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Medientyp:
E-Artikel
Titel:
Nickel and low CO2-controlled motility in Chlamydomonasthrough complementation of a paralyzed flagella mutant with chemically regulated promoters
Beteiligte:
Ferrante, Paola;
Diener, Dennis R;
Rosenbaum, Joel L;
Giuliano, Giovanni
Erschienen:
Springer Science and Business Media LLC, 2011
Beschreibung:
<jats:title>Abstract</jats:title>
<jats:sec>
<jats:title>Background</jats:title>
<jats:p>
<jats:italic>Chlamydomonas reinhardtii</jats:italic> is a model system for the biology of unicellular green algae. Chemically regulated promoters, such as the nickel-inducible <jats:italic>CYC6</jats:italic> or the low CO<jats:sub>2</jats:sub>-inducible <jats:italic>CAH1</jats:italic> promoter, may prove useful for expressing, at precise times during its cell cycle, proteins with relevant biological functions, or complementing mutants in genes encoding such proteins. To this date, this has not been reported for the above promoters.</jats:p>
</jats:sec>
<jats:sec>
<jats:title>Results</jats:title>
<jats:p>We fused the <jats:italic>CYC6</jats:italic> and <jats:italic>CAH1</jats:italic> promoters to an HA-tagged <jats:italic>RSP3</jats:italic> gene, encoding a protein of the flagellar radial spoke complex. The constructs were used for chemically regulated complementation of the <jats:italic>pf14</jats:italic> mutant, carrying an ochre mutation in the <jats:italic>RSP3</jats:italic> gene. 7 to 8% of the transformants showed cells with restored motility after induction with nickel or transfer to low CO<jats:sub>2</jats:sub> conditions, but not in non-inducing conditions. Maximum complementation (5% motile cells) was reached with very different kinetics (5-6 hours for <jats:italic>CAH1</jats:italic>, 48 hours for <jats:italic>CYC6</jats:italic>). The two inducible promoters drive much lower levels of RSP3 protein expression than the constitutive <jats:italic>PSAD</jats:italic> promoter, which shows almost complete rescue of motility.</jats:p>
</jats:sec>
<jats:sec>
<jats:title>Conclusions</jats:title>
<jats:p>To our knowledge, this is the first example of the use of the <jats:italic>CYC6</jats:italic> or <jats:italic>CAH1</jats:italic> promoters to perform a chemically regulated complementation of a <jats:italic>Chlamydomonas</jats:italic> mutant. Based on our data, the <jats:italic>CYC6</jats:italic> and <jats:italic>CAH1</jats:italic> promoters should be capable of fully complementing mutants in genes whose products exert their biological activity at low concentrations.</jats:p>
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