Hph-1: a Mouse Mutant with Hereditary Hyperphenylalaninemia Induced by Ethylnitrosourea Mutagenesis

hph-1: A Mouse Mutant With Hereditary Hyperphenylalaninemia Induced by Ethylnitrosourea Mutagenesis

Vernon C. Bode,* J. David McDonald,* Jean-Louis Guenetzf and Dominique Simonzf *Division of Biology, Kansas State University, Manhattan, Kansas 66506, and fUnilC de GCnCtique des Mummifires, Institut Pasteur de Paris, 75105 Paris, France Manuscript received August 5, 1987 Revised copy accepted October 22, 1987

ABSTRACT Ethylnitrosourea mutagenesisof spermatogonial stem cells and a three-generationbreeding scheme were used to screen for recessive mutations that cause defects in phenylalanine metabolism leading to elevated serum levels of this amino acid. This paper describes the isolation of such a mutation, hph-1, causing a heritable hyperphenylalaninemia in the neonate and weanling and an inability to effectively clear a phenylalanine challenge in the adult. Micro-pedigree analysis of the original mutant mouse and data obtained from crosses of affected and unaffected animals indicate that the mutation segregates in an autosomal recessive manner. An interspecies mouse backcross mapping experiment places the mutant gene locus on mouse chromosome 14 very near Np-1 and a backcross experiment with a conventional inbred mouse strain involving a nearby locus confirms the chromosome 14 assignment. The initial symptomatology of the mutant phenotype suggests this mutant may represent a useful animal model for the study of hyperphenylalaninemia in man.

HE availability of mutants and the possibility of The most common HPA syndrome in humans is T generating additional ones is an extremely use- referred to as classical phenylketonuria (PKU) and is ful, if not essential, element of any research program a heritable metabolic disorder that results in a defi- aimedat understanding particular biological path- ciency of the enzyme phenylalanine hydroxylase ways. The spectrum of mammalian mutants currently (PAH) (EC 1.14.16.1) (SCRIVER and Ctow1980). In available is however relatively limited and such mu- untreated patients,the clinical picture usually in- tants are complex and poorly understood. cludesirreversible impairment of postnatalbrain The report of an unusually efficient chemical mu- development and mental retardation of variable se- tagen of male mouse spermatogonial cells, N-ethyl- verity. The disease is transmitted as an autosomal N-nitrosourea (ENU) (RUSSELLet al. 1979) led several recessive trait whose frequency varies considerably investigators to initiate experiments designed ateval- between populations. In the United States the allele uating its potential for obtaining specific mutants frequency has been estimated at 0.01 (TOURIANand needed for bothbasic and applied research. An initial SIDBLRY1983). Other HPA syndromes, though less set of experiments demonstrated that this approach frequentlyencountered in thehuman population, was useful forinducing mutations throughout the also are accompanied by neurological disorders. mousegenome (JOHNSON andLEWIS 1981). Later Due to the absence of a genetically based animal experiments demonstrated the efficacy of ENU mu- model for HPA, considerable efforts have gone into tagenesis for producing mutants in specific areas of developing chemically induced models (BERRYet al. the mouse genome (k,T/t region of mouse chro- 1975; DELVALLE,DIENEL and GREENGARD1978; WAP- mosome 17) (BODE 1984; SHEDLOVSKYet al. 1986; NIR and MOAK1979; COTTON1986). Although help- JUSTICE and BODE1986). ful in some regards, the use of chemical inhibitors This paper further documents thepracticality and leads to some uncertainty in data interpretation due power of ENU mutagenesis. It describes the induc- to possible secondary effects. We think thatthe tion, isolation and genetic characterization of a mu- mutation we report here may provide a useful model tation causing hyperphenylalaninemia (HPA) in the for studies in comparative pathology, therapeutics mouse that may provide an animalmodel for the and disease prevention without these inherent study of HPA in humans (for a general reviewof uncertainties. human HPA syndromes see TOURIANand SIDBURY 1983). This recessive mutation has been designated MATERIALS ANDMETHODS hyperphenylalaninemia, symbol hph and, since several other genes are able to produce similar pheno- Mouse strains: Mutagen-treated males were (C57BW6 x CBA/Ca)FI animals and were crossed to females of the types due to the rather complex metabolic pathway same genotype. While there are advantages for subsequent involved, we have provisionally ranked this mutation physiological and molecular studies accruing from inducing hph-1 . mutations in a pure inbred genetic background, we reluc-

