A promoter trap for Chlamydomonas reinhardtii: development of a gene cloning method using 5' RACE-based probes.
A promoterless radial spoke protein RSP3 gene has been used to identify promoter regions in the genome of Chlamydomonas reinhardtii. The acceptor strain pf-14 arg7 was transformed with a linearized vector containing the ARG 7.8 gene as a selection marker and a promoterless RSP3 gene. The frequency at which the motility was restored in transformants varied from 2-3%. Several of these were motile only in ammonium-free medium, indicating that the procedure could be used to select inducible promoters. Transformation of nitrogen-starved cells produced about twice as many transformants which were only motile in ammonium-free medium. Since one of the tagging vectors contained an RSP3 gene with a hybridization flag in its 3' untranslated region, it was possible to estimate the size of the new RSP3 transcripts in transformants. The results suggested that in most cases a hybrid RNA was generated consisting of the tagged gene transcript and reporter gene RNA. By 5' RACE, these parts of the new transcripts were amplified and it was shown that the generated DNA fragments could be used to clone a tagged gene. One such example, gene 2BC9, is predicted to code for a mitochondrial matrix protein. The tagging procedure will be optimized for cloning genes induced by nitrogen starvation, the cue for gametogenesis.
Haring
M A
MA
Department of Molecular Cell Biology, BioCentrum Amsterdam, The Netherlands. haring@bio.uva.nl
Beck
C F
CF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010940:Plant Proteins; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000235:genetics; D015800:Protozoan Proteins; D019105:Random Amplified Polymorphic DNA Technique; D015183:Restriction Mapping
D010940:Plant Proteins; D011506:Proteins; D015800:Protozoan Proteins; C060324:radial spoke protein, Chlamydomonas