polylinker sequences  (Integrated DNA Technologies)

Journal: Gene Expression

Article Title: Role of Saccharomyces cerevisiae Rap1 protein in Ty1 and Ty1-mediated transcription

doi:

Figure Lengend Snippet: Factor binding to a Ty1 fragment I (approximately 50 bp) containing either a wild-type (I; lanes 1–4) or mutant (I*R; lane 5) RAP1 binding site and to a 22 bp oligonucleotide containing the Ty1 RAP1 binding site (lanes 8–11). Binding reaction mixtures contained 2–4 μg of protein from wild-type (JF820; lanes 1–5, 9) or rap1-1 (JF1165; lane 10) extracts. The rap1-1 strain was transformed with the RAP1 deletion plasmid pBG51 (see Materials and Methods), and extracts were prepared from cultures grown at room temperature and shifted to 37°C (lane 10). Reactions were performed in the absence of specific competitor (none) or with a 50-fold molar excess of fragment I (lane 2), oligonucleotide duplex containing the RAP1-binding site from the TEF2 gene (TEF2; lane 3), or a 465 bp Pvu II restriction fragment containing polylinker (PL) sequences from plasmid pGEM5Zf (Promega; lane 4). Positions of free probe and complex 1 (I-1 or RBSTy1-1) are indicated to the left of the autoradiogram. In lanes 6, 7, and 8, the extract was omitted. Lanes 1–7 and lanes 8–10 are taken from separate gels. Note also that a different radiolabeled probe was used in the two experiments.

Article Snippet: Reactions were performed in the absence of competitor (–; lanes 1, 5, and 8); or in the presence of a 50-fold molar excess of fragment D (lane 2); oligonucleotide duplex containing the RAP1 binding site from the TEF2 gene (TEF2; lanes 3, 9); oligonucleotide duplex containing the palindromic Mcm1 binding site (PAL; lane 4); a 465 bp Pvu II restriction fragment containing polylinker (PL) sequences from plasmid pGEM5Zf (Promega; lanes 7, 10); or fragment D* R containing a mutation in the RAP1 binding site (lane 6).

Techniques: Binding Assay, Mutagenesis, Transformation Assay, Plasmid Preparation

Journal: Gene Expression

Article Title: Role of Saccharomyces cerevisiae Rap1 protein in Ty1 and Ty1-mediated transcription

doi:

Figure Lengend Snippet: Factor binding to Ty1 fragment D containing either a wild-type (D) or a mutant (D*R) RAP1 binding site. Binding reaction mixtures contained 2–4 μg of protein from wild-type (JF820) or mcm1-1 (JF1096) strains. Reactions were performed in the absence of competitor (–; lanes 1, 5, and 8); or in the presence of a 50-fold molar excess of fragment D (lane 2); oligonucleotide duplex containing the RAP1 binding site from the TEF2 gene (TEF2; lanes 3, 9); oligonucleotide duplex containing the palindromic Mcm1 binding site (PAL; lane 4); a 465 bp Pvu II restriction fragment containing polylinker (PL) sequences from plasmid pGEM5Zf (Promega; lanes 7, 10); or fragment D*R containing a mutation in the RAP1 binding site (lane 6). In lanes 11 and 12, extract was omitted. Positions of the free probe, complex D-1, D-2, and D-3 are indicated on each side of the autoradiogram. To the right is a schematic of the proteins known to be present in each complex.

Article Snippet: Reactions were performed in the absence of competitor (–; lanes 1, 5, and 8); or in the presence of a 50-fold molar excess of fragment D (lane 2); oligonucleotide duplex containing the RAP1 binding site from the TEF2 gene (TEF2; lanes 3, 9); oligonucleotide duplex containing the palindromic Mcm1 binding site (PAL; lane 4); a 465 bp Pvu II restriction fragment containing polylinker (PL) sequences from plasmid pGEM5Zf (Promega; lanes 7, 10); or fragment D* R containing a mutation in the RAP1 binding site (lane 6).

Techniques: Binding Assay, Mutagenesis, Plasmid Preparation