The transformation-related p53 protein: a potential regulator of cell proliferation.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
R Baserga
Publications and source records attributed to R Baserga.
Expression of histone genes in a G1-specific temperature-sensitive mutant of the cell cycle.
The expression of genes coding for the four core histones (H2A, H2B, H3, and H4) was studied in tsAF8 cells. These baby hamster kidney-derived cells are a temperature-sensitive (ts) mutant of the cell cycle that arrest in G1 at the restrictive temperature. When serum-deprived tsAF8 cells are stimulated with serum, they enter the S phase at the permissive temperature of 34 degrees C, but are blocked in G1 at the nonpermissive temperature of 39.6 degrees C. Northern blot analysis using cloned human histone DNA probes detected only very low levels of histone RNA either in quiescent tsAF8 cells or in cells serum stimulated at the nonpermissive temperature for 24 h. Cellular levels of histone RNA were markedly increased in cells serum stimulated at 34 degrees C for 24 h. Temperature shift-up experiments after serum stimulation of quiescent populations showed that the amount of histone RNA was related to the number of cells that entered the S phase. Those cells that synthesized histone RNA and entered the S phase were capable of dividing. This is the first demonstration in a mammalian G1-specific ts mutant that the expression of H2A, H2B, H3, and H4 histone genes depends on the entry of cells into the S phase of the cell cycle.
Animals↗
Transforming potential of deletion mutants of the SV40 T antigen coding gene in Syrian hamster cells.
Thymidine kinase-deficient syrian hamster cells were cotransfected with recombinant plasmids containing the thymidine kinase (TK) gene of Herpes Simplex Virus Type 1, and either intact or partially deleted SV40 T antigen-coding genes. The transformants were selected by their ability to grow in gHAT medium. After selection and cloning, the TK-positive transformants that also expressed T antigen were tested for the extent of their transformation with respect to a number of characteristics, which included saturation density, ability to grow in soft agar, resistance to butyrate and to dibutyryl-cAMP, and plating efficiency. The combined results of these various tests indicate that cells containing partially deleted SV40 T antigen-coding genes are less transformed than cells containing an intact SV40 T antigen-coding gene. However, the amounts of T antigen are lower in cells transformed by deletion mutants than in cells transformed by wild-type T antigen-coding gene. Our data indicate that both the quantity and the quality of T antigen may be important in determining the degree of transformation in Syrian hamster cells.
Agar↗
Growth in size and cell DNA replication.
Explore the source record for details and available documents.
Animals↗
Microinjection of RNA polymerase II corrects the temperature-sensitive defect of tsAF8 cells.
tsAF8 cells are a temperature-sensitive (ts) mutant of BHK cells that arrest in the G1 phase of the cell cycle at the non-permissive temperature of 40.6 degrees C. Previous reports had suggested that the temperature-sensitivity of these cells was based on a defect in either the synthesis, assembly or turnover of RNA polymerase II. We now show that the direct microinjection of purified RNA polymerase II into nuclei of tsAF8 cells corrects the ts defect and allows these cells to enter the S phase of the cell cycle.
Animals↗
Cellular DNA replication is independent of the synthesis or accumulation of ribosomal RNA.
We have used an antibody against RNA polymerase I to investigate the role of rRNA synthesis and/or accumulation in the control of cell proliferation. The antibody was microinjected directly into the nuclei of quiescent Swiss 3T3 cells that were subsequently stimulated with serum. Under the experimental conditions used, the microinjection of the antibody against RNA polymerase I (RNA pol I) caused a 50-70% decrease in nucleolar RNA synthesis that lasted for at least 17 h, a greater than 90% inhibition in the accumulation of nucleolar RNA, and a 70% inhibition in the accumulation of total cellular RNA. A control IgG, similarly microinjected into Swiss 3T3 cells had no inhibitory effect on either the synthesis or accumulation of nucleolar and cellular RNA. Despite the dramatic effect on the synthesis and accumulation of ribosomal RNA (rRNA) the antibody against RNA (rRNA) the antibody against RNA pol I was totally ineffective in inhibiting the entry into S phase of serum-stimulated Swiss 3T3 cells. Cells depleted of cellular RNA by metaphase arrest also entered S phase with subnormal amounts of cellular RNA. The results of these experiments clearly indicate that a normal rate of nucleolar RNA synthesis, and a normal rate of accumulation of total cellular RNA are not a prerequisite for the entry of cells into S phase.
