Species differences in hepatic peroxisome proliferation, cell replication and transforming growth factor-beta1 gene expression in the rat, Syrian hamster and guinea pig.

The objective of this study was to evaluate species differences in the hepatic effects of three potent rodent peroxisome proliferators, namely methylclofenapate (MCP), ciprofibrate (CIP) and Wy-14,643 (WY), particularly with respect to effects on replicative DNA synthesis and transforming growth factor-beta1 (TGF-beta1) gene expression. Male Sprague-Dawley rats, Syrian hamsters and Dunkin-Hartley guinea pigs were given daily oral doses of 0 (corn oil) and 75 mg/kg MCP for periods of 6 and 21 days. Syrian hamsters and guinea pigs were also treated with 25 mg/kg CIP and 25 mg/kg WY. Relative liver weights were significantly increased in peroxisome proliferator-treated rats and Syrian hamsters, but not in guinea pigs. Hepatic peroxisomal (palmitoyl-CoA oxidation) and microsomal (lauric acid 12-hydroxylase) fatty acid oxidising enzyme activities and CYP4A isoform mRNA levels were significantly increased in rats and Syrian hamsters, whereas only minor effects were observed in the guinea pig. Replicative DNA synthesis was studied by implanting 7-day osmotic pumps containing 5-bromo-2'-deoxyuridine during study days -1 to 6 and 14 to 21. Hepatocyte labelling index values were increased by MCP in the rat, but neither MCP, CIP nor WY produced any significant effect on replicative DNA synthesis in the Syrian hamster and guinea pig. MCP treatment increased TGF-beta1 and insulin-like growth factor II/mannose-6-phosphate (IGFII/Man6P) receptor gene expression in the rat. In the Syrian hamster, effects on TGF-beta1 and IGFII/Man6P receptor gene expression were also observed in some instances, whereas TGF-beta1 mRNA levels were essentially unchanged in the guinea pig. These results provide further evidence for marked species differences in response to rodent peroxisome proliferators. While peroxisome proliferators produce a wide spectrum of effects in rat liver, other species such as the Syrian hamster and guinea pig are less responsive and in the case of some endpoints (e.g., cell replication) may be refractory.

Effects of soy or rye supplementation of high-fat diets on colon tumour development in azoxymethane-treated rats.

Evidence is accumulating that a diet high in plant-derived foods may be protective against cancer. One class of plant component under increasing investigation is the phytoestrogens of which there are two main groups: the isoflavones, found mainly in soy products, and the lignans, which are more ubiquitous and are found in fruit, vegetables and cereals with high levels being found in flaxseed. In this study, we have used carefully balanced high-fat (40% energy) diets: a control diet (containing low isoflavone soy protein as the sole protein source), a rye diet (the control diet supplemented with rye bran) and a soy diet (containing as protein source a high isoflavone soy protein). The effect of these diets on the development of colonic cancer was studied in F-344 rats treated with the carcinogen, azoxymethane (two doses of 15 mg/kg given 1 week apart). Colons from treated animals were examined for aberrant crypt foci (ACF) and tumours after 12 and 31 weeks. Results after 12 weeks showed no differences in the total number of ACF in the control, soy or rye bran groups. However, the soy group had increased numbers of small ACF (less than four crypts/focus) while the rye group had decreased numbers of large ACF (greater than six crypts/focus). Examination of colons after 31 weeks gave similar low numbers of ACF in each group with no differences in multiplicity. There were no differences in the number of tumours between the control (1.36 tumours/rat) and soy (1.38 tumours/rat) groups. However, there was a significant decrease in the number of tumours in the rye group (0.17 tumours/rat). These results suggest that soy isoflavones have no effect on the frequency of colonic tumours in this model while rye bran supplementation decreases the frequency of colon cancer. This effect is due not to a decrease in early lesions but in their progression to larger multi-crypt ACF. The study also supports the hypothesis that larger ACF are more predictive of subsequent tumorigenicity.

Investigation of mutant frequency at the HPRT locus and changes in microsatellite sequences in healthy young adults.

