Cyclin A is involved in the control of S phase and mitosis in mammalian cells. Expression of the cyclin A gene in nontransformed cells is characterized by repression of its promoter during the G1 phase of the cell cycle and its induction at S-phase entry. We show that this mode of regulation is mediated by the transcription factor E2F, which binds to a specific site in the cyclin A promoter. It differs from the prototype E2F site in nucleotide sequence and protein binding; it is bound by E2F complexes containing cyclin E and p107 but not pRB. Ectopic expression of cyclin Dl triggers premature activation of the cyclin A promoter by E2F, and this effect is blocked by the tumor suppressor protein p16INK4.Progression through the mammalian cell cycle is controlled by cyclins and cyclin-dependent kinases (cdk) (1). Cyclin gene expression is tightly regulated in a phase-specific manner. Expression of cyclin Dl precedes that of cyclin E in the G1 phase of the cell cycle (2); both proteins are required and are rate-limiting for passage through G, (3)(4)(5)(6)(7). Cyclin A is first expressed at the GI/S transition; it is required for S and M phases (8-10). Cyclin A may be a component of the DNA replication machinery (11,12) and may have a role in transcriptional control during S phase (13,14). Constitutive expression of cyclin A has been associated with tumorigenesis (15, 16); inversely, abolishment of cyclin A gene expression was recently found to cause the growth arrest of adhesiondependent cells grown in suspension (17). Overexpression of cyclin Dl (18) as well as of its partner kinase cdk4 (19) is linked to tumorigenesis, and the gene MTS1, coding for p16INK4, a cellular kinase inhibitor for cdk4, is found inactivated in a large variety of human tumor cell types (20,21). We report here a regulatory link between the expression of cyclins Dl and A. Phase-specific transcription of the human cyclin A gene (22) is mediated by a binding site for the transcription factor E2F (23). Cyclin Dl can activate cyclin A transcription through this element, and this signal is antagonized by p16INK4. MATERIALS AND METHODSReporter Plasmids and Expression Vectors. cDNAs encoding human cyclin A (15), cyclin Bi (24), cyclin Dl (3), cyclin E (25), cdk4 (26), cdc2 (27), and p16'NK4 (28) were subcloned by standard techniques in the cytomegalovirus (CMV)-based expression vector pX (10). Cyclin A promoter/reporter genes were constructed as described (22). Point mutation of the E2F site was performed by PCR and verified by sequence analysis after cloning. The inducible expression vector CMV/T was constructed by inserting the simian virus 40 polyadenylylation sequence upstream of the tetracycline-controlled promoter of plasmid pUHD10-3 (29). Insertion of cDNA coding for firefly luciferase (30), cyclin Dl, and cyclin A into CMV/T yielded plasmids luc/T, cycDl/T, and cycA/T, respectively.Cell Culture and Transfection. NIH 3T3 cells and human diploid fibroblasts from foreskin were cultured and starvation synchronized as described (22). Trans...

We have previously isolated and sequenced Nicotiana cytoplasmic tRNA(Tyr) with G psi A anticodon which promotes readthrough over the leaky UAG termination codon at the end of the 126 K cistron of tobacco mosaic virus RNA and we have demonstrated that tRNA(Tyr) with Q psi A anticodon is no UAG suppressor. Here we show that the nucleotide in the middle of the anticodon (i.e., psi 35) also contributes to the suppressor efficiency displayed by cytoplasmic tRNA(Tyr). A tRNA(Tyr) with GUA anticodon was synthesized in vitro using T7 RNA polymerase transcription. This tRNA(Tyr) was unable to suppress the UAG codon, indicating that nucleotide modifications in the anticodon of tRNA(Tyr) have either stimulating (i.e., psi 35) or inhibitory (i.e., Q34) effects on suppressor activity. Furthermore, we have shown that the UAA but not the UGA stop codon is also efficiently recognized by tobacco tRNA(G psi ATyr), if placed in the TMV context. Hence this is the first naturally occurring tRNA for which UAA suppressor activity has been demonstrated. In order to study the influence of neighbouring nucleotides on the readthrough capacity of tRNA(Tyr), we have established a system, in which part of the sequence around the leaky UAG codon of TMV RNA was inserted into a zein pseudogene which naturally harbours an UAG codon in the middle of the gene. The construct was cloned into the vector pSP65 and in vitro transcripts, generated by SP6 RNA polymerase, were translated in a wheat germ extract depleted of endogenous mRNAs and tRNAs. A number of mutations in the codons flanking the UAG were introduced by site-directed mutagenesis. It was found that changes at specific positions of the two downstream codons completely abolished the readthrough over the UAG by Nicotiana tRNA(Tyr), indicating that this tRNA needs a very specific codon context for its suppressor activity.

To investigate E7-dependent biochemical changes which are involved in cellular transformation, we analyzed the influence of human papillomavirus type 16 (HPV-16) E7 on the expression of cell cycle regulatory proteins. Expression of E7 in established rodent fibroblasts (NIH 3T3), which was shown to be sufficient for transformation of these cells, leads to constitutive expression of the cyclin E and cyclin A genes in the absence of external growth factors. Surprisingly, expression of the cyclin D1 gene, which encodes a major regulator of G 1 progression, is unaltered in E7-transformed cells. In transient transfection experiments, the cyclin A gene promoter is activated by E7 via an E2F binding site. In 14/2 cells, which were used as a model system to analyze the role of HPV-16 E7 in the transformation of primary cells, we observed rapid E7-dependent activation of cyclin E gene expression, which can be uncoupled from activation of the cyclin A gene, since the latter requires additional protein synthesis. E7-driven induction of cyclin E and cyclin A gene expression was accompanied by an increase in the associated kinase activities. Two domains of the E7 oncoprotein, which are designated cd1 and cd2, are essential for transformation of rodent fibroblasts. It is shown here that growth factor-independent expression of the cyclin E gene requires cd2 but not cd1, while activation of cyclin A gene expression requires cd1 function in addition to that of cd2. These data suggest that cyclin A gene expression is controlled by two distinct negative signals, one of which also restricts expression of the cyclin E gene. The ability of E7 to separately override each of these inhibitory signals, via cd1 and cd2, cosegregates with its ability to fully transform rodent fibroblasts. Unlike serum growth factors, E7 induces S-phase entry without activating cyclin D1 gene expression, in keeping with the finding that cyclin D1 function is not required in cells transformed by DNA tumor viruses.