We have constructed a nuclear genomic library from the cruciferous plant Arabidopsis thaliana ecotype Columbia in a cosmid vector, pLZO3, and a host organism, Agrobacterium tumefaciens AGL1, which can directly DNA-transform the parent organism, Arabidopsis. The broad host range cosmid pLZO3 carries a gentamicin acetyltransferase gene as bacterial selective marker and tandem, chimeric neomycin and streptomycin phosphotransferase genes as plant selective markers. Agrobacterium AGL1 carries the hypervirulent, attenuated tumor-inducing plasmid pTiBo542 from which T-region DNA sequences have been precisely deleted, allowing optimal DNA transformation of many dicotyledonous plants. Agrobacterium AGL1 also carries an insertion mutation in its recA general recombination gene, which stabilizes the recombinant plasmids. The Arabidopsis genomic library consists of some 21,600 clones gridded onto 96-well microtiter dishes and, if random, carries at least three genomic equivalents. When probed for the presence of several Arabidopsis low copy-number genes, the genomic library seems representative. As with the unicellular organisms Escherichia coli and Saccharomyces cerevisiae, this DNA transformation competent genomic library should expedite gene isolation, by gene rescue, in multicellular organisms like Arabidopsis.

In higher plants, photorespiratory Gly oxidation in leaf mitochondria yields ammonium in large amounts. Mitochondrial ammonium must somehow be recovered as glutamate in chloroplasts. As the first step in that recovery, we report glutamine synthetase (GS) activity in highly purified Arabidopsis thaliana mitochondria isolated from light-adapted leaf tissue. Leaf mitochondrial GS activity is further induced in response to either physiological CO2 limitation or transient darkness. Historically, whether mitochondria are fully competent for oxidative phosphorylation in actively photorespiring leaves has remained uncertain. Here, we report that light-adapted, intact, leaf mitochondria supplied with Gly as sole energy source are fully competent for oxidative phosphorylation. Purified intact mitochondria efficiently use Gly oxidation (as sole energy, NH3, and CO2 source) to drive conversion of l-Orn to l-citrulline, an ATP-dependent process. An A. thaliana genome-wide search for nuclear gene(s) encoding mitochondrial GS activity yielded a single candidate, GLN2. Stably transgenic A. thaliana ecotype Columbia plants expressing a p35S∷GLN2∷green fluorescent protein (GFP) chimeric reporter were constructed. When observed by laser scanning confocal microscopy, leaf mesophyll and epidermal tissue of transgenic plants showed punctate GFP fluorescence that colocalized with mitochondria. In immunoblot experiments, a 41-kD chimeric GLN2∷GFP protein was present in both leaf mitochondria and chloroplasts of these stably transgenic plants. Therefore, the GLN2 gene product, heretofore labeled plastidic GS-2, functions in both leaf mitochondria and chloroplasts to faciliate ammonium recovery during photorespiration

On the basis of the physiotherapists series, the reliability was acceptable for a number of clinical tests that are used in the evaluation of patients with low back pain. The results suggest that clinical tests should be standardized to a much higher degree than they are today.

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Sixty-five independent, N2 fixation-defective (Nif-) vector insertion (Vi) mutants were selected, cloned, and mapped to the ORS571 genome. The recombinant Nif::Vi plasmids obtained in this way were used as DNA hybridization probes to isolate homologous phages from a genomic library of ORS571 constructed in XEMBL3. Genomic maps were drawn for three ORS571 Nif gene loci. Forty- Rhizobium sp. strain ORS571, uniquely among characterized members of the family Rhizobiaceae, conducts N2 fixation during active bacterial growth (16) as well as during plant nodule symbiosis, in which it is a nonproliferating endosymbiont. ORS571 forms classical, synchronous nodules in roots and in stem lateral root primordia with the host leguminous plant Sesbania rostrata (32). As such, ORS571 appears to be a chimeric N2-fixing bacterium with properties of both orthodox rhizobia and diazotrophic bacteria. Operationally, the chimeric nature of ORS571 N2 fixation facilitates a genetic analysis of rhizobial N2 fixation processes because ORS571 N2 fixation-defective (Nif) mutants may be directly selected and screened ex planta.We have recently developed the vector-insertion (Vi) mutagenesis-cloning strategy and have applied it to ORS571 (9). This method allows the direct molecular cloning of any ORS571 genes in which mutants can be selected or screened. Vi mutagenesis-cloning consists of the following protocol. (Table 1). ORS571 recipients carrying ISSOR-mediated plasmid-genome cointegrates were obtained after platings on rich media containing kanamycin and streptomycin as described previously (9). From this collection, candidate strains were screened by replica plating on ORSNif medium, a defined, twofold-diluted M9 minimal medium containing 0.4% D-glucose, 0.4% succinic acid titrated to pH 6.0 with KOH, 1 mM MgSO4, 0.5 mM CaCl2, 1 ,ug of FeCl3 per ml, 1 ,ug of NaMoO4 per ml, 1 ,ug of pantothenate per ml, 1 ,ug of nicotinate per ml, and 0.3 jig of D-biotin per ml, with and without 15 mM (NH4)2SO4 added as a nitrogen source. Plates were solidified with 0.85% agarose (Clonetech) and incubated in a growth chamber at 30°C under a continous flow atmosphere of 98% N2-1% C0O-1% 02-ORS571 Nif::Vi mutants were identified by their inability to utilize N2 as a nitrogen source on solid media (9). Putative 72 on May 11, 2018 by guest Tn5 is first introduced into