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Molecular cloning in yeast by in vivo homologous recombination of the yeast putative α1 subunit of the voltage‐gated calcium channel
Author(s) -
Iida Kazuko,
Tada Tomoko,
Iida Hidetoshi
Publication year - 2004
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2004.09.021
Subject(s) - protein subunit , saccharomyces cerevisiae , homologous recombination , yeast , mutant , cloning (programming) , molecular cloning , escherichia coli , chemistry , biochemistry , two hybrid screening , biology , microbiology and biotechnology , gene , peptide sequence , computer science , programming language
Saccharomyces cerevisiae has only one gene encoding a putative voltage‐gated Ca 2+ channel pore‐forming subunit, CCH1 , which is not possible to be cloned by conventional molecular cloning techniques using Escherichia coli . Here, we report the successful cloning of CCH1 in yeast by in vivo homologous recombination without using E. coli . Overexpression of the cloned CCH1 or MID1 alone, which encodes a putative stretch‐activated Ca 2+ channel component, does not increase Ca 2+ uptake activity, but co‐overexpression results in a 2‐ to 3‐fold increase. Overexpression of CCH1 does not substantially complement the lethality and low Ca 2+ uptake activity of a mid1 mutant and vice versa. These results indicate that co‐overproduction of Cch1 and Mid1 is sufficient to increase Ca 2+ uptake activity.