Tissue culture-specific expression of a naturally occurring tobacco feedback-insensitive anthranilate synthase.
A cDNA and corresponding promoter region for a naturally occurring, feedback-insensitive anthranilate synthase (AS) alpha-subunit gene, ASA2, has been isolated from an unselected, but 5-methyl-tryptophan-resistant (5MTr), tobacco (Nicotiana tabacum) cell line (AB15-12-1). The ASA2 cDNA contains a putative transit peptide sequence, and Southern hybridization shows that more than one closely related sequence is present in the tobacco genome. The ASA2 cDNA complemented a trpE nonsense mutant Escherichia coli strain, allowing growth on 300 microm 5MT-containing minimal medium without tryptophan, and cell extracts contained feedback-insensitive AS activity. The 5MTr was lost when the E. coli strain was transformed with an ASA2 site-directed mutant (phenylalanine-107-arginine-108 --> serine-107-glutamine-108). Identical nucleotide sequences encoding the phenylalanine-107-arginine-108 region have been found in polymerase chain reaction-amplified 326-bp ASA2 genomic fragments of wild-type (5-methyl-tryptophan-sensitive [5MTs]) tobacco and a progenitor species. High-level ASA2 transcriptional expression was detected only in 5MTr-cultured cells, not in 5MTs cells or in plants. Promoter studies indicate that tissue specificity of ASA2 is controlled by the promoter region between -2252 and -607. Since the ASA2 promoter sequences are not substantially different in the 5MTr and 5MTs lines, the increased levels of ASA2 mRNA in the 5MTr lines are most likely due to changes in a regulatory gene affecting ASA2 expression.
Song
H S
HS
Department of Crop Sciences, University of Illinois, Urbana, Illinois 61801, USA.
Brotherton
J E
JE
Gonzales
R A
RA
Widholm
J M
JM
D000595:Amino Acid Sequence; D000878:Anthranilate Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D005246:Feedback; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D010944:Plants / Q000201:enzymology; D010947:Plants, Toxic; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D014026:Nicotiana / Q000166:cytology / Q000201:enzymology*; D014364:Tryptophan / Q000494:pharmacology
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D014364:Tryptophan; D000878:Anthranilate Synthase