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Article Title: Molecular Cloning and Characterization of ATP-Phosphoribosyl Transferase from Arabidopsis, a Key Enzyme in the Histidine Biosynthetic Pathway

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Figure Lengend Snippet: Alignment of ATP-PRT protein sequences. The primary structures of Arabidopsis AtATP-PRT1 (At ATP-PRT1) and AtATP-PRT2 (At ATP-PRT2) proteins deduced from the corresponding cDNA sequences are aligned with other ATP-PRT proteins. T. goesingense ATP-PRT, T.g ATP-PRT (GenBank accession no. AF003347; X.H. Yan, U. Krämer, I. Raskin, R.D. Smith, and D.E. Salt, unpublished data); S. cerevisiae HIS1, S.c HIS1 (GenBank accession no. J01329; Hinnebusch and Fink, 1983); C. albicans HIS1, C.a HIS1 (GenBank accession no. X83871; Pla et al., 1995); E. coli hisG, E.c hisG (GenBank accession no. V00284; Carlomagno et al., 1988); M. jannaschii hisG, M.j hisG (GenBank accession no. U67562; Bult et al., 1996); and Synechocystis sp. PCC6803 hisG, S.y hisG (GenBank accession no. D64006; Kaneko et al., 1996). Dashes indicate gaps inserted to allow optimal sequence alignment. Conserved amino acid residues were shaded. Single-letter codes for amino acid residues are used, and asterisks indicate termination codons for translation. Intron positions determined from the gene structures for AtATP-PRT1 and AtATP-PRT2 are indicated by arrows above the sequence.

Article Snippet: Isolation of Arabidopsis ATP-PRT1 Gene Approximately 5 × 10 5 recombinant phages of an Arabidopsis Lambda ZAP II genomic library (Stratagene) were screened using the AtATP-PRT1 cDNA as a probe.

Techniques: Sequencing

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Article Title: Molecular Cloning and Characterization of ATP-Phosphoribosyl Transferase from Arabidopsis, a Key Enzyme in the Histidine Biosynthetic Pathway

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Figure Lengend Snippet: Genomic DNA-blot hybridization analysis. Arabidopsis genomic DNA (10 μg) was digested with EcoRI, PstI, or SalI, and subjected to hybridization analysis using the full-lengths of the AtATP-PRT1 and AtATP-PRT2 cDNAs as probes under low- or high-stringency conditions. The restriction enzymes used are indicated at the top of the figure: E, EcoRI; P, PstI; and S, SalI. Molecular size markers are shown on the right.

Article Snippet: Isolation of Arabidopsis ATP-PRT1 Gene Approximately 5 × 10 5 recombinant phages of an Arabidopsis Lambda ZAP II genomic library (Stratagene) were screened using the AtATP-PRT1 cDNA as a probe.

Techniques: Hybridization

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Article Title: Molecular Cloning and Characterization of ATP-Phosphoribosyl Transferase from Arabidopsis, a Key Enzyme in the Histidine Biosynthetic Pathway

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Figure Lengend Snippet: Steady-state levels of Arabidopsis AtATP-PRT gene transcripts. Total RNA (10 μg) was extracted from 1-week-old germinating seeds (lane 1), roots and leaves from 2-week-old seedlings (lane 2), roots and leaves of 3-week-old seedlings (lane 3), and leaves (lane 4) and inflorescence stems (lane 5) from 4-week-old plants. For hybridization, probes specific for either AtATP-PRT1 or AtATP-PRT2 were amplified by PCR (see “Materials and Methods”). The ethidium bromide (EtBr) staining of the rRNA bands for each RNA preparation is also shown.

Article Snippet: Isolation of Arabidopsis ATP-PRT1 Gene Approximately 5 × 10 5 recombinant phages of an Arabidopsis Lambda ZAP II genomic library (Stratagene) were screened using the AtATP-PRT1 cDNA as a probe.

Techniques: Hybridization, Amplification, Staining

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Article Title: Molecular Cloning and Characterization of ATP-Phosphoribosyl Transferase from Arabidopsis, a Key Enzyme in the Histidine Biosynthetic Pathway

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Figure Lengend Snippet: Suppression of a S. cerevisiae his1 null mutant, BY1001, with the Arabidopsis AtATP-PRT cDNAs. A, Construction of a his1::LEU2 null allele on chromosome 5 (Chr.V). Restriction enzyme sites are designated: B, BamHI; Bg, BglII; Sa, SalI; and Xh, XhoI. B, Growth of a S. cerevisiae his1 mutant (BY1001). Strain BY1001 was transformed with either pYES2 (empty plasmid), pKF110 (plasmid carrying S. cerevisiae HIS1 coding region), pKF251 (plasmid harboring AtATP-PRT1 cDNA truncated at the chloroplast transit peptide portion), or pKF252 (plasmid containing AtATP-PRT2 cDNA without the chloroplast transit peptide region). After the transformation, the cells were cultivated on a minimal-Gal plate supplemented with an amino acids mixture without l-His, Leu, and Ura (SC/Gal-His- Leu-Ura).

Article Snippet: Isolation of Arabidopsis ATP-PRT1 Gene Approximately 5 × 10 5 recombinant phages of an Arabidopsis Lambda ZAP II genomic library (Stratagene) were screened using the AtATP-PRT1 cDNA as a probe.

Techniques: Mutagenesis, Transformation Assay, Plasmid Preparation

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Article Title: Molecular Cloning and Characterization of ATP-Phosphoribosyl Transferase from Arabidopsis, a Key Enzyme in the Histidine Biosynthetic Pathway

doi:

Figure Lengend Snippet: Analysis of Arabidopsis ATP-PRT proteins. A, Purification of the recombinant AtATP-PRT1 protein. Sample protein (10 μg for each lane) was analyzed by SDS-PAGE and Coomassie Brilliant Blue staining. A protein size marker was applied in lane 1. Whole-cell extract from the E. coli after the isopropylthio-β-galactoside induction (lane 2) was applied to an amylose resin column. The expressed fusion protein was eluted with 10 mm maltose (lane 3). After digesting with factor Xa (lane 4), the sample was sequentially purified by a hydroxylapatite (lane 5) and a second amylose resin chromatography to remove MBP (lane 6). B, Crude extracts (containing 10 μg of protein) from Arabidopsis seedlings were separated by 10% to 20% SDS-PAGE, and the proteins were electrophoretically transferred to a PVDF membrane for immunodetection with anti-AtATP-PRT1 polyclonal antibodies. Lane 1, Leaves from 1-week-old seedlings; lane 2, leaves from 2-week-old seedlings; lane 3, leaves from 3-week-old seedlings; lane 4, leaves from 4-week-old seedlings; lane 5, roots from 2-week-old seedlings; lane 6, roots from 3-week-old seedlings; lane 7, roots from 4-week-old seedlings; and lane 8, the purified recombinant AtATP-PRT1. Molecular size markers are shown on the left. Six independent experiments were carried out, and one of the representative results is shown.

Article Snippet: Isolation of Arabidopsis ATP-PRT1 Gene Approximately 5 × 10 5 recombinant phages of an Arabidopsis Lambda ZAP II genomic library (Stratagene) were screened using the AtATP-PRT1 cDNA as a probe.

Techniques: Purification, Recombinant, SDS Page, Staining, Marker, Chromatography, Immunodetection