Isolation of Human Fibroblast Catalase cDNA Clones

Vol. 259, No. 22, Issue of November 25, pp , 13819-13823.1984 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1984 by The American Society of Biological Chemists, Inc.

Isolation of Human Fibroblast Catalase cDNA Clones SEQUENCE OF CLONESDERIVED

FROM SPLICED AND UNSPLICED mRNA* (Received for publication, June 18, 1984)

Robert G. KornelukSO, Frank QuanSll, William H. Lewis11 , Kevin S. Guise$, Huntington F. Willard$(, Maria Tzetis HolmesT, and Roy A. Gravel$( From the iResearch Institute., Hosoital . .for Sick Children, Departments of llMedicaZ Genetics and IISurgery, University of Toronto, Toronto, Canada

pected, to the short armof chromosome 11 and describe the We report the isolation and sequence of partial cDNA clones coding for human catalase. These clones were occurrence in one cDNA clone of a 462-bp‘ insertion interrecovered from a humau fibroblast cDNA library by rupting the expected protein sequence. Analysis of cDNA screening with mixturesof oligonucleotide probes de- clones lacking this insertion suggests that it corresponds to duced from the amino acid sequence of human eryth- an intron. rocyte catalase. A comparison of their nucleotide sequence with the known protein sequence and mapping MATERIALS AND METHODS ofhomologousDNA sequences to theshort armof A human cDNA library constructed from SV40-transformed fibrochromosome 11 in somaticcell hybrids confirmed that they coded for catalase.One of these clones contained blast mRNA in the vector pcD was generously provided by Drs. H. a 462-base insertion interrupting the coding sequenceOkayama and P. Berg (4). Procedures for plating and blotting the library to nitrocellulose filters, DNA preparation, restriction mapwith stop codons in all three reading frames. The 5’ ping, and Southern blotting and autoradiography were described and 3’ ends of the insertion correspond to the donor previously (7) except that rapid plasmid preparations wereby the and acceptor consensus sequences of introns. Inspecalkaline lysis method (8). tion of clones lacking the insertion confirm the locationDNA Probes-Oligonucleotide probes were synthesized by Pharof thesplice sites. We suggest this clone correspondsto macia-P-L Biochemicals and were radiolabeled at their 5’ ends using [y3*P]ATP(specific activity 3000 Ci/mmol, DuPont-New England the product of reverse transcription of an unspliced mRNA species. The catalase geneis the closest genetic Nuclear) and T4 polynucleotide kinase (Pharmacia-P-L Biochemimarker mapped to Wilms tumor, one the of most prev- cals) to a specific activity of1-5 x 10’ cpm/pg. Prehybridization, alent of childhood cancers. Catalase cDNA probes will hybridization, and washing of filters was done at 42 “C for the 17mer probe and 28 “C for the 12-mer probe. Filters were prehybridized be useful to the examinationof mitotic recombination overnight in 6 X SSC (1 X SSC = 0.15 M NaCl, 0.015 M trisodium in theetiology of this disease and may provide a usefulcitrate), 10 x Denhardt’s (1x Denhardt’s = 0.02% polyvinylpyrrolistarting pointtothesearchfortheputative Wilms done, 0.02% bovine serum albumin, 0.02% Ficoll), 0.05 M sodium tumor gene. phosphate, pH 7.0. Hybridization was done overnight in prehybridization buffer containing 