A cDNA, Cys1ACr, encoding an isoform of O-acetylserine(thiol) lyase has bee
n 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 wit
h 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 structu
rally more similar to higher plant O-acetylserine(thiol)lyase than to the c
orresponding prokaryotic enzymes. The M-terminal extension present in Cys1A
Cr showed several characteristics of an organellar transit peptide, with a
length typical for C. reinhardtii. Southern blot analysis suggested that th
e C. reinhardtii genome may contain a single copy of the organellar O-acety
lserine(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 tr
anscript abundance a longer period of sulfur limitation was required, reach
ing a maximum level of approximate to threefold Cys1ACr mRNA when compared
with the level of a sulfate-grown culture.