Genetics 118: 299-305 (February, 1988) 300 V. C. Bode et al. tantly sacrificed these in this experiment to profit from the differ at many coat color, biochemical, and antigenic loci greater fecundity thatmutagen-treated F, males exhibit in addition to restriction enzyme cleavage sites it is possible compared to thecomponent inbred strains. Such males to explore a large portion of the mouse chromosome map also tolerate higher doses of ENU without being perma- using a single backcross (BONHOMME, MARTINand THALER nently sterilized or killed. In terms ofDNA sequence 1978). Seventy offspring were typed for 44 segregating loci analysis, however this means that the mutation reported (BONHOMMEet al. 1984) as wellas for theirserum PHE here could have been induced in either C57BL/6 or CBA/ concentration (GUTHRIE andSusr 1963) allowing their clas- Ca chromatin. sification as either homozygotes or heterozygotes for the Mutagenesis: Stock ENU (purchasedfrom Bioclinical M. m. domesticus variant of the polymorphic loci and for the Laboratories, Bohemia, New York) was assayed for activity hph-f mutation. Also, blots of genomic restriction enzyme by the colorimetric method of PACHOLECand CHAPMAN digests of interspecific backcross animals were hybridized (1985) (data not shown). Males were treated with mutagen with a rat PAH cDNA clone (DAHLand MERCER1986) by intraperitoneal injection of either a single dose of 250 according to the method of SOUTHERN(1975) to determine mg ENU/kg body weight or with multiple injections of 100 thesegregation of an EcoRI restriction fragment length mg/kg given at weekly intervals over a period of 3 or 4 polymorphism associated with the PAH structural gene. weeks. Other details of the mutagenesis procedure are as A subsequent mapping study was done using a similar reported in BODE(1984). Briefly summarizing, immediately backcross to a conventional inbred mouse strain (ie., BUB/ prior to injection sufficient 0.1 M (pH4.5) potassium BnJ) carrying a polymorphic esterase-10 enzyme on chro- phosphate buffer was added to ENU crystals to give a final mosome I4 near the Np-f gene (WOMACKet al. 1977). hph- concentration of 15 mg/ml. All operations were done in an f homozygotes were mated to BUB/BnJ animals. The F, efficient chemical hood andthe injected animals were progeny were backcrossed to the hph-f homozygous parent maintained in the hood for several days after treatment. and the backcross progeny were assessed with respect to Mutagen-treated males were caged with females 7-8 weeks their hph-f and Es-IO phenotypes. after treatment and on regaining fertility were caged with new females on a weekly basis. Screening: To identify animals with a higher than normal blood level of phenylalanine (PHE), we used the RESULTS bacterial growth inhibition method of Gu-IHRIE and SUSI Carrier screening: It was unlikely a priori that the (1963). The amount of PHE in a dried blood sample is estimated from its ability to support the growth of Bacillus GUTHRIEscreen would detect the usual heterozygote subtilis in minimal medium containing an inhibitor of PHE carrier of a mutation causing HPA, since, at least in synthesis. [The intermediates of PHE metabolism, phen- humans, the levels of PHE in the blood of carriers ylpyruvate and phenyllactate, also permit bacterial growth are near normal. However, since