0.5-1.0 X lo6 cpm/ml of probe. The filters were washed for 2 h in 6 X SSC with 2-3 changes. cDNA probes were nick translated using the Amersham nicktranslationkit and [cx-~’P]~CTP(specific activity 3000 Ci/mmol, Wilms tumor, one of the most common of childhood neo- DuPont-New England Nuclear) to a specific activity of 1-2 X 10’ plasias, occurs in both inherited and spontaneous forms. The cpm/pg. Hybridization conditions were as described (7) except that discovery that it can be associated with deletions of chromo- solutions for Southern blotting of genomicDNA contained 1 M some 11 centering around band p13 has led to the finding of sodium chloride, 1%sodium dodecyl sulfate, and 10%dextran sulfate. reduced catalase activity in some individuals (1-3) and to the The washing steps were as described except that in some experiments, additional wash at 65 “C in 0.1 X SSC, 0.1% sodium dodecylsulfate suggestion that the catalase gene might serve as a unique an was performed. marker for a gene or genes located in llp13 which predispose DNA Sequencing-Restriction fragments for sequencing were puindividuals to formation of the tumor. We therefore set out rified from agarose gels by electroelution and NACS (Bethesda Reto isolate cDNA clones for human catalase for use as molec- search Laboratories) column chromatography according to their protocol. All sequencing was done by the Sanger dideoxy chain ular probes to thisregion of the humangenome. In this paper, we describe the isolation and sequence of termination method (9, 10). Dideoxynucleotide triphosphates, deoxtriphosphates, and DNA polymerase (Klenow fragment) cDNA clones coding for human catalase. The clones were ynucleotide were from Bethesda Research Laboratory. The BamHI-KpnI, KpnIrecovered from a cDNA library constructed by Okayama and SphI, and SphI-Hind111fragments of pCAT 1 were sequenced using Berg (4)from SV40-transformed human fibroblasts by screen- the 15-mer universal primer (New England Biolabs) after subcloning ing with oligonucleotide probes deduced from the amino acid into thephages M13mp18 and mp19 (Bethesda Research Laboratory) sequence of human erythrocyte catalase (5,6). We report the (11). The 5’ ends of pCAT 1, 30, 38,41,47, 67, 77, 80, 106, and 110 localization of homologous genomic DNA sequences, as ex- were sequenced directly as double-stranded restriction fragments (12) using a pcD specific 15-mer primer, 5’-CAGTGGATGTTGCCT-3’ * This work was supported by grants from the Medical Research (synthesized by Pharmacia-P-L Biochemicals). The primer is comCouncil, Department of National Health and Welfare, and the Na- plementary to a pcD sequence adjacent to the 5’ end of the cDNA tional Cancer Institute of Canada. The costs of publication of this inserts and allows the synthesis of the mRNA strand of the cDNA article were defrayed in part by the payment of page charges. This insert. Restriction fragments were chosen for sequencing that inarticle must therefore be hereby marked “advertisement” in accord- cluded the 15-mer primer binding site and up to 700 bp of cDNA insert. ance with 18 U.S.C. Section 1734 solely to indicate this fact. Recipient of a Medical Research Council of Canada postdoctoral fellowship. ’ The abbreviations used are: bp, base pair; kb, kilobase pair.

