Proc. Natl. Acad. Sci. USAVol. 83, pp. 6337-6341, September 1986Biochemistry

Molecular cloning of the human esterase D gene, a genetic markerof retinoblastoma

(esterase D cDNA/retinoblastoma marker/DNA sequence)

EVA Y.-H. P. LEE AND WEN-HWA LEEDepartment of Pathology, School of Medicine, University of California at San Diego, La Jolla, CA 92093

Communicated by Russell F. Doolittle, May 12, 1986

ABSTRACT Retinoblastoma, the most common intraocu-lar tumor, represents one of the prototypes of inheritablecancers. To elucidate the mechanisms that give rise to thistumor, the retinoblastoma gene (RB) must be molecularlycloned. The difficulty encountered in cloning the gene is thatlittle of its function or structure is known. The human esteraseD gene, on the other hand, has been localized cytogenetically tothe same sub-band of chromosome 13q14:11 as the RB gene.The esterase D gene thus provides a convenient starting pointfor cloning theRB gene. In this communication, we describe theisolation of the esterase D cDNA clone. Its identification isbased on three lines of evidence. (') This cDNA encodes aprotein immunologically related to the esterase D protein. (il)The deduced amino acid sequences of this clone containsequences identical to the three CNBr-cleaved peptides of theesterase D protein. (iii) This clone is mapped to the chromo-some 13q14 region by Southern genomic blotting using differ-ent deletion mutants. The availability of this clone should allowfor the cloning of the RB gene by chromosome walking; thediagnosis of genetic defects such as retinoblastomas and Wilsondisease, whose genes are closely linked to the esterase D gene;and the exploration of the large family ofhuman esterase genes.

Human esterase D is one member of a group of nonspecificesterases. The polymorphic nature of this enzyme has beena valuable marker in studies of population genetics (1, 2).Recently, the genetic locus of esterase D was mapped to thechromosome 13q14:11 region by correlating the loss ofenzyme activity with deletions ofchromosome 13 (3, 4). Thisregional assignment coincides with the location of a gene(RB) involved in the tumorigenesis of retinoblastomas (5-7).The molecular mechanism of the formation of this tumor isunknown. Inactivation of gene(s) (RB) mapped to the chro-mosome 13q14:11 region is believed to be the primary causeof this inheritable childhood cancer (7-10). However, cloningthe RB gene is difficult since little of its structure or functionis known. This situation is similar to that encountered incloning the muscular dystrophy or the cystic fibrosis genes(11, 12). The localization of the esterase D gene and the RBgene to the same sub-band of chromosome 13q14:11, there-fore, provides an advantageous approach for cloning the RBgene by chromosomal walking using the esterase D gene asthe starting point. In addition to its usefulness in cloning theRB gene, the tight linkage between these two genes couldallow the esterase D gene to serve as a crucial marker inelucidating the behavior of the RB gene (13, 14). Moreover,the defective gene in Wilson disease was found to be linkedto the esterase D gene (15). Esterase D should provide avaluable marker in the diagnosis of these inheritable geneticdiseases.

In addition to serving as a genetic marker of retinoblasto-mas, esterase D may play a role in detoxification (16). Wehave recently observed that the esterase D protein is distrib-uted at the highest level in liver and kidney and that it isinducible by phenobarbital but not phorbol myristate estertreatment. To further study the function and regulation of thisenzyme, it would be extremely helpful to obtain the esteraseD gene clone.

In this communication, we describe the cloning of theesterase D cDNA by screening a Xgtll expression libraryusing our newly prepared anti-esterase D antibody. We havealso sequenced both the esterase D protein and the cDNAclone. The deduced amino acid sequences of the cDNA clonewere identically matched to the protein sequences. Further-more, this gene was mapped to chromosome 13q14 bySouthern genomic blotting using different deletion mutants.The availability of the esterase D cDNA clone shouldtherefore facilitate future studies of retinoblastomas.

