Characterization of the poly(adenylic acid) sequences in RNA synthesized in vitro by mouse myeloma nuclei

Abstract

The size and quantity of poly(A)sequences made by mouse myeloma nuclei in vitro are dependent on the concentration of KCI, ATP, other ribonucleoside triphosphates, as well as the nature of the divalent cation in the reaction medium. Reduction of the KC1 concentration from 120 mM to 5 mM, for example, stimulates poly(A) synthesis 10- to 20-fold. These poly (A) sequences are similar in size to cellular nuclear poly(A), but the RNA molecules to which they are attached are much shorter than poly(A) containing RNA molecules made at 120 mM KC1. Presence of Mn2+ in the medium led to a much more heterogenous population of poly(A) sequences. From such observations we have found reaction conditions in which nuclei synthesize molecules that resemble native nuclear poly(A) + RNA. Not only are the lengths and amounts of the poly(A) sequences similar, but they also undergo a terminal turnover like that of the poly(A) in hnRNA. An oligo(A) sequence that resembles the oligo(A) found in non-poly(A) containing hnRNA of mouse myeloma and HeLa cells is also synthesized in vitro. These observations suggest that some processing functions are retained during the in vitro incubation of these nuclei.