PMC:4502370 / 13115-14457 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/4502370","sourcedb":"PMC","sourceid":"4502370","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4502370","text":"Rpo26 mutants\nAn intron-less RPO26 ORF was PCR-amplified with sense-strand primers designed to introduce a BamHI restriction site immediately upstream of the translation start codon and an antisense primer that introduced an XhoI site downstream of the stop codon. The PCR product was digested with BamHI and XhoI and then inserted into a yeast expression vector pRS425-TPI (2 μ LEU2) to yield pRS425-TPI-RPO26, in which expression of RPO26 is driven by the yeast TPI1 promoter, contained in a 2.2-kb PvuII fragment from pYX132 (Novagen).\nTruncated RPO26 alleles were constructed by PCR amplification with: (i) sense strand primers that introduced a new start codon at the positions specified plus a flanking BamHI site and/or (ii) antisense strand primers that introduced a new stop codon after the positions specified plus a flanking XhoI site. Single-alanine mutations R79A, E89A, R97A, E124A, R135A, R136A, D145A, and E150A were introduced into RPO26-(78-155) by two-stage PCR overlap extension with mutagenic primer oligonucleotides. The PCR products containing the mutated RPO26 ORFs were digested with BamHI and XhoI and inserted into BamHI/XhoI-cut pRS425-TPI-RPO26 in lieu of the wild-type RPO26 gene. The inserts of all plasmid clones were sequenced to exclude the acquisition of unwanted mutations during amplification and cloning.","divisions":[{"label":"title","span":{"begin":0,"end":13}},{"label":"p","span":{"begin":14,"end":538}}],"tracks":[]}