MATERIALS AND METHODSCells, DNA, and RNA. Human mutant fibroblasts,

GM1142, GM2718, and GM3887 were obtained from theHuman Genetic Mutant Cell Repository (Camden, NJ) andcharacterized as described (17). Human retinoblastoma cellline Y79, neuroblastoma cell line LA-N-5, and Chinesehamster-human hybrid cell line 34-2-3 were provided asdescribed (18, 19). All these cells were grown in Dulbecco'smodified Eagle's medium (GIBCO) supplemented with 10%fetal calf serum. Genomic DNA was extracted from thesecells as described (20). Cellular messenger RNA was pre-pared by the guanidine isothiocyanate/cesium chloride meth-od and enriched by oligo(dT)-Sepharose column chromatog-raphy (20).

Partial Determination ofAmino Acid Sequence of Esterase DProtein. Purified human esterase D protein (16) was treatedwith cyanogen bromide and the product was purified byreversed-phase HPLC (Brownlee RP 300). After the elutedpolypeptides were dried, their amino acid sequences weredetermined by solid-phase Edman degradation with HPLCanalysis of the phenylthiohydantoin derivative as described(21).

Construction of Oligonucleotide Probes. Mixed oligonucle-otide probes were synthesized on a synthesizer usingphosphotriester chemistry (R. Doolittle's laboratory, Dept.of Chemistry, University of California at San Diego). Threesets of oligonucleotide mixtures corresponding to the possi-ble coding sequences of each peptide were constructed. Theoligonucleotide mixtures were purified by gel electrophoresison 20% polyacrylamide/8 M urea gels and subsequentlylabeled at the 5' end with [y-32P]ATP by using T4 polynu-cleotide kinase (20).Antibody Screening of the Agtll cDNA Library. Rabbit

anti-human esterase D antibodies were prepared against

Abbreviations: bp, base pair(s); kb, kilobase(s).

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A B0.5 Peptide I N-terminus blockedPeptide II Met-Tyr-Ser-Tyr-Val-Thr-Glu-Glu-Leu-Pro-Gln-Leu-Ile-Asn

IV Sequence 5t'ATTAC-TCCNTAC.T-ACN-GAA-GAA-CT 3'26mer Probe 5'AG-CTC-CTC-NGT-NAC-GTA- GA-GTA-CAT 3'T T A A A

III l Peptide III l4et-Lys-Phe-Ala-Val-Tyr.-Leu-Pro-Pro-Lys-Ala-Glu-Thr-GlySequence 5'ATG-AA -TT -GCN-M-TA: 3'G T18mer Probe 5'ATA-NAC-NGCGM- TT-CAT 3'II "'s C ~ ~~~~~~~A CPeptide IV Met-Gly-Gly-His-Gly-Ala-Leu-Ile-Cys-Ala-Leu-Lys-Asn-Pro-Gl

Sequence ~~~~~~~AT..GN..T A C1u-Sequence 5'ATC-TG _GCN-CTN-ANAAGAAiCCN-GG 3'T

G

0 C C A T15 20 23mer Probe 5'CC-NGG- TT- TT-NAG-NGC- CA-GAT 3'A T C ATime, min

FIG. 1. Profile of CNBr-cleaved esterase D protein separated by reversed-phase HPLC (Brownlee RP 300) (A) and partial amino acidsequences of three CNBr-cleaved peptides and the corresponding sequences used to construct the synthetic oligonucleotide probe mixture (B).

homogenous enzyme as described (16). The IgG fraction ofthis antibody was further purified by protein A Sepharosecolumn chromatography and absorbed with boiled Esche-richia coli strain Y1090 to remove any nonspecific binding.Optimum antibody concentrations and binding were deter-mined by dot analysis with purified esterase D protein. TwoXgtl1 cDNA libraries constructed using human hepatomamRNA (22) and human placenta mRNA (23), respectively,were plated on E. coli strain Y1090 at a density of 1 x 105phage per 150-mm Petri dish and screened as described (24).After 3-5 hr of incubation at 420C, the bacterial lawns werecovered with a 137-mm nitrocellulose filter that had beensoaked in 10 mM isopropyl f3-D-thiogalactoside and dried.Incubation was continued but shifted to 370C for 5 hr. Thefilters were first soaked with 3% nonfat dry milk and thenincubated with the prepared anti-esterase D IgG at a con-centration of 1 ,ug/ml overnight. After briefly washing with0.1% Nonidet P-40, they were incubated for 2 hr with'25I-labeled protein A, washed, and dried. XAR-5 film wasexposed to the filters overnight at -80°C using an intensifyingscreen. Positive phage were picked and repeatedly rescreenedat lower density until a pure population was obtained.DNA Sequencing. The XEL22 clone containing a 1100-base-

