Edwin H. McConkey

Stability of nuclear RNA in mammalian cells.

Abstract The kinetics of entry of [3H]adenosine into ATP, cellular RNA, and nuclear RNA of mouse L cells were determined and analyzed. A molar accumulation curve for RNA was estimated from the specific radioactivities of RNA and ATP; this curve was resolved graphically into stable and unstable components. The stability of the unstable component (mostly heterogeneous nuclear RNA) was estimated by applying first-order decay analysis. Heterogeneous, nuclear RNA decays with an apparently uniform half-life of 23 minutes, considerably greater than some previous estimates. It is synthesized at an instantaneous rate of 5.4 × 10−2 pg/cell per minute and reaches a steady-state level of 1.8 pg/cell in the nucleus, or 7% of the total cellular RNA. Only about 2% of the heterogeneous RNA synthesized in L cells enters polysomes as messenger RNA. The implications of these values are discussed with reference to similarly determined values for sea urchin embryos.

Proposed Uniform Nomenclature for Mammalian Ribosomal Proteins

SummaryThe numbering systems for mammalian ribosomal proteins used in several laboratories have been correlated and a proposal for a standard system is presented.

Phosphorylation of ribosomal protein S6 in suspension cultured HeLa cells.

SummaryHeLa cell ribosomal protein S6, and the increase in its phosphorylation level that occurs after resuspending cells in fresh medium plus serum, were studied using two-dimensional gel electrophoresis. The maximum level of S6 phosphorylation occurs about 2 h after adding fresh medium and serum to cells that have been allowed to grow to high density; this results in an almost complete shift of the spot representing S6 in two-dimensional polyacrylamide gels to a new location. Mixing experiments showed that the differences in the level of phosphorylation occur in vivo and are not an artifact of in vitro sample preparation. This method of stimulating S6 phosporylation provides a convenient system for studying the functional significance of the phenomenon. Only one other ribosomal protein was detectably phosphorylated using [32P]-labeling and autoradiography of dried two-dimensional gels. The level of phosphorylation of this protein, L14, does not change after serum stimulation.

Double-label autoradiography for comparison of complex protein mixtures after gel electrophoresis

Abstract Comparison of complex protein mixtures by high-resolution, two-dimensional polyacrylamide gel electrophoresis is simplified by coelectrophoresis of 3 H-labeled and 14 C-labeled preparations, followed by two-step autoradiography. Both qualitative and quantitative comparisons are possible. Applications to genetics, molecular evolution, and metabolism are described.

Control of ribosomal protein phosphorylation in HeLa cells

Abstract The effects of a large series of hormones, cyclic nucleotides and metabolic inhibitors on phosphorylation of ribosomal protein S6 in HeLa cells suggest that at least two metabolic pathways are involved. One responds to insulin and epidermal growth factor; the other responds to adenosine 3′, 5′-cyclic monophosphate. Some phosphodiesterase inhibitors can suppress the phosphorylation of S6 that ordinarily is stimulated by insulin.

Completion of nascent hela ribosomal proteins in a cell-free system

Abstract HeLa cell 3H-labeled proteins completed in vitro and 14C-labeled 60S subunit ribosomal proteins isolated from cells labeled in vivo were processed together and applied to a carboxymethyl-cellulose column. A linear gradient of NaCl was used to elute the adsorbed proteins. Prominent peaks of both isotopes eluted together at 0.1M and from 0.175M to 0.25M NaCl. Polyacrylamide gel electrophoresis of the doubly-labeled proteins confirmed the identity of the ribosomal proteins completed in vitro with the ribosomal proteins labeled in vivo .

Vimentin-derived proteins: Differences between normal human fibroblasts and transformed human cells☆

Abstract Two-dimensional gel electrophoresis revealed a quantitative difference in a series of polypeptides ranging in MW from 45 000 to 51 000 and of lower isoelectric pH than vimentin, when comparing normal human fibroblasts with a virally transformed subline and with HeLa cells. Re-extraction of purified [35S]vimentin with cold whole cell homogenates and peptide mapping showed that these polypeptides are derivatives of vimentin. They may be natural components of a normal fibroblasts architecture or they may arise from a pool of vimentin that is not structured within intermediate filaments at the time of extraction. Furthermore, we show that vimentin from the two transformed cell types is more resistant to proteolysis by whole cell homogenates than vimentin from normal fibroblasts. Structural alteration of vimentin may play an important role in the expression of transformation.

Crosslinking of proteins to ribosomal RNA in HeLa cell polysomes by sodium periodate.

Abstract HeLa cell polysomes were oxidized with sodium periodate and reduced with sodium borohydride to induce covalent crosslinks between ribosomal RNA and nearby proteins. We proved that RNA was tryly crosslinked to protein in oxidized, and not in control, samples using denaturing cesium trichloroacetate density gradients and phenol extraction. By both one- and two-dimensional gel analysis, we found that protein S3a can be crosslinked to 18S RNA, protein L3 to 28S RNA, and proteins L7′ and L23′ to 5.8S RNA. Because of the specificity of the periodate reaction, and since we were able to crosslink protein S1 to 16S RNA in Escherichia , coli 30S ribosomal subunits, it is likely that we have crosslinked proteins to the 3′OH ends of HeLa polysomal RNAs.

Nucleolar protein metabolism in actinomycin D treated HeLa cells

Abstract Nucleolar proteins from actinomycin D treated HeLa cells and controls were compared after labeling with radioactive amino acids for one to four hours. Measurements of specific activity showed suppression of labeling in treated nucleoli, with no effect in cytoplasm or nucleoplasm. Electrophoresis on sodium dodecyl sulfate-polyacrylamide gels indicated extensive similarity between rapidly labeled nucleolar proteins of control cells and proteins from cytoplasmic ribosomes. Actinomycin D selectively suppressed labeling of nucleolar proteins that co-migrate with ribosomal proteins, although synthesis of ribosomal proteins continued on cytoplasmic polysomes.

Identification of human gene products from hybrid cells: A new approach

A method is described for detection of human polypeptides from hybrid cells, following high-resolution two-dimensional polyacrylamide gel electrophoresis of total cellular protein.3H-labeled hybrid cell proteins are mixed with14C-labeled parental cell proteins, electrophoresis is carried out, and polypeptides that occur only in the3H-labeled preparation are detected by double-label autoradiography. The possibility of developing a standard human polypeptide two-dimensional gel map, which would allow identification of electrophoretically separable components as specific proteins, is discussed.