Proc. Natl. Acad. Sci. USAVol. 85, pp. 1883-1887, March 1988Genetics

Gene encoding the human 13-hexosaminidase 13 chain: Extensivehomology of intron placement in the a- and 13-chain genes

(lysosomal enzyme/gene duplication/Sandhoff disease)

RICHARD L. PROIAGenetics and Biochemistry Branch, National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda, MD 20892

Communicated by Elizabeth F. Neufeld, November 16, 1987 (received for review September 11, 1987)

ABSTRACT Lysosomal (3-hexosaminidase (EC 3.2.1.52)is composed of two structurally similar chains, a and (3, thatare the products of different genes. Mutations in either genecausing .8-hexosaminidase deficiency result in the lysosomalstorage disease GM2-gangliosidosis. To enable the investiga-tion of the molecular lesions in this disorder and to study theevolutionary relationship between the a and (3 chains, the(3-chain gene was isolated, and its organization was character-ized. The (3-chain coding region is divided into 14 exons dis-tributed over -40 kiobases of DNA. Comparison with the a-chain gene revealed that 12 of the 13 introns interrupt the cod-ing regions at homologous positions. This extensive sharing ofintron placement demonstrates that the a and (3 chains evolvedby way of the duplication of a common ancestor.

Lysosomal p-hexosaminidase (EC 3.2.1.52) is composed oftwo chains, a and p, that are encoded by separate genes ondifferent chromosomes. The a-chain gene, on chromosome15, and the p-chain gene, on chromosome 5, specify precur-sor glycoproteins that associate noncovalently to give rise top-hexosaminidase A (ap) and p-hexosaminidase B (JIB).Either during or after transit to lysosomes, the chains areproteolytically processed to their mature forms (reviewed inrefs. 1-3).The a and , chains are structurally and functionally related.

Comparison of their amino acid sequences, derived fromcDNA clones, reveals an identity of >50o (4, 5). In accord-ance with the similarity in primary sequence, each chaincarries a catalytic site involved in the hydrolysis of plinkedN-acetylhexosamine residues (6). There are, however, signif-icant differences in their substrate specificities. The a-chaincatalytic site, as expressed in p-hexosaminidase A (al8), hy-drolyzes sulfated N-acetyl-p-D-glucosamine residues and gan-glioside GM2, provided an activator protein is also present.The 8-chain catalytic site, expressed in both p-hexosami-nidase A (ap) and B (,8), functions to degrade mainlywater-soluble N-acetylglucosaminides and -galactosaminides.The GM2-gangliosidoses are caused by inherited defects

in either the a-chain gene (Tay-Sachs disease) or the ,8-chaingene (Sandhoff disease) (1, 2). As a result of the deficit inp-hexosaminidase A, ganglioside GM2 accumulates in thenervous system causing the clinical manifestations observedin the diseases. In general, both diseases occur infrequentlyand in a variety of different ethnic groups. The notable ex-ceptions are Ashkenazi Jews and a French-Canadian popula-tion from Eastern Quebec that have a significantly highercarrier frequency for Tay-Sachs disease than the generalpopulation (7, 8). Tay-Sachs and Sandhoff diseases are clin-ically similar; both display heterogeneity with regard to se-verity and age of onset (9).

To understand the molecular defects that underlie Sand-hoff disease and to investigate further the relationship be-tween the a and p chains, the gene encoding the 8 chain of/-hexosaminidase has been isolated, and its intron-exonorganization has been characterized. Comparison with thea-chain gene (10) reveals a striking similarity in structure,demonstrating that both genes were derived from a commonancestor.

EXPERIMENTAL PROCEDURESIsolation of Clones. A Hep G2 cDNA-Agt 11 library (11)

was obtained from M. Spiess and H. Lodish (MassachusettsInstitute of Technology) and was screened with an oligonu-cleotide probe (5'-CTGGCATATTCAATCACCAT-3') cor-responding to a portion of the published p-chain sequence(12). A clone was isolated that contained a 1.7-kilobase (kb)insert. After restriction fragments were subcloned into M13vectors, the cDNA was sequenced by the Sanger method(13) with M13 and ,-chain-specific oligonucleotides as prim-ers.For the isolation of clones containing 8-chain gene se-

quences, three human genomic libraries constructed in bac-teriophage A vectors were used-a fetal liver library in Cha-ron 4A (14) obtained from T. Maniatis (Harvard University),an EMBL3 library from R. Myerowitz (National Institute ofDiabetes, Digestive and Kidney Diseases, National Institutesof Health) prepared from the DNA of the cultured fibroblastsfrom a Tay-Sachs patient (15), and an EM13L3-lymphocyteDNA library purchased from Clontech (Palo Alto, CA). Thelibraries were screened with restriction fragments of thep-chain cDNA insert. Positive clones were purified throughthree rounds of screening.

