The origin of human chromosome 18 from a human/ape ancestor.

Karyotype analysis by previous investigators demonstrated that human chromosome 18 differs from homologous chromosomes in the great apes by a pericentric inversion. The present study uses fluorescence in situ hybridization on human and pygmy chimpanzee chromosomes to confirm the inversion and to delimit the regions where the breakpoints must have occurred in the human/ape ancestor.

Assignment of the gene for beta-casein (CSN2) to 4q13 --> q21 in humans and 3p13 --> p12 in chimpanzees.

The human beta-casein gene (CSN2) has been mapped by fluorescence in situ hybridization to human chromosome 4q13 --> q21 and to the homologous chimpanzee chromosome at 3p13 --> p12. This confirms the presence of a pericentric inversion that distinguishes the two species, as first observed by chromosomal banding studies.

S6 phosphorylation accompanies recruitment of ribosomes and mRNA into polysomes in response to dichlororibofuranosyl benzimidazole.

Dichlororibofuranosyl benzimidazole (DRB), a potent inhibitor of nuclear RNA synthesis and messenger RNA (mRNA) accumulation, produces a paradoxical mobilization of rRNA and mRNA from the subpolysomal pool into polysomes in HeLa cells during the first 40 min of treatment. S6 is phosphorylated concurrently with polysome accumulation, and ribosomal subunits containing phosphorylated S6 are preferentially localized in polysomes, indicating that they form initiation complexes more readily than their non-phosphorylated counterparts.

Molecular evolution, intracellular organization, and the quinary structure of proteins.

High-resolution two-dimensional polyacrylamide gel electrophoresis shows that at least half of 370 denatured polypeptides from hamster cells and human cells are indistinguishable in terms of isoelectric points and molecular weights. Molecular evolution may have been more conservative for this set of proteins than sequence studies on soluble proteins have implied. This may be a consequence of complexities of intracellular organization and the numerous macromolecular interactions in which most polypeptides participate. It is suggested that the term "quinary structure" be used to refer to macromolecular interactions that are transient in vivo. Such interactions will not be evident from the composition of purified proteins, but they may constitute an important source of constraints on changes in primary structure.

How many proteins are there in a typical mammalian cell?

There is a major disparity between the number of polysomal mRNA species found in mammalian cells and the number of polypeptides detected by high-resolution two-dimensional polyacrylamide gel electrophoresis. Here we show that technical factors are not responsible for the relative paucity of proteins, and that the translation products of rare mRNAs would be easily detectable if all mRNAs were translated in proportion to their abundance. We conclude that a large majority of rare mRNAs are translated at no more than a tenth the average translation rate, if they are translated at all. There may be no more than 2000 physiologically significant primary gene products (polypeptides) in a typical mammalian cell.

Preferential utilization of phosphorylated 40-S ribosomal subunits during initiation complex formation.

HeLa cells grown to a high density in spinner culture contain little or no phosphorus associated with ribosomal protein S6. When cells are transferred to fresh medium containing 10% calf serum, S6 becomes rapidly and multiply phosphorylated. Ribosomal proteins were extracted from subpolysome and polysome fractions, displayed on two-dimensional gels, and the distribution of phosphorylated S6 was quantified. Polysomal ribosomes have a higher percentage of phosphorylated S6 than subpolysomes at all times after transfer and the difference becomes more pronounced as the extent of phosphorylation increases. This difference cannot be explained by preferential phosphorylation of polysomal ribosomes, since kinase activity is equally distributed between polysomes and subpolysomes. Likewise, preferential dephosphorylation of subpolysomal ribosomes during cell fractionation does not occur. We interpret our results to mean that phosphorylated 40-S subunits form initiation complexes more efficiently than non-phosphorylated 40-S subunits.

Rapid alterations in initiation rate and recruitment of inactive RNA are temporally correlated with S6 phosphorylation.

