The Pto gene in tomato confers resistance to races of Pseudomonas syringae pv. tomato that carry the avirulence gene avrPto. A yeast artificial chromosome clone that spans the Pto region was identified and used to probe a leaf complementary DNA (cDNA) library. A cDNA clone was isolated that represents a gene family, at least six members of which genetically cosegregate with Pto. When susceptible tomato plants were transformed with a cDNA from this family, they were resistant to the pathogen. Analysis of the amino acid sequence revealed similarity to serine-threonine protein kinases, suggesting a role for Pto in a signal transduction pathway.

The nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) has two zinc fingers, each containing the invariant metal ion binding residues CCHC. Recent reports indicate that mutations in the CCHC motifs are deleterious for reverse transcription in vivo. To identify reverse transcriptase (RT) reactions affected by such changes, we have probed zinc finger functions in NC-dependent RT-catalyzed HIV-1 minusand plus-strand transfer model systems. Our approach was to examine the activities of wild-type NC and a mutant in which all six cysteine residues were replaced by serine (SSHS NC); this mutation severely disrupts zinc coordination. We find that the zinc fingers contribute to the role of NC in complete tRNA primer removal from minus-strand DNA during plus-strand transfer. Annealing of the primer binding site sequences in plus-strand strong-stop DNA [(؉) SSDNA] to its complement in minus-strand acceptor DNA is not dependent on NC zinc fingers. In contrast, the rate of annealing of the complementary R regions in (؊) SSDNA and 3 viral RNA during minus-strand transfer is approximately eightfold lower when SSHS NC is used in place of wild-type NC. Moreover, unlike wild-type NC, SSHS NC has only a small stimulatory effect on minus-strand transfer and is essentially unable to block TAR-induced self-priming from (؊) SSDNA. Our results strongly suggest that NC zinc finger structures are needed to unfold highly structured RNA and DNA strand transfer intermediates. Thus, it appears that in these cases, zinc finger interactions are important components of NC nucleic acid chaperone activity.Reverse transcription, a critical event in the retrovirus life cycle, consists of a complex series of reactions that culminate in synthesis of a linear, double-stranded DNA copy of the viral RNA genome (27; reviewed in references 4 and 14). This process is catalyzed by the virus-encoded reverse transcriptase (RT) enzyme. However, it is known that in addition to RT, host and other viral factors play important roles in viral DNA synthesis.One of these accessory factors is the viral nucleocapsid protein (NC), a small basic, single-stranded nucleic acid binding protein, which is tightly associated with genomic RNA in the interior of the mature virus particle (for reviews, see references 14, 16, and 59). Studies on the solution structure of free human immunodeficiency virus type 1 (HIV-1) NC indicated that this protein consists of a flexible polypeptide chain and two rigid zinc-binding domains connected by a short basic peptide linker (55-57, 67, 69, 70). Recently, De Guzman et al. (19) solved the three-dimensional nuclear magnetic resonance structure of NC bound to the SL3 RNA stem-loop in the HIV-1 packaging signal. They showed that the N-terminal basic residues of NC in the complex form a helix that binds to the major groove of the RNA stem largely by nonspecific electrostatic interactions, whereas the zinc fingers are involved in specific interactions with the G residues in the GGAG tetraloop (19).The zinc fingers are in cl...

We designed and characterized a two-dimensional, gradient-index phononic crystal ͑GRIN PC͒ to control the propagation of acoustic waves. The GRIN PC was composed of solid cylinders arranged in a square lattice and immersed in an epoxy. The refractive index along the direction transverse to the phononic propagation was designated as a hyperbolic secant gradient distribution. This distribution was modulated by means of the density and elastic moduli of the cylinders. The effective refractive indices in each row of the GRIN PC were determined from band diagrams obtained via a plane-wave expansion method. The acoustic wave propagation was numerically investigated by a finite-difference time-domain method, and the results were compared to the analytical beam trajectories derived from the hyperbolic secant profile. These results show that the GRIN PC allows acoustic focusing over a wide range of working frequencies, making it suitable for applications such as flat acoustic lenses and couplers.

In this paper, we numerically and experimentally demonstrate the existence of complete band gaps and resonances in a plate with a periodic stubbed surface. Numerical results show that a complete band gap forms as the stub height reaches about three times the plate thickness. In the experiment, we used a pulsed laser to generate broadband elastic waves and used optical devices as well as point piezoelectric transducers to detect wave signals. The results show that the numerical predictions are in very good agreement with those measured experimentally. Remarkable resonances on the top surface of the stubs are found and discussed.

Successful application of photonic crystals has led recently to a rapidly growing interest in the analogous acoustic effects in periodic elastic structures called phononic crystals. This study is aimed at developing a theory for two-dimensional phononic crystal consisting of materials with general anisotropy. Explicit formulations of the plane harmonic bulk wave and the surface wave dispersion relations in such a general phononic structure are derived based on the plane wave expansion method. Two-dimensional phononic structures with either the square or the hexagonal lattice are considered in the numerical examples. Band gap characteristics of the phononic structures with different anisotropic background materials ͑isotropic, cubic, hexagonal, and ortho-rhombic͒ are calculated and discussed.