Analysis of Genonic Clones. The genomic clones weremapped with restriction endonucleases by hybridizing par-tial digests to oligonucleotides complementary to the A COSends (16). Restriction endonuclease fragments containingexonic sequences were identified by Southern blotting andhybridization with oligonucleotides corresponding to thep-chain coding sequence. These fragments were isolated byelectrophoresis through low-melting agarose gels and sub-cloned into pUC-13 or -18. The sequences of the intron-exonjunctions were determined by sequencing the plasmid DNAby the dideoxy chain-termination method (13) with a seriesof B-chain-specific oligonucleotide primers. To allow recog-nition of sequence artifacts, each sequence was determinedby using both the Klenow fragment of DNA polymerase Iand reverse transcriptase.The localization of the exons was accomplished by restric-

tion endonuclease mapping of the plasmid inserts withdouble digestions and subsequent Southern blot analysis thatused exon-specific oligonucleotide probes. In cases wherepaucity of restriction sites made the positioning of exonsdifficult, the localization was accomplished with a methodthat used exonuclease III generated fragments. Briefly,DNA fragments were directionally digested with exonu-

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a ~~~~~~~~~~~~~~~~~~~~~ATGACA AGC TCC AGG CTT TGG TTT TCG CTG CTG CTG GCG GCA GCG TTC GCA IGA CGG 57Met Thr Ser Ser Arg Leu Trp Phe Ser Leu Leu Leu Ala Ala Ala Phe Ala Gly Arg 19

Met Lou Lou Ala Leu Leu Leu Ala Thr Leu Leu Ala Ala Met Leu Ala Leu Leu Thr Gln Val Ala Leu Val Val Gbn Val Ala Glu Ala Ala Arg Ala Pro Ser Val Ser Ala Lys Pro 40I ATG CTG CTG GCG CTG CTG TTG GCG ACA CTG CTG GCG GCG ATG TTG GCG CTG CTG ACT CAG GTG GCG CTG GTG GTG CAG GTG GCG GAG GCG GCT CGG GCC CCG AGC GTC TCG GCC AAG CCG 120

a GCG ACG ICC CTC TGG CCC TGG CCT CAG AAC TTC CMA ACC TCC GAC CAG CGC TAC GTC CTT TAC CCG AAC AAC TTT CMA TTC CAG TAC GAT GTC AGC TCG GCC GCG CAG CCC GGC TGC TCA 177Ala Thr Ali Lou Top Pro Trp Pro Gln Asn Phe Gln Thr Ser Asp Gln Arg Tyr Val Lou Tyr Pro Asn Aen Pb. Gbn Phe Gbn Tyr Asp Val Ser Ser Ala Ala Gbn Pro Gly Cys Ser 59

(3 Gly Pro Ala Lou Top Pro Leu Pro Leu Ser Val Lys Met Thr Pro Ase Leu Leu His Lou Ala Pro Glu Aen Pies Tyr Ilb Sr His Ser Pro Asn Her Tier Ala Gly Pro Ser Cys Thr 80GIG CCG GCG CTG TGG' CCC CTG CCG CTC TCG GTG MAG ATG ACC CCG MAC CTG CTG CAT CTC GCC CCG GAG MAC TTC TAC ATC AGC CAC AGC CCC MAT TCC ACG GCG GGC CCC TCC TGC ACC 240

INTRON 1

a GTC CTC GAC GAG ICC TTC CAl CGC TAT CIT GAC CTG CTT TTC lIT TCC GIG TCT TGG CCC CIT CCT TAC CTC ACA GIG MAA CII CAT ACA CTG GAG MAG MT GTG TTG ITT GTC TCT GTA 297a

Val Lou Asp Glu Ala Ph. Glb Arg Tyr Arg Asp Leu Leu Pb. Gly Ser Gly Ser Top Pro Arg Pro Tyr Leo Thr G~ly Lys Arg His Tier Leu Glb Lys Ase Val Leou Val Val Ser Val 99Leo Leou lb Glu Ala Pb. Arg Ar; Tyr His Gly Tyr Ile Phe. Gly Phe Tyr Lys Top His His Ito Pro Ala Glb Phe Glb Ala Lys Tier Gln Val Gln Glb Leu Lou Val Ser Ito' Ther 120CTG CTG GAG GMA GCG TTT CIA CIA TAT CAT GGC TAT ATT TTT lIT TTC TAC MAG TGG CAT CAT GMA CCT WIC GMA TTC CAl GCT AAA ACC CAl ITT CAl CMA CTT CTT GTC TCA ATC ACC 360

1 ~~~~INTRON 2 INTRON 3INTRON 1

a GTC ACA CCT GGA TGT MAC CAl CTT CCT ACT TTG GAG TCA GTG GAG MAT TAT ACC CTG ACC ATA MAT GAT GAC CAl TGT TTA CTC CTC TCT GAG ACT GTC TGG GGA GCT CTC CIA lIT CTG 417Val Thr Pro Gly Cys Asn Gln Leo Pro Thr Lou Glb Her Val Olu Asn T1yr Pier Leou Thr Ile Asn Asp Asp Glb Cys Leu Lou Leo Ser Glb Thr Val Poip Oly Ala Leo Anrg +~y Lu139

