Jeffrey N. Davidson

Irradiation-reduced human chromosome 21 hybrids

Rodent-human somatic cell hybrids have been constructed which contain fragments of human chromosome 21 as their only human material. This was done by irradiating rodent-human somatic cell hybrids containing a complete chromosome 21 to fragment the genome and then rescuing human GAR synthetase and various amounts of flanking chromosome 21 DNA by fusing with GAR synthetase-deficient hamster cells and selecting for growth in purine-free medium. Four irradiation-reduction hybrids were produced by this method and contain the distal, proximal, and central portions of the long arm of human chromosome 21, all centered about GAR synthetase. These irradiation-reduction hybrids were used as a panel to regionally map single-copy and individual copies of repetitive sequences. Using these hybrids along with another independently constructed hybrid, the GAR synthetase gene was mapped distal to SOD-1 and proximal to CP21G1(D21S60). Of special interest is the regional mapping of the gene for the amyloid β-protein distal to pPW236B(D21S11) and proximal to SOD-1.

The aspartate transcarbamylase domain of a mammalian multifunctional protein expressed as an independent enzyme in Escherichia coli

SummaryAlthough aspartate transcarbamylase (ATCase) is an independent, monofunctional enzyme in Escherichia coli, mammalian ATCase is one of the globular enzymatic domains of the multifunctional CAD protein. We subcloned fragments of the hamster CAD cDNA and assayed polypeptide products expressed in E. coli for ATCase activity in order to isolate a stretch of cDNA which encodes only the ATCase domain. Three such expression constructs contain fragments of hamster CAD cDNA similar in length to the gene encoding the E. coli ATCase catalytic subunit (pyrB). These constructs yield stable proteins with ATCase activity, ascertained by both in vivo and in vitro assays; the clones also possess sequence homology with the pyrB gene at both the 5′ and 3′ ends. The clone producing the most active ATCase contains cDNA which is analogous to the entire pyrB gene, plus a small amount of CAD sequence upstream of this region. Because these constructs produce independently folded, active ATCase from a piece of cDNA the size of the E. coli pyrB gene, they open the door for the in-depth investigation of the isolated mammalian enzyme domain utilizing recombinant DNA technology. This approach is potentially useful for the analysis of domains of other multifunctional proteins.

Complete hamster CAD protein and the carbamylphosphate synthetase domain of CAD complement mammalian cell mutants defective in de novo pyrimidine biosynthesis

The mammalianCAD gene codes for a 240-kDa multifunctional protein that catalyzes the first three steps of de novo pyrimidine biosynthesis. Previously, the longest cDNA construct available was missing approximately 500 bp of coding sequence at the 5′ end, thereby lacking the sequence to encode the entire carbamylphosphate synthetase (CPSase) domain. Here, a completeCAD hamster cDNA is constructed, placed into a mammalian expression vector, and transfected into hamster cells deficient in CAD. Transfectants show coordinately restored levels of all three enzyme activities and the presence of full-length CAD protein. A derivative construct of theCAD cDNA was generated that should encode only the CPSase domain. When transfected into mammalian cells, a protein was synthesized that had significant CPSase activity both in vivo and in vitro. The two constructs generated in this study will facilitate the study of CAD structure, function, and allosteric regulation.

Mapping of the gene encoding the multifunctional protein carrying out the first three steps of pyrimidine biosynthesis to human chromosome 2.

SummaryThe CAD gene encodes a trifunctional protein that carries the activities of the first three enzymes (carbamyl phosphate synthetase II, aspartate transcarbamylase, and dihydroorotase) of de novo pyrimidine biosynthesis. Genomic fragments of the human CAD gene have been obtained by screening a human genomic library in bacteriophage lambda using a Syrian hamster cDNA clone as a probe. These human genomic clones have been used to assign the CAD gene to human chromosome 2 using in situ hybridization to human metaphase chromosomes and Southern blot hybridization analysis of DNA isolated from a panel of Chinese hamster/human hybrid cells. In situ hybridization analysis has allowed further localization of this gene to the chromosomal region 2p21-p22.