In an attempt to understand the inter-individual variation that occurs in in vivo mutant frequency at the HPRT locus, we have examined the effect of polymorphisms in genes for metabolic enzymes on the mutation rate. In the same population of human volunteers, the background variant frequency in a number of microsatellite sequences was studied to determine individual variation in the capacity to repair mismatches in these sequences. The HPRT mutant frequency of T-cells isolated from a group of 49 healthy, non-smoking adults varied from 0.25 to 9.64 x 10(-6). The frequency of polymorphisms in CYP1A1, GSTM1 and NAT2 among these individuals was similar to those published, and when subjected to univariate analysis these polymorphisms showed no influence on the HPRT mutant frequency. However, there was a significant interaction between the GSTM1 null genotype and the slow acetylator status in NAT2 (P < 0.05) which was associated with higher mutant frequency. Analysis of 30 microsatellite sequences in 20 HPRT proficient clones per individual showed only six alterations in total, giving an overall mutation rate per allele of 0.01%, whilst three alterations were found in five HPRT deficient clones per individual examined for changes in 10 microsatellites, giving an overall mutation rate per allele of 0.3%. Thus, the alterations detected are probably due to background mutations and not to differences in mismatch repair capacity.

Long-term analysis of colonic aberrant crypt formation after treatment of Sprague-Dawley rats with azoxymethane.

Human colon cancer is a multistage disease which has been shown to have a number of well-defined histological and genetic events. This knowledge has identified a series of stages in the development of colon cancer in which dietary components and chemicals may play either a beneficial or detrimental role. Azoxymethane-induced colon cancer in the rat represents a way of investigating such effects on the temporal development of the disease. To assess the stages involved in the long-term development of colon cancer in this animal model, Sprague-Dawley rats were treated with either one or two (given 24 hours apart) doses of azoxymethane (15 mg/kg). These low doses were chosen in an attempt to mimic the slow development of the human disease. At varying time intervals (5-84 weeks) after treatment, animals were killed and their colons were examined for lesions. Evidence was found in the distal region of the colon of a progression from early alterations (aberrant crypt foci) to microadenomas and polyps. This progression occurs in the region where carcinomas were found. The best correlation with tumorigenicity was the multiplicity of the crypts in each focus rather than simply the number of aberrant crypt foci. The aberrant crypts were microdissected from the colon and DNA was prepared. The following genes were screened for mutation using polymerase chain reaction with single-strand conformation polymorphism, oligonucleotide hybridisation, restriction site changes and sequencing: Ki-ras (exons 1 and 2), p53 (exons 5, 6, and 7 which correspond to exons 5-8 in humans), and APC (exon 15 corresponding to the mutation cluster region in humans). Extensive studies of the aberrant crypt foci formed revealed no mutations in these lesions. These results suggest that the aberrant crypt focus may be a useful short-term preneoplastic marker. However, it is clear from this and other studies that the genetic progression in the rat may vary according to the treatment regimen used and differs from that found in human. Key genes in the development of colon cancer in the rat remain to be elucidated.

Detection of CYP1A1 mRNA levels and CYP1A1 Msp polymorphisms as possible biomarkers of exposure and susceptibility in smokers and non-smokers.

It is important to determine sensitive biomarkers for both exposure and susceptibility since differences in individual susceptibility to potentially hazardous chemicals may represent a major variable in the assessment of risk. Induction of cytochrome P450IA1 (CYP1A1) may be a measure of environmental exposure to aromatic hydrocarbons in cigarette smoke. This study investigated the use of reverse transcriptase-polymerase chain reaction (RT-PCR) to detect constitutive levels of CYP1A1 mRNA in the peripheral lymphocytes of a population of smokers and non-smokers as a potential marker of exposure. In addition, the presence of an Msp 1 restriction fragment length polymorphism was analyzed using a simple PCR method as a biomarker for susceptibility. DNA and RNA were isolated from the peripheral lymphocytes of 20 smokers and a matched group of non-smokers. RT-PCR was used to detect the endogenous levels of CYP1A1 mRNA with glyceraldehyde-3-phosphate dehydrogenase as a control gene. The 3'-region of CYP1A1 gene was amplified by PCR and underwent restriction digestion with Msp 1 to detect the polymorphism. The endogenous CYP1A1 expression as detected by RT-PCR was very low and variable and there was a slight but not significant increase in the smokers by comparison with non-smokers. Thirty-two of the volunteers were homozygous for the normal allele while 8 were heterozygous for the uncommon Msp 1 allele and none was homozygous for the polymorphism. The allele frequency (0.1) was found to be in Hardy-Weinberg equilibrium. Since only a slight increase was seen in endogenous CYP1A1 mRNA levels in the peripheral lymphocytes of smokers by comparison with non-smokers, the effect may have been diluted by variation in sensitivity to dose, a threshold of exposure effect, or the return of mRNA to baseline between exposures. The wide variation in mRNA levels may reflect the influence and exposure of different environmental factors. The sensitivity of PCR-based methods suggests that they may have an important role in future overall biomonitoring of exposure and susceptibility to environmental chemicals.