13819

13820

Human Catalase cDNA

Chromosomal Mapping-Cells from seven unrelated human and three different rodent cell lines were fused and somatic cell hybrids isolated as described (13). For this study, hybrid clones were isolated from 10 separate fusion experiments designated W4 (GM 74 X Chinese hamster wg3H);W44(cell strain 44 X wg3H);W19 (cell strain 19 X wg3H); W53 (cell strain 53 X wg3H); A54 (cell strain 54 X mouse A9); A23, L23, or LT23 (cell strain 23 X mouse A9 or LTK); and A48 or W48 (GM 2859 X A9 or wg3H). Hybrid clones were harvested for DNA preparation and for chromosome analysis at the same cell passage. Human chromosome composition of cell hybrids was determined by detailed karyotyping of a minimum of 25 cells after trypsin-Giemsa banding (14). In most cases, chromosomes were scored both before and after DNA harvest. For this study, the presence or absence of human chromosome 11was confirmed in each instance by Cellogel electrophoresis of lactate dehydrogenase A activity, a known isozyme marker for chromosome ll(15).

B: B

S

H

I I

FIG.2. A , composite restriction endonuclease map of human pCAT 41 and pCAT 1 catalase cDNA clones. Enzymes represented are: Ap, ApaI; Av, AvaII; B, BamHI; H, HindIII; K, KpnI; N , NaeI; RESULTS Ps, PstI; Pv,PuuII; S, SphI; T,TthlllI; X, XhoI. The 5' PstI cloning The intron found in pCAT of the pCD vector is indicated by (Ps). Isolation of Human Catalase cDNA Clones-A mixture of site 1 is the separate KpnI-containingfragment. Restriction enzymes not 32P-labeled17-mer oligonucleotides corresponding to amino cutting the cDNA inserts are: AccI, BgA, BglII, ChI, EcoRI, HaeII, acids 390-395 of human erythrocyte catalase (Fig. 1)was used HincII, HpaI, MluI, NcoI, NdeI, NruI, PuuI, SaA, S e d , Smd, PstI, to screen 3.2 x 10' of the estimated 1.4 x lo6 independently SstI, SstII, StuI, Xbal. B and C, strategy used to determine the derived recombinants of the pcD library. Thirty probe binding nucleotide sequence of the cDNA clones. B , K, and S are correspondclones wererecovered, and these fell intoas many as 25 ing sites in the two maps. B , sequencing using the pcD 15-mer primer. internal BamHI (B), SphI ( S ) ,and HindIII (H) sites of pCAT distinct groups by preliminary restriction mapping. This The 41 are shown. The numbers indicate the clones used. The arrows group of isolates was reduced further after digestion with show the 5' end of each clone and the length of nucleotide sequence HincII or AuaI by Southern blottingusing either a mixture of obtained. Where two clones are shown, this indicates that two inde32P-labeled12-mer oligonucleotides corresponding to amino pendent clones of the same length were isolated and sequenced. C, acids 182-185 of catalase (Fig. 1) or the 32P-labeled17-mer sequencing using the universal M13 15-mer primer. The internal probe. Onlythree of the clones hybridized significantly to the BamHI ( B ) ,SphI ( S ) ,KpnI ( K ) , and HindIII ( H ) sites of pCAT 1are shown. Intron sequences are represented by dashed lines. The BamHI12-mer probe (data not shown) and the clone showing the KpnI, KpnI-SphI, and SphI-HindIII fragments were subcloned into strongest hybridization to both probes, pCAT 1, was selected M13mp18 and mp19 and sequenced in both orientations.The arrows for further study. indicate the direction of Sequencing and the length of sequence The identity of pCAT 1 was established by comparing its obtained.

nucleotide sequence with the published amino acid sequence of human catalase. The 2100-bp insert of pCAT 1was extensively mapped using a variety of restriction endonucleases in preparation for sequencing (Fig.2). An 800-bp BglI-KpnI fragment of pCAT 1 containing approximately 250 bp of the pcD vector was sequenced using the 15-mer primer. The resulting nucleotide sequencewas found to correspond to amino acids 164-267 of human catalase with few exceptions (see below). This result indicates that pCAT 1 is a cDNA clone encoding human catalase. To isolate clones of varying lengths, the cDNA library (1.2 X IO6clones) was rescreened with the BamHI-SphI fragment of pCAT 1. Sixteen new isolates were obtained, their inserts varying in lengths from 900 to 2200 bp. When the restriction map of pCAT 1 was compared to that of the new isolates, pCAT 1 appeared to have an insertion of approximately 450 bp containing a unique KpnI site (Fig. 2). A final screen of lo6 additional clones using the 5' PstI-PuuII fragment of pCAT 41 (Fig. 2) as the probe failed to reveal any longer clones. 12 mer Pmino Acid

Sequence

182 -Trp- Asp

17 mer

-

Phe

-

390 Trp-

-Pro-Met-

Cys- Met- Gln

-

Asp-

mRNA Sequence

5'-UGG-GA(U)-UU(U)-UGG-3'

~'-CCN-AUG-UG[U)-AUG-CA(A)-GA(U)-~' C G C

Probe Sequence

5-'TGG-GA(T)-TT(T)-TGG-3'

3'-GGN-TAC-AC(A)-TAC-GT[T)-CT-5' G C

c

c

c

c

FIG. 1. Mixedoligonucleotide probes. The amino acid sequences used to specify the 12-mer (residues 182-185) and the 17mer (residues 390-395), the possible codons for those amino acids, and the sequences of the oligonucleotide probes designed to account for all possible codon combinations are shown.

