We have been interested in understanding more about the sequences that constitute the polyomavirus late promoter. Our approach has been to target specific deletions to the viral intergenic region by oligonucleotidedirected mutagenesis. Wild-type and mutant promoter cassettes with defined deletions were then inserted into a promoterless expression vector containing the bacterial chloramphenicol acetyltransferase (CAT) gene (cat). Plasmids were introduced into mouse NIH 3T3 cells by transfection, and promoter activities were assessed by quantitation of both CAT enzyme and cat mRNA levels. In this report, we present the results of experiments designed to map promoter elements which affect late transcription in the absence of early viral proteins and viral DNA replication. Using this approach, we mapped two major cis-acting elements (a positive and a negative one) which affect transcription in our transient expression system. The first, positive, element coincided with the enhancer A element, which is known to be important for early transcription and viral DNA replication. Removal of this element reduced late transcription by 50to 100-fold. The second element was a negative one; removal of 89 base pairs that included two high-affinity large-T-antigen-binding sites just to the early side of the inverted repeat structure within the replication origin resulted in a 5to 10-fold increase in late promoter activity. The implications of these findings for late promoter function and regulation are discussed.

Background: Most PDE5-selective inhibitors also potently inhibit photoreceptor PDE6. Results: Evolutionary trace analysis predicted amino acids responsible for the selectivity of tadalafil binding to the PDE6 catalytic site without altering vardenafil binding. Conclusion: A limited number of amino acid residues account for drug selectivity of PDE inhibitors. Significance: This work will help identify more selective PDE5 inhibitors lacking adverse side effects on vision.

Following the sexual phase of its life cycle, the hypotrichous ciliate Oxytricha nova transforms a copy of its chromosomal micronucleus into a macronucleus containing short, linear DNA molecules with an average size of 2.2 kilobase pairs. In addition, more than 90% of the DNA sequences in the micronuclear genome are eliminated during this process. We have examined the organization of macronuclear DNA molecules in the micronuclear chromosomes. Macronuclear DNA molecules were found to be clustered and separated by less than 550 base pairs in two cloned segments of micronuclear DNA. Recombinant clones of two macronuclear DNA molecules that are adjacent in the micronucleus were also isolated and examined by DNA sequencing. The two macronuclear DNA molecules were found to be separated by only 90 base pairs in the micronuclear genome.Hypotrichous ciliated protozoa, such as Oxytricha nova, undergo a drastic genome reorganization process as part of their normal life cycle (25,26). The ability of these unicellular organisms to alter their DNA stems from the fact that each cell contains two types of nuclei. The micronucleus contains an unrearranged genome composed of chromosome-sized DNA molecules, but is transcriptionally inactive. The second type of nucleus in the cell, the macronucleus, is responsible for nuclear transcription during vegetative growth of the organism, despite having an unusual genetic constitution. The macronuclear genome consists entirely of multiple copies of short, linear, gene-sized DNA molecules with an average size of 2.2 kilobase pairs (kbp) (33). Since most macronuclear DNA molecules are transcribed (23; J. Heumann, Ph.D. thesis, University of Colorado, Boulder, 1977), and current evidence is consistent with each molecule's specifying a single product (11), they are often referred to as macronuclear genes.Following each sexual phase of the life cycle, the macronucleus is destroyed and a new one develops from a mitotic copy of the micronucleus. This process of macronuclear development involves a complex series of events that dramatically alter the micronuclear genome (1,25). At the cytological level, the micronuclear chromosomes are first replicated a number of times to form polytene chromosomes. Vesicles then form within the developing macronucleus in association with the fragmentation of the polytene chromosomes. Most of the DNA within each vesicle is subsequently destroyed. Finally, the vesicle disappear and the remaining low-molecular-weight DNA molecules undergo multiple rounds of replication to form the mature macronucleus. Comparative studies on the macronuclear and micronuclear genomes indicate that macronuclear development does not simply involve fragmenting the chromosomes to generate the gene-sized DNA molecules, but also entails the elimination of more than 90% of the sequence complexity of the micronuclear genome (17). In addition, studies on the chromosomal organization of particular macronuclear genes indicate that two additional types of rearrangement events occur during developm...