Molecular evolution of enzyme structure: construction of a hybrid hamster/Escherichia coli aspartate transcarbamoylase

SummaryAspartate transcarbamoylase (ATCase, EC 2.1.3.2) is the first unique enzyme common to de novo pyrimidine biosynthesis and is involved in a variety of structural patterns in different organisms. InEscherichia coli, ATCase is a functionally independent, oligomeric enzyme; in hamster, it is part of a trifunctional protein complex, designated CAD, that includes the preceding and subsequent enzymes of the biosynthetic pathway (carbamoyl phosphate synthetase and dihydroorotase). The complete complementary DNA (cDNA) nucleotide sequence of the ATCase-encoding portion of the hamster CAD gene is reported here. A comparison of the deduced amino acid sequences of the hamster andE. coli catalytic peptides revealed an overall 44% amino acid similarity, substantial conservation of predicted secondary structure, and complete conservation of all the amino acids implicated in the active site of theE. coli enzyme. These observations led to the construction of a functional hybrid ATCase formed by intragenic fusion based on the known tertiary structure of the bacterial enzyme. In this fusion, the amino terminal half (the “polar domain”) of the fusion protein was provided by a hamster ATCase cDNA subclone, and the carboxyl terminal portion (the “equatorial domain”) was derived from a clonedpyrBI operon ofE. coli K-12. The recombinant plasmid bearing the hybrid ATCase was shown to satisfy growth requirements of transformedE. coli pyrB− cells. The functionality of thisE. coli-hamster hybrid enzyme confirms conservation of essential structure-function relationships between evolutionarily distant and structurally divergent ATCases.

SITE-DIRECTED SUBSTITUTION OF SER1406 OF HAMSTER CAD WITH GLUTAMIC ACID ALTERS ALLOSTERIC REGULATION OF CARBAMYL PHOSPHATE SYNTHETASE II

Ser1406 of the allosteric region of the hamster CAD enzyme, carbamyl phosphate synthetase II (CPSase), is known to be phosphorylatedin vitro by cAMP-dependent protein kinase (PKA). Metabolic labeling experiments described here demonstrate that CAD is phosphorylated in somatic cells in culture. Phosphorylation is stimulated by treating cells with 8-bromo-cAMP, a PKA activator. The stimulation is essentially prevented by pretreatment with H-89, a PKA specific inhibitor. Substitution of Ser1406 with alanine results in an enzyme with kinetics and allosteric regulation indistinguishable from unsubstituted CAD. However, substitution to glutamic acid increases CPSase activity by reducing the apparent Km (ATP). The UTP concentration required to give 50% inhibition is increased rendering this altered enzyme significantly less sensitive to feedback inhibition, but allosteric activation by PRPP is unaffected. While these data do not prove that Ser1406 is phosphorylatedin vivo, they do indicate that a specific alteration at this residue can affect allosteric regulation.

Transcriptional regulation of the human CAD gene during myeloid differentiation.

CAD codes for a trifunctional protein involved in the catalysis of the first three enzymatic activities in the de novo pyrimidine biosynthetic pathway, namely, carbamoyl-phosphate synthetase II (EC 6.3.5.5), aspartate transcarbamylase (EC 2.1.3.2), and dihydroorotase (EC 3.5.2.3). CAD regulation was studied in the human promyelocyte leukemic line HL-60 as it differentiated into monocytic or granulocytic lineages after induction by 12-O-tetradecanoylphorbol-13-acetate or trans-retinoic acid and dibutyryl cyclic AMP, respectively. Within 12 h of induction of HL-60 cells with either inducer, total cellular levels of CAD RNA essentially disappeared. On the other hand, no apparent decreases in beta-actin RNA levels were seen even 48 h after HL-60 cells were induced, as compared with untreated cells. With nuclear runoff assays, it was clearly shown that the inactivation of CAD gene expression during the induction of HL-60 cells with either inducer was at the transcriptional level. The nuclear runoff experiments also demonstrated that the CAD gene expression was shut down in less than 4 h after induction, well before morphological changes were observed in these cells. At the enzymatic level, the activity of aspartate transcarbamylase, one of the three enzymes encoded by the CAD gene, decreased by about half within 24 h of induction, suggesting a CAD protein half-life of 24 h in differentiating HL-60 cells. Nevertheless, this means that significant levels of aspartate transcarbamylase activity remained even after the cells have stopped proliferating. From the RNA data, it is clear that CAD gene expression is rapidly turned off as promyelocytes begin to terminally differentiate into macrophages and granulocytes. We suspect that the inactivation of the CAD gene in induced HL-60 cells is a consequence of the differentiating cells leaving the cell cycle and becoming nonproliferating.