Nucleotide Sequence of Human Cataluse cDNAs-The nucleotide sequence of the catalase cDNA isolates was determined by the stategy shown in Fig.2. Where possible, the pcD 15-mer primer was used to determine the sequence of different clones from their 5' ends. For these experiments, suitable 5' restriction fragments were isolated and sequenced as already described. In addition, the BamHI-KpnI, KpnISphI,and SphI-Hid11 fragments of pCAT 1 were each subcloned into bothM13mp18 and mp19 and sequenced using the M13 universal 15-mer primer. The nucleotide sequence coding for amino acids 75 to the COOH terminus (residue 526) is shown in Fig. 3. The amino acid sequence in the single open reading frame corresponds to the partial amino acidsequence of human erythrocyte catalase reported by Schroeder et al. (5, 6) with only a small number of exceptions. Amino acid residues 158-159 should read Ile-Leu instead of Leu-Ile. Residue 169should read Arg as reported for the bovine erythrocyte sequence, instead of Lys (5). The two Asp/Asn ambiguities at residues 212 and 225 are Asn residues. The remainder of the deduced amino acid sequence is identical to the erythrocyte sequence. In addition, 17 amino acids and 2 Glx or Asx ambiguities not determined in that study can now be assigned. This includes the assignment of Phe to residue 278 in contrast to Leu in the bovine erythrocyte amino acid sequence. The other undetermined amino acid residues are unchanged from the bovine sequence. The protein coding sequenceterminates with a TGA codon after amino acid residue 526, leavinga 3' noncoding regionof approximately 700 bp in all of the pCAT clones. The stop codon was confirmed by sequencing the sense strand of two independent clones (pCAT 38 and 67), and the antisense

13821

Human Catalase cDNA 76 1

L y s G l y A l a G l y A l a Phe G l y Tyr Phe G l u V a l Thr His A s p I l e Thr L y s Tyr Ser L y s A l a L y s V a l Phe G l u His Ile GGGCGGGGCC AAA GGA GCA GGG GCC TIT GGC TAC TIT GAG GTC ACA CAT GAC ATT ACC AAA TAC TCC AAG GCA AAG GTA TIT GAG CAT ATT

103 90

GlyLysLys GGAAACAAG

133 180

ValLys

Phe A l a Thr Pro I l e A l a V a l A r g Phe Ser Thr V a l A l a C l y G l u Ser G l y Ser A l a A s p Thr V a l A r g A s p P r o A r g G l y GTP CGG GAC CCT CGT GGG TIT CCA ACT CCC ATC GCA GPT CGG TIT TCC ACT CTT CCT GGA GAA TCG GGT TCA CCT GACACA

* * * * * * * * Leu Ile G l yA s nA s n Thr Pro I l e Phe Phe I l e A r gA s p Pro I l e L e uP h e Pro Ser Phe Tyr Thr G l u A s p G l y A s n Trp A s p L e u V a l GGA AAT AAC ACC CCC ATP GAT CTC OIT T l T TTC ATC AGG GAT CCC ATA Tpc TIT CCA TCT G'PC AAA TIT TAC ACA GAA GAT GGT AAC TGG

163 270

L y s A * A * * 12- mer Phe I l e His Ser C l n L y s A r g A s n Pro G l n Thr His L e u L y s A s p Pro A s p Met V a l T r p A s p Phc Trp Ser L e u A r g Pro G l u Ser L e u TXT A X CAC AGC CAA AAG AGA AAT CCT CAG ACA CAT CTG AAG GAT CCGGAC A n GTC "2 CAC TTC TGG AGC CTA CGT CCT GAG TCT CPC

193

His G l n V a l

I l e Pro A s p G l y

Asp

Hi5 A r g His Met A s n G l y

Tyr G l y Ser His Thr Phe L y s Leu V a l AAT GGA TAT GGA TCA CAT ACT TIT AAG CM; CTT

360

Ser Phe L e u Phe Ser A s p A r g G l y CAT CAG GTT TCT 'ITC TI1; TIT ACT GAT CGGGGG

