When added to a culture medium of resting confluent chick embryo fibroblasts in the absence of serum, thrombin (EC 3.4.21.5) is able to stimulate DNA synthesis 12 hr later and to cause a substantial increase in cell number over a period of 4 days. As compared to thrombin, prothrombin exhibits low mitogenic activity. However, in the presence of purified Factor Xa (EC 3.4.21.6) and Factor V, prothrombin is converted to thrombin by "thromboplastin activity" supplied by the fibroblasts.Prothrombin, either purified or as a constituent of plasma or serum, may thus be considered to be a reservoir of mitogenic activity in tissue culture unless antithrombin is present in the culture medium in amounts sufficient to neutralize the thrombin formed. By use of a specific inhibitor of proteases, and by separation of prothrombin by absorption on BaSO4, we estimate that the potential mitogenic activity of prothrombin is approximately 30-50% of the total activity that can be obtained by treatment of fibrinogen-free plasma with thromboplastin. In addition to its mitogenic activity, thrombin can also stimulate the migration of cells. These experiments with thrombin illustrate that well-characterized proteases of blood can act as potent mitogens and suggest that they may play a role in the process of wound healing.Many lines of evidence suggest that the control of cell growth either in vivo or in culture is mediated by agents in the extracellular fluid. Even though there are several agents capable of stimulating growth of a variety of cell types, such as serum (1-3), plasma (4), hormones (5-7), antigens (9), plant lectins (10, 11), and proteases (12, 13), very little progress has been made in any instance on the elucidation of the mechanism of cell growth, in particular the initial interaction of mitogens with cell surface components.In some ways the proteases offer a unique opportunity for the study of changes on the cell surface during mitogenic stimulation since (1) 50% ethanol, 6.7 Ci/mmol, New England Nuclear Co.) were added, and the incubation was continued for 1 hr. The acid-insoluble fraction was collected and the radioactive DNA was measured.When cell growth was measured by counting the number of cells as a function of time, cell cultures were prepared as described above except that the number of cells seeded was reduced to 3 to 4 X 105 per 35-mm culture plate. After the medium was replaced with Dulbecco modified Eagle's medium containing the materials to be tested, the cultures were incubated at 370 for 4 days and the cell count measured each day by a Coulter counter.Treatment of Fibrinogen-Free Plasma with Phenyl Methyl Sulfonyl Fluoride (PhMeSO2F). Fibrinogen-free plasma was prepared by dialysis of oxalated plasma (16) against water and by further heat treatment for 30 min at 560. One milliliter of a standard solution of brain thromboplastin was mixed with 9 ml of a solution containing 10% fibrinogen-free bovine plasma in Dulbecco modified Eagle's medium. After incubation at 370 for 2 hr, the precipitate was re...

We have prepared cytoskeletons from normal and Rous sarcoma virus-transformed cells by extraction with nonionic detergents in a buffered salt solution designed to preserve the structure as it exists in vivo. Virtually all of the phosphoprotein pp6Osrc in the cell is bound to such cytoskeletons. Furthermore, when these cytoskeletons are incubated in situ with pp6Osrc is phosphorylated. Labeling of other apparently transformation-specific cytoskeletal phosphoproteins is also observed. These results directly demonstrate an association between pp6Osrc and elements ofthe cytoskeleton and suggest that pp60src may exert at least some of its effects as a consequence of its interaction with this cellular framework. Rous sarcoma virus (RSV) efficiently infects and transforms fibroblasts in culture, and it causes sarcomas when injected into chickens. The transformation of cultured fibroblasts results in a number of changes affecting cell morphology, motility, metabolism, and growth regulation. All of these alterations from normal cell behavior appear to be due to the expression in infected cells of a single viral genetic element, the src gene (1). This gene codes for a phosphoprotein of molecular weight (Mr) 60,000 (pp608rc), which can be immunoprecipitated from RSV-infected cells with serum taken from rabbits bearing RSV-induced tumors (TBR serum) (2). A growing body of evidence indicates that pp6Osrc is a protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) (3-7) with the unusual property of phosphorylating tyrosine, rather than serine or threonine, residues (8). Although some cellular polypeptides have been identified that may be substrates for pp60src kinase activity (8,9), their function in the cell remains unknown. Immunofluorescent (10) and electron microscopic (11) studies have indicated a cytoplasmic localization of pp6Osrc, with some concentration at the inner surface of the plasma membrane, particularly at gap junctions between cells, and under ruffles. Evidence has also been presented (12) for the existence of a "signal sequence" (13, 14) in pp60src translated in vitro from virion RNA. Although this observation cannot predict the distribution of pp6Osrc between the particulate and soluble fractions of the cell in vivo, it does indicate that the mRNA for pp6O"rc may code for a protein with the capacity to interact with membranes. In fact, substantial amounts of pp6osrc are found to be membrane-associated when cells are ruptured by homogenization or sonication (15). Nevertheless, these fractionation procedures result in the release of roughly half of pp6osrc in soluble form (6). Thus, it has appeared there might be two forms of pp6Osrc in the cell: one soluble form free in the cytoplasm and a second form associated with the inner surface of the plasma membrane, perhaps as an integral membrane protein. We report here a finding that provides an alternative explanation for this behavior.It has become apparent in recent years that the cytoplasm of mammalian cells is organized by a cytoskeletal fram...

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The "amber" (am) mutants of bacteriophage T4 are a recently discovered class of mutants that can replicate in certain derivatives of Escherichia coli strain K-12 but not in E. coli B. ' The bacterial strains that support growth of the am mutants, thus making their isolation and propagation possible, are called the "permissive" hosts. The biochemical basis of this permissiveness has not yet been clarified. With some of the am mutants, infection of E. coli B leads to a subnormal production of phage DNA; with others, DNA is formed but infection is abortive because of failure of late stages of phage development.2It is known that infection of E. coli with a T-even bacteriophage leads to the appearance of several new enzyme activities and to an increase in several other enzyme activities, all related to synthesis of phage DNA ("early enzymes").312 These Downloaded by guest on

MATERIALS AND METHODSNormal human serum was obtained from healthy young adult males as previously described (1).Hog kidney conjugase (9, 10) was prepared as described to us by Wood and Hitchings (11) with several minor modifications. Fresh hog kidneys (285 g) were defatted and minced in 200 ml of distilled water in a Waring Blendor for 45 seconds. This suspension was incubated under toluene at 37'C for 4 hours and then at room temperature overnight. Thirty ml of distilled water was added, and the coarse aggregates of fat were