Construction of a cDNA to the hamster CAD gene and its application toward defining the domain for aspartate transcarbamylase.

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. coil 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.The six-step pathway of pyrimidine biosynthesis is common to organisms ranging from bacteria to mammals. In Escherichia coli, each step is catalyzed by a separate protein or protein complex, and aspartate transcarbamylase (ATCase; EC 2.1.3.2), the second enzyme, is the major point of allosteric control of the pathway (24). In mammals, the six enzymes are associated with only three proteins, and carbamoylphosphate synthetase (CPSase; EC 6.3.5.5), the first rather than the second enzyme of the pathway, is affected through allosteric regulation (20). The first three enzymes, i.e., CPSase, ATCase, and dihydroorotase (DHOase; EC 3.5.2.3), are found on a single trifunctional protein designated CAD. The CAD protein of hamster occurs as an oligomer of identical monomers of Mr 220,000 (8,10,25). Within each monomer, the protein appears to be organized into a series of independent functional domains separated from one another by proteolysis-sensitive peptide bridges (11,25,39). Each of the three enzyme activities is associated with a different domain.The CAD monomer of Syrian hamsters is encoded by an mRNA of 7.9 kilobases (kb; 45). The gene, on the other hand, spans more than 25 kb and is interrupted by over 30 introns (35). Previously, Wahl and coworkers (45) constructed a plasmid clone (pCAD41) containing a 2.3-kb insert complementary to the 3' region of CAD mRNA. However, for purposes of analyzing the organization of the CAD gene, a more complete CAD cDNA would be invaluable. In this paper we describe the construction of much larger cDNA inserts by the highly efficient cloning technique developed by Okayama and Berg (32). This paper also shows how the CAD cDNA can be used in conjunction with a bacterial * Corresponding author. mutant defective in pyrimidine biosynthesis as a powerful expression system (9) that can defin...

Gradient expression of Cdx along the rat intestine throughout postnatal development

“…At this stage, epithelial cell renewal is accelerated. Consistently, we found a rise in the concentration of mRNAs coding for CAD and PCNA, two proteins respectively involved in pyrimidine biosynthesis [17,18] and in DNA replication [15]. This study also confirms the profile of GLUC mRNA [23].…”

Mammalian aspartate transcarbamylase (ATCase): Sequence of the ATCase domain and interdomain linker in the CAD multifunctional polypeptide and properties of the isolated domain

“…3 determined. We also confirmed the published sequence (17), which extends 456 nucleotides further downstream to the stop codon. We sequenced all of this region but not both strands.…”

MYC Abrogates p53-Mediated Cell Cycle Arrest in N-(Phosphonacetyl)-l-Aspartate-Treated Cells, Permitting CAD Gene Amplification

“…Selections were performed as described by Perry et al (42). Briefly, 5 ϫ 10 4 cells, plated on 10-cm dishes, were grown in medium containing dialyzed fetal calf serum in the presence of PALA or MTX at concentrations three times the 50% inhibitory concentration (IC 50 ). Drug-resistant colonies, detectable after 4 to 5 weeks, were cloned or, alternatively, fixed, stained, and counted.…”

“…This fragment was recloned into pSP72 (Promega). The inserts from pSV2dhfr (47) and pCAD142 (50), which contain the mouse dhfr and hamster cad cDNAs, respectively, were used as probes. Radioactive bands were quantified with a PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.).…”