Journal: Journal of Experimental Botany

Article Title: Induction of vacuolar invertase inhibitor mRNA in potato tubers contributes to cold-induced sweetening resistance and includes spliced hybrid mRNA variants

doi: 10.1093/jxb/err043

Figure Lengend Snippet: INH forms isolated from potato genotypes 1021/1 and 937/3 using a combination of cDNA library screening and PCR amplification from genomic DNA and cDNA.

Article Snippet: PCR amplification using the primer pair, cDNA template, and PCR Extender proofreading Taq DNA polymerase (5 Prime Co., Gaithersburg, MD, USA) resulted in a band of ∼800 bp, which was ligated into pBluescript and six clones sequenced for each cultivar.

Techniques: Isolation, cDNA Library Assay, Amplification

Journal:

Article Title: Saccharomyces cerevisiae DNA Polymerase ? and Polymerase ? Interact Physically and Functionally, Suggesting a Role for Polymerase ? in Sister Chromatid Cohesion

doi: 10.1128/MCB.23.8.2733-2748.2003

Figure Lengend Snippet: (A) Pol2 coimmunoprecipitates with Trf4. Coexpression of FLAG-Pol2 and His-Trf4 and immunoprecipitation protocols are described in Materials and Methods. Western blots of crude extract (labeled “I” for input protein) from insect cells expressing various combinations of Trf proteins and FLAG-Pol2 were probed with antibody against Trf4 or anti-FLAG Pol2 as indicated. Lane 1, no recombinant protein; lane 2, His-Trf4; lane 3, His-Trf4 plus FLAG-Pol2. These extracts were incubated with anti-FLAG beads. After washing of the beads, proteins that bound from extracts (labeled “B” for bound protein) were eluted by boiling. The proteins were analyzed on Western blots probed with antibody against Trf4 or anti-FLAG Pol2 as indicated on the right. Lane 4, no recombinant protein; lane 5, His-Trf4; lane 6, His-Trf4 plus FLAG-Pol2. (B) Pol2 coimmunoprecipitates with Trf5. Coexpression of FLAG-Pol2 and Trf5-His is described in Materials and Methods. Western blots of crude extract from insect cells are represented in the same order as those in Fig. ​Fig.2A.2A. (C) Recombinant Trf4 prepared in E. coli stimulates Pol ɛ holoenzyme. Trf4 was purified exactly as described previously (70). The oligo(dT)12-18 primer extension assay is described in Materials and Methods. Reaction mixtures contained 680 ng of Trf4, the amount required to observe Trf4 DNA polymerase activity (lanes 2 and 3), and/or 0.15 U of Pol ɛ, as indicated, are shown. The high level of Trf4 is saturating for stimulatory activity (see panel D). Lane 1, no protein; lane 2, Trf4-His with 0.1 mM dTTP; lane 3, Trf4-His with 1 mM dTTP; lane 4, Pol ɛ with 0.1 mM dTTP; lane 5, Pol ɛ with 1 mM dTTP; lane 6, Pol ɛ plus Trf4-His with 0.1 mM dTTP; and lane 7, Pol ɛ plus Trf4-His with 1 mM dTTP. (D) Titration of stimulatory activity of scTrf4 made in bacteria. The indicated amounts of scTrf4 were assayed for stimulation of [3H]dTMP incorporation by 0.15 U of Pol ɛ on an oligo(dT)-poly(dA) substrate as described in Materials and Methods. (E) scTrf4-His expressed in insect cell cochromatographs with Pol ɛ-stimulatory activity. scTrf4-His was expressed in insect cells. Silver staining of Trf4-His after purification through Ni2+-nitrilotriacetic acid and Mono Q columns, as described in Materials and Methods, and gel electrophoresis is shown at the top. Numbers refer to MonoQ fraction numbers. The same fractions are assayed for stimulation of primer extension by pol ɛ. Each fraction from the Mono Q column was dialyzed, and 2 μl of each fraction was used in a 20-μl reaction. Fraction 13 contained 17 ng of Trf4 protein (13 nM); but 2.7 nM Trf4 gave equivalent stimulation (not shown). The first lane shows no primer extension, the second lane shows activity of 0.15 U of Pol ɛ (0.5 nM) alone, and the subsequent lanes are the Mono Q fractions of the Trf4 purification assayed with 0.15 U of Pol ɛ (0.5 nm). The fraction numbers are identified above. (F) scTrf4-His and Pol ɛ-stimulatory activity copurify. [3H]dTMP incorporation assay: the same fractions from the Mono Q column were assayed for [3H]dTMP incorporation on an oligo(dT)-poly(dA) substrate as described in Materials and Methods in the presence of 0.15 U of Pol ɛ (0.5 nm). (G) Trf4 from the MonoQ column is highly purified. Coomassie-stained gel of Trf4 from fraction 14 of the MonoQ column. (H) scTrf4-His does not efficiently stimulate Pol2-140 lacking the C-terminal 1,000 amino acids. Three levels (0.075, 0.15, or 0.3 U) of either Pol ɛ (solid dots) or truncated Pol2 protein (open dots) were assayed with saturating amounts (40 ng) of Trf4-His purified from insect cells. Similar results were obtained with scTrf4 prepared in E. coli.

Article Snippet: As controls that the stimulation was not due to contaminating E. coli DNA polymerase I (the most abundant DNA polymerase in the bacterial extracts) in the scTrf4 preparation, we showed that DNA polymerase I of E. coli (Gibco BRL) did not stimulate Pol ɛ and antibody to E. coli Pol I (gift of S. Linn, U.C.

Techniques: Immunoprecipitation, Western Blot, Labeling, Expressing, Recombinant, Incubation, Purification, Primer Extension Assay, Activity Assay, Titration, Silver Staining, Nucleic Acid Electrophoresis, Staining