chromatin was resuspended in Buffer A, and respun through 60% sucrose as described, only 7% of the radioactivity was found in the supernatant
Summary
Chromatin and nucleai ribonucleoprotein (nRNP) have been prepared from a human carcinoma cell line. Following a l-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. Experiments involving short pulses of [3H]tryptophan, and pulse-chase, suggested that the rapidly turning over proteins of nRNP were not complexed radioactivity with RNA while still associated with chromatin. It has been reported that all of the hnRNA can be isolated free of chromatin in the form of nRNP [5, 7], we have found that in cultured colonic carcinoma cells a considerable proportion of uridine pulse-labeled nuclear RNA is associated with chromatin, and some of this appears to be precursor to species isolated as nRNP. Because it has been demonstrated that protein is complexed to the RNA transcript while still associated with chromatin (8-ll), we investigated whether protein isolated as nRNP is found first associated with the Contract NO1 CP 43366 and Grants CA14991 and 08748
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