PHE metabolism is (BIXBY,PALLAIAO and PRYLES1963) but their presence at complex it was considered possible that mutagenesis high levels in blood would also suggest a mutation worthy of further study.] To perform a test, a drop of blood was could induce rare “spoiler” type mutations acting in collected from each animal on a piece of filter paper. A a dominant manner that could therefore be detected discof suitable diameter was punchedfrom this and in the heterozygous state. Since little is known about incubated on agar containing minimal medium, the inhib- themouse enzymes or theirsubunit organization, itor and B. subtilis spores. The PHE concentration of the and theassay is relatively easy to perform,we decided blood sample was estimated by comparing the bacterial growth ringaround the test discswith those observed to screen the FI progeny of mutagenized males. We around control discs containing known amounts of PHE. tested animals at 5-6 weeks of age when they are We utilized a commercially available test kit (Sigma kit 160- ready for discardafter screening associated with B) allowing 48 samples to be analyzed on one 13 x 18 x 3 othermutagenesis experiments. No positivetests cm plastic dish. The plates were incubated for 16-18 hr at were observed in the testing of 7015 first generation 37” prior to measuring growth zone diameters. To photograph the assay plate the surface of the agar offspring. We concludethat, if hph mutants are was flooded with a freshly prepared 0.5% (w/v) solution of producedat the average per locusfrequency of 1 N,N,N’,N’-tetramethyl-$-phenylenediaminedihydrochlor- mutation in 1500 gametes seen at other genetic loci ide (Sigma Chemical).After 5 min the solution was de- (BODE1984), the test cannot readily allow carrier canted, the surface rinsed with distilled water and a pho- detection. We alsoconclude that when screening tograph taken. The agar gel is stained a dark purple but whole mouse elevated PHE with the the bacterial growth rings remain unstained and are seen blood for GUTH- as clear halos. Because the clear halos diffuse outward with RIE assay, false positive readingsare very rare in mice time, recorded growth measurements were those from the in this age group. unstained plates. Generation of homozygous mutants: Having es- Blood from normal mice gives growth rings correspond- tablished that it was necessary to produce homozy- ing to 2-4 mg/dl (0.12-0.24 mM) PHE. When blood from gous mutant animals, the three-generation breeding a member of a litter gave a growth ring larger than the 4 mg/dl control disc this identified the parents as putative scheme outlined in Figure1 was undertaken. A given carriers of an hph mutation and further litters from them ENU-mutagenizedmale was caged with wild-type were examined. females to produce approximately 25 male progeny, Linkage analysis: To map the hph-f gene we used an generation-1 (G-1). Each G-1 male was matedto a interspecific mouse backcrossof the type (Mus musculus wild-type female to produce 2-4 G-2 daughters that domesticus X Mus spretus) F1 female X M. musculus domesticus male segregating for hph-f. Since the two species, M. m. werethen mated back to their fathers (backcross). domesticus on the one hand and M. spretus on the other, For any given mutant locus(mq), half of the daughters ENU Induction of hph-l 30 I

