Lennart Philipson

Genes specifically expressed at growth arrest of mammalian cells

A subtraction cDNA library enriched for RNA sequences preferentially expressed in growth-arrested cells was prepared. Six cDNA clones were identified, varying in abundance from 2% to 0.0002% of the library and in size from 0.8 to 10 kb. The corresponding mRNAs are downregulated with different kinetics upon induction of growth by serum. The kinetics of induction after serum starvation and density-dependent inhibition of two of these growth-arrest-specific (gas) genes were investigated in more detail. Two cell lines transformed by viral onc genes did not express the two gas genes. The full-length cDNA for one gene has been sequenced and the protein product preliminarily characterized by in vitro translation.

The division cycle and RNA-synthesis in diploid human cells at different passage levels in vitro

Abstract The division cycle and RNA synthesis were analysed in early and late passage cells of a human fibroblastic strain (WI-38). Cells were exposed to tritium labeled thymidine or uridine. Cultures after the 40th passage were found to be very heterogeneous regarding the length of the division cycle. The fraction of cells involved in DNA synthesis after a short pulse was smaller and the generation time was prolonged in late passage cells. Evidence is presented that the cause for a prolonged generation time was mainy due to retardation in the G1 and G2 periods. The rate of synthesis of RNA was found to be decreased in late passage cells. However, the pattern of incorporation of labeled precursor was the same in both types of cells and active RNA synthesis occurred in 100 per cent of the phase III cells.

The growth arrest-specific gene, gas1, is involved in growth suppression

This report describes the structure of the mRNA, the protein product, and the growth-regulating activity of one of the growth arrest-specific genes, gas1. From the predicted amino acid sequence, in vitro translation of gas1 mRNA, and immunofluorescence of cells in culture, it appears that the gas1 protein is an integral plasma membrane protein whose expression is linked to growth arrest. When gas1 is overexpressed from a constitutive promoter in quiescent cells, the serum-induced transition from the G0 to the S phase of the cell cycle is inhibited without affecting the normal early serum response. Ectopic expression of the gas1 gene by microinjection in normal and transformed NIH 3T3 cell lines with the notable exception of SV40-transformed 3T3 cells leads to inhibition of DNA synthesis. Thus, gas1 appears to be one component of a negative circuit that governs growth suppression. Its effect is, however, abolished in SV40-transformed cells.

Structural proteins of adenoviruses

Abstract The penton antigen from adenovirus type 2 has been purified by DEAE chromatography, agarose chromatography and preparative polyacrylamide electrophoresis. The final product is homogeneous by electron microscopy, immunoelectrophoresis, polyacrylamide electrophoresis, and analytical ultracentrifugation. Penton antigen so prepared has a sedimentation coefficient of 10.5 and a molecular weight around 400,000. A minimum of three antigenic specificities can be demonstrated with immunodiffusion. Trypsin selectively inactivates the cytopathic effect of the type 2 penton. At high trypsin concentrations the main part of the vertex capsomer antigenicity and morphology is also lost. Rabbit antisera to pure pentons contain low titers of neutralizing antibodies when assayed by inhibition of plaque formation. The use of the fluorescent focus assay reveals, in contrast, high neutralizing activity in these sera. Treatment of penton antigen with pyridine releases free vertex capsomers which are immunologically and morphologically intact and have the ability to induce cytopathic changes in KB-cell cultures.

Inhibition of Influenza Virus Ribonucleic Acid Polymerase by Ribavirin Triphosphate

Ribavirin 5′-triphosphate (RTP), derived from the broad-spectrum antiviral compound ribavirin (Virazole), can selectively inhibit influenza virus ribonucleic acid polymerase in a cell-free assay. Ribavirin and its 5′-monophosphate have no effect on the polymerase. The inhibition is competitive with respect to adenosine 5′-triphosphate and guanosine 5′-triphosphate. RTP also inhibits ApG- and GpC-stimulated influenza virus ribonucleic acid polymerase. Since ribavirin is phosphorylated in the cell, the inhibition of influenza multiplication in the cell may also be caused by RTP.

Translation of MuLV and MSV RNAs in nuclease-treated reticulocyte extracts: Enhancement of the gag-pol polypeptide with yeast suppressor tRNA

Abstract The virion RNAs from Moloney murine leukemia virus (MuLV) and Moloney murine sarcoma virus (MSV) were translated in a micrococcal nuclease-treated cell-free system from rabbit reticulocytes. The predominant polypeptides formed from 35S MuLV RNA were 78,000 and 65,000 daltons in molecular weight, and minor components with molecular weights of 180,000,110,000, 52,000 and 40,000 daltons were also observed. The 30S MSV RNA yielded two predominant polypeptides of 62,000 and 43,000 daltons, and minor components about 72,000, 40,000 and 18,000 daltons in molecular weight. The predominant polypeptides generated by both MuLV and MSV RNA were found to be precursors of the core proteins by immunoprecipitation with specific antisera. The 180,000 dalton molecular weight polypeptide encoded by MuLV RNA was immunoprecipitated both by antisera to the core protein (p30) and reverse transcriptase. The major products therefore appear to be Pr65 gag and Pr78 gag ; an important minor product is Pr180 gag-pol . Most of the products of When purified yeast suppressor tRNA was added to the translation mixture directed by 35S MuLV RNA, the amount of the Pr78 gag was reduced, while the yield of the Pr180 gag-pol was enhanced. Amber suppressor tRNA was about 3 times as effective as ochre suppressor tRNA and nonsuppressor tRNA. This pattern of suppression was also seen for an established amber mutation (UAG) in the synthetase gene of Qβ (Qβ aml), suggesting that it is a UAG codon which terminates synthesis of Pr78 gag . In the MSV system, the amber suppressor tRNA, and to a lesser extent the ochre suppressor tRNA, markedly increased the synthesis of the 72,000 dalton molecular weight polypeptide with a slight reduction of the 62,000 dalton protein. Since read-through between the core protein and reverse transcriptase genes occurs to a low level both in vivo and in vitro and can be enhanced in vitro by amber suppressor tRNA, these results suggest that a suppression mechanism may control the relative amounts of core protein and reverse transcriptase synthesized from 35S mRNA. Such a mechanism might be used more generally by mRNAs from mammalian cells.

