PLANT PHYSIOLOGY , Vol 105, Issue 1 377-384, Copyright © 1994 by American Society of Plant Biologists
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MOLECULAR BIOLOGY AND GENE REGULATION |
Molecular Cloning and Characterization of the pyrB1 and pyrB2 Genes Encoding Aspartate Transcarbamoylase in Pea (Pisum sativum L.)
C. L. Williamson and R. D. Slocum
Department of Biological Sciences, Goucher College, Baltimore, Maryland 21204-2794
We cloned cDNAs encoding two different pea (Pisum sativum L.) aspartate
transcarbamoylases (ATCases) by complementation of an Escherichia coli
[delta]pyrB mutant. The two cDNAs, designated pyrB1 and pyrB2, encode
polypeptides of 386 and 385 amino acid residues, respectively, both of
which exhibit typical chloroplast transit peptide sequences. Wheat germ
ATCase antibody recognizes a 36.5-kD polypeptide in pea leaf and root
tissues that is similar in size to other plant ATCase polypeptides and to
the catalytic polypeptides of bacterial ATCases. Northern analyses indicate
that the pyrB1 and pyrB2 transcripts are 1.6 kb in size and are
differentially expressed in pea tissues. The small transcript size and data
from biochemical studies indicate that plant ATCases are simple homotrimers
of 36- to 37-kD catalytic subunits, rather than part of a multifunctional
enzyme containing glutamine-dependent carbamoylphosphate synthetase and
dihydroorotase activities, as is seen in other eukaryotes. In the pea
ATCases, the carbamoylphosphate- and aspartate-binding domains are highly
homologous to those of other prokaryotic and eukaryotic ATCases and
critical active-site residues are completely conserved. The pea ATCases
also exhibit a putative pyrimidine-binding site, consistent with the known
allosteric regulation of plant ATCases by UMP in vitro.