Excitatory amino acid-induced cytotoxicity in primary cultures of mouse cerebellar granule cells correlates with elevated, sustained c-fos proto-oncogene expression.

An elevated, sustained expression of c-fos mRNA was found in primary cultures of mouse cerebellar granule cells following exposure to toxic concentrations of the excitatory amino acids, L-glutamate, L-homocysteate, S-sulpho-L-cysteine and N-methyl-D-aspartate (NMDA), using leakage of lactate dehydrogenase (LDH) as an indicator of cytotoxicity. In contrast, when used at non-toxic concentrations these compounds induced a rapid and transient increase in c-fos mRNA levels. Both LDH release and elevated, sustained c-fos mRNA induction were blocked (in the case of L-homocysteate) or reduced (in the case of L-glutamate and S-sulpho-L-cysteine) by the selective NMDA receptor antagonist (DL(+/-)-2-amino- 5-phosphonopentanoic acid) whereas 6-cyano-7-nitroquinoxaline-2,3-dione (a selective antagonist at non-NMDA ionotropic receptors) had no effect. These data suggest a role for altered c-fos mRNA expression in excitotoxic mechanisms.

Molecular analysis of chemically-induced mutations in mammalian cell assays.

Two mammalian cell mutation assays, the HPRT/V79 assay and the TK/mouse lymphoma assay, were compared for their ability to respond to the genotoxic chemicals ethyl methanesulfonate (EMS) and mitomycin C (MMC). Whereas EMS induced a high mutant frequency at both loci, MMC produced few mutants at the hprt locus, but induced a large number of mutants at the tk locus. Southern blotting analysis showed that this difference was due to the type of genetic damage induced by the two chemicals. Intragenic changes ranging from point mutations to loss of the entire gene were recovered as viable mutants at both the hprt and tk loci. Thus, EMS which causes mainly intragenic mutations induced similar mutant frequencies at both loci. The large multilocus deletions induced by MMC, in which the damage was assumed in many cases to extend into a gene essential for growth since most TK mutants were slow-growing, could not be recovered at the hprt locus. Whereas both loci will detect intergenic mutations, mutants carrying large-scale damage are recoverable only at the heterozygous tk locus. At the hemizygous hprt locus no homologous chromosome exists to provide the function of essential genes if these are lost along with hprt in multilocus deletions. Most human cancers develop as a highly complex process involving both gene and multilocus mutations in oncogenes and tumour suppressor genes. Thus the TK/mouse lymphoma assay is a more appropriate in vitro test for the detection of chemicals capable of causing the types of DNA lesions important in human cancer.

Growth characteristics and Ha-ras mutations of cell cultures isolated from chemically induced mouse liver tumours.

Cells have been isolated from liver tumours that have arisen in control C3H/He mice, in mice given 10 micrograms diethylnitrosamine (DEN) during the neonatal period or in mice given a diet containing phenobarbitone (PB) to allow a daily intake of 85 mg/kg/day. The cells were grown to the 8 degrees subculture when their growth characteristics were investigated in monolayer culture and following suspension in soft agar and on transplantation into nude mice. In addition, DNA was isolated from the cultures and from tumours that grew in nude mice and analysed for mutations at codon 61 of the Ha-ras oncogene. All cells derived from DEN-induced hepatocellular carcinomas (HCC) demonstrated a lack of density inhibition of growth in monolayer culture, grew in soft agar and formed tumours in nude mice with an average mean latency of 29 days. Three of the seven lines showed mutations in Ha-ras: two were CAA-->AAA transversions and one showed a CAA-->CTA transversion. In contrast, cells isolated from eosinophilic nodules in mice given PB showed inhibition of growth at confluence, did not grow in soft agar and only four of eight formed tumours in nude mice with a mean average latent period of 181 days. Cells grown from HCC in mice given PB showed a lack of density inhibition of growth, however, they did not grow in soft agar nor did they form tumours in nude mice. A single spontaneous HCC from a control mouse showed a similar growth pattern to HCC cells isolated from mice given PB. Cells from a basophilic nodule, taken from a control untreated mouse grew vigorously in culture and in soft agar and formed tumours in nude mice with a latency of 6 days. None of the cells isolated from control mice or from mice given PB showed evidence of mutations at codon 61 of Ha-ras. These data confirm that there are fundamental differences in the biology of cells grown from tumours that develop in mice under different treatment regimes. These studies also demonstrate the utility of cell culture and molecular biology in addressing the fundamental mechanism of mouse hepatic neoplasia.

