Selective determination of mRNA specific radioactivity in HeLa cells without the use of inhibitors |
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Authors: | U Wiegers G Kramer H Hilz |
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Affiliation: | Institut für Physiologische Chemie der Universität Hamburg 2 Hamburg 20, Martinistraße 52, Germany |
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Abstract: | Polysomes from (3H)-uridine pulse-labeled HeLa cells were isolated and the specific radioactivity of polysome-associated mRNA was determined by selective enzymic hydrolysis at 0°C of the ribosomal mRNA sections. ribosomal mRNA protected from hydrolysis during ribonuclease treatment and subsequently isolated by the proteinase K method (1) exhibited the same specific radioactivity as the interribosomal mRNA split products.When labeled polysomes were subjected to ribonuclease treatment at 25°C instead of 0°C a higher specific radioactivity of the interribosomal split products resulted, while intraribosomal sections still exhibited the same values as after 0°C treatment. The labeled polysomes used as substrate exhibited one single A260 and radioactivity peak in CsCl density gradients. No RNP material banding at ? = 1.35 ? 1.45 could be detected. However, the radioactivity maximum banded at slightly lower densities than the A260 peak (? = 1.55 versus 1.57). The shift appears to be caused by a contaminant RNA. These findings as well as the radioactivity pattern of pulse-labeled polysomes in sucrose gradients may indicate the presence of newly synthesized mRNA associated with monosomes (and oligosomes) protected from ribonuclease action at 0°C by (transport?) proteins. |
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