HeLa cells propagated in spinner culture for 3-4 days without replenishing medium or serum progressively decrease the amount of mRNA and rRNA in polysomes, as well as the elongation rate. Treatment of these cells with low doses of cycloheximide shifts at least two thirds of the subpolysomal ribosomal particles into polysomes, indicating that the rate of ribosome attachment limits translation in these cells. Transfer of serum factor-depleted cells to fresh medium containing 10% calf serum likewise results in an extensive translocation of mRNA and rRNA into polysomes. Polysome absorbance profiles and sizes suggest that serum stimulation causes these changes by enhancing initiation rate. Newly recruited mRNAs derive from both subpolysomal translocation and recent nuclear RNA export, and contain a greater proportion of poly(A)-deficient mRNA molecules than the pre-stimulated polysomal mRNA population. Kinetic measurements show that these events occur principally within 20 min after serum addition, suggesting rapid modifications of preexisting components are involved. The phosphorylation kinetics of ribosomal protein S6, which closely parallel the alterations in translational activity, suggest that this modification may influence ribosome function.

Phosphorylation of ribosomal protein S6. Relationship to protein synthesis in HeLa cells.

The time course of S6 phosphorylation and several aspects of protein synthesis have been studied in suspension cultures of HeLa cells, following transfer to fresh medium and serum. The phosphorylation of S6 is not temporally correlated with changes in polypeptide initiation and elongation rates, as judged from polysome profiles. Phosphorylation of S6 can be maximal within 30 min after transfer; elongation and initiation rates increase coordinately and more slowly, becoming maximal about 6 h after transfer, a time at which the net phosphorylation of protein S6 is greatly reduced or negligible. Recruitment of messenger RNA into polysomes is another response to fresh medium and serum; this response occurs almost as rapidly as the phosphorylation of S6. We suggest that the phosphorylation of S6 may play a role in messenger RNA recruitment.

Identification of homologous ribosomal proteins in HeLa cells and in Tetrahymena pyriformis. A study of proteins binding 5-S RNA and acidic proteins released from 40-S subunits by EDTA.

Tetrahymena pyriformis 60-S ribosomal subunits treated with EDTA release a 7-S particle containing 5-S RNA and a 36000-Mr protein that is similar to mammalian 5-S-RNA-binding protein L5 in molecular weight, in two-dimensional acrylamide gel mobility, and in peptide pattern as generated by a simple, one-dimensional acrylamide gel technique. Human and T. pyriformis 40-S ribosomal subunits, treated with buffers lacking magnesium or containing EDTA, release varying amounts of two large acidic proteins. We have identified these released proteins by two-dimensional gel electrophoresis.

HeLa ribosomal protein S6. Insulin and dibutyryl cyclic AMP affect different phosphopeptides.

Tryptic phosphopeptides from HeLa ribosomal protein S6 have been separated by chromatography, followed by electrophoresis. Three phosphopeptides were found in S6 from cells treated in vivo with dibutyryl cAMP and at least four different phosphopeptides were found from cells treated with insulin plus the essential amino acids. Only phosphoserine was found in S6 isolated from insulin-treated cells.

Evidence for control of protein synthesis in HeLa cells via the elongation rate.

The effects of fresh medium and serum on protein synthesis in suspension-cultured HeLa cells after growth to high cell density (> 5 x 10(5) cells/ml) were studied. Cells which were resuspended in fresh medium plus serum and grown for 24 hours (control) were compared with cells grown for 2 hours after resuspension (stimulated). The spectrum of proteins being synthesized by control and stimulated cells does not appear to be grossly different; that is, the weight and number average molecular weights of newly synthesized whole-cell protein are about the same in both cultures. Also, no significant differences were observed in the number of ribosomes per polysome or in the fraction of total ribosomes in polysomes. However, the transit times (combined elongation and termination times) were found to differ significantly; the average transit time for control cells was 2.24 minutes, while the average transit time for stimulated cells was 1.26 minutes. (An appendex evaluating the methodology involved in measuring the transit time is included.) In aggreement with the difference in transit time, the absolute rate of protein synthesis in stimulated cells was approximately 1.8 times the rate measured in control cells. These data are taken as evidence that under certain conditions, the rate of elongation and/or termination of polypeptide chains limits the overall rate of translation, and that cells can respond to growth conditions by changing the elongation and/or termination rate of protein synthesis.

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 with 14C-labeled parental cell proteins, electroporesis is carried out, and polypeptides that occur only in the 3H-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.

Human heterozygosity: a new estimate.

Several hundred polypeptides from four human diploid fibroblast cell lines were compared by high-resolution two-dimensional polyacrylamide gel electrophoresis and double-label autoradiography under conditions where allelic products that differ by a single charged amino acid would be distinguished. The average heterozygosity represented by this set of gene products appears to be less than 1% for changes involving charged amino acids.