BWU-treated (C57B1/6 X CBA/Ca)Pl male (m/+)

X (C57B1/6 X CBA/Ca)Pl female (+/+)

G-1 (mp/+) + (mq/+)1 + (mr/+) .. . mate males only

FIGURE1 .-Outline of the three-generation x (c57B1/6 x CBA/Ca)P1 (4') breeding scheme utilized to produce homozygous mutant animals.

G-2 (mq/+) + (+/+I mate 2-4 females

X G-1 father (mq/+)

6-3 (q/4)+ (-/+I + (+/+I will be mutant heterozygotes, like their fathers, and litters can be grownto weaning and tested. One half will be wild type. If a daughter is heterozygous, quarter of the offspring from a carrier daughterwill one quarter of her G-3 progeny will be homozygous be homozygous mutant.Those daughters that are for mutation y and will express the particular reces- not carriers produce litters in which one-half of the sive trait associated with adefect in gene y. The animals are carriers, so the risk of identifying but occurrence of suchanimals indicates thatthe G-1 then losing a mutant allele is low. male andthe G-2 daughterare carriers for the The average litter size remained very constant in mutationin y. Given that aparticular G-1 male is G-1 and G-2 litters, with theformer being 7.18 heterozygous for a desired recessive mutation, that (t2.26) and the latter 7.02 (k 1.82). The average G- litters from three G-2 daughters are analyzed and 3 litter size of 4.56 ( t 2.26) indicates that nearly three that their average G-3 litter size is 5 there is a 77% members of an average litter are never born, presum- probability thata G-1 male carrierfor a recessive ably due to homozygosity of newly-induced recessive mutation will be detected by the outlined procedure. embryonic lethal mutations. The most common visi- Because our previous studies (BODE1984) indicated ble mutant phenotypes seen in G-1 and G-2 animals thatan ENU-mutagenized male producessperm are dominantcataract formation and dominantwhite- from at least 100 independently mutagenized clones spotting. Because G-3 litters are sacrificed at 1 week of spermatogonial cells, we analyzed about 25 sons of age, there is little opportunity to observethese (G-1 males) from each. A sample of this size from animals for the development of visible phenotypes. the sperm repertoire of a given male should include Therefore the most notable feature with regard to very few identicalmembers. Although thethree- G-3 litters is thereduction in averagelitter size generation breeding scheme depicted in Figure 1 is mentioned above. applicable to recessive mutations in general and thus G- 1 sons (105) from 19 mutagen-treated males the G-3 progenycould have beenscreened for a were screened by the protocol described above. largenumber of different mutations, we chose to Among the 105 was one male that carried a recessive focus on thosecausing high blood levels of PHE. mutationcausing HPA. This initial hph-I carrier The G-2 litter from each G-1 male was culled to 2- animal was one of 26 male offspring that were tested 4 daughters at weaning. These remained caged with from aparticular mutagenized male. The positive their father (backcross) and their G-3 progeny were GUTHRIEreading that identifiedthe hph-I carrier sacrificed and tested at 1 week of age. Since we male was the only abnormal reading (Le., outside the expected most animals to be negative, this is a rela- normal 2-4 mg PHE/dl whole blood range) seen in tively efficient way of obtaininga single type of screening the progeny of the 105 G-1 males. mutant. Only one cage is requiredper G-1 male A typical assay plate with a positive sample is shown tested. When the rare positive is encountered, other in Figure 2. When assays are performed 1 week after FIGURE2.-Representative GUTHRIEassay plate usedin screening for hyperphenylalaninemic mice. The numbered growth halos represent the results from control discs impregnated with whole blood containing the stated number of mg PHE per dl of blood. The (*) symbol indicates a growth halo of sufficient diameter to be considered a positive reading (ie., greater than the diameter of the growth halo from the 4 mg PHUdl whole blood control disc).

FIGURE3.-A partial pedigree of the original ENU-treated male (symbolized by the letter M) depicting the induction, identification and transmission of the mutant allele hph-I. Circles represent females and squares males. The designated genotypes homozygous mutant (closed circles and squares), heterozygous mutant (halfclosed circles and squares) and wild type (open circles and squares), were assigned on the basis of phenotype and breeding data in the same manner as described in the legend to Table 1. In some cases, for simplification purposes or where animals were not progeny tested, the total number of animals in a litter is depicted within a circle and a square with the number of affected animals beneath next to an arrow pointing up indicating that this is the number of animals that were found with elevated blood PHE levels. Three positive tests were observed in a litter of ten animals born to one of the G-2 daughters. Two of the daughters gave no hyperphenylalaninemic offspring when mated to their father (not shown in graphic) and were thus presumed to be noncarriers. The third, was a heterozygous mutant animal and 17 out of 62 (27.4%) of her offspring exhibited elevated blood PHE levels. One affected male from the second litter of the G-2 carrier female was mated to a carrier sister and 13 out of 21 (61.9%) the progeny were affected. ENU Induction of hph-l 303