Animals↗
Cell-cycle-specific cDNAs from mammalian cells temperature sensitive for growth.
A library of double-stranded cDNA was constructed from ts13 cells, a G1-specific temperature-sensitive hamster cell line. The cDNAs, cloned into pBR322, were prepared from poly(A)+ mRNA isolated from ts13 cells 6 hr after serum stimulation at the permissive temperature of 34 degrees C. Differential screening of the library with G1-specific and G0-specific single-stranded cDNA probes prepared from the same cells identified five cDNA clones whose sequences were preferentially expressed in G1. Levels of RNA complementary to these clones were 3- to 6-fold higher in G1 than in other phases of the cell cycle. When ts13 cells were arrested in G1 at the restrictive temperature of 39.6 degrees C, the levels of RNA complementary to p13-2A9 and p13-4F1 were as high as 10 times that found in a resting population, while the expression of sequences complementary to p13-2A8 did not significantly change from levels found in G0. RNA and Southern gel blot analysis suggest that these cell-cycle-specific clones represent either low copy or moderately repetitive gene sequences. Results with another ts mutant of the cell cycle, tsAF8, which is a ts mutant of RNA polymerase II, showed that these cell-cycle-specific sequences have a rapid turnover. The use of G1-specific ts mutants of the cell cycle provides an approach to determine which cell-cycle-dependent genes are most relevant to cell cycle progression.
Animals↗
Microinjected pBR322 stimulates cellular DNA synthesis in Swiss 3T3 cells.
When pBR322 is manually microinjected into the nuclei of quiescent Swiss 3T3 cells it stimulates the incorporation of [3H]thymidine into DNA. The evidence clearly shows that this increased incorporation that is detected by in situ autoradiography in microinjected cells represents cellular DNA synthesis and not DNA repair or plasmid replication. The effect is due to pBR322 and not due to impurities, mechanical perturbances due to the microinjection technique, or aspecific effects. This stimulation is striking in Swiss 3T3 cells. Some NIH 3T3 cells show a slight stimulation, but hamster cells, derived from baby hamster kidney (BHK) cells, are not stimulated when microinjected with pBR322. The preliminary evidence seems to indicate that the integrity of the pBR322 genome is important for the stimulation of cellular DNA synthesis in quiescent Swiss 3T3 cells. These results, although of a preliminary nature, are of interest because they indicate that a prokaryotic genome may alter the cell cycle of mammalian cells. From a practical point of view the stimulatory effect of microinjected pBR322 on cellular DNA synthesis has a more immediate interest, because pBR322 is the vector most commonly used for molecular cloning and 3T3 cells are very frequently used for gene transfer experiments.
Animals↗
Role of the p53 protein in cell proliferation as studied by microinjection of monoclonal antibodies.
Two monoclonal antibodies against the p53 protein, PAb 122 and 200-47, were microinjected into mammalian cells as a probe to determine the role of the p53 protein in cell proliferation. PAb 122 recognizes the p53 proteins of mouse and human cells but not of hamster cells, whereas 200-47 recognizes the p53 proteins of mouse and hamster cells but not of human cells. The ability of these antibodies to inhibit serum-stimulated DNA synthesis of cells in culture correlates with their ability to recognize the species-specific antigenic determinants. More important, however, is the observation that microinjected PAb 122 inhibits the transition of Swiss 3T3 cells from G0 to S phase, but has no effect on the progression of these cells from mitosis to the S phase.
Acridine Orange↗
Molecular biology of cell division.
Explore the source record for details and available documents.
Animals↗
Effect of butyrate on adenovirus infection in semipermissive cells.