(J Leu Glb Ser Glb Cy& Asp Ala Ph. Pro Asn Ilb Ser Her Asp 01u Ser Tyr Pier Leou Lou Val Lys Gbu Pro Val Ala Val Leou Lys Ali Ase Arg Val Pop Oly Ala Leou Ag dly Leo 160OCTT CMe TCA GAG TGT GAT GCT TTC CCC MAC ATA TCT TCA GAT GAG tCT TAT ACT TTA CTT GTG MAA GMA CCA GTG GCT GTC CTT MAG ICC MC AlA ITT TGG GGA ICA TTA CIA lT TTA 480

INTRON 4INTRON 2 INTRON 3

a GAG ACT TTT AGC CAl CTT ITT TGG MAA TCT GCT GAG GGC ACA TTC TTT ATC MAC MAG ACT GAG ATT GAG GAC TTT CCC CGC TTT CCT CAC CII GGC TTG CTG TTG GAT ACA TCT CGC CAT 537lT1hi, Pb. Her GIn Leo VaIi Trp Lys Set Ala Glu Gly Tier Pb. Ph. Ii1. Aso Lys Tier Gi Xie Glu Asp Phe Pro Ar; Pb. Pro lie Arg Oly Lou Leou Lou Asp Tier Her Arg His 179

.Gl Tier Pb. Her Gin Lou Val Tyr Ito Asp Ser Tyr Gly Tier Pb. Thr XiC Ason Glb Sr Thr II. Ile Asp Ser Pro Ar; Pb. Sor His Ar; Giy Ile Lou Ile Asp Tier Her Ar; His 20(GAG ACC TTT AGC CAl TTA ITT TAT CMA GAT TCT TAT GGA ACTITTC ACC ATC MAT GMA TCC ACC ATT ATT GAT TCT CCA All TTT TCT CAC AlA IdA ATT TTI ATT GAT ACA TCC AlA CAT 600

INTRON 5INTRON 4

a TAC CTG CCA CTC TCT AGC ATC CTG GAC ACT CTG GAT GTC ATG GCG TAC MAT MAA TTG MAC GTG TTC CAC TGG CAT CTI ITA GAT GAT CCT TCC TTC CCA TAT GAG AGC TTC ACT TTT CCA 657Tr Leo Pro Leu Ser Ser Ilie Leou A~sp Tbr Ljo Asp Val Het Ala Tyr Asn Lye Lou Aen Val Phe Bid Pop Hi. Lou Val Asp Asp Pro Her Pb. Pro Tyr Glb Her Pho Thr Pb. Pro 219Tyr Leo Pro Val Lys Ilie Ilie Leou Lys Tier Leo Asp Ala Net Ala Ph. ken Lys Ph. Asn Vai Leu His Top Hi. Iie Val Asp Asxp Gte Her Pb. Pro Tyr Gin Her I1e Tier Pb. Pro 240TyAT CTIG CCA ITT MAG ATT ATT CTT MAA ACT CTI GAT ICC ATG GCT TTT MAT MIG TTT MAT ITT CTT CAC TGG CAC ATA ITT GAT GAC CAG TCT TTC CCA TAT CAl AGC ATC ACT TTT CCT 720

iNTRON 6INTRON 5

a GAG CTC ATG AlA MIG GIG TCC TAC MAC CCT GTC ACC CAC ATC TAC ACA GCA CAl GAT GTG MAG GAG GTC ATT GMA TAC GCA CII CTC CII lIT ATC CIT GTG CTT GCA GAG TTT GAC ACT77Gbl Leo met Arq Lye Gip Her Tyr Asn Pro Val Thr HIls Ile Tyr Tier Ala Gin Asp Val Lys Glb Val Ile Glu Tyr Ala Ar;9 Leou Ar; Giy Ile Ar; Val Lou Ala 010 Pb. Asp Tier 259Glu Leo Ser Asn Lye Gip Her Tyr Ser Lou Ser H-is Val Tyr Tier Pro Ace Asp Val Arg Met Val lie Glu Tyr Ala Ar; Leou Ar; 01 Xlb. Ar; Vki Lou Pro Gbu Pb. Asp Tier 279GAG TTA AGC MT MAA GGA AIC TAT TCT TTG TCT --CAT ITT TAT ACA CCA MAT GAT GTC CIT ATG GTG ATT GMA TAT ICC AlA TTA CIA GGA ATT CIA GTC CTG CCA GMA TTT GAT ACC 837