TABLE 1 TABLE 2 Inheritance of hyperphenylalaninemic phenotype Mapping the hph-I locus relative to Np-I

Offspring phenotype Genotype Frequency

Parentalgenotypes PHEHigh Normal PHE Total Parental classes Np-I"hph-IlNp-I" hph-1 34 1) hph-llhph-1 X hph-11 71 0 71 Np-I"hph-llNp-lh + 24 hph-1 2) hph-llhph-I x hph-11+ 132(45.4%) 159(54.6%) 291 Recombinant classes 3) hph-ll+ X hph-ll+ 203(25.2%)603(74.8%) 806 Np-1"hph-llNp-lh hph-1 0 0 4) hph-Ilhph-1 X +I+ 0 28 28 Np-1" hph-llNp-I" + Total examined 47 Animals that tested positive onthe GUTHKIEassay gave all affected progeny when mated with similar animals and were thus Total recombinandtotal examined 0147 inferred to be homozygous for the hph-1 mutation (see cross 1). Animals that were negative when tested by the GUTHKIEassay but which gave positive progeny when mated to positive animals were TABLE 3 inferred to be heterozygous for the hph-1 mutation (see cross 2). Animals identified as heterozygous, when mated to other animals Mapping the hph-1 locus relative to Es-IO with the same characteristics gave positive offspring in a proportion consistent with autosomal recessive segregation (see cross 3). Ani- mals that were negative when tested by the GUTHRIEassay and Genotype Frequency that gave no positive progeny when mated to positive animals were Parental classes inferred to be wild type with respect to the hph-l locus (see cross 4). Es-lO" h~h-llE.\-l~Ykph-1 25 Es-l(Y' hph-llE,\-l(Y Es-l(Y' f 33 Recombinant classes birth, the size of the growth zones indicates homo- Es-IO" hph-llE.~-l~Yhph-1 9 zygotes have blood levels of PHE between 10 and 20 Es-IO" Itph-1IE.\-IO" i 5 mg/dl (0.61 and 1.21 mM), comparedexamined to 2-4 mg/dl Total 72

(0.12-0.24 mM) for both heterozygotes and normal Total recombinantiTotal examined = 14/72 = 0.19 mice. This was verified by quantitative spectropho- tometric determination of serum PHE concentration by the method of SHEN and ABEL (1977)(MCDONALD were examined for cosegregation with hph-1. hph-1 and BODE1987). was found to be closely linked to theNp-1 (Nucleoside Segregation and symptomatology of hph-I. The phosphorylase-1) locus on mousechromosome 14. inheritance of HPA is as expected for a single Men- The M. spretus mouse species (SPE/Pas) that we used delian gene acting in a recessive manner (see Figure carries a rare electrophoretical variant Np-lb (BON- 3 andTable 1). Inpreparing the partialpedigree HOMME et al. 1984) that neither C57BW6 nor CBA/ represented in Figure 3, one homozygous male was Ca strains carry. In all instances we found cosegre- misclassified at weaning. An analysis of its progeny gation of hph-1 with the C57BW6 and CBA/Ca Np-1 stronglysuggested that it was a homozygote even allele (0 recombinants out of 47 backcross progeny though the PHE level in its blood was normal when examined) (see Table 2). This establishes the distance it was tested at 6 weeks of age. When this male was of hph-1 from Np-1 as less than 2 (& less than 2.0) challenged with PHE in its drinking water (5 mg/ml) cM. Later the hph-1 locus was mapped relative to the its blood level rose to 20 mg/dl (1.21 mM). This is the Es-10 (esterase-10) locus on mouse chromosome 14 only instance of a false negative Guthrie assay result usinga backcross toa conventional inbred mouse that we have observed.After this misclassification strain (see Table 3). The mouse strain BUB/BnJ used occurred we began to challenge all potential hph-1 for this backcross carries an esterase-10 enzyme (Es- homozygotes older than 3 weeks of age. Prolonged lor)that migrates slower than either the C57BL/6 or exposure to PHEin the drinkingwater has frequently CBA/Ca enzyme (Es-1On) during electrophoresis led to the deathof homozygotes but not heterozygotes (WOMACKet al. 1977). These results show clearly that or wi!d type animals. Death occurs after a chronic hph-1 is also linked toEs-10; the two loci are separated wasting syndrome that is more prolonged the greater by 19 (k4.6) cM. WOMACKet al. (1977) reported a the age of the homozygous animal at the onset of recombination distance between Es-10 and Np-1 of' PHE loading (data not shown). Unchallenged homo- 10.2 (k 1.9) cM. While this suggests a gene order of zygotes are normally healthy and fertile in both sexes. Es-10-Np-1 -hph-1 we are reluctant to proposethis due Linkage of hph-I. The chromosomal location of to the possibility of error in recombination interval hph-1 was initially determined by interspecies back- calculation caused by heterogeneity in recombination cross. A total of 44 loci (including 15 of the 19 mouse between crosses involving conventionalstrains of autosomes, the X chromosome andsome polymorphic mice and interspecific crosses involving M. spretus as loci not yet mapped to a particularchromosome) reported by NADEAUet al. (1986). 304 V. C. Bode et al.

