Construction of a CDNA to the Hamster CAD Gene and Its Application Toward Defining the Domain for Aspartate Transcarbamylase

Overview

Journal Mol Cell Biol

Specialty Cell Biology

Date 1985 Jul 1

PMID 2862577

Citations 17

Authors

K Shigesada

G R Stark

J A Maley

L A Niswander

J N Davidson

Affiliations

Soon will be listed here.

Abstract

cDNA complementary to hamster mRNA encoding the CAD protein, a multifunctional protein which carries the first three enzymes of pyrimidine biosynthesis, was constructed. The longest of these recombinants (pCAD142) covers 82% of the 7.9-kilobase mRNA. Portions of the cDNA were excised and replaced by a lac promoter-operator-initiation codon segment. The resultant plasmids were transfected into an Escherichia coli mutant defective in aspartate transcarbamylase, the second enzyme of the pathway. Complementation of the bacterial defect was observed with as little as 2.2 kilobases of cDNA sequence, corresponding to the 3' region of the mRNA. DNA sequencing in this region of the hamster cDNA reveals stretches which are highly homologous to the E. coli gene for the catalytic subunit of aspartate transcarbamylase; other stretches show no homology. The highly conserved regions probably reflect areas of protein structure critical to catalysis, while the nonconserved regions may reflect differences between the quaternary structures of E. coli and mammalian aspartate transcarbamylases, one such difference being that the bacterial enzyme in its native form is allosterically regulated and the mammalian enzyme is not.

Citing Articles

Aspartate-90 and arginine-269 of hamster aspartate transcarbamylase affect the oligomeric state of a chimaeric protein with an Escherichia coli maltose-binding domain.

Qiu Y, Davidson J Biochem J. 1998; 329 ( Pt 2):243-7.

PMID: 9425105 PMC: 1219037. DOI: 10.1042/bj3290243.

MYC abrogates p53-mediated cell cycle arrest in N-(phosphonacetyl)-L-aspartate-treated cells, permitting CAD gene amplification.

Chernova O, Chernov M, Ishizaka Y, Agarwal M, Stark G Mol Cell Biol. 1998; 18(1):536-45.

PMID: 9418900 PMC: 121521. DOI: 10.1128/MCB.18.1.536.

Multiple mechanisms of N-phosphonacetyl-L-aspartate resistance in human cell lines: carbamyl-P synthetase/aspartate transcarbamylase/dihydro-orotase gene amplification is frequent only when chromosome 2 is rearranged.

Smith K, Chernova O, Groves R, Stark M, Martinez J, Davidson J Proc Natl Acad Sci U S A. 1997; 94(5):1816-21.

PMID: 9050862 PMC: 20000. DOI: 10.1073/pnas.94.5.1816.

Human chromosome 3 mediates growth arrest and suppression of apoptosis in microcell hybrids.

Speevak M, Chevrette M Mol Cell Biol. 1996; 16(5):2214-25.

PMID: 8628288 PMC: 231209. DOI: 10.1128/MCB.16.5.2214.

Aspartate transcarbamoylase genes of Pseudomonas putida: requirement for an inactive dihydroorotase for assembly into the dodecameric holoenzyme.

Schurr M, Vickrey J, Kumar A, Campbell A, Cunin R, Benjamin R J Bacteriol. 1995; 177(7):1751-9.

PMID: 7896697 PMC: 176802. DOI: 10.1128/jb.177.7.1751-1759.1995.

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