The chloroplast gene encoding ribosomal protein S4 in Chlamydomonas reinhardtii spans an inverted repeat--unique sequence junction and can be mutated to suppress a streptomycin dependence mutation in ribosomal protein S12.
The ribosomal protein gene rps4 was cloned and sequenced from the chloroplast genome of Chlamydomonas reinhardtii. The N-terminal 213 amino acid residues of the S4 protein are encoded in the single-copy region (SCR) of the genome, while the C-terminal 44 amino acid residues are encoded in the inverted repeat (IR). The deduced 257 amino acid sequence of C. reinhardtii S4 is considerably longer (by 51-59 residues) than S4 proteins of other photosynthetic species and Escherichia coli, due to the presence of two internal insertions and a C-terminal extension. A short conserved C-terminal motif found in all other S4 proteins examined is missing from the C. reinhardtii protein. In E. coli, mutations in the S4 protein suppress the streptomycin-dependent (sd) phenotype of mutations in the S12 protein. Because we have been unable to identify similar S4 mutations among suppressors of an sd mutation in C. reinhardtii S12 obtained using UV mutagenesis, we made site-directed mutations [Arg68 (CGT) to Leu (CTG and CTT)] in the wild-type rps4 gene equivalent to an E. coli Gln53 to Leu ribosomal ambiguity mutation (ram), which suppresses the sd phenotype and decreases translational accuracy. These mutants were tested for their ability to transform the sd S12 mutation of C. reinhardtii to streptomycin independence. The streptomycin-independent isolates obtained by biolistic transformation all possessed the original sd mutation in rps12, but none had the expected donor Leu68 mutations in rps4. Instead, six of 15 contained a Gln73 (CAA) to Pro (CCA) mutation five amino acids downstream from the predicted mutant codon, irrespective of rps4 donor DNA. Two others contained six- and ten-amino acid, in-frame insertions at S4 positions 90 and 92 that appear to have been induced by the biolistic process itself. Eight streptomycin-independent isolates analyzed had wild-type rps4 genes and may possess mutations identical to previously isolated suppressors of sd that define at least two additional chloroplast loci. Cloned rps4 genes from streptomycin-independent isolates containing the Gln73 to Pro mutation and the 6-amino acid insertion in r-protein S4 transform the sd strain to streptomycin independence.
Randolph-Anderson
B L
BL
Department of Botany, Duke University, Durham, NC 27708, USA.
Boynton
J E
JE
Gillham
N W
NW
Huang
C
C
Liu
X Q
XQ
D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D016825:Chlamydomonas reinhardtii / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D002736:Chloroplasts / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010944:Plants / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D015183:Restriction Mapping; D012269:Ribosomal Proteins / Q000096:biosynthesis* / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013307:Streptomycin / Q000494:pharmacology*; D014170:Transformation, Genetic
D017931:DNA Primers; D012269:Ribosomal Proteins; C034212:ribosomal protein S12; C031798:ribosomal protein S4; D013307:Streptomycin