Effect of some peroxisome proliferators on transforming growth factor-beta 1 gene expression and insulin-like growth factor II/mannose-6-phosphate receptor gene expression in rat liver.

Male Sprague-Dawley rats were given daily oral doses of either corn oil (control), 80 mg/kg nafenopin (NAF), 50 mg/kg methylclofenapate (MCP), 50 mg/kg Wy-14,643 (WY) or 250 mg/kg clofibric acid (CA) for 7 days. All four compounds increased relative liver weight and produced hepatic peroxisome proliferation as assessed by induction of both peroxisomal (palmitoyl-CoA oxidation) and microsomal (lauric acid 12-hydroxylase) fatty acid oxidising enzyme activities. RNA was extracted from liver samples and analysed for expression of transforming growth factor-beta 1 (TGF-beta 1) and the insulin-like growth factor II/mannose-6-phosphate (IGFII/Man6P) receptor (which may be involved in transporting latent TGF-beta 1 into hepatocytes). TGF-beta 1 mRNA levels were increased to 151-178% of control by all four compounds, whereas NAF, MCP and WY, but not CA, increased IGFII/Man6P receptor mRNA levels to 195-209% of control. The induction of TGF-beta 1 and IGFII/Man6P receptor expression by short term treatment with peroxisome proliferators may represent an adaptive response to limit the initial hyperplastic effects of such compounds.

Screening for p53 mutations in C3H/He mouse liver tumors derived spontaneously or induced with diethylnitrosamine or phenobarbitone.

Distinct differences have been described in the development of C3H/He mouse liver tumors induced by the genotoxic carcinogen diethylnitrosamine (DEN) and by the nongenotoxic agent phenobarbitone (PB) in terms of pathology and the frequency of mutation at codon 61 of the Ha-ras oncogene. To further define the mechanisms involved, we screened the tumor suppressor gene p53 for mutations in exons 5, 7, and 8 using polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analysis. Nearly all the mutations so far described have been found within these three exons. In this study a total of six spontaneous tumors, eight tumors induced by PB, 14 tumors induced by DEN, and five samples of normal liver tissue were screened, and no mutations were found in any of the tumors examined. The positive control, the plasmid LTRp53cG (val), had a point mutation in exon 5 that was detected by PCR-SSCP. Since many of the tumors were late-stage hepatocellular carcinomas, we concluded that mutations in exons 5, 7, and 8 of the p53 gene do not play an important role in the development of chemically induced liver tumors in the C3H/He mouse.

Molecular analysis of mutations at the tk locus of L5178Y mouse-lymphoma cells induced by ethyl methanesulphonate and mitomycin C.

Mutations at the tk locus of mouse-lymphoma L5178Y cells were induced by treatment with ethyl methanesulphonate (EMS), primarily a point mutagen and mitomycin C (MMC), a potent clastogen. Mutant colony size was distinctly bimodal with 35% of spontaneous mutants growing as small colonies and 65% large. The proportion of small colonies increased only slightly to 41% in EMS-treated cultures but to 64% after MMC treatment. Mutations were analysed by Southern and Northern blotting. Digestion of DNA with the restriction enzyme, Nco I, revealed that many mutants had lost a 6.3-kb fragment which constituted the loss of the entire tk gene. Almost all of the EMS-induced large-colony mutants analysed (9/10) retained the tk+ allele suggesting the presence of an intragenic mutation. Of the small-colony mutants, half (6/12) had lost the tk+ gene and presumably other genes affecting growth and half retained the tk+ allele suggesting point mutations in both the tk gene and other sites in the genome affecting growth. A very different spectrum of mutation was induced with MMC. Only 1/12 of the large-colony mutants were due to intragenic mutation, the remaining large-colony mutants having lost the tk+ allele while all the small-colony mutants had lost the tk+ gene presumably with the deletion extending to genes essential for normal growth. Northern blot analysis showed no changes in the size of tk transcript in any mutants. Alterations in the amount of tk mRNA were not detectable since all mutants produced an mRNA of similar size and amount, which may indicate the production of an abnormal mRNA from the tk- allele. Unlike cell-mutation assays that use hemizygous loci (such as hprt+/0) for detecting potential chemical carcinogens, the mouse-lymphoma tk+/- assay allows the recovery of both intragenic and intergenic mutations thus enabling the detection of both point mutagens such as EMS and potent clastogens like MMC.

