Isolation of Nicotiana plumbaginifolia cDNAs encoding isoforms of serine acetyltransferase and O-acetylserine (thiol) lyase in a yeast two-hybrid system with Escherichia coli cysE and cysK genes as baits.
We applied the yeast two-hybrid system for screening of a cDNA library of Nicotiana plumbaginifolia for clones encoding plant proteins interacting with two proteins of Escherichia coli: serine acetyltransferase (SAT, the product of cysE gene) and O-acetylserine (thiol)lyase A, also termed cysteine synthase (OASTL-A, the product of cysK gene). Two plant cDNA clones were identified when using the cysE gene as a bait. These clones encode a probable cytosolic isoform of OASTL and an organellar isoform of SAT, respectively, as indicated by evolutionary trees. The second clone, encoding SAT, was identified independently also as a "prey" when using cysK as a bait. Our results reveal the possibility of applying the two-hybrid system for cloning of plant cDNAs encoding enzymes of the cysteine synthase complex in the two-hybrid system. Additionally, using genome walking sequences located upstream of the sat1 cDNA were identified. Subsequently, in silico analyses were performed aiming towards identification of the potential signal peptide and possible location of the deduced mature protein encoded by sat1.
Liszewska
Frantz
F
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland.
Gaganidze
Dali
D
Sirko
Agnieszka
A
D000123:Acetyltransferases / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D019757:Carbon-Oxygen Lyases / Q000737:chemistry / Q000235:genetics*; D017931:DNA Primers; D018076:DNA, Complementary / Q000302:isolation & purification*; D018744:DNA, Plant / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid; D051050:Serine O-Acetyltransferase; D014026:Nicotiana / Q000235:genetics*; D020798:Two-Hybrid System Techniques
D001426:Bacterial Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D018744:DNA, Plant; D029968:Escherichia coli Proteins; D007527:Isoenzymes; D000123:Acetyltransferases; C070785:CysE protein, E coli; D051050:Serine O-Acetyltransferase; C018725:O-acetylhomoserine (thiol)-lyase; D019757:Carbon-Oxygen Lyases