Site-specific incorporation of phosphotyrosine using an expanded genetic code.

Access to phosphoproteins with stoichiometric and site-specific phosphorylation status is key to understanding the role of protein phosphorylation. Here we report an efficient method to generate pure, active phosphotyrosine-containing proteins by genetically encoding a stable phosphotyrosine analog that is convertible to native phosphotyrosine. We demonstrate its general compatibility with proteins of various sizes, phosphotyrosine sites and functions, and reveal a possible role of tyrosine phosphorylation in negative regulation of ubiquitination

A Genome-Wide Analysis of FRT-Like Sequences in the Human Genome

Efficient and precise genome manipulations can be achieved by the Flp/FRT system of site-specific DNA recombination. Applications of this system are limited, however, to cases when target sites for Flp recombinase, FRT sites, are pre-introduced into a genome locale of interest. To expand use of the Flp/FRT system in genome engineering, variants of Flp recombinase can be evolved to recognize pre-existing genomic sequences that resemble FRT and thus can serve as recombination sites. To understand the distribution and sequence properties of genomic FRT-like sites, we performed a genome-wide analysis of FRT-like sites in the human genome using the experimentally-derived parameters. Out of 642,151 identified FRT-like sequences, 581,157 sequences were unique and 12,452 sequences had at least one exact duplicate. Duplicated FRT-like sequences are located mostly within LINE1, but also within LTRs of endogenous retroviruses, Alu repeats and other repetitive DNA sequences. The unique FRT-like sequences were classified based on the number of matches to FRT within the first four proximal bases pairs of the Flp binding elements of FRT and the nature of mismatched base pairs in the same region. The data obtained will be useful for the emerging field of genome engineering

Zinc Finger Recombinases with Adaptable DNA Sequence Specificity

Site-specific recombinases have become essential tools in genetics and molecular biology for the precise excision or integration of DNA sequences. However, their utility is currently limited to circumstances where the sites recognized by the recombinase enzyme have been introduced into the DNA being manipulated, or natural ‘pseudosites’ are already present. Many new applications would become feasible if recombinase activity could be targeted to chosen sequences in natural genomic DNA. Here we demonstrate efficient site-specific recombination at several sequences taken from a 1.9 kilobasepair locus of biotechnological interest (in the bovine β-casein gene), mediated by zinc finger recombinases (ZFRs), chimaeric enzymes with linked zinc finger (DNA recognition) and recombinase (catalytic) domains. In the "Z-sites" tested here, 22 bp casein gene sequences are flanked by 9 bp motifs recognized by zinc finger domains. Asymmetric Z-sites were recombined by the concomitant action of two ZFRs with different zinc finger DNA-binding specificities, and could be recombined with a heterologous site in the presence of a third recombinase. Our results show that engineered ZFRs may be designed to promote site-specific recombination at many natural DNA sequences

Genetic Code Mutations: The Breaking of a Three Billion Year Invariance

The genetic code has been unchanging for some three billion years in its canonical ensemble of encoded amino acids, as indicated by the universal adoption of this ensemble by all known organisms. Code mutations beginning with the encoding of 4-fluoro-Trp by Bacillus subtilis, initially replacing and eventually displacing Trp from the ensemble, first revealed the intrinsic mutability of the code. This has since been confirmed by a spectrum of other experimental code alterations in both prokaryotes and eukaryotes. To shed light on the experimental conversion of a rigidly invariant code to a mutating code, the present study examined code mutations determining the propagation of Bacillus subtilis on Trp and 4-, 5- and 6-fluoro-tryptophans. The results obtained with the mutants with respect to cross-inhibitions between the different indole amino acids, and the growth effects of individual nutrient withdrawals rendering essential their biosynthetic pathways, suggested that oligogenic barriers comprising sensitive proteins which malfunction with amino acid analogues provide effective mechanisms for preserving the invariance of the code through immemorial time, and mutations of these barriers open up the code to continuous change

Time-integrated luminosity recorded by the BABAR detector at the PEP-II e+e- collider

This article is the Preprint version of the final published artcile which can be accessed at the link below.We describe a measurement of the time-integrated luminosity of the data collected by the BABAR experiment at the PEP-II asymmetric-energy e+e- collider at the ϒ(4S), ϒ(3S), and ϒ(2S) resonances and in a continuum region below each resonance. We measure the time-integrated luminosity by counting e+e-→e+e- and (for the ϒ(4S) only) e+e-→μ+μ- candidate events, allowing additional photons in the final state. We use data-corrected simulation to determine the cross-sections and reconstruction efficiencies for these processes, as well as the major backgrounds. Due to the large cross-sections of e+e-→e+e- and e+e-→μ+μ-, the statistical uncertainties of the measurement are substantially smaller than the systematic uncertainties. The dominant systematic uncertainties are due to observed differences between data and simulation, as well as uncertainties on the cross-sections. For data collected on the ϒ(3S) and ϒ(2S) resonances, an additional uncertainty arises due to ϒ→e+e-X background. For data collected off the ϒ resonances, we estimate an additional uncertainty due to time dependent efficiency variations, which can affect the short off-resonance runs. The relative uncertainties on the luminosities of the on-resonance (off-resonance) samples are 0.43% (0.43%) for the ϒ(4S), 0.58% (0.72%) for the ϒ(3S), and 0.68% (0.88%) for the ϒ(2S).This work is supported by the US Department of Energy and National Science Foundation, the Natural Sciences and Engineering Research Council (Canada), the Commissariat à l’Energie Atomique and Institut National de Physique Nucléaire et de Physiquedes Particules (France), the Bundesministerium für Bildung und Forschung and Deutsche Forschungsgemeinschaft (Germany), the Istituto Nazionale di Fisica Nucleare (Italy), the Foundation for Fundamental Research on Matter (The Netherlands), the Research Council of Norway, the Ministry of Education and Science of the Russian Federation, Ministerio de Ciencia e Innovación (Spain), and the Science and Technology Facilities Council (United Kingdom). Individuals have received support from the Marie-Curie IEF program (European Union) and the A.P. Sloan Foundation (USA)