Photoreceptor phosphodiesterase 6 (PDE6) is the central effector of the visual excitation pathway in both rod and cone photoreceptors, and PDE6 mutations that alter PDE6 structure or regulation can result in several human retinal diseases. The rod PDE6 holoenzyme consists of two catalytic subunits (P␣␤) whose activity is suppressed in the dark by binding of two inhibitory ␥-subunits (P␥). Upon photoactivation of rhodopsin, the heterotrimeric G protein (transducin) is activated, resulting in binding of the activated transducin ␣-subunit (Gt ␣ ) to PDE6, displacement of P␥ from the PDE6 active site, and enzyme activation. Although the biochemistry of this pathway is understood, a lack of detailed structural information about the PDE6 activation mechanism hampers efforts to develop therapeutic interventions for managing PDE6-associated retinal diseases. To address this gap, here we used a cross-linking MS-based approach to create a model of the entire interaction surface of P␥ with the regulatory and catalytic domains of P␣␤ in its nonactivated state. Following reconstitution of PDE6 and activated Gt ␣ with liposomes and identification of cross-links between Gt ␣ and PDE6 subunits, we determined that the PDE6 -Gt ␣ protein complex consists of two Gt ␣ -binding sites per holoenzyme. Each Gt ␣ interacts with the catalytic domains of both catalytic subunits and induces major changes in the interaction sites of the P␥ subunit with the catalytic subunits. These results provide the first structural model for the activated state of the transducin-PDE6 complex during visual excitation, enhancing our understanding of the molecular etiology of inherited retinal diseases.

Photoreceptor cGMP phosphodiesterase (PDE6) is the central enzyme in the visual transduction cascade. The PDE6 catalytic subunit contains a catalytic domain and regulatory GAF domains. Unlike most GAF domain-containing cyclic nucleotide phosphodiesterases, little is known about direct allosteric communication of PDE6. In this study, we demonstrate for the first time direct, inter-domain allosteric communication between the GAF and catalytic domains in PDE6. The binding affinity of PDE6 for pharmacological inhibitors or for the C-terminal region of the inhibitory ␥ subunit (P␥), known to directly inhibit PDE6 catalysis, was increased ϳ2-fold by ligands binding to the GAF domain. Binding of the N-terminal half of P␥ to the GAF domains suffices to induce this allosteric effect. Allosteric communication between GAF and catalytic domains is reciprocal, in that drug binding to the catalytic domain slowed cGMP dissociation from the GAF domain. Although cGMP hydrolysis was not affected by binding of P␥1-60, P␥ lacking its last seven amino acids decreased the Michaelis constant of PDE6 by 2.5-fold. P␥1-60 binding to the GAF domain increased vardenafil but not cGMP affinity, indicating that substrate-and inhibitor-binding sites do not totally overlap. In addition, prolonged incubation of PDE6 with vardenafil or sildenafil (but not 3-isobutyl-1-methylxanthine and zaprinast) induced a distinct conformational change in the catalytic domain without affecting the binding properties of the GAF domains. We conclude that although P␥-mediated regulation plays the dominant role in visual excitation, the direct, inter-domain allosteric regulation described in this study may play a feedback role in light adaptational processes during phototransduction. The photoreceptor cyclic nucleotide phosphodiesterase (PDE6)3 is the central enzyme in the vertebrate visual signaling pathway in rods and cones. Phototransduction is initiated when light induces the isomerization of the 11-cis-retinal chromophore of rhodopsin, which leads to activation of the photoreceptor-specific G-protein, transducin. Activated transducin then causes activation of PDE6, which results in rapid lowering of cGMP levels, closure of cGMP-gated ion channels, and hyperpolarization of the cell membrane (1-3). Hydrolysis of cGMP by PDE6 must be precisely regulated to control the amplitude and kinetics of the photoresponse. Furthermore, each of these parameters undergoes additional modulation in response to ever-changing conditions of ambient illumination. The PDE6 holoenzyme consists of a catalytic dimer of ␣ and ␤ subunits (P␣␤) and two inhibitory ␥ subunits (P␥) that are tightly bound to P␣␤. Transducin activation of PDE6 results from displacement of the inhibitory constraint of P␥ upon activated transducin binding to PDE6. The affinity of P␥ for the P␣␤ catalytic dimer is also modulated in a reciprocal manner by noncatalytic cGMP binding to PDE6 at sites distinct from the catalytic site (Ref. 4 and reviewed in Ref. 5).Photoreceptor PDE6 is one of five members of the cla...