Adenovirus 2 stimulated cellular DNA synthesis in quiescent cultures of semipermissive tsAF8 cells and 3T3 cells. Such stimulation was inhibited by Na-butyrate, which also inhibited viral DNA replication in tsAF8 cells. In addition, butyrate inhibited the expression of early regions E1A and E2 of adenovirus 2 in both tsAF8 and 3T3 cells, while it had little effect on permissive HeLa cells.
Adenoviruses, Human↗
Inhibition of SV40-induced cellular DNA synthesis by microinjection of monoclonal antibodies.
The region of the SV40 large T-antigen molecule recognized by a panel of monoclonal antibodies has been determined using hybrid Adeno-SV40 viruses, and manual microinjection of cloned deletion mutants. In addition, an investigation was made of how monoclonal antibodies microinjected into the nucleus can affect the ability of the T-antigen coding gene to stimulate cell DNA synthesis. The monoclonal antibody Pab 14, that recognized the -COOH terminal half of large T, was comicroinjected into quiescent cells together with plasmid pCl-1. This plasmid contains only that part of the T-antigen coding gene that extends from nucleotide residue 120, counterclockwise to nucleotide residue 4002, and makes a truncated T antigen 33,000 in molecular weight and missing the last 435 amino acids on the -COOH terminal side. Monoclonal antibody Pab 14 did not inhibit the stimulation of cellular DNA synthesis caused by microinjection of pCl-1, although it did inhibit cell DNA synthesis induced by microinjection of pSV2G, a recombinant plasmid that contains the entire T-antigen coding gene of SV40.
Adenoviruses, Human↗
Mutational analysis of simian virus 40 T antigen: stimulation of cellular DNA synthesis and activation of rRNA genes by mutants with deletions in the T-antigen gene.
The biological activity of several deletion mutants of simian virus 40, cloned in pBR322, was determined. Three functions of the simian virus 40 A gene were studied: (i) the ability to express T antigen; (ii) the ability to induce cell DNA replication; and (iii) the ability to reactivate silent rRNA genes in hybrid cells. Recombinant plasmid DNA was introduced into cells by manual microinjection or by transfection. The results (together with previous reports) indicate that the critical sequences for these three functions are located separately on the simian virus 40 A gene, as follows: (i) the sequences necessary for the detection of the common antigenic determinant of T antigen extend from nucleotide 4147 to nucleotide 4001 (map units 0.45 to 0.42); (ii) the sequences critical for the stimulation of cell DNA synthesis extend from nucleotide 4327 to nucleotide 4001 (map units 0.49 to 0.42); and (iii) those critical for the reactivation of rRNA genes extend approximately from nucleotide 3827 to nucleotide 3526 (map units 0.39 to 0.33).
Animals↗
Molecular biology of cell division.
Different domains of the SV40 A gene have different functions, such as viral DNA replication, cell DNA replication, and stimulation of cellular RNA synthesis. The sequences in the SV40 A gene that are critical for the induction of cell DNA synthesis lie on the map between nucleotide 4360 and nucleotide 4001, a stretch of 360 nucleotides coding for 120 of the 708 amino acids of the large T antigen. The sequences critical for stimulation of rRNA synthesis lie on the map further downstream, between nucleotides 3827 and 3506, thus indicating that the signals for growth in size and for cell DNA replication can be dissociated. Methylation of the SV40 A gene at multiple ECoRI* sites has no effect on its expression. However, methylation of the HSV-TK gene at one single ECoRI site 70 base pairs upstream from the cap site inhibits its expression. The results indicate that methylation of genes affects their expression, but only when methylation occurs at specific sites.
Amanitins↗
Microinjection of cloned SV40 DNA fragments in the study of cell proliferation.
DNA recombinant technology and the manual microinjection technique were used to study the base sequences in the SV40 early gene coding for cell DNA replication and nucleolar activation. Sequences critical for rRNA gene activation are located between nucleotides 3826 to 3506 (0.39-0.33 m.u.). Base sequences from nucleotide 4350 to 4190 (0.49-0.46 m.u.) are required for cellular DNA replication. Major T antigen determinant is coded by a sequence extending from nucleotide 4190 to 3880 (0.46-0.42 m.u.). Considering that an increase in cell size is regularly accompanied by an increase in the cellular amount of rRNA, nucleolar genes should be a target for growth-in-size signals. Therefore, the SV40 early gene presents a domain responsible for cell growth in size, and another separate but proximate region coding for cellular DNA replication.
