Molecular cloning in yeast by in vivo homologous recombination of the yeast putative α1 subunit of the voltage‐gated calcium channel | Zendy

Iida Kazuko | Zendy; Tada Tomoko | Zendy; Iida Hidetoshi | Zendy

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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.

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