Journal: Journal of Biological Chemistry

Article Title: The Caenorhabditis elegansmRNA 5′-Capping Enzyme

doi: 10.1074/jbc.m212101200

Figure Lengend Snippet: FIG. 5. The addition of the Ceg1-interacting region from Cet1 onto CEL-1 RTPase domain allows it to function in the presence of Ceg1. YSB719 was transformed with (left) pRSH-CEG1 or (right) pDB20H-MCE-(211–597). His isolates were subsequently transformed with the following LEU2/2 m plasmids: vector (pAD5); CET1 (pAD5- CET1); CET1-(1–225)-CEL-1-(13–221) (pAD5-CET1 (1–225)-CEL-1-(13– 221), expressing a fusion protein containing residues 1–225 of Cet1 and CEL-1-(13–221) tagged with the HA epitope); CET1-(1–265)-CEL-1-(13– 221) (pAD5-CET1 (1–265)-CEL-1-(13–221)); cet1–446-(1–265)-CEL-1-(13– 221) (pAD5-cet1–446-(1–265)-CEL-1-(13–221), expressing a fusion pro- tein consisting of residues 1–265 from the cet1–446 mutant (11) and CEL-1-(13–221) tagged with the HA epitope); and CEL-1-(13–221) (pAD5- CEL-1-(13–221)). Leu His transformants were grown in the presence of 5-FOA, and the plates were incubated for 3 days at 30 °C.

Article Snippet: Leu His transformants were grown in the presence of 5-FOA, and the plates were incubated for 3 days at 30 °C. at T E X A S U N IV ctober 16, 2014 http://w w w .jbc.org/ D ow nloaded from of the chimeric RTPases support viability of the same strain if MCE-(211–597) replaces Ceg1 as the GTase (Fig. 5, right), showing that these proteins are functional.

Techniques: Transformation Assay, Plasmid Preparation, Expressing, Mutagenesis, Incubation

Journal: Journal of Biological Chemistry

Article Title: The Caenorhabditis elegansmRNA 5′-Capping Enzyme

doi: 10.1074/jbc.m212101200

Figure Lengend Snippet: FIG. 6. RTPase activities of CEL-1-(13–221) mutants. A, purification of recombinant polyhistidine-tagged CEL-1-(13–221) protein. The peak fractions of the heparin-Sepharose CL-6B column were analyzed by SDS-PAGE and visualized by Coomassie Brilliant Blue staining (top panel) or immunoblotting with monoclonal anti-polyhistidine antibody (bottom panel). 400 and 50 ng of protein were loaded for the top and bottom panels, respectively. Lane 1, wild-type CEL-1-(13–221); lane 2, C136S; lane 3, R142K; lane 4, D76N; lane 5, E111Q; lane 6, D112N. For reasons that are not clear, the D76N mutant shows slightly altered mobility. B, RTPase assay. The wild-type and mutated CEL-1-(13–221) proteins were incubated at 30 °C for 10 min with 1 M termini of a [-32P]GTP-labeled 65 nucleotide RNA. Reaction mixtures were analyzed by thin layer chromatography (TLC) on polyethyleneimine-cellulose plates. Released phosphate was detected by autoradiography, and radioactive spots were cut out and quantitated by liquid scintillation counting. Relative amounts of released phosphate were plotted versus protein amount. C, in vivo analysis of CEL-1-(13–221) mutants by plasmid shuffling. YSB719 carrying pDB20H-MCE-(211–597) was transformed with the vector pAD5 or derivatives expressing the indicated CEL-1 alleles. Leu His transformants were tested for growth in the presence of 5-FOA. Plates were incubated for 4 days at 30 °C. D, immunoblot analysis of S. cerevisiae whole-cell extracts. YSB719 cells carrying pDB20H-MCE-(211–597) and CEL-1-(13–221) derivatives were grown in selective media but without shuffling out the CEG1/CET1 plasmid. Extracts were prepared, and 10-g protein was analyzed by SDS-PAGE and immunoblotting with the anti-HA antibody12CA5. Lane 1, pAD5; lane 2, pAD5-CEL-1-(13–221); lane 3, pAD5-CEL- 1-(13–221)C136S; lane 4, pAD5-CEL-1-(13–221)C136A; lane 5, pAD5-CEL-1-(13–221)R142K; lane 6, pAD5-CEL-1-(13–221)R142A; lane 7, pAD5- CEL-1-(13–221)D76N; lane 8, pAD5-CEL-1-(13–221)E111Q; lane 9, pAD5-CEL-1-(13–221)D112N.