223 450

Asp A s n A l a A s n G l y Glu A l a V a l Qv C y s L y s Phe His Tyr L y s Thr A s p G l n C l y I l e L y s A s n L e u Ser V a l G l u A s p A l a AlaArgLeu AAT CCA AAT GGG GAG GCA OIT TAT TGC AAA TIT CAT TAT AAG ACT GAC CAG GGC ATC A M AAC CIT TCT GTT GAA GAT GCG GCGAGA C'IT

253 540

Ser G l n G l u A s p Pro A s p Tyr G l y I l e A r g A s p L e u Phe A s n A l a I l e A l a Thr G l y L y s TCC CAG G M GAT CCT GAC TAT GGC ATC CGG GAT CIT TIT AAC GCC ATT GCCACAGGAAAG

253 630

Met Thr Phe A s n G l n A l a G l u Thr Phe Pro Phe A s n Pro Phe A s p L e u Thr L y s A n ACA TIT AAT CAGGCA GAA ACT TIT CCA TIT AAT CCA TIT GAT CTC ACC AAG CTGAGT

720

AG-CT

ATT CCA CAT GGA CAT CCC CACA'PC

*

TACCTAATTA

GMAAAAAAT

CTAGTCAAAC

*

Tyr Pro Ser T r p Thr Phe Tyr I l e G l n V a l TAC CCC TCC TGG ACT TIT TAC ATC CAG GTC

AATTATAATA ATGGCCAAGT

510

GAGTGTCCTA

AGCTCTC"

GAGTGCTTAC

CAGCATClTA

CITCCACGlT

C CA T "CCI TAETXl ' G A

900

ATATIGITAT

TAACAGGGAA

CAGATTATCA

AAAGCTGATC

TACITPPTCC

TGGGGAAACT

990

ATITAACl"

CCCACTCATC

TTAAA"lT

ATCTIYTATT

GGATITAAAA

ATPAT?TKA TEGClTGAT

CAGTAAACAA

CTATAlTGTT

TElTIT3A

CATATACMA

ATACAGAGGG

TACCACTICA

CTCAGTATIT

ACCAClTACT ATl'GTCMAG

GTAETCTAT CPCGCITCAT

TGTA-AA

ATCTGGTATT

8

V a l Trp Pro His L y s A s p WAG GTP Tcc CCT CAC AAG GAC

Tyr Pro TAC CCT

301 1050

m G T c GGGcTmAc A T I T

309 1170

L e u I l e Pro V a l G l y L y s L e u V a l L e u A s n A r q A s n P r o CPC ATC CCA GTT GGT AAA CPC GTC TTA AACCGGAATCCA

339 1260

P r o Pro G l y I l e G l u A l a Ser P r o A s p L y s Met L e u C l n G l y A r g L e u Phe A l a Tyr Pro A s p Thr His A r g His A r 9 L e u G l y P r o A s n CCA CCT GGC ATP GAG GCC ACT CCT GAC AAA A n : C'IT CAG GGC CGC C l T TlT GCC TAT CCT GAC ACT CAC CGC CAT CGC CTC GGA CCC AAT

369

Tyr L e u His I l e Pro V a l A s n

CCITCAGTPG ATECCTGGTA

ATl'GTCAATAT

GACATCATIT

V a l A s n Tyr Phe A l a C l u V a l G l u G l n GTP AAT TAC TlT K T GAG GlT CAACAG

I l e A l a Phe A s p Pro S e r A s n ATA GCC TIT GACCCAAGCAAC

*

Met A n

*

1350

17-ner ValAlaAsnTyr G l n A r g A s p G l y Pro Met C y s Met G l n A s p A s n G l n G l y C y s Pro Tyr A r g A l a A r g TGC ATG CAG GAC AAT CAG GGT TAT CIT CAT ATA CCT GTC AAC TGT CCC TAC CGT GCT CGA CTG GCC AAC TAC CAA CCT GAC GGCCCGATG

399 1440

Glx G l y A l a Pro A s n Tyr Tyr Pro A s n Ser Phe G l y A l a Pro G l u G l n G l n CAA CAG GGT GCT CCA AAT TAC TAC CCC AAC AGC TIT GGT GCT CCG GAA

