A high-throughput Arabidopsis reverse genetics system

A collection of Arabidopsis lines with T-DNA insertions in known sites was generated to increase the efficiency of functional genomics. A high-throughput modified thermal asymmetric interlaced (TAIL)-PCR protocol was developed and used to amplify DNA fragments flanking the T-DNA left borders from approximately 100000 transformed lines. A total of 85108 TAIL-PCR products from 52964 T-DNA lines were sequenced and compared with the Arabidopsis genome to determine the positions of T-DNAs in each line. Predicted T-DNA insertion sites, when mapped, showed a bias against predicted coding sequences. Predicted insertion mutations in genes of interest can be identified using Arabidopsis Gene Index name searches or by BLAST (Basic Local Alignment Search Tool) search. Insertions can be confirmed by simple PCR assays on individual lines. Predicted insertions were confirmed in 257 of 340 lines tested (76%). This resource has been named SAIL (Syngenta Arabidopsis Insertion Library) and is available to the scientific community at www.tmri.org.     

Mutants of the CuA site in cytochrome c oxidase of Rhodobacter sphaeroides: II. Rapid kinetic analysis of electron transfer

The function of the binuclear Cu(A) center in cytochrome c oxidase (CcO) was studied using two Rhodobacter sphaeroides CcO mutants involving direct ligands of the Cu(A) center, H260N and M263L. The rapid electron-transfer kinetics of the mutants were studied by flash photolysis of a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine-55. The rate constant for intracomplex electron transfer from heme c to Cu(A) was decreased from 40000 s(-1) for wild-type CcO to 16000 s(-1) and 11000 s(-1) for the M263L and H260N mutants, respectively. The rate constant for electron transfer from Cu(A) to heme a was decreased from 90000 s(-1) for wild-type CcO to 4000 s(-1) for the M263L mutant and only 45 s(-1) for the H260N mutant. The rate constant for the reverse reaction, heme a to Cu(A), was calculated to be 66000 s(-1) for M263L and 180 s(-1) for H260N, compared to 17000 s(-1) for wild-type CcO. It was estimated that the redox potential of Cu(A) was increased by 120 mV for the M263L mutant and 90 mV for the H260N mutant, relative to the potential of heme a. Neither mutation significantly affected the binding interaction with cytochrome c. These results indicate that His-260, but not Met-263, plays a significant role in electron transfer between Cu(A) and heme a.     

A rapid and noninvasive method for detecting tissue-limited mosaicism: detection of i(12)(p10) in buccal smear from a child with Pallister-Killian syndrome

Pallister-Killian syndrome (PKS), a rare disorder, is characterized by tissue-limited or tissue-specific mosaicism. The characteristic chromosome abnormality associated with PKS is i(12p), which is seen predominantly in skin fibroblast cultures. Diagnosis of i(12p) has been carried out on buccal smears before and was shown to be an easy and feasible method. All previously published studies used alpha-satellite probes for the diagnosis and as such have several pitfalls. Our approach, using dual-color, locus-specific probes, has high specificity and sensitivity for the diagnosis of i(12p). Using statistical analysis, we have also confirmed that the signal pattern in interphase nuclei is consistent with isochromosome 12p.     

T-DNA recombination and replication in maize cells

T-DNA recombination and replication was analyzed in 'black mexican sweet' (BMS) cells transformed with T-DNAs containing the replication system from wheat dwarf virus (WDV). Upon recombination between the T-DNA ends, a promoterless marker gene (gusA) was activated. Activation of the recombination marker gene was delayed and increased exponentially over time, suggesting that recombination and amplification of the T-DNA occurred in maize cells. Mutant versions of the viral initiator gene (rep), known to be defective in the replication function, failed to generate recoverable recombinant T-DNA molecules. Circularization of T-DNA by the FLP/FRT site-specific recombination system and/or homologous recombination was not necessary to recover circular T-DNAs. However, replicating T-DNAs appeared to be suitable substrates for site-specific and homologous recombination. Among 33 T-DNA border junctions sequenced, only one pair of identical junction sites was found implying that the population of circular T-DNAs was highly heterogenous. Since no circular T-DNA molecules were detected in treatments without rep, it suggested that T-DNA recombination was linked to replication and might have been stimulated by this process. The border junctions observed in recombinant T-DNA molecules were indicative of illegitimate recombination and were similar to left-border recombination of T-DNA into the genome after Agro-mediated plant transformation. However, recombination between T-DNA molecules differed from T-DNA/genomic DNA junction sites in that few intact right borders were observed. The replicating T-DNA molecules did not enhance genomic random integration of T-DNA in the experimental configuration used for this study.     

The C. elegans apoptotic nuclease NUC-1 is related in sequence and activity to mammalian DNase II

The Caenorhabditis elegans nuc-1 gene has previously been implicated in programmed cell death due to the presence of persistent undegraded apoptotic DNA in nuc-1 mutant animals. In this report, we describe the cloning and characterization of nuc-1, which encodes an acidic nuclease with significant sequence similarity to mammalian DNase II. Database searches performed with human DNase II protein sequence revealed a significant similarity with the predicted C. elegans C07B5.5 ORF. Subsequent analysis of crude C. elegans protein extracts revealed that wild-type animals contained a potent endonuclease activity with a cleavage preference similar to DNase II, while nuc-1 mutant worms demonstrated a marked reduction in this nuclease activity. Sequence analysis of C07B5.5 DNA and mRNA also revealed that nuc-1(e1392), but not wild-type animals contained a nonsense mutation within the CO7B5.5 coding region. Furthermore, nuc-1 transgenic lines carrying the wild-type C07B5.5 locus demonstrated a complete complementation of the nuc-1 mutant phenotype. Our results therefore provide compelling evidence that the C07B5.5 gene encodes the NUC-1 apoptotic nuclease and that this nuclease is related in sequence and activity to DNase II.     

