Nickel and low CO₂-controlled motility in Chlamydomonas through complementation of a paralyzed flagella mutant with chemically regulated promoters.
BACKGROUND
Chlamydomonas reinhardtii is a model system for the biology of unicellular green algae. Chemically regulated promoters, such as the nickel-inducible CYC6 or the low CO₂-inducible CAH1 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.


RESULTS
We fused the CYC6 and CAH1 promoters to an HA-tagged RSP3 gene, encoding a protein of the flagellar radial spoke complex. The constructs were used for chemically regulated complementation of the pf14 mutant, carrying an ochre mutation in the RSP3 gene. 7 to 8% of the transformants showed cells with restored motility after induction with nickel or transfer to low CO₂ conditions, but not in non-inducing conditions. Maximum complementation (5% motile cells) was reached with very different kinetics (5-6 hours for CAH1, 48 hours for CYC6). The two inducible promoters drive much lower levels of RSP3 protein expression than the constitutive PSAD promoter, which shows almost complete rescue of motility.


CONCLUSIONS
To our knowledge, this is the first example of the use of the CYC6 or CAH1 promoters to perform a chemically regulated complementation of a Chlamydomonas mutant. Based on our data, the CYC6 and CAH1 promoters should be capable of fully complementing mutants in genes whose products exert their biological activity at low concentrations.
ENEA, Casaccia Research Center, Via Anguillarese 301, 00123 Rome, Italy.
Paola
P
Ferrante
Dennis R
DR
Diener
Joel L
JL
Rosenbaum
Giovanni
G
Giuliano
D015153:Blotting, Western; D002245:Carbon Dioxide / Q000494:pharmacology*; D002696:Chlamydomonas / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D005407:Flagella / Q000187:drug effects / Q000502:physiology*; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D005816:Genetic Complementation Test; D007700:Kinetics; D009068:Movement / Q000187:drug effects; D009154:Mutation / Q000235:genetics*; D009532:Nickel / Q000494:pharmacology*; D010641:Phenotype; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014170:Transformation, Genetic / Q000187:drug effects; D019076:Transgenes / Q000235:genetics
D010940:Plant Proteins; D002245:Carbon Dioxide; D009532:Nickel
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