Pro Ser A l a L e u G l u His Ser I l e G l n Tyr Ser G l y Glu V a l CCT TCT GCC lTG GAGCAC AGC ATC CAA TAT Tcp GGA GAA CTC

429 1530

A r g A r g Phe A s n Thr A l a A s n A s p A s p A s n V a l Thr G l n Val A r g A l a CGGACA T I C AAC ACT GCC AAT GAT GAT M C GlT ACT CAG G K CGGGCA

Asx Phe Tyr V a l A s n V a l L e u A s n G l u G l u G l n A r g TIT TAT GTG AAC GTC CTC AAT GAG GAA CAGAGG

459 1620

TCT GAGAAC

489 1710

G l y Ser His I l e G l n A l a L e u L e u A s p L y s GGG AGC CAC ATC CAG GCT CIT Crc GAC AAG

519 1000

A l a A l a Arg G l u Lys A l a A s n Leu GCG GCA AGG GAG AAC GCA AAT CTC TCA

C y sG l uA s n

L y s Arg L e u AAA CGT CIY;

I l e A l a G l y His L e u L y s A s p A l a G l n I l e Phe I l e G l n L y s L y s A l a V a lL y s A s n Phe Thr G l u V a l His P r o A s p Tyr ATT GCC GGC CAC CTG AAG GAT GCA CAA ATT TM: ATC CAGAAG AAA GCG GTC AAG AAC TTC ACT GAG GTC CAC CCT GAC TAC

...

( I l e His Thr:Phe V a l C l n Ser G l y Ser His) Tyr A s n A l a G l u L y s Pro L y s A s n A l a I l e His Thr Phe V a l G l n Ser G l y S e r His L e u TAC AAT GCT GAG AAG CCT AAG AAT GCG ATP CACACC TlT GTG CAG TCC GGA TCT CAC TIC.

GGCCGGGGCC

CTCCACCTGT

GCATGAAGCT

FIG. 3. The nucleotide sequence of human catalase cDNA including the intron found in pCAT 1. The nucleotide sequence of the coding strand and the deduced amino acid sequence are shown. The translation stop codon is indicated by . . ..Amino acids are numbered according to the human erythrocyte catalase protein sequence of Schroeder et al. (5). Residues determined by Schroeder et ol. (5) that differ from the deduced amino acids sequence are indicated above. Asterisks indicate residues not previously determined. Brackets indicate amino acid sequences of unassigned peptides. The oligonucleotide binding sites are underlined.

strand of another (pCAT 1).This result extends the carboxyl terminus of human erythrocyte catalase beyond residue 507 (Ala) identified by Schroeder et al. (6). Ten of the additional amino acids, residues 508-517, can be accounted for by the sequence of two extra peptides from the erythrocyte enzyme that could not be assigned by those authors (Fig. 3). This leaves the human fibroblast coding sequence nine amino acids longer at the carboxyl end than human erythrocyte catalase. The KpnI insertion present in pCAT 1 was revealed by DNA sequencing to be 462 bp long beginning after amino acid residue 300 (Ala) (Fig. 3). This insertion did not appear in any other clones spanning thisregion. The insertion contains numerous stop codons in all three reading frames, the first interrupting the open reading frame of the apparent coding sequence 10 bp after residue 300. The possibility that the

KpnI insertion corresponds to an intron is considered under "Discussion." Chromosomal Mapping of the Catalase Gene-The human catalase gene, CAT, has previously been mapped to chromosome 11, band p13 upon analysis of the protein product (13). To confirm that theisolated cDNAs do,in fact, correspond to human catalase, we examined a series of human-rodent somatic cell hybrids to correlate the presence of DNA sequences homologous to pCAT 1 with a specific human chromosome. A 9-kb PstI band in genomic DNA digests revealed by hybridization with the BamHI-SphI fragment of pCAT 1 correlated with the presence of human chromosome 11 in 18 hybrids tested (Table I). No other human chromosomes cosegregated with human catalase in these hybrids. Examples of human catalase positive and negative hybrids are shown in