Evidence that mammalian glutamine-dependent carbamyl phosphate synthetase arose through gene fusion

SummaryOn the basis of homology, the mammalian CAD (glutamine-dependent carbamyl phosphate synthetase-aspartate transcarbamylase-dihydroorotase) gene appears to have arisen from the fusion of four separate ancestral genes. Evidence for two of these precursor genes is found in the carbamyl phosphate synthetase (CPSase) domain of CAD. In prokaryotes, such as Escherichia coli CPSase is encoded by two distinct cistrons of the carAB operon. Whereas carA and carB are separated by a short noncoding intercistronic region, the homologous sequences of the CAD gene encode an amino acid bridge. This bridge connects the subdomains of the CAD CPSase. We constructed a bacterial carAB fusion gene in which the intercistronic region codes for a hamster bridgelike sequence. The fused carAB gene directs the synthesis of a stable bifunctional polypeptide whose glutamine-dependent CPSase activity is comparable to the E. coli CPSase holoenzyme. The fusion in E. coli of the single gene counterparts of CAD demonstrates a potential model system to study the genetic events that lead to gene fusion and the creation of multienzymatic proteins.

Posttranscriptional regulation of the expression of CAD gene during differentiation of F9 teratocarcinoma cells by induction with retinoic acid and dibutyryl cyclic AMP

We have studied the regulation of expression of the carbamoyl‐phosphate synthetase II‐aspartate transcarbamylase‐dihydroorotase gene in F9 teratocarcinoma cells during their differentiation into parietal endoderm cells by induction with a combination of retinoic acid and dibutyryl cyclic AMP. Steady‐state levels of CAD mRNA decreased by 7‐fold in F9 cells following 120 h of retinoic acid and dibutyryl cyclic AMP induction as compared to levels in uninduced cells. Conversely, no apparent changes were found in the steady‐state levels of β‐actin mRNA between induced and uninduced cells. Despite a 7‐fold decrease in the steady‐state levels of CAD mRNA, its rate of transcription remained the same between induced and uninduced cells, indicating a role for posttranscriptional mechanisms for its down regulation during retinoic acid‐ and dibutyryl cyclic AMP‐induced differentiation of F9 cells. The cellular growth rate of F9 cells as determined by [3H]thymidine uptake and parallel cell counting decreased markedly during their induction with retinoic acid and dibutyryl cyclic AMP. Taken together, it is apparent that the expression of the CAD gene is cell‐growth‐dependent and its regulation in this system is at the posttranscriptional level.

Substitutions in hamster CAD carbamoyl-phosphate synthetase alter allosteric response to 5-phosphoribosyl-α-pyrophosphate (PRPP) and UTP

CPSase (carbamoyl-phosphate synthetase II), a component of CAD protein (multienzymic protein with CPSase, aspartate transcarbamylase and dihydro-orotase activities), catalyses the regulated steps in the de novo synthesis of pyrimidines. Unlike the orthologous Escherichia coli enzyme that is regulated by UMP, inosine monophosphate and ornithine, the mammalian CPSase is allosterically inhibited by UTP, and activated by PRPP (5-phosphoribosyl-a-pyrophosphate) and phosphorylation. Four residues (Thr974, Lys993, Lys954 and Thr977) are critical to the E. coli inosine monophosphate/UMP-binding pocket. In the present study, three of the corresponding residues in the hamster CPSase were altered to determine if they affect either PRPP activation or UTP inhibition. Substitution of the hamster residue, positionally equivalent to Thr974 in the E. coli enzyme, with alanine residue led to an enzyme with 5-fold lower activity and a near loss of PRPP activation. Whereas replacement of the tryptophan residue at position 993 had no effect, an Asp992-->Asn substitution yielded a much-activated enzyme that behaved as if PRPP was present. The substitution Lys954-->Glu had no effect on PRPP stimulation. Only modest decreases in UTP inhibitions were observed with each of the altered CPSases. The results also show that while PRPP and UTP can act simultaneously, PRPP activation is dominant. Apparently, UTP and PRPP have distinctly different associations within the mammalian enzyme. The findings of the present study may prove relevant to the neuropathology of Lesch-Nyhan syndrome