A new silver stain for electrophoretically separated polypeptides can be rapidly and easily used and can detect as little as 0.01 nanogram of protein per square millimeter. When employed with two-dimensional electrophoresis, it should permit qualitative and quantitative characterization of protein distributions in body fluids and tissues. It has been used to demonstrate regional variations in cerebrospinal fluid proteins.

The wild-type E6 and E7 genes of human papillomavirus type 16 (HPV16) can cooperate to immortalize normal human keratinocytes in culture. The E6 open reading frame of HPV16 and other HPV types highly associated with cervical cancer has the potential of encoding both full-length E6 and two truncated E6* proteins, the latter being generated via splicing within the E6 open reading frame portion of the E6-E7 polycistronic transcript. Those types, such as HPV6, that are infrequently associated with cervical carcinoma lack the splice site and encode only a full-length E6. We have now found that, in addition to cooperating with E7 to immortalize keratinocytes, HPV16 E6 can induce anchorage-independent growth in NIH 3T3 cells and trans-activate the adenovirus E2 promoter. HPV6 E6 was also able to trans-activate the adenovirus E2 promoter, although it was inactive in both cell transformation assays. An HPV16 splice site mutant which expressed only the full-length HPV16 E6 was active in all three assays, indicating that the E6* proteins are not required for these activities. The plasmid which encodes the E6* proteins was inactive and did not potentiate the activity of the HPV16 splice site mutant. The mutation that prevented splicing in E6-E7 mRNA severely reduced the level of E7 protein and increased E6 protein. Taken together, the results suggest that the primary function of the splice within E6 is to facilitate the translation of E7 and reduce translation of full-length E6, rather than to generate biologically active E6* proteins.

Activation of CDK4 is regulated, in part, by its association with a D-type cyclin. Conversely, CDK4 activity is inhibited when it is bound to the cyclin-dependent kinase inhibitor, p16 INK4A. To investigate the molecular basis of the interactions between CDK4 and cyclin D1 or p16INK4A we performed site-directed mutagenesis of CDK4. The interaction was examined using in vitro translated wild type and mutant CDK4 proteins and bacterially expressed cyclin D1 and p16 fusion proteins. As mutational analysis of CDC2 suggests that its cyclin binding domain is primarily located near its amino terminus, the majority of the mutations constructed in CDK4 were located near its amino terminus. In addition, CDK4 residues homologous to CDC2 sites involved in Suc1 binding were also mutated. Our analysis indicates that cyclin D1 and p16 binding sites are overlapping and located primarily near the amino terminus. All CDK4 mutations that resulted in decreased p16 binding capability also diminished cyclin D1 binding. In contrast, amino-terminal sequences were identified, including the PSTAIRE region, that are important for cyclin D1 binding but are not involved in p16 binding.The eukaryotic cell cycle is controlled by a series of regulatory "checkpoints," two of which occur at the G 1 /S and G 2 /M phase transitions. The cyclin-dependent kinases (CDKs) 1 are key regulators of these checkpoints (1-4). The CDKs are serine/threonine protein kinases that are composed of a catalytic CDK subunit and a regulatory cyclin subunit. Compelling evidence suggests that D-type cyclins and their major catalytic partners, CDK4 and CDK6, are key regulators of the G 1 /S phase checkpoint. Cyclin D-CDK holoenzymes accumulate when resting cells are stimulated to proliferate by addition of growth factors. Their associated kinase activities are first detected in mid G 1 phase and increase as cells approach the G 1 /S boundary (5, 6). Removal of growth factors early in the G 1 phase results in rapid decay of cyclin D-CDK activity and correlates with failure of the cells to enter the S phase. Removal of growth factors in late G 1 also results in decay of cyclin D-CDK activity; however, it does not inhibit progression through the cell cycle (7). Furthermore, microinjection of cyclin D1 antibodies into cells during G 1 prevents entry into S phase whereas injections at or after S phase have no effect (8, 9). Thus, cyclin D-CDK complexes are essential for the G 1 to S phase transition.Activation of individual CDKs is dictated in part by the temporal expression of their cognate cyclins and by stagespecific phosphorylation and dephosphorylation events (10). An additional layer of CDK regulation has emerged with the discovery of the CDK inhibitor proteins (CKIs) p15 (11, 12), p16 (13), p18 (14), p19 (14), p21 (15), p27 (16), and p57 (17). Binding of these inhibitory proteins to specific CDKs or specific CDKcyclin complexes inhibits kinase activity and thus blocks cell cycle progression. The human p16INK4A/MTS1/CDKN2 (hereafter referred to as p16) gene encodes a...

The E6 proteins of the high-risk human papillomaviruses (HPVs) have been shown to form a complex with and induce the degradation of human p53 in vitro. To determine whether p53 is degraded more rapidly in cells expressing E6 in vivo, the half-life of p53 was determined by pulse-chase analysis in early-passage normal human keratinocytes and fibroblasts, human keratinocytes immortalized with HPV type 16 (HPV16) E6 plus E7, and nonimmortal keratinocytes transfected with E6. The results of these experiments indicate that (i) the half-life of newly synthesized p53 is relatively long (4 h) in early-passage human keratinocytes and fibroblasts but short in keratinocytes expressing E6 (15 to 30 min), (ii) a similar increased rate of p53 degradation was measured in lines immortalized with HPV16 E6 plus E7 and senescent cells expressing E6, indicating that this increase is not simply the result of selection in the immortalized lines, and (iii) very low levels of expression of E6 result in a greatly decreased half-life of p53, suggesting that E6 acts in a catalytic manner.

The major late 16S mRNA species of simian virus 40 encodes both a 61-amino-acid protein, LP1, and the major virion protein, VP1. Although the initiation signal GCCAUGG is usually utilized at high efficiency, at least one-third of 40S ribosomal subunits bypass it when it is present on the major 16S mRNA of simian virus