Understanding Disputes: The Politics of Argument

Understanding Disputes: The Politics of Argument. Pat Caplan. ed. Oxford, England: Berg Publishers, 1995. 248 pp.     

The Potential of Thiarubrine C as a Nematicidal Agent against Plant- parasitic Nematodes

Thiarubrine C, a polyacetylenic 1,2-dithiin isolated from the roots of Rudbeckia hirta (Asteraceae), exhibited strong nematicidal activity in in vitro and growth chamber assays. Thiarubrine C was toxic, in the absence of light, to the plant-parasitic nematodes Meloidogyne incognita and Pratylenchus penetrans at LC₅₀s of 12.4 ppm and 23.5 ppm, respectively. A minimum exposure time between 12 and 24 hours was the critical period for nematode mortality due to thiarubrine C. Although thiarubrine C was not totally dependent on light for toxicity, activity was enhanced in the presence of light, especially with the microbivorous nematode, Teratorhabditis dentifera. Upon exposure of M. incognita juveniles to 20 ppm thiarubrine C for 1 hour, infection of tomato plants was greatly reduced compared to untreated checks. Thiarubrine C was also effective in reducing plant infection when mixed with soil 24 hours prior to or at planting, unlike other related compounds such as δ-terthienyl.     

Synthesis and characterization of the asymmetrically coordinated dinuclear complex [{L(Ph2acac)FeIII}(μ-O){FeIII(Cl4-cat)L}](ClO4) and its one-electron reduced and oxidized forms. Models for dinuclear non-heme active sites

The asymmetrically coordinated complex [{L(Ph₂acac)Fe^III}(μ-O){Fe^III(Cl₄-cat)L}](BPh₄)·1.5toluene has been synthesized and structurally characterized (Ph₂acac^–=1,3-diphenylpropane-1,3-dionate, Cl₄-cat^2–=tetrachlorocatecholate, L=1,4,7-trimethyl-1,4,7-triazacyclononane). This species can be electrochemically oxidized and reduced by one electron, respectively, yielding two species which both have an S=1/2 ground state. It is shown that the oxidation is ligand-centered, affording a coordinated semiquinonate(1–) ligand with S=1/2 which is antiferromagnetically coupled to a high-spin Fe^III ion (S=5/2) yielding an S=2 state which, in turn, is antiferromagnetically coupled (through the oxo bridge) to the second high-spin Fe^III ion (S=5/2) yielding the observed S=1/2 ground state. In contrast, the reduction is metal-centered generating a mixed-valent species with an [Fe^III-O-Fe^II]³⁺ core; intramolecular antiferromagnetic coupling again produces an S=1/2 ground state. The symmetrical complex [{LFe^III(Ph₂acac)}₂(μ-O)](ClO₄)₂ has also been synthesized, as have the mononuclear species [LFe^II(Ph₂acac)Cl] and [LFe^III(aacac)Cl](ClO₄)·1 mesitylene [aacac^–=3-(9-anthryl)acetylacetonate(1–)], all of which have been characterized by X-ray crystallography. The magnetism, the Mössbauer-, EPR-, and UV-VIS-spectra and the electrochemistry of complexes are reported.     

Isoproterenol Acts as a Biased Agonist of the Alpha-1A-Adrenoceptor that Selectively Activates the MAPK/ERK Pathway

The α_1A-AR is thought to couple predominantly to the Gα_q/PLC pathway and lead to phosphoinositide hydrolysis and calcium mobilization, although certain agonists acting at this receptor have been reported to trigger activation of arachidonic acid formation and MAPK pathways. For several G protein-coupled receptors (GPCRs) agonists can manifest a bias for activation of particular effector signaling output, i.e. not all agonists of a given GPCR generate responses through utilization of the same signaling cascade(s). Previous work with Gα_q coupling-defective variants of α_1A-AR, as well as a combination of Ca²⁺ channel blockers, uncovered cross-talk between α_1A-AR and β₂-AR that leads to potentiation of a Gα_q-independent signaling cascade in response to α_1A-AR activation. We hypothesized that molecules exist that act as biased agonists to selectively activate this pathway. In this report, isoproterenol (Iso), typically viewed as β-AR-selective agonist, was examined with respect to activation of α_1A-AR. α_1A-AR selective antagonists were used to specifically block Iso evoked signaling in different cellular backgrounds and confirm its action at α_1A-AR. Iso induced signaling at α_1A-AR was further interrogated by probing steps along the Gα_q /PLC, Gα_s and MAPK/ERK pathways. In HEK-293/EBNA cells transiently transduced with α_1A-AR, and CHO_α_1A-AR stable cells, Iso evoked low potency ERK activity as well as Ca²⁺ mobilization that could be blocked by α_1A-AR selective antagonists. The kinetics of Iso induced Ca²⁺ transients differed from typical Gα_q- mediated Ca²⁺ mobilization, lacking both the fast IP₃R mediated response and the sustained phase of Ca²⁺ re-entry. Moreover, no inositol phosphate (IP) accumulation could be detected in either cell line after stimulation with Iso, but activation was accompanied by receptor internalization. Data are presented that indicate that Iso represents a novel type of α_1A-AR partial agonist with signaling bias toward MAPK/ERK signaling cascade that is likely independent of coupling to Gα_q.