Identification of CDK4 sequences involved in cyclin D1 and p16 binding.

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 p16(INK4A) 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.

Structure and developmental regulation of the murine ctk gene.

Csk-type protein tyrosine kinase (Ctk) is a protein tyrosine kinase with structural and functional similarities to C-terminal Src kinase (Csk). These enzymes catalyze the phosphorylation of C-terminal regulatory tyrosine residues of Src family enzymes. Here we describe the structure of the ctk gene and characterize the pattern of ctk expression. Ctk and a closely related enzyme, nervous tissue and T-lymphocyte kinase (Ntk), are assembled by selective use of two of the exons. Expression of ctk was found to be restricted primarily to the brain, ctk and csk exhibit a different expression profile in the developing mouse brain. The expression of the ctk mRNA and mature polypeptide increase postnatally, while the expression of csk decreases with age. Our results thus show that despite their structural and functional similarities, ctk and csk differ markedly in their patterns of expression.

Electrooptic polymer materials and devices for global optical interconnects.

Global optical interconnects can provide high data rate parallel communication capability through access to the internal nodes of very large scale integrated circuits. Topographic arrays of polymeric electrooptic multilayer devices such as etalons or multilayer mirrors broadcast the data stored on the surface of the chip. Differential detection of this image permits interconnection without the need for high contrast ratios. An experimental demonstration of a point-to-point interconnection using a Fabry-Perot etalon with a polymeric thin film spacer is presented.

Infectious circular DNA of human adenovirus type 5: regeneration of viral DNA termini from molecules lacking terminal sequences.

A series of plasmids containing the entire human adenovirus genome with viral DNA termini joined 'head to tail' has been isolated. Several plasmids were able to generate infectious virus following transfection of human cells in spite of having small deletions and rearrangements at the junctions of termini. One plasmid has lost 2 bp of DNA from one end of the viral genome and 11 bp from the other end yet produced viruses with complete wild-type sequences at both ends of the genome. We propose a model for replication of viral DNA off circular templates in which regeneration of terminal information involves translocation of primer and polymerase during initiation of DNA replication. The model suggests a novel mechanism for extension of the 5' ends of linear DNA molecules which could be applicable to chromosomal telomeres.

Liquid crystal adaptive lens: beam translation and field meshing.

The liquid crystal adaptive lens (LCAL) is an electrooptic device that emulates a variable focal length lens. The LCAL focuses light by creating a parabolic refractive-index profile across the aperture of a liquid crystal cell. The focal length is electronically controlled by applying appropriate voltages to the array of independent electrodes, thus grading the refractive index of the liquid crystal material across the aperture. Beam translation perpendicular to the optical beam path is described theoretically and demonstrated. This capability coupled with the LCAL's programmable focal length allows 3-D beam control. Meshing, the smoothing of the refractive index between adjacent electrodes, is a critical parameter in achieving near diffraction-limited optical performance. Using two planar electrodes and a ground plane immersed in an isotropic dielectric as a model, a steady-state dc theoretical computer simulation is compared with experiment. Improvements in the liquid crystal cell design demonstrate improved performance over previous LCALs. A larger number of electrodes creates an image without spatial aliasing within the aperture.

Human adenovirus cloning vectors based on infectious bacterial plasmids.

By making use of the fact that human adenovirus DNA circularizes in infected cells, and that circular forms of the viral genome are infectious, we have developed an improved adenovirus-based cloning system. A deletion mutant of adenovirus type 5 (Ad5) with deletions in early regions 1 (E1) and 3 (E3) was converted to a bacterial plasmid which can regenerate infectious virus following transfection into human 293 cells. A single XbaI recognition site in the deleted E3 region serves as a site for the insertion of foreign DNA. We have used this system to clone a number of genes into the Ad5 genome and describe the insertion of the neomycin/G418 resistance marker into Ad5 as an example.