A new species of virus-coded low molecular weight RNA from cells infected with adenovirus type 2

A virus-coded low molecular weight RNA (5.2S), which migrates slightly faster on polyacrylamide gels than the well characterized adenovirus-specific 5.5S RNA, has been isolated from cells infected with adenovirus type 2. Hybridization-competition experiments and RNA fingerprints indicate that the two virus-associated (VA) RNAs differ in their primary structures. The gene for 5.2S RNA is located to the right of the gene for 5.5S RNA, on the I strand of a DNA segment which extends between positions 30.3 and 32.2 on the map of adenovirus type 2 DNA. Both 5.5S and 5.2S RNA can be detected early after infection and also in the presence of cytosine-arabinoside or cycloheximide. After the onset of viral DNA replication, the synthesis of 5.2S RNA levels off, whereas 5.5S RNA is synthesized in increasing amounts. Both 5.2S and 5.5S RNAs are synthesized in isolated nuclei by an enzyme which resembles RNA polymerase III in its sensitivity to alpha-amanitin. In isolated nuclei, both RNA species are labeled with beta-32P-labeled GTP, which suggests that they are initiated at separate promotor sites.

Gene fusion vectors based on the gene for staphylococcal protein A

Two plasmid vectors, containing the gene coding for staphylococcal protein A and adapted for gene fusion, have been constructed. These vectors will allow fusion of any gene to the protein A gene, thus giving hybrid proteins which can be purified, in a one-step procedure, by IgG affinity chromatography. As an example of the practical use of such vectors, the protein A gene has been fused to the lacZ gene of Escherichia coli. E. coli strains containing such plasmids produce hybrid proteins with both IgG binding and beta-galactosidase activities. The hybrid protein(s) can be immobilized on IgG-Sepharose by its protein A moiety with high efficiency without losing its enzymatic activity and they can be eluted from the column by competitive elution with pure protein A. The fused protein(s) also binds to IgG-coated microtiter wells which means that the in vivo product can be used as an enzyme conjugate in ELISA tests.

A gene fusion system for generating antibodies against short peptides

A novel method to obtain specific antibodies against short peptides is described, involving synthesis of the corresponding oligodeoxynucleotides followed by cloning into a new set of fusion vectors, pEZZ8 and pEZZ18, based on two synthetic IgG-binding domains (ZZ) of Staphylococcus aureus protein A. The soluble gene fusion product thus obtained, can be collected from the culture medium of Escherichia coli and rapidly recovered in a one-step procedure by IgG affinity chromatography. The system was used to express a fusion protein consisting of the two Z fragments and the C-terminal part [amino acids (aa) 57-70] of human insulin-like growth factor I (IGF-I). This 16-kDa protein was purified by affinity chromatography on IgG Sepharose and antibodies were raised in rabbits. The fusion protein elicited peptide-specific antibodies, as measured by solid-phase radioimmuno assay and Western blotting, reactive with both synthetic C-terminal peptide and the native human IGF-I protein. The results suggests that the gene fusion system can be used for efficient antibody production against short peptides encoded by synthetic oligodeoxynucleotides.

Structural proteins of adenoviruses: X. Isolation and topography of low molecular weight antigens from the virion of adenovirus type 2☆

Abstract With high resolution SDS-polyacrylamide gel electrophoresis ( Maizel, 1971 ) and a new method to extract the basic proteins, it was ascertained that adenovirus type 2 contains at least 10 distinct polypeptides (II, III, IIIa, IV–X) and possibly more. Five proteins (V, VI, VII, VIII, and X) were purified by selective extraction in urea at high ionic strength, and low pH followed by preparative polyacrylamide electrophoresis toward the cathode at pH 3.4. Antisera, produced against proteins V, VI, and VII were used to reveal that these proteins were antigenically distinct and unrelated to hexons, pentons, and fibers. The location of the polypeptides was investigated by two methods of virion degradation ( Prage et al. , 1970 ). Polypeptides V and VII were associated with the DNA-containing core. Polypeptide VI appeared to be associated with all hexons of the virion. Hexons from the triangular facets of the capsid contained in addition to polypeptides II and VI, polypeptide IX, and possibly also polypeptide VIII. Hexons purified from infected cells contained only polypeptide II. Polypeptide IIIa was preferentially released together with the peripentonal hexons.