Construction and identification of cDNA clones for mouse ribosomal proteins: Application for the study of r-protein gene expression

Abstract

A set of recombinant DNAs with inserts of mRNA sequences coding for various mouse ribosomal proteins (r- proteins) were isolated. The recombinants were selected from a library of cDNA clones representing the small (⩽12S) poly(A) + mRNA of mouse L cells, which is relatively enriched for the r-protein mRNAs. Selection consisted of testing the various recombinant plasmids for their ability to hybridize selectively with an mRNA that directs the synthesis of a recognizable r-protein in a cell-free translational system. Translation products were subjected to a preliminary screen by electrophoresis on one-dimensional Triton X-100/urea slab gels and then more definitively identified by the two-dimensional gel analyses conventionally used for ribosomal proteins. The set of recombinants represents the mRNAs specifying ribosomal proteins S16, L7, L13, L18, L19, L30, L32/33 and probably L10. The recombinant plasmids were used as probes to determine the size and abundance of the corresponding mRNAs and their presumptive nuclear precursors. In general, there is a good correlation between mRNA size and the size of the encoded proteins, each rp-mRNA having a relatively uniform proportion (29 + 5%) of non-coding sequence. The pattern of poly(A) + nuclear components was very distinctive for each of the rp-mRNAs, indicating that the majority of rp-mRNAs are probably not derived from common polycistronic transcripts. The largest detectable nuclear components were about 3- to 7-fold larger than their respective mature mRNAs. The individual rp-mRNAs appear to constitute about 0.06 to 0.12% of the poly(A) +mRNA in L cells and about 0.06 to 0.38% in MPC-11 myeloma cells; for L cells this corresponds to about 300–400 copies of each mRNA per cell. The content of large nuclear molecules containing rp-mRNA sequences is estimated to be very low, on the order of only a few molecules per cell. Preliminary Southern blot analysis of EcoRI restriction fragments of mouse embryo DNA revealed very complex patterns, suggesting that a family of homologous sequences for the individual rp-mRNAs may exist in the mouse genome.