NRO 6INTRON 7

a CCT GGC CAC ACT TTG TCC TGG GGA CCA II T ATC CCT GGA TTA CTG ACT CCT TIC TAC TCT GGG TCT GMG CCC TCT GGC ACC TTT GGA CCA GTG MI CCC AlT CTC MAT MAT ACC TAT GAG 897Pro Giy His Thr Leo Her Pop Giy' Pro 47yIl Pro Gly Lou Leo Tier Pro Cye Tyr Her Gly Ser Ito Pro Ser Gly Tier Pb. sly Pro Val Awn Pro6 Ser Lou Asm Asn Tier Tyr Ito29

P Pro Giy His Tier Leou Her Tvp Giy Lys Gylie Lys Asp Leou Leo Tier Pro Cys Tyr Her Arg Gle Ace Lys Leu Asp Ser Pb. Giy Pro Iie Asn Pro Thr Led Asn Thr Pier Tyr Ser 319CCT GGG CAT ACA CTA TCT TGG GGA MAA GfT CAl MAA GAC CTC CTG ACT CCA TGT TAC AlT AlA CM MAC MIG TTG GAC TCT TTT GGA CCT'ATA AMC CCT ACT CTG MAT ACA ACA TAC AGC 957

I ~~~~~~~~~~~~~~INTRONSINTRON 7

aTTC ATG AIC ACA TTC TTC TTA GMA GTC AGC TCT GTC TTC CCA GAT TTT TAT CTT CAT CtT GGA GGA GAT GAG ITT GAT ±TC ACC TIC TI MIG TCC MAC CCA GAl ATC CMG GAC TTT ATG 1017Pie. Met Ser Tier Pb. Pies Litu 010 Vat Her Ser Val Pb. Pro Asp Phe Tyr Leo His Lai Gly OLy Asp GSb Val Asp Pb. Thr Cys Pr Lys Her Asm Pro Ito Il. GIn Asp Pbi Not39

(3Pb. Leo Thr Tier Pie. Pb. Lys 010 Ile Her Ito Val Pb. Pro Asp Gle Ph. Ite Hie Leo Giy 017 Asp Gio Val Ito Pb. Lye Cys T GlpItoHr Asn Pro Lys Ilie Gin Asp Pb. net 359TTC CTT ACT ACA TTT TTC MAA GMA ATT AlT GAG GTI TTT CCA GAT CM TTC ATT CAT TTG GGA GGA GAT GMA GTG GM TTT MAA TGT TI GMA TCA MAT CCA MAA ATT CM GAT TTC ATG 1077

INTRON 9INTRON 8

aGAll Ml AA GGC TTC lIT GAG GAC TTC MIG CAl CTG GAG TCC TTC TAC ATC CAl ACG CTG CTG GAC ATC GTC TCT TCT TAT GGC MIG GIC TAT GTG GTG TGG CAl GMG GTG TTT GAT MAT 1137Ar; Lys Lys Gily Pb. Giy Ito Asp Pb. Lye Gle Leo Glu Her Pb. Tyr Ile Gin Th!r Leo Leou Asp XIi Vat Ser Sbr Tyr Gly Lys Giy tyr Vat val Pop Gin Gab Val Pb. Asp Aen379AspGl Lye Gly Pb. Gip Thr Asp Pb. Lye Lys Leo Gio Her Pb. Tyr Ile Gin Lys Vat Leou Asp II* Ite Ala Thr Ile Ace Lye Gly Ser Gte Vai Pop Gin 010i Val Pb. Aksp Asp39All MA MAA GGC TTT GGC ACA GAT TTT MI MAA CTA GMA TCT TTC TAC ATT CMA MG ITT TTG GAT ATT ATT GCA ACC ATA MC MI GGA TCC ATT GTC TGG CAl GAG ITT TTT GAT GAT 1197

INTRON'10IINTRON 9

AAMA ITA Ml; TT CAl CCA GAC ACA ATC ATA CAl GTG TGG CIA GAG GAT ATT CCA GTG MAC TAT ATI MI GAG CTG GMA CTG GTC ACC MlG ICC GGC TTC CII ICC CTT CTC TCT ICC CICC 1257Lys Vat Lye It. Gte Pro Aso Tier 1ie It. Gte Vai Pop Arg Ito Asp It. Pro' Vat Ace Pyr Net Lye 010 Leou Glu Leo Val Tier Lys Ala Gi1 Pb. Arg Ala Leo Leo Her Ala Pro 419

(3 Lys Ata Lye Leo Ata Pro Glp Tier II* Vat Ito Vai Pop Lys --Asp Ser Ala-----Tyr Pro Ito Gic Lou Ser Arq Vab Tier Ala Ser Gly Pb. Pro Vat It. Lou Her Ala Pro 436MAA ICAAMI CTT GCG CCI tIC ACA ATA ITT GMA GTA TGG MAA --- GAC MGC ICA-----TAT CCT GAG GMA CTC MGT AlA GTC ACA GCA TCT GGC TTC CCT GTA ATC CT'T TCT GCT CCT 1308