Search for Ha-ras codon 61 mutations in liver tumours caused by hexachlorobenzene and Aroclor 1254 in C57BL/10ScSn mice with iron overload.

C57BL/10ScSn mice administered iron--dextran and fed the environmental pollutants hexachlorobenzene (HCB) and polychlorinated biphenyls (PCBs) develop hepatic nodules and carcinomas within 18 months. A range of lesions from the livers were analysed for the presence of mutations in the Ha-ras proto-oncogene at codon 61 using the polymerase chain reaction to amplify DNA from formalin-fixed sections, followed by oligonucleotide hybridization. Only two mutations from 23 preneoplastic and neoplastic lesions induced by HCB were detected (a focus of altered cells and a trabecular cell carcinoma). With Aroclor 1254 no mutations were detected in 28 areas at various stages of carcinogenesis analysed. Sequencing of the two mutations generated by HCB showed a C-->T transversion at the first base of codon 61 (carcinoma) and an A-->T transversion at the second base (proliferative focus). Thus, in marked contrast to some other systems of mouse liver tumour induction, hepatocarcinogenesis caused by HCB and PCBs in C57BL/10ScSn mice is an example of carcinogenesis which does not involve a high frequency of Ha-ras gene mutation at codon 61.

Analysis of the Ha-ras oncogene in C3H/He mouse liver tumours derived spontaneously or induced with diethylnitrosamine or phenobarbitone.

In a study of the mechanisms involved in the induction of tumours by chemicals, the Ha-ras oncogene was analysed in liver tumours induced by the genotoxic carcinogen diethylnitrosamine (DEN), or the non-genotoxic agent phenobarbitone (PB) in C3H/He mice. Mutations were detected using the polymerase chain reaction and oligonucleotide hybridization. Codon 61 mutations were detected in 41% of DEN-induced tumours (19/46), either in the first base (CG----AT, 12/19), a transversion, or the second base (AT----GC, 7/19), a transition. Codon 61 mutations were also found in 29% of spontaneous tumours (all CG----AT, 6/21) but none were detected in PB-induced tumours (0/15) or in normal liver tissue of untreated mice (0/30). No mutations were detected at codon 12. Low and variable expression of the Ha-ras gene was detected in all liver tissues with moderately raised levels (175-200%) in spontaneous, DEN and PB-induced tumours as compared to normal liver tissue. The H-ras gene was methylated to some extent in all liver tissues, with no discernible difference between the treatments. The frequency of the Ha-ras mutation at codon 61 in DEN-induced tumours is greater than in spontaneously arising tumours. This increase is not accompanied by any specific alteration in the expression or methylation of the gene. Since PB-induced tumours do not possess mutations in the Ha-ras gene at codons 12 or 61, the data suggest that the non-genotoxic agent PB induces tumours in the C3H/He mouse liver with a mechanism distinct from that of spontaneous tumours or those that result from treatment with a potent genotoxic carcinogen such as DEN.

An increase in plasminogen activator mRNA occurs at an early stage in ethylnitrosourea-induced transformation of rat brain cells.

