In a search for genes that regulate circadian rhythms in mammals, the progeny of mice treated with Nethyl-N-nitrosourea (ENU) were screened for circadian clock mutations. A semidominant mutation, Clock, that lengthens circadian period and abolishes persistence of rhythmicity was identified. Clock segregated as a single gene that mapped to the midportion of mouse chromosome † To whom correspondence should be addressed. * Present address: Department of Biological Sciences, Wichita State University, Wichita, KS 67260, USA.Published as: Science. 1994 April 29; 264(5159): 719-725. HHMI Author Manuscript HHMI Author Manuscript HHMI Author Manuscript5, a region syntenic to human chromosome 4. The power of ENU mutagenesis combined with the ability to clone murine genes by map position provides a generally applicable approach to study complex behavior in mammals.Progress has been made at the physiological and cellular levels in our understanding of circadian systems (1), yet the molecular mechanism of circadian clocks has not been fully elucidated (2). The isolation of "clock mutants" and the widespread requirement for protein synthesis in circadian clock systems imply that gene expression is an integral component of the oscillator (2). Recent molecular work with the Drosophila period (per) and Neurospora frequency (frq) genes suggests that a circadian cycle of per and frq transcription, respectively, may lie at the heart of the oscillator mechanism in these species (3). However, no information exists concerning the molecular elements of the clock system in mammals. In the absence of specific mechanistic information, genetics has been a powerful approach to uncover unknown elements. We report here the isolation of a mutation in the mouse that changes two central properties of circadian rhythms: the intrinsic period length and the persistence of rhythmicity. Taken together, our results define a gene, named Clock (for circadian locomotor out-put cycles kaput) that is essential for normal circadian behavior.Because the majority of clock mutants isolated in other organisms have been semidominant (4), we screened heterozygotes directly in the mouse. With the mutagen ENU, average forward mutation frequencies of 0.0015 per locus per gamete (1 in 700) can be achieved in the mouse (5). Male mice of the inbred strain C57BL/6J (B6) were treated with a single injection of ENU and after recovery of fertility were mated with untreated B6 females (6). First generation (G1) offspring would be heterozygous for any induced mutations but otherwise possess an isogenic B6 background (Fig. 1A). Normal B6 mice exhibit a robust circadian rhythm of wheel-running activity; we used this behavioral assay to screen for circadian mutants (Fig. 1B). Activity rhythms were monitored during exposure to a lightdark cycle (LD) to assess synchronization or entrainment behavior and in constant darkness (DD) to determine the circadian period of the locomotor activity rhythm (7). Laboratory mice typically have circadian periods of less than 24 hours, w...

Treatment with N-ethyl-N-nitrosourea (ENU) efficiently generates single-nucleotide mutations in mice. Along with the renewed interest in this approach, much attention has been given recently to large screens with broad aims; however, more finely focused studies have proven very productive as well. Here we show how mutagenesis together with genetic mapping can facilitate the rapid characterization of recessive loci required for normal embryonic development. We screened third-generation progeny of mutagenized mice at embryonic day (E) 18.5 for abnormalities of organogenesis. We ascertained 15 monogenic mutations in the 54 families that were comprehensively analyzed. We carried out the experiment as an outcross, which facilitated the genetic mapping of the mutations by haplotype analysis. We mapped seven of the mutations and identified the affected locus in two lines. Using a hierarchical approach, it is possible to maximize the efficiency of this analysis so that it can be carried out easily with modest infrastructure and resources.