INTRON 10ITO

a TGG TAC CTG MAC CIT ATA TCC TAT CGC CCT GAC TGG MI GAT TTC TAC GTA ITI GMA CCC CTI GCA TTT GMA lIT ACC CCT GAG CMG MI GCT CTG GTG ATT lIT GGA GAG GCT TGT ATG 1377Pop Tyr Leo Ann Arg Ilie Her Tyr Gly Pro Asp Pop Lye Asp Phe Tyr Vat Val 010 Pro Leo Aba Pb. Ito 0,ly Tier Pro Glti GIn Lye Ala Leo Vat Ile Oly Oly Gio Ala Cys met 459

(3 Pp Tyr Leou Asp Leo 1ie Her Tyr Gly Gin Asp Pop Arg Lys Tyr Tyr Lye Val Gio Pro Leo Asp Pb. Glp GiyT Pier Gin Lys GIn Lye Gin Leou Ph. Ile 017 Gly 010 Ala Cye Lou 476TGG TAC TTA GAT TTG ATT AGC TAT GGA CMA GAT TGG All MAA TAC TAT MAA GTG GMA CCT CTT GAT TTT GGC IfT ACT CAl MAA CAl MAA CMA CTT TTC ATT lIT GGA GMA ICT TGT CTA 1428

INTRON 12INTRON 1 1

a TGG GGA GMA TAT GTG GAC MAC ACA MAC CTG GTC CCC All CTC T9GI CCC AlA GCA Ill GCT ITT ICC GMA All CTG TGG AGC AMC Ml TTG ACA TCT GAC CTG ACA TTT IC'C TAT GMA CIT 1497Pop Glp Glu Tyr Val Asp Asn Tier Asn Leo Vab Pro Arp Leo Prr Pro Ar; Ala Glp Ala Val Aba 010 Ar; Leou Pop Her Ace' Lye Leo Tier Ser Asp Leo Tier Ph. Ala Tyr Ito Ar; 499

(3 Top rlp Gio Tyr Val 'Asp Ala Tier Asn Leou Tir Pro A~r; Leou Pr Pro Arp Ala Ser Ala Val Gby 010 Avg Leo Pop Her Ser Lye Asp Vat Arg Asp Met Asp Asp Ala Tyr Asp Ar; 516TGG GGA GMA TAT GTG bAT GCA ACT MAC CTC ACT CCA AlA TTA TGG CCT CII GCA AlT GCt ITT lIT GAG AlA CTC TGG AlT TCC MAA GAT GTC AlA GAT ATG GAT GAC ICC TAT GAC AlA 1548

INTRON 13IINTRON 12

a TTG TCA CAC TTC CGC TGT GAG TTG CTG All CIA lIT GTC CAl ICC CMA CCC CTC MAT GTA GIC TTC TGT GAG CAl GAG TTT GMA CAl ACC TGA 1590Leo ber His Phe Ar; Cys Gbo Leo Leo Argi Ar; Gtp Vab Gbe Ala Gin Pro Leou Asn Vab Gly Phe Cye Gbo Gin Glu Pie. Ito Gbe Thr --- 529

(3 Leo Thr Arg His Arq Cys Arg Met Vab ltj Ar; Gty Ibe Ata Aba Gle Pro Leo Tyr Ala Glt Tyr Cys Ace His 11l5 Asn Met --- 544CTG ACA All CAC CIC TIC All ATI ITC GT CIT GGA ATA GCT GCA CMA CCT CTT TAT GCT GGA TAT TGT MAC CAT GAG MC ATG TMA 1632

INTRON 13

FIG. 1. Relative position of introns in the a- and f3-protein coding regions. a-Chain nucleotide and deduced amino acid sequences are fromMyerowitz et al. (4), and the f3-chain nucleotide and deduced amino acid sequences were derived in this study The nucleotide and amino acidnumbering is in the right margin. The two amino acid sequences were aligned to maximize similarity of amino acids by the PRTALN programof Wilber and Lipman (21). Identical amino acids at the same position are in bold type. The positions of intronic interruption are indicated. Theintron positions of the a-chain gene are from Proia and Soravia (10).

clease III for various times, treated with S1 nuclease, and fragments were hybridized to the exon-specific oligonucleo-electrophoresed in agarose gels. After Southern blotting the tide probes. The exon position was easily identified by de-

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termining the length of DNA removed before the hybridiza-tion signal was eliminated.

General Methods. Oligonucleotides were prepared by C.Camerini-Otero (National Institute of Diabetes, Digestiveand Kidney Diseases, National Institutes of Health) on an

Applied Biosystems (Foster City, CA) model 381A DNAsynthesizer. Double-stranded DNA fragments were labeledby nick-translation with [a-32P]dCTP according to Rigby et al.