Animals↗
Microinjection of monoclonal antibody to protein p53 inhibits serum-induced DNA synthesis in 3T3 cells.
Monoclonal antibody directed against the transformation-related protein p53 was microinjected manually into the nuclei of quiescent Swiss 3T3 mouse cells. The cells were subsequently stimulated with 10% fetal calf serum. Microinjection of p53 antibody at or around the time of serum stimulation clearly inhibited the subsequent entry of Swiss 3T3 cells into the S phase of the cell cycle. p53 antibody had no effect on serum-stimulated DNA synthesis when it was microinjected 4 hr or later after serum stimulation. Monoclonal antibody to an unrelated antigen, Lyt-2.2, had no effect on serum-stimulated DNA synthesis regardless of the time it was microinjected. Under similar experimental conditions, p53 antibody had no effect on simian virus 40- or adenovirus 2-induced DNA synthesis. These experiments add strength to the suggestion that p53 is involved in the regulation of cell proliferation.
Adenoviruses, Human↗
Effect of methylation on expression of microinjected genes.
The cloned genes for the simian virus 40 large tumor antigen and for herpes simplex virus (HSV) thymidine kinase (TK) were methylated with EcoRI methylase. The genes were microinjected into the nuclei of TK-deficient (tk-) cells, and expression of the genes was determined by immunofluorescence staining for the simian virus 40 large tumor antigen and by [3H]thymidine incorporation followed by autoradiography for HSV TK. We found that methylation of the simian virus 40 gene, under EcoRI or EcoRI* conditions, resulting in methylation at sites within the gene and in the surrounding sequences, has no effect on expression of the large tumor antigen when the gene is manually microinjected into mammalian nuclei. However, methylation of the HSV tk gene at the two EcoRI sites markedly reduces or abolishes the expression of this gene. One of the EcoRI sites of HSV tk is approximately 1.1 kilobases downstream from the 3' end of the gene and is believed to have no regulatory function in the expression of the tk gene. The other EcoRI site is 79 base pairs upstream from the 5' end of the gene and has considerable homology to the regulatory sequence proposed by [Benoist C., O'Hare, K., Breathnach, R., & Chambon, P. (1980) Nucleic Acids Res. 8, 127-142]. Our results are direct proof that methylation can alter gene expression and also that the effect depends strictly on the sites that are methylated.
Antigens, Neoplasm↗
Gene transfer: DNA microinjection compared with DNA transfection with a very high efficiency.
We have developed a procedure that gives a very high efficiency of transfection in mammalian cells with low-molecular-weight DNA (approximately 10(4) base pairs). The procedure uses cells in suspension that are shocked with polyethylene glycol 4 h after replating. We compared this transfection technique to the standard technique involving manual microinjection of DNA into the nuclei of mammalian cells, using recombinant plasmids containing the simian virus 40 A gene or the herpes simplex virus thymidine kinase gene or both. The efficiency of transfection depends on a number of variables, the most important of which is the difference in transfectability of different cell lines. In our laboratory, the cell line that had the highest efficiency of transfection was tk-ts13, which is derived from baby hamster kidney cells that are deficient in thymidine kinase and temperature sensitive for growth. Under the appropriate conditions, as many as 70% of these cells can be transfected so that transient gene expression can be detected. With the manual microinjection technique, gene expression is independent of the cell line used and occurs faster than after transfection. The results suggest that the critical stage in transfection is the delivery of DNA molecules to the nucleus. Our experiments also indicate that an enzymatic function, in our case, thymidine kinase activity, gives a higher percentage of positive transfectants than when proteins are visualized only by indirect immunofluorescence. The transfection procedure described in this paper is simple and reproducible and, although less efficient than microinjection, ought to be useful in phenotypic and genotypic studies in which transfer of genes to a large number of cells is desirable.
Animals↗