Human Catalase cDNA

13822 TABLE I

Segregation of cloned catalase gene in 18 human-rodent hybrids Segregation of human 9-kb PstI band (see Fig. 4) in 18 humanrodent hybrids (see “Materials and Methods”). +/+ and -/- indicate number of cell hybrids in which the probe segregated with the indicated chromosome (concordant). +/- indicates the number of cell hybrids inwhich the human 9-kb band was present, but the indicated chromosome was not. -/+ indicates number of cell hybrids in which the human 9-kb band was absent, but the indicated chromosome waspresent. Discordancy is the percentage of scored hybrids in which the probe did not segregate with the indicated chromosome. Chromosomes 11 and X were not scored in one hybrid which contained only a portion of each chromosome due to a translocation. Chromosome

Concordant +I+

-1-

Discordant -I+ +I-

1

2

3

4

5

8

7

8

9 kb-

Discordancy %

3

2

1 2 3 4 5 6 7 8 9 10 11 712 13 214 15 16 17 18 719 20 21 22 X

4 2 5

8 6 4

5 3 4 1

6 8 6 8 8 8 8

4 9 4 2 5 7 5 3 3 5 4 5 7 1 9 0

5 9 6 4 6 4 4 8 2 9

5 7 4 6 4 6 5 8 7 5 0 5 7 4

1 3 5 2 3 1 3 1 1 1 0 2 4

4

0

6 6 4 5 4 2 8 0 9

3 5 3 2 5 5 1 6 0

33 56 50 44 39 39 44 50 44 33 0 39 61 61 22 50 61 39 39 50 39 50 35 50

FIG.4. soutnern blot anaysls of numan Catalase gene in somatic cell hybrids. DNAs were digested with PstI, separated on a 1% agarose gel, transferred to nitrocellulose, and hybridized with 32P-labeled BarnHI-SphIfragment from pCAT 1. Filter was not washed in 0.1 X SSC, 65 “C. DNA was from: lanes 1 and 2, two different human fibroblast strains; lane 3, mouse LTK-; lane 4, Chinese hamster wg3H; lanes 5-7 mouse-human hybrids, lane 8, hamster-human hybrid. Arrow indicates prominent humanband a t 9 kb. Hybrids in l a n e s 5-8 segregate products of an X/11 translocation (see text). None of these hybrids contains thenormal 11 homologue. Lane 5 , hybrid A48-1G, contains Xp/llp (:llpll+pter) and Xq/llq (llqter+llpll:) chromosomes; lane 6,hybrid A48-1Gaz43,contains X p / l l p chromosome only; lane 7, hybrid A48-1F, contains Xq/llq chromosome only; lane 8, hybrid W48-12cAz61, contains Xp/llp chromosome only. Human DNA (lanes 1 and 2) also contains two PstI fragments of 1.7 and 1.4 kb which hybridize faintlyto theprobe. These bands were not scored in hybrids and their relationship to the catalase gene is unknown.

