Altered control of the rat cell cycle induced by adenovirus requires expression of transformation region ElA, but not of E1B, E2A, E2B, or late genes. We show here that neither E3 nor E4 is required, so the effect results directly from an ElA product. Mutants with defects in the 289-amino-acid (aa) ElA product had little or no effect on the rat cell cycle even at 1,000 IU The adenovirus transformation gene ElA has distant sequence homology with the retrovirus oncogenes v-myc and v-myb and can replace myc in transformation of primary cells (24,26). ElA induces expression of other viral early genes (2,12,27), and at least two cell genes, thymidine kinase and the 70,000-molecular-weight (70K) heat shock protein (1,3,8,11,21). The function of ElA in transformation appears to be "immortalization" of the cell and reduction of requirements for serum growth factors. The ability of adenovirus to induce a growth cycle in rodent cells arrested in the Gi phase by deprivation of serum or reduction of Ca2+ concentration, and to alter cycle progression in growing cells (1,5,8,18), may be related to the ElA transformation functions. Experiments with mutant viruses showed that alteration of control of the rat cell growth cycle requires expression of ElA, but not of E1B, E2A, E2B, or late genes (1,5,8,19). However, because ElA induces all of the other early regions, it remained possible that the cell cycle effects were actually due to E3 or E4, under the control of ElA. In this paper, we show that expression of human adenovirus type 5 (AdS) regions E3 and E4 is not necessary for alteration of rat cell cycle progression, which must therefore be a direct effect of ElA. Experiments with six mutants having different defects in the ElA 289-amino-acid (aa) product showed complete or almost complete defects in alteration of cell cycle progression even at 1,000 IU per cell. In this respect, these mutants lack the "multiplicity-dependent leakiness" reported for ElA mutants in human cells (20,27 ElA function in rat cells. The leakiness of some ElA mutants at high multiplicity in rat cells appears to be due to residual function of the mutant proteins. Additional leak in HeLa cells may be due to a cellular ElA-like function. MATERIALS AND METHODSViruses and cells. Ad2/5 pm975 (17) was a generous gift from A. J. Berk, University of California, Los Angeles; AdS hrA (28) was from D. Solnick, Yale University, New Haven, Conn.; and inSOO (6) was from N. C. Jones, Purdue University, West Lafayette, Ind. The origins of other AdS ElA mutants have been described previously (3). Locations of the ElA mutations are shown in relation to the early El mRNA species in Fig. 1. ElA mutants were grown in 293 cells and titrated by methods described previously (4,5,18,19), and with the exception of pm975 had a ratio of titer on 293 cells to that on HeLa cells of 103 to 104.Mutant d1808, deleted from between 91.4 and 92.0 to between 97.2 and 98.4 map units (m.u.) (7), and thus defective in subregions 1 or 2 through 7 of early region E4, was kindly supplied by G...

Rat embryo cells were infected with adenovirus type 5 mutants that code for only one of the two early ElA proteins, mutants with defects in one of the two conserved regions common to the two proteins, or mutants with defects in the 46-amino-acid region unique to the 289-amino-acid ElA protein. Cells were scored for altered cell cycle progression, disruption of actin stress fibers, and activation of E2A expression. Mutants lacking either ElA protein were able to cause all of these effects; but mutants lacking a 243-amino-acid protein had less effect, and mutants lacking a 289-amino-acid protein much less effect, than wild-type virus. A mutation in any of the * Corresponding author. growth of rat embryo fibroblasts have been described previously (7). Baby rat kidney (BRK) cells were prepared as described by Ruley (35). Detection of viral gene activation and actin stress fiber reorganization. Viral gene activation was detected by indirect immunofluorescence with an antiserum that reacts with the E2A gene product, and reorganization of stress fibers

1. We have examined methods necessary for preparing post-mitochondrial supernatants from Tetrahymena pyriformis strain HSM that are capable of efficient cell-free protein synthesis. 2. The requirements for optimum synthesis in these extracts are described. 3. Data relating to the kinetics of protein synthesis and the initiation capacity of these supernatants are presented.

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Altered control of the rat cell cycle induced by adenovirus requires expression of transformation region E1A, but not of E1B, E2A, E2B, or late genes. We show here that neither E3 nor E4 is required, so the effect results directly from an E1A product. Mutants with defects in the 289-amino-acid (aa) E1A product had little or no effect on the rat cell cycle even at 1,000 IU per cell. A mutant (pm975) lacking the 243-aa E1A product altered cell cycle progression, but less efficiently than did wild-type virus. The 289-aa E1A protein is therefore essential for cell cycle effects; the 243-aa protein is also necessary for the full effect but cannot act alone. Mutants with altered 289-aa E1A proteins showed different extents of leak expression of viral early region E2A as the multiplicity was increased; each leaked more in human than in rat cells. dl312, with no E1A products, failed to produce E2A mRNA or protein at 1,000 IU per cell in rat cells but did so in some experiments in human cells. There appears to be a very strict dependence of viral early gene expression on E1A in rat cells, whereas dependence on E1A is more relaxed in HeLa cells, perhaps due to a cellular E1A-like function. Altered cell cycle control is more dependent on E1A function than is early viral gene expression.