The nucleocapsid protein (NC) of human immunodeficiency virus type 1 has two zinc fingers, each containing the invariant CCHC zinc-binding motif; however, the surrounding amino acid context is not identical in the two fingers. Recently, we demonstrated that zinc coordination is required when NC unfolds complex secondary structures in RNA and DNA minus-and plus-strand transfer intermediates; this property of NC reflects its nucleic acid chaperone activity. Here we have analyzed the chaperone activities of mutants having substitutions of alternative zinc-coordinating residues, i.e., CCHH or CCCC, for the wild-type CCHC motif. We also investigated the activities of mutants that retain the CCHC motifs but have mutations that exchange or duplicate the zinc fingers (mutants 1-1, 2-1, and 2-2); these changes affect amino acid context. Our results indicate that in general, for optimal activity in an assay that measures stimulation of minus-strand transfer and inhibition of nonspecific self-priming, the CCHC motif in the zinc fingers cannot be replaced by CCHH or CCCC and the amino acid context of the fingers must be conserved. Context changes also reduce the ability of NC to facilitate primer removal in plus-strand transfer. In addition, we found that the first finger is a more crucial determinant of nucleic acid chaperone activity than the second finger. Interestingly, comparison of the in vitro results with earlier in vivo replication data raises the possibility that NC may adopt multiple conformations that are responsible for different NC functions during virus replication.The nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) is a small, basic, nucleic acid-binding protein which associates with genomic RNA in the mature virion core (14,15,54); the mature protein is generated by proteolytic cleavage of the Gag precursor (36,47,63). Structural studies have revealed that free HIV-1 NC in solution has two rigid zinc-binding domains or zinc fingers, each containing the invariant CCHC metal ion-binding motif (30,37,59,61). The two fingers are covalently linked to each other by a short flexible basic amino acid region and are flanked by flexible Nor C-terminal "tails" (49-51, 59, 60, 62). The Summers group has recently solved the three-dimensional structures of HIV-1 NC bound to the SL2 (3, 4) and SL3 (18) RNA stem-loops that form part of the larger HIV-1 packaging signal, by nuclear magnetic resonance (NMR) analysis.The two NC zinc fingers are located in close proximity (45, 46, 49, 50) but exhibit only weak interactions with one another (13,43,46,49,66). Interestingly, their structures are similar (58), despite differences in the amino acid sequences surrounding the CCHC motifs (37, 54). Moreover, the biochemical properties (8, 45) and biological activities of the two fingers are not equivalent, and the presence of both fingers is critical for production of replication-competent virus (9,21,26,28,29,48,72); in addition, the positions of the zinc fingers cannot be exchanged (21,26).NC function in vir...

Human APOBEC3B (A3B) is a member of the APOBEC3 (A3) family of cytidine deaminases, which function as DNA mutators and restrict viral pathogens and endogenous retrotransposons. Recently, A3B was identified as a major source of genetic heterogeneity in several human cancers. Here, we determined the solution NMR structure of the catalytically active C-terminal domain (CTD) of A3B and performed detailed analyses of its deaminase activity. The core of the structure comprises a central five-stranded β-sheet with six surrounding helices, common to all A3 proteins. The structural fold is most similar to that of A3A and A3G-CTD, with the most prominent difference found in loop 1. The catalytic activity of A3B-CTD is ~15-fold less than that of A3A, although both exhibit similar pH dependence. Interestingly, A3B-CTD with an A3A loop 1 substitution had significantly increased deaminase activity, while a single residue change (H29R) in A3A loop 1 reduced A3A activity to the level seen with A3B-CTD. This establishes that loop 1 plays an important role in A3-catalyzed deamination by precisely positioning the deamination-targeted C into the active site. Overall, our data provide important insights into the determinants for the activities of individual A3 proteins and facilitate understanding of their biological function.

In this paper, we investigate the frequency band-gap features of micromachined air/silicon phononic band structures using layered slanted finger interdigital transducers (SFIT). In order to achieve the applications of phononic crystals on the microelectromechanical system related components, the frequency band-gap widths of surface waves are studied both theoretically and experimentally in micrometer scale phononic crystals. For further integration with the complementary metal-oxide semiconductor processing techniques, silicon is chosen as the base material of the two-dimensional phononic crystals in this study. To cover the frequency band-gap width of the phononic crystal, the wideband SFIT- and the SFIT∕ZnO∕Si-layered structures in the measurement are analyzed and discussed. For layered structures, the dispersive relation is calculated by the effective permittivity approach, and the frequency response of the layered SFIT is then simulated by the coupling-of-modes model. The frequency band-gap width and the frequency range of two-dimensional air/silicon phononic crystals in micrometer scale are measured, and the result agrees well with the theoretical evaluation.