Journal: Molecular and Cellular Biology

Article Title: A Human Sodium-Dependent Vitamin C Transporter 2 Isoform Acts as a Dominant-Negative Inhibitor of Ascorbic Acid Transport

doi: 10.1128/MCB.24.8.3150-3156.2004

Figure Lengend Snippet: Cloning and distribution of full-length hSVCT2 and its short isoform. (A) Identification of full-length and short isoforms of hSVCT2. cDNA from fetal brain tissue was amplified using hSVCT2-3 and hSVCT2-5 primers (see Materials and Methods). The PCR products were separated in a 1% agarose gel, and bands at 2 kb were excised and cloned into the T/A vector. DNA isolated from the clones was digested with EcoRI. Lane 1, molecular weight marker; lane 2, empty T/A; lanes 3 and 4, T/A vector with full-length hSVCT2; lane 5, T/A vector with hSVCT2-short. Arrows, short and long isoforms. (B) Sequencing analysis of full-length hSVCT2 and its short isoform. (C) Northern blot analysis of hSVCT2 distribution in cell lines. A poly(A + ) RNA master blot prepared from different cancer cell lines was probed with a DNA fragment obtained by BamHI digestion of full-length hSVCT2. Arrow, hSVCT2 RNA product.

Article Snippet: Clones that had homology to hSVCT2 were subcloned into the EcoRI site of the mammalian expression vectors pcDNA4/HisMaxC and pCMV-Tag4A (Invitrogen).

Techniques: Clone Assay, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Plasmid Preparation, Isolation, Molecular Weight, Marker, Sequencing, Northern Blot

Journal: Infection and Immunity

Article Title: Plasmodium vivax Ookinete Surface Protein Pvs25 Linked to Cholera Toxin B Subunit Induces Potent Transmission-Blocking Immunity by Intranasal as Well as Subcutaneous Immunization ▿

doi: 10.1128/IAI.00306-10

Figure Lengend Snippet: Construction and expression analysis of Pvs25H in Pichia pastoris . (a) The 5′- and 3′-coding regions of the Pvs25 gene sequence with its predicted amino acid sequence (Ala 23 to Leu 195 ). The Pvs25 gene fused to a hexahistidine tag at its C terminus was inserted into the SnaBI and EcoRI sites of pPIC9K, and the gene was integrated into the chromosomal DNA of P. pastoris strain GS115 by homologous recombination. (b) After selection of a high-producer clone that secreted the recombinant protein, nickel-affinity chromatography-purified Pvs25H was analyzed by SDS-PAGE (lane 1). M, molecular marker. (c) The affinity-purified Pvs25H was fractionated by size exclusion chromatography from which at least five chromatographic peaks were observed. (d) Size exclusion chromatography fractions 14-26 were subjected to SDS-PAGE (15% acrylamide). Based on the apparent molecular mass of each protein band, the fractions 16, 17, 18-19, 21-22, and 22-23 were defined as the tetramer, trimer, dimer, monomer B, and monomer A, respectively. (e) Selected chromatography peaks were subjected to 5 to 20% acrylamide gradient SDS-PAGE under nonreducing or reducing (10% 2-ME and boiling) conditions. (f) Hydrophobic interaction chromatography of the Pvs25H-A and Pvs25H-B monomeric isoforms. Elution fractions (A) are eluates of 2 M NH 4 SO 4 (Pvs25H-A) and elution fractions (B) are eluates of PBS (Pvs25H-B). (g) Each isoform (M, a mixture of dimer, trimer and tetramer; B, B isoform; A, A isoform) was subjected to 15% acrylamide SDS-PAGE under various conditions, as indicated. (h) The N-terminal protein sequence of each isoform. Positively numbered amino acid residues (Ala 1 and the following) are residues of the Pvs25 protein; negatively numbered amino acids are derivatives of the pPIC9K α-factor secretion signal. The A monomer revealed a single, unique sequence, but the B monomer and the multimers showed a mixture of different N termini.

Article Snippet: The fragment was subcloned into the SnaBI and EcoRI sites of the P. pastoris expression vector pPIC9K (Life Technologies, Carlsbad, CA) to construct the plasmid pPvs25H, which was designed to express an α-factor signal-Pvs25-hexahistidine fusion protein (see Fig. ).

Techniques: Expressing, Sequencing, Homologous Recombination, Selection, Recombinant, Affinity Chromatography, Purification, SDS Page, Marker, Affinity Purification, Size-exclusion Chromatography, Chromatography, Hydrophobic Interaction Chromatography

Journal: Eukaryotic Cell

Article Title: Use of the Plant Defense Protein Osmotin To Identify Fusarium oxysporum Genes That Control Cell Wall Properties ▿ Genes That Control Cell Wall Properties ▿ ‡

doi: 10.1128/EC.00316-09

Figure Lengend Snippet: Disruption of the FOR3 gene in F. oxysporum f. sp. nicotianae . (A) Schematic view of the FOR3 gene disruption strategy. The DNA inserts of p FOR3 and p for3 ::HYG are shown. The FOR3 coding region is indicated by the gray striped box and noncoding regions by the gray line. The vector sequences are indicated by the black line. Restriction sites on the cloning vector are indicated in black, and those in FOR3 are indicated in gray. The sizes of cloned DNA fragments (double-headed arrows), as well as the probes used for Southern (dotted line) or Northern (dashed line) blot analyses, are indicated. Restriction site abbreviations are as follows: E, EcoRV; E1, EcoRI; H, HindIII; K, KpnI; P, PstI; S, SalI; Xb, XbaI; Xh, XhoI. (B) Southern blot of restriction-digested genomic DNA of the wild-type ( FOR3 ) and Δ for3 mutant strains. Restriction site abbreviations are as in panel A. (C) Northern blot of total RNA of the wild-type ( FOR3 ) and Δ for3 mutant strains with a FOR3 probe ( FOR3 ) and the corresponding ethidium bromide signal (RNA) shown.

Article Snippet: To construct p for3 ::HYG, a 0.7-kb fragment containing the 3′ part of the FOR3 ORF (+3205 bp to +3941 bp) was amplified from F. oxysporum genomic DNA by PCR using the primer pair 5′-ATCGAGTCTTGCCGACGAT-3′/5′-TGAGATCCGTCTTCAGGATC-3′ and inserted into the EcoRV site of the pSTblue-1 vector (Novagen, Madison, WI).

Techniques: Plasmid Preparation, Clone Assay, Northern Blot, Southern Blot, Mutagenesis