Maize glutamine synthetase cDNAs: isolation by direct genetic selection in Escherichia coli.
Maize glutamine synthetase cDNA clones were isolated by genetic selection for functional rescue of an Escherichia coli delta glnA mutant growing on medium lacking glutamine. The Black Mexican Sweet cDNA library used in this study was constructed in pUC13 such that cDNA sense strands were transcribed under the control of the lac promoter. E. coli delta glnA cells were transformed with cDNA library plasmid DNA, grown briefly in rich medium to allow phenotypic expression of the cDNAs and the pUC13 ampr gene, and challenged to grow on agar medium lacking glutamine. Large numbers of glutamine synthetase cDNA clones have been identified in individual 150-mm Petri dishes; all characterized cDNA clones carry complete coding sequences. Two cDNAs identical except for different 5' and 3' termini have been sequenced. The major open reading frame predicts a protein with an amino acid sequence that exhibits striking similarity to the amino acid sequences of the predicted products of previously sequenced eukaryotic glutamine synthetase cDNAs and genes. In addition, the maize glutamine synthetase cDNAs were shown to contain a 5' mini-ORF of 29 codons separated by 37 nucleotide pairs from the major ORF. This mini-ORF was shown not to be essential for the functional rescue of the E. coli delta glnA mutant. Expression of the cDNAs in E. coli is presumed to be due to the function of a polycistronic hybrid lac messenger RNA or translational fusions encoded by the pUC plasmids. Proteins of the expected sizes encoded by two different pUC clones were shown to react with antibodies to tobacco glutamine synthetase.
Snustad
D P
DP
Department of Genetics and Cell Biology, University of Minnesota, St. Paul 55108.
Hunsperger
J P
JP
Chereskin
B M
BM
Messing
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D003062:Codon; D004247:DNA / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics*; D005974:Glutamate-Ammonia Ligase / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D010944:Plants / Q000235:genetics; D010957:Plasmids; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014169:Transformation, Bacterial; D003313:Zea mays / Q000235:genetics*
D003062:Codon; D011993:Recombinant Fusion Proteins; D004247:DNA; D005974:Glutamate-Ammonia Ligase