Both the inhibitory effect of aphidicolin on the replicative alpha-polymerase and the reversibility of its action in vivo (Pedrali-Noy & Spadari, 1979, Biochem. Biophys. Res. Commun. 88, 1194-2002) allow the synchronization of cells in culture. Aphidicolin prevents G1 cells from entering the DNA synthetic period, blocks cells in "S" phase, allows G2, M and G1 cells to continue the cell cycle and to accumulate at the G1/S border. Aphidicolin is a more useful reagent than hydroxyurea and thymidine because it does not affect cell viability or "S" phase duration and does not interfere with the synthesis of dNTPs or DNA polymerases. In fact cells exposed to the drug continue to synthesize all three DNA polymerases alpha, beta and gamma as well as all dNTPs which, when the block is removed, are present at levels optimal for DNA initiation and replication. The technique is simple and can be applied to cells growing in suspension or monolayers and allows one to harvest large quantities of synchronized cells.

Chimeric proteins were constructed to define the nuclear localization signals (NLSs) of human ribosomal protein S6. The complete cDNA sequence, different cDNA fragments and oligonucleotides of the human ribosomal proteins S6, respectively, were joined to the 5' end of the entire LacZ gene of Escherichia coli by using recombinant techniques. The hybrid genes were transfected into L cells, transiently expressed, and the intracellular location of the fusion proteins was determined by their ,B-galactosidase activity. Three NLSs were identified in the C-terminal half of the S6 protein. Deletion mutagenesis demonstrated that a single NLS is sufficient for targeting the corresponding S6-,Bgalactosidase chimera into the nucleus. Removal of all three putative NLSs completely blocked the nuclear import of the resulting S6-,3-galactosidase fusion protein, which instead became evenly distributed in the cytoplasm. Chimeras containing deletion mutants of S6 with at least one single NLS or unmodified S6 accumulated in the nucleolus. Analysis of several constructs reveals the existence of a specific domain that is essential but not sufficient for nucleolar accumulation of S6.

Several procedures were used to disassemble rat liver rough microsomes (RM) into ribosomal subunits, mRNA, and ribosome-stripped membrane vesicles in order to examine the nature of the association between the mRNA of bound polysomes and the microsomal membranes. The fate of the mRNA molecules after ribosome release was determined by measuring the amount of pulse-labeled microsomal RNA in each fraction which was retained by oligo-dT cellulose or by measuring the poly A content by hybridization to radioactive poly U. It was found that ribosomal subunits and mRNA were simultaneously released from the microsomal membranes when the ribosomes were detached by: (a) treatment with puromycin in a high salt medium containing Mg ++, (b) resuspension in a high salt medium lacking Mg ++, and (c) chelation of Mg ++ by EDTA or pyrophosphate.Poly A-containing mRNA fragments were extensively released from RM subjected to a mild treatment with pancreatic RNase in a medium of low ionic strength. This indicates that the 3' end of the mRNA is exposed on the outer microsomal surface and is not directly bound to the membranes. Poly A segments of bound mRNA were also accessible to [aH]poly U for in situ hybridization in glutaraldehyde-fixed RM.Rats were treated with drugs which inhibit translation after formation of the first peptide bonds or interfere with the initiation of protein synthesis. After these treatments inactive monomeric ribosomes, as well as ribosomes bearing mRNA, remained associated with their binding sites in microsomes prepared in media of low ionic strength. However, because there were no linkages provided by nascent chains, ribosomes, and mRNA, molecules were released from the microsomal membranes without the need of puromycin, by treatment with a high salt buffer containing Mg ++.Thus, both in vivo and in vitro observations are consistent with a model in which mRNA does not contribute significantly to the maintenance of the interaction between bound polysomes and endoplasmic reticulum membranes in rat liver hepatocytes. 414

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Infection of BHK 21 cells by vesicular stomatitis virus (VSV) results in the intracellular synthesis of the five viral proteins which are easily detectable in polyacrylamide gels after short labeling periods with [35S]methionine. In addition, a 6th prominent radioactive protein band appears intracellularly in VSV‐infected BHK cells. This additional polypeptide is also coded by the viral genome, because it is immunoprecipitated by antibodies against viral particles and more specifically by antibodies against purified G‐protein. We propose to call this derivative of the G‐protein Gsi‐protein (short intracellular G‐protein). It is associated with intracellular membranes and has an apparent mol. wt. of 58 000. Both G‐ and Gsi‐protein have the same kinetics of appearance in the cell. The ratio of G‐:Gsi‐protein in BHK 21 cells is approximately 85:15. The mol. wt. difference of approximately 6000 daltons between G‐ and Gsi‐protein is not due to variations in the degree of glycosylation because trypsin digestions of both [3H]mannose‐labeled glycoproteins gave rise to identical glycopeptide patterns. Incubation of microsomes with trypsin demonstrates that Gsi‐protein is protected in its full length by intracellular membranes. Gsi‐protein is lacking an extended carboxy‐terminal region of the viral G‐protein sequence because it is not modified by palmitic acid and is not immunprecipitated by specific antibodies against a C‐terminal peptide of the G‐protein. Limited proteolysis by endoproteinase arg C indicates that the structure of Gsi‐protein is very similar to the shedded form of the G‐protein which has been previously described in the literature.(ABSTRACT TRUNCATED AT 250 WORDS)