TABLE 4 mouse is an excellent subject for minor surgery and Mapping the Pah locus relative to c-myb locus (in addition to blood) a single kidney or testis or a liver biopsy can easily be removed for biochemical or Genotype Frequency immunological studies with minimal risk of compro- mising thesubsequent fertility or viability of the Parental classes Pah" c-myb"1Pah"c-myb" 20 animal. Mutant isolation could be carried out as an Pah" c-rnyb"lPah"c-myb.' 19 ongoing activity in a modest-sized animal colony. Recombinant classes Although it is more efficient to screen G-3 progeny Pah" c-myb"lPah"c-myb' 8 for a larger number of traits, there are advantages c-myb"1PaKc-myb" 10 Pah" c-myb"1PaKc-myb" in undertaking a highly focused search such as the Total examined 57 one reported here. In addition to the inherent value Total recombinantltotal examined 18/57 = 0.32 of themutant and the information that maybe obtained from it, we hope that this study will alert mammalian biologists working in a variety of fields to the availability of this method for obtaining mu- Linkage of the PAH structural gene: The EcoRI tants, and will stimulate consideration of which spe- restriction fragment length polymorphism between cific mouse mutant phenotypes would be informative M. m. domesticus and M. spretw at the phenylalanine in particular systems, and lead to the design of new hydroxylase structuralgene locus (provisionally ways for detecting and using such mutants. called Pah) allowed for the determination of possible The enzyme PAH has been studied more exten- cosegregation of this locus with other polymorphic sively in therat than in the mousebut thereare loci in thesame interspecificmouse backcross as many reasonsto believe thatthe mouse system is described in the mapping of the hph-l locus. The M. similar to the rat. Hybridization of mouse genomic m. domesticus allele is referred to as Pah'" and the M. DNA with a rat PAH cDNA probe suggests a high spretw allele as Pah'. Of all the polymorphic loci degree of sequencesimilarity between the mouse and examined, only one showed detectable cosegregation rat gene. Thus, we were able to use a cDNA probe with Pah (see Table 4). Pah cosegregated with the c- of therat PAH geneto probe restrictionenzyme myb oncogene on mouse chromosome 10 in 39 out digests of genomic DNA from interspecific backcross of 57 backcross progeny for a recombination interval offspring. On thebasis of the segregation of an EcoRI of 32 (26.2) cM. restriction site polymorphism discovered between M. m. domesticus and M. spretus, we found no evidence of DISCUSSION linkage between the PAH structural gene and Np-1. The rate-limiting step in the catabolism of phen- In humans, PAH is encoded by a single gene. This ylalanine is catalyzed by the enzyme PAH and a has been shown by reconstituting PAH enzyme activ- deficiency of this hepatic enzymeis the most common ity from a cDNA molecule (LEDLEYet al. 1986) and cause of HPA in humans (SCKIVEK andC~.ow 1980). by cloning a unique chromosomallocus for this cDNA The reaction catalyzed by PAH requires the cofactor (DILLELAet al. 1986). The allelic variations of PAH 5,6,7&tetrahydrobiopterin that is maintained in the in rats (MERCERet al. 1984) suggests the same