Molecular cloning in yeast by in vivo homologous recombination of the yeast putative alpha1 subunit of the voltage-gated calcium channel.
Saccharomyces cerevisiae has only one gene encoding a putative voltage-gated Ca2+ 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 Ca2+ channel component, does not increase Ca2+ uptake activity, but co-overexpression results in a 2- to 3-fold increase. Overexpression of CCH1 does not substantially complement the lethality and low Ca2+ uptake activity of a mid1 mutant and vice versa. These results indicate that co-overproduction of Cch1 and Mid1 is sufficient to increase Ca2+ uptake activity.
Iida
Kazuko
K
Medical R&D Center, The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan.
Tada
Tomoko
T
Iida
Hidetoshi
H
D001483:Base Sequence; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000235:genetics*; D002470:Cell Survival; D003001:Cloning, Molecular; D017931:DNA Primers; D004274:DNA, Recombinant / Q000235:genetics; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism
C108765:CCH1 protein, S cerevisiae; D015220:Calcium Channels; D017931:DNA Primers; D004274:DNA, Recombinant; C090463:MID1 protein, S cerevisiae; D008562:Membrane Glycoproteins; D029701:Saccharomyces cerevisiae Proteins; D002118:Calcium