Large neutral amino acids (LNAAs) have been used on a limited number of patients with phenylketonuria (PKU) with the purpose of decreasing the influx of phenylalanine (Phe) to the brain. In earlier studies on mice with PKU (ENU(2)/ENU(2)), LNAAs were given and a surprising decline in blood Phe concentrations was observed. The formula used in the mouse experiment (PreKUnil) lacked lysine. Therefore, a new formulation of LNAAs (NeoPhe) was developed, introducing changes in the concentration of some amino acids and adding lysine, so that such a mixture could be used in humans. The new formula was found to be effective in reducing blood Phe concentration in mice by about 50% of the elevated levels. Patients with PKU were given LNAAs and blood Phe concentrations were determined in an open-label study. Three centers--in Russia, the Ukraine and the USA--took part in the study. NeoPhe was given at 0.5 g/kg per day in three divided doses to eight subjects with PKU and at 1.0 g/kg per day to three patients, for one week. The NeoPhe resulted in decrease of elevated blood Phe by 50% in both groups. The preliminary data from this study are encouraging and a double blind placebo-controlled trial will be required to show long-term efficacy and tolerance of LNAAs in the treatment of PKU.

Mutant mice exhibiting heritable hyperphenylalaninemia have been isolated after ethylnitrosourea mutagenesis of the germ line. We describe one mutant pedigree in which phenylalanine hydroxylase activity is severely deficient in homozygotes and reduced in heterozygotes while other biochemical components of phenylalanine catabolism are normal. In homozygotes, injection of phenylalanine causes severe hyperphenylalaninemia and urinary excretion of phenylketones but not hypertyrosinemia. Severe chronic hyperphenylalaninemia can be produced when mutant homozygotes are given phenylalanine in their drinking water. Genetic mapping has localized the mutation to murine chromosome 10 at-or near the Pah locus, the structural gene for phenylalanine hydroxylase. This mutant provides a useful genetic animal model affected in the same enzyme as in human phenylketonuria.Disorders ofphenylalanine catabolism, resulting in phenylketonuria (PKU) and hyperphenylalaninemia (HPH), were among the first heritable errors of metabolism discovered in the human (1). The rate-limiting step in mammalian phenylalanine catabolism is hydroxylation to produce tyrosine. This reaction, catalyzed by phenylalanine hydroxylase (PAH) (2), requires the reduced pteridine cofactor tetrahydrobiopterin (3), which is synthesized from GTP (4) through a number of intermediates and is maintained in its reduced form by quinonoid dihydropteridine reductase (q-DHPR) (5). Mutations reducing the activity of PAH, q-DHPR, or the enzymes involved in tetrahydrobiopterin synthesis result in HPH because of a block in phenylalanine hydroxylation (6). In humans, PKU is defined as a condition resulting from mutations that abolish or severely reduce PAH activity (7). Other defects in phenylalanine catabolism are termed HPH. Extensive research has been undertaken to characterize these disorders (early work is reviewed in refs. 8 and 9 with recent summations in refs. 10 and 11). Laboratory mice with defined PKU and HPH mutations would be helpful in evaluating features of these diseases by permitting investigations not acceptable with human subjects. To produce such mutants we have used the alkylating agent N-ethyl-N-nitrosourea, which induces mutations in the mouse germ line at a frequency near 10-3 per locus (12). We refer to all mutants with deficiencies in phenylalanine catabolism by their common phenotype, HPH. We have screened the progeny of 347 gametes and have isolated four mutant pedigrees exhibiting the HPH phenotype. One of these, HPH-1, was detected by its neonatal HPH phenotype and is deficient in GTP cyclohydrolase activity (13)(14)(15). The others were detected by their impaired ability to clear a phenylalanine challenge (described in ref. 13). One of these, HPH-5, we now report to be deficient in PAH. MATERIALS AND METHODSBiochemical Determinations. Liver homogenates were prepared for PAH assay as described in ref. 16 methyltetrahydropterin, and the enzyme extract. The background rates of NADH oxidation were determined in the absence ofphenylalanine and ind...

