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THE PLANT CELL, Vol 7, Issue 4 447-461, Copyright © 1995 by American Society of Plant Biologists
Arabidopsis Phosphoribosylanthranilate Isomerase: Molecular Genetic Analysis of Triplicate Tryptophan Pathway Genes
J. Li, J. Zhao, A. B. Rose, R. Schmidt and R. L. Last
Boyce Thompson Institute for Plant Research and Section of Genetics and Development, Cornell University, Tower Road, Ithaca, New York 14853-1801
Phosphoribosylanthranilate isomerase (PAI) catalyzes the third step of the
tryptophan biosynthetic pathway. Arabidopsis PAI cDNAs were cloned from a
cDNA expression library by complementation of an Escherichia coli trpC- PAI
deficiency mutation. Genomic DNA blot hybridization analysis detected three
nonallelic genes encoding PAI in the Arabidopsis genome. DNA sequence
analysis of cDNA and genomic clones indicated that the PAI1 and PAI2 genes
are virtually identical with only a single conservative amino acid
difference throughout the deduced coding region as well as extensive
conservation of introns and flanking regions. PAI3 shows less identity
(90%) with PAI1 and PAI2. All three PAI polypeptides possess an N-terminal
putative plastid target sequence, suggesting that these enzymes all
function in plastids. The PAI1 gene is flanked by nearly identical direct
repeats of ~350 nucleotides. Our results indicate that, in contrast to most
microorganisms, the Arabidopsis PAI protein is not fused with
indole-3-glycerolphosphate synthase, which catalyzes the next step in the
pathway. Yeast artificial chromosome hybridization studies indicated that
the PAI2 gene is tightly linked to the anthranilate synthase [alpha]
subunit 1 (ASA1) gene on chromosome 5. PAI1 was mapped to the top of
chromosome 1 using recombinant inbred lines, and PAI3 is loosely linked to
PAI1. cDNA restriction mapping and sequencing and RNA gel blot
hybridization analysis indicated that all three genes are transcribed in
wild-type plants. The expression of antisense PAI1 RNA significantly
reduced the immunologically observable PAI protein and enzyme activity in
transgenic plants. The plants expressing antisense RNA also showed two
phenotypes consistent with a block early in the pathway: blue fluorescence
under UV light and resistance to the anthranilate analog
6-methylanthranilate. The extreme nucleotide conservation between the
unlinked PAI1 and PAI2 loci suggests that this gene family is actively
evolving.
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