expla- appropriate state ofreduction by the enzymequi- nation for rats. While there is no direct evidence of nonoid dihydropterin reductase (EC 1.6.99.7) (KAUF- a single PAH locus in the mouse this is the logical MAN 1976; AMAKEGO,RANDLES and WAKING 1984). inference. Thus, the HPA that we are considering in Defects in the biosynthesis of tetrahydrobiopterin or hph-l mutant mice is probably not the direct conse- in the activity of the reductase can also lead to the quence of a mutation within the structural gene of hyperphenylalaninemicstate. In terms of mutage- PAH. The PAH gene itself exhibitssome linkage nesis, one can thus expect at least three direct ways with the c-myb oncogene on mouse chromosome 10, to generate a hyperphenylalaninemic mouse: (1) mu- an observation consistentwith a similar recent finding tations reducing PAH apoenzyme activity, (2) muta- by LEDLEYet al. (1987). tions interrupting cofactor synthesis and(3) muta- PKU in humans results frommutations in the tions reducingthe reductase enzyme activity. The structural gene for PAH (LEDLEY, DILELLAWOO and biochemical nature of the hph-l mutation will require 1985). Thus the hph-l mutation is not analogous to more investigation prior to its classification into one PKU in humans. However, the ease with which blood of these general areas. PHE levels can be manipulated in this mutant and Althoughthe hph-l mutantrequires additional the pathology that ensues should provide an inter- study in order to evaluate its specific use as an animal estingmodel for the studyof chronic HPA in a model for PKU, its isolation provides a paradigm for mammalian system. In addition, because the etiology obtaining mice having other metabolic defects. The ofthe hph-1 mutationappears to stem from an ENU Induction of hph-l 305 abberration of biopterin metabolism (MCDONALDand in a mouse &haplotype using ethylnitrosourea mutagenesis. BODE1987), this mutation should prove valuable in Genet. Res. 47: 187- 192. characterizing more atypical forms of HPA. KAUFMAN,S., 1976 Phenylketonuria, biochemical mechanisms. Adv. Neurochem. 2: 1-132. Research at Kansas State University byV. B. and J. M.was LEDLEY,F. D., A. G. DILELLA andS. L. C. WOO, 1985 Molecular supported by National Institute of Health grant 5 R01 HD5354- biology of phenylalanine hydroxylase and phenylketonuria. 06. Research at the PasteurInstitute byV. B. was partially Trends Genet. 11: 309-313. supported by an American Cancer Society Eleanor Roosevelt LEDLEY,F. D., H. E. GRENNET,M. MCGINNIS-SHELNUT,S. and L. International Cancer Fellowship. H. M. DAHL andJ. F. B. MERCER, C. Woo,1986 Retroviral-mediated genetransfer of human Royal Children's Hospital Research Foundation, Victoria, Aus- phenylalanine hydroxylase into NIH 3T3 andhepatoma cells. trailia, are thanked for graciously providing the rat cDNA clone Proc. Natl. Acad. Sci. USA 83: 409-413. used in mapping the PAH gene. SYLVIETUTOIS and MAKCCAS- LEDLEY,F. D., S. A. LEDBETTER,D. H. LEDBETTERand S. L. C. TELLAZZI are thanked for their help in typing mouse blood samples WOO, 1987 Localization of mouse phenylalanine hydroxylase for nucleoside phosphorylase- I. gene to chromosome 10. Cytogenet. 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