Improved Limits on B0B^{0} decays to invisible (+γ)(+\gamma) final states

We establish improved upper limits on branching fractions for B0 decays to final States 10 where the decay products are purely invisible (i.e., no observable final state particles) and for final states where the only visible product is a photon. Within the Standard Model, these decays have branching fractions that are below the current experimental sensitivity, but various models of physics beyond the Standard Model predict significant contributions for these channels. Using 471 million BB pairs collected at the Y(4S) resonance by the BABAR experiment at the PEP-II e+e- storage ring at the SLAC National Accelerator Laboratory, we establish upper limits at the 90% confidence level of 2.4x10^-5 for the branching fraction of B0-->Invisible and 1.7x10^-5 for the branching fraction of B0-->Invisible+gammaComment: 8 pages, 3 postscript figures, submitted to Phys. Rev. D (Rapid Communications

Measurement of B(B-->X_s {\gamma}), the B-->X_s {\gamma} photon energy spectrum, and the direct CP asymmetry in B-->X_{s+d} {\gamma} decays

The photon spectrum in B --> X_s {\gamma} decay, where X_s is any strange hadronic state, is studied using a data sample of (382.8\pm 4.2) \times 10^6 e^+ e^- --> \Upsilon(4S) --> BBbar events collected by the BABAR experiment at the PEP-II collider. The spectrum is used to measure the branching fraction B(B --> X_s \gamma) = (3.21 \pm 0.15 \pm 0.29 \pm 0.08)\times 10^{-4} and the first, second, and third moments = 2.267 \pm 0.019 \pm 0.032 \pm 0.003 GeV,, )^2> = 0.0484 \pm 0.0053 \pm 0.0077 \pm 0.0005 GeV^2, and )^3> = -0.0048 \pm 0.0011 \pm 0.0011 \pm 0.0004 GeV^3, for the range E_\gamma > 1.8 GeV, where E_{\gamma} is the photon energy in the B-meson rest frame. Results are also presented for narrower E_{\gamma} ranges. In addition, the direct CP asymmetry A_{CP}(B --> X_{s+d} \gamma) is measured to be 0.057 \pm 0.063. The spectrum itself is also unfolded to the B-meson rest frame; that is the frame in which theoretical predictions for its shape are made.Comment: 37 pages, 19 postscript figures, submitted to Phys. Rev. D. No analysis or results have changed from previous version. Some changes to improve clarity based on interactions with Phys. Rev. D referees, including one new Figure (Fig. 13), and some minor wording/punctuation/spelling mistakes fixe

Precise Measurement of the e+ e- --> pi+ pi- (gamma) Cross Section with the Initial-State Radiation Method at BABAR

A precise measurement of the cross section of the process $e^+e^-\to\pi^+\pi^-(\gamma)$ from threshold to an energy of 3GeV is obtained with the initial-state radiation (ISR) method using 232fb$^{-1}$ of data collected with the BaBar detector at $e^+e^-$ center-of-mass energies near 10.6GeV. The ISR luminosity is determined from a study of the leptonic process $e^+e^-\to\mu^+\mu^-(\gamma)\gamma_{\rm ISR}$, which is found to agree with the next-to-leading-order QED prediction to within 1.1%. The cross section for the process $e^+e^-\to\pi^+\pi^-(\gamma)$ is obtained with a systematic uncertainty of 0.5% in the dominant $\rho$ resonance region. The leading-order hadronic contribution to the muon magnetic anomaly calculated using the measured $\pi\pi$ cross section from threshold to 1.8GeV is $(514.1 \pm 2.2({\rm stat}) \pm 3.1({\rm syst}))\times 10^{-10}$.Comment: 58 pages, 56 figures, to be submitted to Phys. Rev.

Search for lepton-number violating processes in B+ -> h- l+ l+ decays

We have searched for the lepton-number violating processes B+ -> h- l+ l+ with h- = K-/pi- and l+ = e+/mu+, using a sample of 471+/-3 million BBbar events collected with the BaBar detector at the PEP-II e+e- collider at the SLAC National Accelerator Laboratory. We find no evidence for these decays and place 90% confidence level upper limits on their branching fractions Br(B+ -> pi- e+ e+) K- e+ e+) pi- mu+ mu+) K- mu+ mu+) < 6.7 x 10^{-8}.Comment: 8 pages, 4 postscript figures, submitted to Phys. Rev. D. R