localization of the catalase gene to chromosome 11, band p13 (1-3). The relatedness of the human erythrocyte and fibroblast Y sequences as well as those of the bovine liverand erythrocyte enzymes affirm that there is a single catalase gene. This is Fig. 4. The probe also hybridized with a series of bands in supported by the presence of a single major genomic DNA mouse and hamster DNA (Fig. 4). In mouseDNA, PstI fragment hybridizing to the pCAT cDNA probe. However,it fragments of 3.5, 2.6, 2.2, and 1.0 (faint) kb hybridized, while is interesting to note the divergence of the apparentcarboxyl in hamster DNA, PstI fragments of 4.4, 2.6, and 2.2 kb were terminus of the different tissue enzymes. Schroeder et al. (5) seen. The intensity of the rodent bands was reduced relative had previously reported that the bovine liver enzyme termito thehuman fragments, but not eliminated, when filters were nated at residue 506 (Asn), while the bovine and human washed in 0.1 X SSC at 65 “C. erythrocyte enzymes now appear to extend to residue 517 The human catalase gene was regionally mapped on chro- (His). The termination codon of human fibroblast catalase mosome 11by analyzing DNA fromsomatic cell hybrids made occurs after amino acid residue526 (Leu). Whether the fibrowithhumancell strain GM2859 containing a reciprocal blast enzyme extends this far or if it is processed back to translocation between chromosomes11and X, with the break- residue 517 is unknown. Nevertheless, the results strongly points near the centromere on both chromosomes(46,X, suggest that processing at the carboxyl end does occur and t(X;ll)(plll-qll1;pll) (16). Hybridcloneswerederived that tissue-specific protein differences appear to prevail, at which contained either theIlp/Xp translocation chromosome least between liver and erythrocytes. or the llq/Xq translocation chromosome in the absence of An unexpected finding was the 462-bp insertion after resithe normal chromosome 11 homologue. As shown in Fig. 4, due 300 (Lys) in one of the clones found spanning this region. the presence of the human 9.0-kb PstI fragment co-segregated Its structure resembles that of an intron (17). At the 5‘ end with the llp/Xp chromosome (and the l l p marker lactate of the insertion site is the sequence5’-AAG/GTGAG-3’ dehydrogenase A), allowing assignment of human CAT DNA (slash = splice site). Thisis a perfect subset of the consensus to llpll+llpter. sequences 5’- A CAG/GT GAG-3‘. A Similarly the sequence 5’TCATTTTCAG/GT-3’ is found at the3’ end of the insertion. DISCUSSION This corresponds well with the consensus sequence, Two lines of evidence prove conclusively that pCAT cDNA G showing a single purine in the pyrimidine clones code for human catalase. First, comparison of the 451 (Py)XCAG/GT, deduced amino acids of pCATwith the published protein (Py) track. sequence showa near perfect match, and second, the assignEvidence that the designated splice sites are indeed operment of human DNA sequences homologous to pCAT 1 to ating assuch inthe catalase gene is apparent upon inspection the short arm of chromosome 11 is consistent with the prior of pCAT clones missingthe inserted sequence. Residues300-

Human Catalase cDNA 301 in these clones have the DNA sequence 5’-AAGGTT-3’, showing that thesplice would have to have occurred between the two G residues. This result, along with the presence of numerous stop codons in all three reading frames, provides compelling evidence that pCAT 1 contains an intron within the cDNA sequence. An analogous 76-bp long sequence, with apparent donor and acceptor splice sites, has also been reported for ahuman adenosine deaminase cDNA (18), although, in that case no clones were found without this extra sequence. It appears that when total cellular RNA is used for the construction of cDNA libraries, nuclear RNA transcripts might also be included among the RNA species subject to reverse transcription. This is true even after oligo(dT)-cellulose chromatography of the RNA, since polyadenylation of primary transcripts occurs before splicing (19). The use of libraries with a bias toward longer cDNAs, such as thelibrary used in thisstudy, probably accounts for the successful recovery of intron-containing cDNAs. DNA probes for the human CAT gene should prove valuable for a number of lines of investigation. They, of course, will allow detailed analysis of gene structure,both in normal individuals and those with inherited acatalasemia (20). In addition, the availability of probes for CAT will contribute to a growing genetic map of the short arm of chromosome 11 (21, 22) if restriction fragment length polymorphisms can be identified. Such CAT restriction fragment length polymorphisms would be of immediate interest for the investigation of families exhibiting the hereditary form of Wilms tumor. Lastly, several laboratories have recently reported that Wilms tumor canbe associated with homozygosity of genetic markers on the short armof chromosome 11in cases where heterozygosity could be demonstrated in normal tissues of the same individual (23-26). As catalase is the genetic marker mapping closest to theWilms tumor region, the cDNA probes described here will be useful not only for examination of mitotic recombination in the etiology of Wilms tumor, but also as a logical starting point in the search for the Wilms tumor gene. Acknowledgments-We are grateful to Paul Bevilacqua for suggesting these studies, to M. Tropak for computer assistance, to V. E. Powers for assistance with hybrids, and to L. Rossiter for assistance with the manuscript.

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