Brain tumor cells cultured after transplacental induction by the nitrosamide, N-ethyl-N-nitrosourea had a higher level of plasminogen activator activity than control cells from adult rat brain. Cultures (BE10) derived 2 days after exposure to the carcinogen showed a rise in this proteolytic activity at the 17th passage but were not able to form colonies in agar or tumours in syngeneic rats until passages 44/45. Cultures (BE11) derived 2 days after exposure to buffer did not show a rise in this enzyme activity nor were they able to grow in agar or animals at comparable passages. Zymography and inhibition studies showed that the enzyme produced by the tumour cells was related to human tissue-type plasminogen activator rather than to urokinase. Northern blot analysis showed a higher level of tissue plasminogen activator related mRNA in tumour cells than control cells. There was an increase in mRNA level during passaging of the carcinogen-exposed culture, BE10, which correlated with the increased enzyme activity. There was no rise in the barely detectable mRNA levels of the comparable buffer-exposed culture, BE11. The results suggest that alteration in the transcriptional control of this proteolytic enzyme occurs at an early stage in the transformation process.

Organization of a multifunctional protein in pyrimidine biosynthesis. A domain hypersensitive to proteolysis.

When the multifunctional protein that catalyses the first three steps of pyrimidine biosynthesis in hamster cells is treated with staphylococcal V8 proteinase, a single cleavage takes place. The activities of carbamoyl-phosphate synthetase (EC 6.3.5.5), aspartate carbamoyltransferase (EC 2.1.3.2) and dihydro-orotase (EC 3.5.2.3) and the allosteric inhibition by UTP are unaffected. One fragment, of Mr 182000, has the first and third enzyme activities, whereas the other fragment, of Mr 42000, has aspartate carbamoyltransferase activity and an aggregation site. A similar small fragment is observed in protein digested with low concentrations of trypsin. A similar large fragment is seen after digestion with trypsin and as the predominating form of this protein in certain mutants defective in pyrimidine biosynthesis. These results indicate that a region located adjacent to the aspartate carbamoyltransferase domain is hypersensitive to proteinase action in vitro and may also be sensitive to proteolysis in vivo.

Effects of caffeine on pyrimidine biosynthesis and 5-phosphoribosyl 1-pyrophosphate metabolism in Chinese hamster cells.

Caffeine, at doses which enhance killing by UV light, inhibits the biosynthesis of pyrimidines in Chinese hamster ovary cells (K1) in culture. This inhibition was measured as a decrease in [14C]UTP and [14C]CTP accumulation after a 3-h incubation with [14C]aspartate or [14C]orotate and a similar decrease in Urd-A cells (which lack the first three enzymes of the pathway) using [14C]orotate as substrate. There was no such inhibition in Urd-C cells (which lack the last two enzymes) using [14C]aspartate as substrate and measuring accumulation of orotate. There is some inhibition of the fifth enzyme of the pathway, orotate phosphoribosyltransferase by caffeine in vitro and this is most striking at low 5-phosphoribosyl 1-pyrophosphate concentrations. The level of 5-phosphoribosyl 1-pyrophosphate is decreased in Chinese hamster ovary K1 cells by about 20% after 3 h and by about 70% after 16 h in the presence of caffeine. It is suggested that inhibition of pyrimidine biosynthesis by caffeine over a 16-h period may be due mainly to decreased intracellular 5-phosphoribosyl 1-pyrophosphate levels but that in the decrease in pyrimidine accumulation over 3 h, direct inhibition of orotate phosphoribosyltransferase by caffeine may also play a role.

Organization of a multifunctional protein in pyrimidine biosynthesis. Analyses of active, tryptic fragments.

The multifunctional protein which catalyzes the first three steps of pyrimidine biosynthesis in hamster cells can be cleaved by trypsin into enzymatically active fragments. When the fragments are separated by nondenaturing polyacrylamide gel electrophoresis, three major polypeptide bands appear. Carbamyl phosphate synthetase (EC 2.7.2.9), aspartate transcarbamylase (EC 2.1.3.2), and dihydroorotase (EC 3.5.2.3) activities are associated with 129,000-, 660,000-, and 94,000-dalton bands, respectively. Further analysis of these fragments by denaturing polyacrylamide gel electrophoresis has shown that the aspartate transcarbamylase band seen on the nondenaturing gel is actually a large aggregate of 39,000-dalton fragments and the dihydroorotase band is a dimer of 44,000-dalton fragments. The data reported here indicate that (i) this multifunctional protein is composed of three enzymatically independent domains, (ii) the sum of the molecular weights of these three domains (129,000 + 39,000 + 44,000 = 212,000) is similar to that of the undigested monomer (220,000 daltons), and (iii) a site important to the formation of the native multimeric protein is probably near the aspartate transcarbamylase domain.