Article Snippet: Leu His transformants were grown in the presence of 5-FOA, and the plates were incubated for 3 days at 30 °C. at T E X A S U N IV ctober 16, 2014 http://w w w .jbc.org/ D ow nloaded from of the chimeric RTPases support viability of the same strain if MCE-(211–597) replaces Ceg1 as the GTase (Fig. 5, right), showing that these proteins are functional.

Techniques: Purification, Recombinant, SDS Page, Staining, Western Blot, Mutagenesis, Incubation, Labeling, Thin Layer Chromatography, Autoradiography, In Vivo, Plasmid Preparation, Transformation Assay, Expressing

Journal: Chemical Genomics

Article Title: Development of a Yeast Two-Hybrid Screen for Selection of Human Ras-Raf Protein Interaction Inhibitors

doi: 10.1007/978-1-59259-948-6_18

Figure Lengend Snippet: In the first step of the integrative transformation strategy used to construct the modified yeast strains SKY51 and SKY194: the HXT9 gene is targeted to the yeast PDR1 locus. (A) A plasmid vector containing upstream (5′) and downstream (3′) flanking regions of the PDR1 gene separated by a unique restriction site (BamHI). (B) An integrative cassette containing hisG-URA3-cadA is inserted at the unique BamHI site between the PDR1 gene fragments. (C) The structure of the recombination intermediate containing a PDR1 gene disrupted by the hisG-URA3-cadA cassette is shown. (D) Recombination between hisG and cadA sequences of the hisG-URA3-cadA cassette on the chromosome and the hisG and cadA sequences on separately prepared linear hisG-HXT9-cadA cassette is detected by growth on 5FOA (i.e., ura3 - phenotype), which reflects insertion of HXT9 into the chromosomal location of the PDR1 gene with loss of PDR1 and URA3. (E) The genomic structure of the PDR1 locus of the modified yeast strains SKY51 and SKY194, derived from SKY48 and SKY191, respectively.

Article Snippet: Cycloheximide (CYH), 4-nitroquinoline-oxide (4-NQO), sulfomethuron methyl (SMM), and 5-fluoro-orotic acid (5FOA) (BioMol, PA).

Techniques: Transformation Assay, Construct, Modification, Plasmid Preparation, Derivative Assay

Journal: Chemical Genomics

Article Title: Development of a Yeast Two-Hybrid Screen for Selection of Human Ras-Raf Protein Interaction Inhibitors

doi: 10.1007/978-1-59259-948-6_18

Figure Lengend Snippet: In a second step of the integrative transformation strategy used to construct the modified yeast strains SKY54 and SKY197 from strains SKY51 and SKY194, the HXT11 gene is targeted to the yeast PDR3 locus. (A) Plasmid vector was constructed containing the upstream (5′) and downstream (3′) flanking regions of the PDR3 gene separated by a unique restriction site (BamHI). (B) An integrative cassette containing hisG-URA3-Int was inserted at the unique BamHI site between the PDR3 gene fragments. This was used for integration by homologous replacement of PDR3 as selected by a ura -phenotype. (C) The structure of recombination intermediate containing a PDR3 gene disrupted by the hisG-URA3-Int cassette is shown. (D) Next, homologous recombination between hisG and Int sequences of the integrated hisG-URA3-Int cassette on the chromosome and a separately prepared, linear hisG-HXT11-Int cassette results in insertion of the HXT11 gene at the endogenous yeast PDR3 1ocus as selected by growth on 5FOA media. (E) The genomic structure of the PDR3 locus of the final yeast strains, SKY54 and SKY197.

Article Snippet: Cycloheximide (CYH), 4-nitroquinoline-oxide (4-NQO), sulfomethuron methyl (SMM), and 5-fluoro-orotic acid (5FOA) (BioMol, PA).

Techniques: Transformation Assay, Construct, Modification, Plasmid Preparation, Homologous Recombination