1. Measurements were made of the nitrogen and energy balances of pigs of 30, 60 and 90 kg given a conventional diet at various daily rates. 2. Body protein synthesis was estimated from the irreversible loss of leucine from the blood following the infusion of [1-14C]leucine, and from the oxidation of the labelled amino acid. 3. Protein synthesis (g/d) increased by 2.17 for each 1 g increase in daily protein accretion and by 1.55 for each 1 g increase in daily protein intake. 4. At 30 kg, pigs close to energy equilibrium synthesized 270 g protein daily compared with 406 g and 512 g when their ration supplied twice and three times their maintenance requirement. 5. There was a close correlation between the daily urinary excretion of urea + ammonia and total amino acid catabolism estimated from the catabolism of leucine, but the latter underestimated the observed excretion by 2.5 g N/d. 6. The results imply that protein turnover accounts for only a proportion of the heat production associated with protein deposition.

1. The relationships between the intakes of protein and of non-protein energy (NPE), nitrogcn retention and body protein synthesis have been studied in female pigs weighmg 30 and 35 kg.2. Four animals were assigned to three regimens and given a conventional (basal) diet supplemented with fat, carbohydrate or protein. After 1 week, measurements of N excretion in urine and faeces (7 d collection) and gaseous exchange (3-4 d) were made. At the end of the balance period a solution of [l-14C]leucine was infused at a constant rate. Body protein synthesis was then calculated as the difference between the apparent irreversible loss of blood leucine and the loss of "C in expired air.The animals were then offered the basal diet without supplement for 10 d and the measurements of N retention, energy retention and protein synthesis were repeated.3. The intakes of metabolizable energy (ME; MJ/kg body-weight (W)0'76 per d) were 1.75 for fat, 1.58 for carbohydrate, 1-25 for protein and 1.18 for the basal diet; corresponding intakes of apparently digestible N (ADN; g N/kgWo'6 per d) were 2.30,2.31,4.35 and 2-17. Daily N retention, which during the period of basal feeding was 13.6 g was increased by between 3.4 and 7.2 g by the supplements. Daily fat deposition was also increased in the animals that received the diets supplemented with carbohydrate and fat.4. The rate of leucine catabolism was significantly reduced in the animals receiving the diets that were supplemented with W E and increased by the addition of protein to the diet.5. When based on the spec& radioactivity of blood leucine both the synthesis and breakdown of body protein (per unit metabolic body-weight) were increased by 30% in the animals receiving the high-protein diet but the increases in protein synthesis associated with the addition of carbohydrate (+ 14%) and fat (+ 12%) were much less marked. Consideration of these results together with previous observations (Reeds et uf. 1980) suggested that body protein synthesis (g N/d) increased by 0.88 for each g increase in daily ADN and by 0.93 for each MJ increase in daily ME intake. 6. Comparison of the results obtained with the animals given high-carbohydrate diets and those given high-protein diets suggested an increase in heat production of 14 KJ/g of additional fat deposition. A similar comparison of animals receiving the high-protein and basal diets suggested a heat increment of 233KJ/g additional protein deposition. The changes in heat production and protein synthesis in the animals given the protein supplement were compatible with a heat increment of 5.3 KJ/g additional protein synthesized. Because of the large proportion of heat production associated with the deposition of fat this could not be confirmed with either of the other supplements, but it is possible that the energy cost of protein accretion varies with the relative proportions of protein and NPE in the diet. In growing pigs (Reeds et al. 1980) and children (Golden et al. 1977) increases in N retention associated with increases in food intake a...

Phenylketonuria (PKU) results from a deficiency in phenylalanine hydroxylase, the enzyme catalyzing the conversion of phenylalanine (PHE) to tyrosine. Although this inborn error of metabolism was among the first in humans to be understood biochemically and genetically, little is known of the mechanism(s) involved in the pathology of PKU. We have combined mouse germline mutagenesis with screens for hyperphenylalaninemia to isolate three mutants deficient in phenylalanine hydroxylase (PAH) activity and cross-reactive protein. Two of these have reduced PAH mRNA and display characteristics of untreated human PKU patients. A low PHE diet partially reverses these abnormalities. Our success in using high frequency random germline point mutagenesis to obtain appropriate disease models illustrates how such mutagenesis can complement the emergent power of targeted mutagenesis in the mouse. The mutants now can be used as models in studying both maternal PKU and somatic gene therapy.

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