(17), and oligonucleotides were end-labeled with [y-32P]ATPand T4 polynucleotide kinase. Phage DNA was isolated byglycerol gradients as described (18). Plasmid DNA was iso-lated by the alkaline lysis method (19) followed by centrifu-gation through a cesium chloride/ethidium bromide stepgradient (20). Plasmid DNA was sequenced by using a kitfrom Promega Biotec (Madison, WI). Other general methodswere essentially as described by Maniatis et al. (18).

RESULTS AND DISCUSSIONScreening of a Hep G2 cDNA library resulted in the isolationof a 1703-base-pair p-chain cDNA. The p-chain protein cod-ing region beginning with the first ATG in the cDNA is shownin Fig. 1. This cDNA contains a 64-base-pair 3'-untranslatedregion and 12 base pairs upstream of the first ATG. Thesequence of this cDNA matches precisely with that reportedby Korneluk et al. (5) except for the presence of one addi-tional cytidine (position 28 in Fig. 1). Furthermore, thisadditional cytidine was also found in the corresponding ge-

nomic sequence. Enzymatically active 8-hexosaminidase Bcan be produced from this cDNA after transfection into COScells (data not shown), implying that the sequence contains a

functional protein-coding region.Three human genomic libraries were screened with the

EcoRI-derived fragments of the 8-chain cDNA. This resultedin the isolation of a series of overlapping genomic clonesencompassing the p-chain gene (Fig. 2). To facilitate the finestructural analysis of the gene, restriction fragments from thegenomic inserts containing exonic sequences were identified,after Southern blotting, by hybridization with ,8-chain-specificoligonucleotides. The exon-containing fragments were sub-cloned into plasmid vectors as indicated in Fig. 2.The strategy for the determination of the intron-exon

structure of the p8 gene was based on the utilization of a

battery of p-chain-specific oligonucleotides. The oligonucle-otides were used as primers to sequence the genomic DNAcontained within the plasmid subclones. The resulting se-quences were then compared to the 8-chain cDNA sequenceto identify the precise intron-exon junctions. The oligonu-cleotides were also used as specific hybridization probes to

KILOBASESEXONS

GENE

EcoRI MAP

PLASMIDSUBCLONES

0 10

ES2

20

locate the position of the exons within the gene. These an-

alyses revealed that the p-chain coding region is divided into14 exons over a stretch of -40 kb of genomic DNA (Fig. 2).The EcoRI restriction map of the cloned DNA is consistentwith Southern blot analysis of EcoRI-digested total genomicDNA (data not shown), indicating that the organization ofthe cloned and the cellular DNA is the same.

The sequences of the intron-exon junctions of the 8-chaingene are compiled in Table 1. All intron sequences conformto the "AG/GT" rule as formulated by Breathnach andChambon (22). The size of the p8-chain introns ranges from-.0.2 to 11 kb, with the introns in the 5' half of the genegenerally larger than those in the 3' half (Fig. 2 and Table 1).In contrast, the exons show the restricted size distributiontypical of most eukaryotic genes.The extent of exon 1 at the 5' end has not yet been

determined so its exact size is not known. Correspondingly,the existence of an additional intron in the 5'-untranslatedregion is possible. Sequence analysis of the 5' genomicregion revealed an in-frame ATG 12 codons upstream fromthe first ATG in the cDNA (data not shown). Since thecDNA contains a functional ATG initiator codon, this raisesthe possibility that there may be more than one initiatorcodon in the gene. Analysis of the 3' end of the gene was

complicated by an incomplete 3'-untranslated region in thecDNA, as suggested by the absence of a poly(A) tract.However, comparison of the sequence reported by Korneluket al. (5), which apparently contains the full 3'-untranslatedregion, with the genomic sequence revealed no intronicinterruptions.Comparison of the derived amino acid sequences of the a

and p chains shown in Fig. 1 reveals an overall identity of>50%o with considerable divergence of the sequences at theamino termini. The overall structural and functional similar-ities suggest a common evolutionary origin for the a and P

chains. To shed light on the evolutionary history of the 8-hexosaminidase subunits, the structural features of the twogenes were compared.The position of the a- and 8-gene intervening sequences

relative to their coding sequences is shown in Fig. 1. Whenthe a- and p-coding sequences are aligned for maximalidentity it is apparent that, with the exception of the firstintron in each gene, the remaining 12 introns interrupt atprecisely the same positions. Within genes derived from a

