The plastocyanin-deficient phenotype of Chlamydomonas reinhardtii Ac-208 results from a frame-shift mutation in the nuclear gene encoding preapoplastocyanin.
Ac-208 is a plastocyanin-deficient mutant of Chlamydomonas reinhardtii that contains only 2-3% of the wild-type level of plastocyanin-encoding mRNA and no detectable plastocyanin. Sequence analysis of the ac-208 plastocyanin-encoding gene reveals a single nucleotide insertion in the first exon compared with the wild-type gene; this alters the reading frame and results in a premature nonsense codon. We have introduced the genomic sequence encoding plastocyanin from a wild-type strain into ac-208 by cotransformation with a selectable marker encoding nitrate reductase. Of 22 nit+ transformants characterized, nine contained additional plastocyanin-encoding sequences (compared with untransformed cells) and each of these nine transformants was found to accumulate the protein. Transformants that do not contain newly introduced plastocyanin sequences retain the plastocyanin-deficient phenotype. The introduced plastocyanin-encoding sequences are stable during mitotic growth in liquid culture over a period of several months, as is expression from the introduced sequences. We suggest that the decreased steady state level of plastocyanin-encoding messages is a consequence of the frame-shift mutation in the structural gene. The ability to complement ac-208 with plastocyanin-encoding sequences will allow the introduction and analysis of in vitro mutagenized plastocyanin sequences in vivo in transgenic C. reinhardtii cells.
Quinn
J
J
Department of Chemistry and Biochemistry, UCLA 90024-1569.
Li
H H
HH
Singer
J
J
Morimoto
B
B
Mets
L
L
Kindle
K
K
Merchant
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000032:analysis / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002467:Cell Nucleus / Q000502:physiology*; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D016368:Frameshift Mutation; D015723:Gene Library; D005796:Genes; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010641:Phenotype; D010970:Plastocyanin / Q000032:analysis / Q000235:genetics*; D011498:Protein Precursors / Q000032:analysis / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D015183:Restriction Mapping
D001059:Apoproteins; D009838:Oligodeoxyribonucleotides; D011498:Protein Precursors; D012333:RNA, Messenger; C040778:apoplastocyanin; D004247:DNA; D010970:Plastocyanin