Cysteine biosynthesis in Chlamydomonas reinhardtii. Molecular cloning and regulation of O-acetylserine(thiol)lyase.
A cDNA, Cys1ACr, encoding an isoform of O-acetylserine(thiol) lyase has been isolated from Chlamydomonas reinhardtii, using a PCR-based approach. The inclusion of dimethylsulfoxide in the PCR reaction has been demonstrated to be essential for the correct amplification of C. reinhardtii templates with complex secondary structures caused by a high G + C content. The deduced amino acid sequence exhibited highest similarity with plant O-acetylserine(thiol)lyase isoforms, indicating that the C. reinhardtii enzyme was structurally more similar to higher plant O-acetylserine(thiol)lyase than to the corresponding prokaryotic enzymes. The N-terminal extension present in Cys1ACr showed several characteristics of an organellar transit peptide, with a length typical for C. reinhardtii. Southern blot analysis suggested that the C. reinhardtii genome may contain a single copy of the organellar O-acetylserine(thiol)lyase gene. O-acetylserine(thiol)lyase activity was strongly induced by sulfur-deficient conditions (up to sevenfold the level observed in a sulfur-repleted cell culture) and required the presence of a nitrogen source. Northern blot analysis showed a different pattern of regulation of Cys1ACr to that observed at the activity level. To obtain an increase of transcript abundance a longer period of sulfur limitation was required, reaching a maximum level of approximately threefold Cys1ACr mRNA when compared with the level of a sulfate-grown culture.
Ravina
C G
CG
Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC y Universidad de Sevilla, Spain.
Barroso
C
C
Vega
J M
JM
Gotor
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016825:Chlamydomonas reinhardtii / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D003545:Cysteine / Q000096:biosynthesis*; D003547:Cysteine Synthase / Q000235:genetics* / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D017125:Genes, Protozoan; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016053:RNA, Protozoan / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013455:Sulfur / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D016054:DNA, Protozoan; D012333:RNA, Messenger; D016053:RNA, Protozoan; D013455:Sulfur; D003547:Cysteine Synthase; D003545:Cysteine