common ancestor, as with the globin (25), serine protease(24, 26), vitellogenin (27), and corticotropin-proenkephalin(28) families, there is conservation of intron location withrespect to coding regions. The striking conservation of intron

~~~~~~~~~I 13

1 2 3 45 6 7 8 9 1011 12 14

l 1 1 11 1 11111I ~~II I I

E4

ES 5

SS 3.6"I-I

EE7

50

SE 5

E30

F 0E6i

30 40

GENOMIC i lA ICLONES A-151 A-17

A-10 A-11 A-20

A-14 A-3

FIG. 2. Map of the human 8-hexosaminidase /8-chain gene. Open bar at the top of the figure depicts the /-chain gene from 5' (on the left)to 3' (on the right). Exons 1-14 are represented by vertical bars of various thicknesses. Introns are represented by the open areas. Below the/3-gene structural map are an EcoRI restriction map of this segment ofDNA and positions of plasmid subclones and of genomic clones. Insertsfrom the bacteriophage genomic clones are A-SA, A-15, A-10, A-14, A-1, A-3, A-20, and A-17. Genomic fragments subcloned into plasmid vectorsare indicated above the primary genomic clones. The fragments were derived with EcoRI (E.7, E7, E4, E.8, E3, and E6), EcoRI and Sal I (ES2,ES5, and SE5), and Sac I and Sal I (SS3.6).

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Table 1. Sequence of the (3-hexosaminidase (3-chain gene intron-exon junctionsExon Sequence of intron-exon junction Intronsize, size,

Exon bp 5' border 3' border kb

1 >263 TTT CGA CG gtgagc.... ttatttctcaaacag A TAT CAT -4.8263 264Arg-88 Arg-88

2 146 GAG TCT T gtaagt .... aaatcctcaatacag AT ACT TTA -4.1409 410Tyr-137 Tyr-137

3 66 TTA CGA G gtaagt .... ttttactttcctcag GT TTA GAG -2.8475 476Gly-159 Gly-159

4 47 TAT GGA ACT gtaagt .... tttattttgtcatag TTC ACC ATC -0.4522 523Thr-174 Phe-175

5 111 AAA ACT CTG gtaagt .... tatgtttgcttgcag GAT GCC ATG -10.7633 634Leu-211 Asp-212

6 102 AGC AAT AAA gtgagt .... atatggcttttacag GGA AGC TAT -8.3735 736Lys-245 Gly-246

7 130 GGA AAA G gtaagg .... aatattttcttctag GT CAG AAA -1.7865 866Gly-289 Gly-289

8 181 AAA TGT TG gtaaga .... cactttttgcttcag G GAA TCA -1.11046 1047Trp-349 Trp-349

9 87 ATT CAA AA gtaagt .... tttttgtaatactag G GTT TTG -1.81133 1134Lys-378 Lys-378

10 73 AAA GCA AAG gtgagc .... taacgttaaattaag CTT GCG CCG -0.51206 1207Lys-402 Leu-403

11 175 TTT GGC G gtaagt .... atgattttaatttag GT ACT CAG -1.81381 1382Gly-461 Gly-461

12 91 AGA TTA TG gtatgg .... tgatttaaattttag G CCT CGG -0.21472 1473Trp-491 Trp-491

13 105 ATG GTC GA gtaaga .... tcatgttatctacag A CGT GGA -0.31577 1578Glu-526 Glu-526

14 169

Exon sequences are shown in upper case letters; intron sequences are in lower case. Numbersimmediately below the sequence refer to the nucleotide positions of the ,B chain in Fig. 1 that flank eachintron. Below these numbers are the amino acids in the (-chain protein sequence interrupted by orflanking each exon. bp, Base pair(s).

positioning in the ,3-hexosaminidase genes is clear evidencethat the duplication of a progenitor gene was a step in theevolution of the a and 3 chains.A comparison of the overall architecture of the a and ,B

a-GENE

P-GENE

11

21

genes is shown in Fig. 3. Given the great size diversity ofeukaryotic genes, the a gene (-z35 kb) and P gene (-40 kb)are of reasonably similar size. Both genes have 13 intronsdividing their protein coding regions into 14 exons. In con-

AATAA T10 12

21 31 41 51 61 71 81 91 1l 1 131 14

11 11 I 11111

11

0

TI I I31 4151

10

+20

KILOBASES

I I I11 I1171 8T 91 1o0111 121 1 14

13AATAAA

30 40

FIG. 3. Comparison of the structural organization of the a- and ,(-chain genes. Exons are represented by vertical bars of various thicknesses,and the introns are represented by open areas. The organization of the a-chain gene is from Proia and Soravia (10). Sequence similarity betweenthe first and second exons of two genes is indicated by dashed lines. The hatched area in exon 14 of the a-chain gene corresponds to theadditional 3'-untranslated sequence found in the larger of the two a-chain mRNAs (4). The polyadenylylation signals, AATAAA and ATTAAAfor the a-chain gene (4, 5) and ATTAAA for the (8-chain gene (5), are indicated.

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trast to the general conservation of exon size, the absolutelength of corresponding introns varies greatly between thetwo genes. In limited comparisons of intron sequences, littlesimilarity is observed except near splice junctions. Thedifferences of intron size and sequence are consistent withintronic divergence by deletional and insertional events ashas been observed in other gene families (27, 29). The roughsimilarity of the genes in their size and their exon spacingat the 3' ends may be a reflection of the primordial genestructure prior to its duplication.

Introns have been demonstrated to demarcate structuraland functional regions of proteins (30). In the case of the achain (10), it was observed that intron 1 interrupts at a gly-cine residue preceding a pair of basic amino acids (Lys-Arg,residues 86 and 87) that serve as a proteolytic processing siteduring the maturation of this polypeptide. The ,8 polypeptideundergoes two major proteolytic cleavages during matura-tion (E. F. Neufeld, personal communication). But in con-trast to the a chain, the new amino termini created byprocessing of the p chain are not located near an intron-exonjunction.The amino acid sequences carried by exons 1 and 2 in the

13-hexosaminidase genes are much more dissimilar (26% iden-tity) than those specified by the remaining exons of the twogenes (63% identity) (Fig. 1). Also, there is variation in thelength of the a and (8 polypeptides at the amino termini (Fig.1). How might this regional sequence diversity and thecorresponding discordance in intron-exon structure be ex-plained? After the duplication of an ancestral gene, thisvariation may have been introduced by a mechanism called"intron sliding." Craik et aL (26, 31) have proposed thatmutations causing splice junctional slippage result in lengthand sequence variability by the extension or contraction ofexons. They suggest that the resulting length variation isexpressed as surface loops on protein structures. Such ahypothesis is attractive because the sliding of intron 1 in oneof the f8-hexosaminidase genes would account for the posi-tional noncoincidence of intron 1 and for the sequence diver-gence and length variation at the amino-terminal end of thechains. The resulting exons could have then diverged towarddifferent functions as new structural domains. An inconsis-tency with an intron sliding model is the apparent absence ofinserted or deleted sequences at the positions of intron 1 whenthe a and (8 chains are aligned (Fig. 1).

Intron deletion or insertion during the evolution of the,B-hexosaminidase genes may also be a plausible explanationfor the difference in intron-exon structures. In this case,however, additional mechanisms would have to be invokedto explain the variation of size and sequence at the aminotermini of the subunits. "Exon shuffling," i.e., the recruit-ment of exonic units from unrelated genes (30), is unlikely tohave occurred due to the diminished but distinct sequenceidentity between the 5' exons of the two genes (Fig. 1).Resolution of the mechanisms involved in the evolution ofthe a and ,Bgenes will require the isolation and examination,from a more primitive organism, of a /-hexosaminidase genethat has not been duplicated.

In addition to contributing to an understanding of 13-hexosaminidase evolution, the clarification of the P-chaingene structure is essential for the studies of the genetic de-fects underlying Sandhoff disease. A heterogeneous group ofmutations have been compiled by examination of the pro-tein, the mRNA, and the gross gene structure of the ( chain(23, 32-34). The availability of the fine gene structure alongwith cloned genomic sequences will permit characterizationof these gene defects at the molecular level.

I am grateful to C. Camerini-Otero for the synthesis of theoligonucleotides, E. Soravia for assistance with the cDNA isolation,and E. F. Neufeld for providing her unpublished data. I also thankL. Eidels, R. Myerowitz, A. Robbins, and S. Sonderfeld-Fresko forhelpful comments on the manuscript.

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eds. Stanbury, J. B., Wyngaarden, J. B., Fredrickson, D. S.,Goldstein, J. L. & Brown, M. S. (McGraw-Hill, New York), pp.945-969.

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Greenwald, S., Lim, J. S. T., Roy, C., Schoenfeld, V., Lowden, J.A. & Kaback, M. M. (1983) Am. J. Hum. Genet. 35, 1259-1269.

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18. Maniatis, T., Fritsch, E. F. & Sambrook, J., eds. (1982) MolecularCloning: A Laboratory Manual (Cold Spring Harbor Laboratory,Cold Spring Harbor, NY)

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Biophys. Res. Commun. 117, 835-845.21. Wilber, W. J. & Lipman, D. (1983) Proc. Natl. Acad. Sci. USA 80,

726-730.22. Breathnach, R. & Chambon, P. (1981) Annu. Rev. Biochem. 50,

349-383.23. O'Dowd, B. F., Klavins, M. H., Willard, H. F., Gravel, R.,

Lowden, J. A. & Mahuran, D. J. (1986) J. Biol. Chem. 261,12680-12685.

24. Rodgers, J. (1985) Nature (London) 315, 458-459.25. Maniatis, T., Fritsch, E. F., Laver, J. & Lawn, R. M. (1980) Annu.

Rev. Genet. 14, 145-178.26. Craik, C. S., Qui-Lim, C., Swift, G. H., Quinto, C., MacDonald, R.

J. & Rutter, W. J. (1984) J. Biol. Chem. 259, 14255-14264.27. Wahli, W., Dawid, I. B., Wyler, T., Weber, R. & Ryffle, G. U.

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