pair (bp) EcoRI insert was subcloned into M13 mpll at theEcoRI site. The single-stranded DNAs of two recombinantswith opposite orientation were isolated and used to construct3' deletion mutants as described (25). Five mutants withdifferent lengths of insert from each parental clone weresequenced by using the dideoxy-nucleotide chain-termina-tion method (26).

Southern Blot Analysis. Human genomic DNA (10 ,ug) fromdifferent cells was digested with endonucleases and subjectedto electrophoresis. Genomic blots were hybridized with32P-labeled EL22 cDNA clone as described (20).RNA Blot Analysis. Poly(A)-enriched mRNA was prepared

and electrophoresed on 1.2% agarose containing formalde-hyde and transferred to a nitrocellulose filter (20). The RNAblot was hybridized with the same probe as described above.

RESULTSScreening for cDNA Clones Containing the Esterase D Gene.

Two complementary approaches were used for cloning theesterase D gene. The first approach involved immunoscreen-ing of expression cDNA libraries using antibodies against theesterase D protein. The second approach entailed the use ofoligonucleotide probes with sequences deduced from partialamino acid sequences of the purified esterase D protein.Since both approaches required a significant amount of thepurified esterase D protein, we have developed a protocol topurify human esterase D protein from erythrocytes to bio-chemical homogeneity (16). The purified esterase D protein

was characterized by sequence analysis starting from theNH2 terminus by Edman degradation. However, no aminoacid residue was obtained, suggesting a blocked NH2 termi-nus. Further amino acid composition analysis of the protein

Aal b c it' b c'

O-G a]-.X_........

B

hkb23.7-

9.5-6.7 -

2.3 -.l -

0.6-

FIG. 2. Identification of positive esterase D clones from XgtllcDNA libraries. (A) Positive clones detected by autoradiography,EL22a and EL22b, were lysogenized in E. coli Y1090 and induced toexpress fusion proteins. Lysate samples of the parental BNN103(Xgtll) (lane a), Y1090(EL22a) (lane b), and Y1090(EL22b) (lane c)were analyzed on a 7.5% NaDodSO4/polyacrylamide gel and stainedwith Coomassie brilliant blue. /-Galactosidase (114 kDa) was clearlyinduced and expressed in BNN103(Xgtll), but the expression of anyfusion protein of EL22 was not apparent. Duplicate samples on thesame polyacrylamide gel were electrotransferred onto a nitrocellu-lose filter. The filter was first preincubated with 3% nonfat dry milkin TBS (Tris-buffered saline; 0.17 M NaCl/0.01 M Tris HCl, pH 7.5)for 1 hr and then incubated with rabbit IgG specific to esterase D in1% gelatin for 12 hr. After washing with TBS, the filter was furtherincubated with 125I-labeled protein A for 2 hr. A fusion protein of 145kDa was detected in the recombinant lysogen of EL22a and EL22b(lanes b' and c') but not in the parental strain (lane a'). (B)Hybridization of 32P-labeled 23-mer to EL22a (lane a) and EL22b(lane b). DNA from recombinants X EL22a and EL22b were digestedwith EcoRI and electrophoresed through a 0.8% agarose gel. Thedigested DNA fragments were then transferred onto a nitrocellulosefilter and hybridized with 32P-labeled 23-mer in 6x SSC at 37°C (lxSSC = 0.15 M NaCl/0.015 M sodium citrate). A DNA fragment of1.1 kb was detected under the washing condition of 3 x SSC at 37°C.

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showed that it contained four or five methionine residues.The esterase D protein was then cleaved by cyanogenbromide (CNBr) and the four resultant peptides were purifiedby HPLC as shown in Fig. 1A. Sequences containing 13, 14,and 14 amino acid residues each were generated frompeptides II, III, and IV, respectively (Fig. 1B). No sequencewas obtained from peptide I, however, suggesting it waslocated at the NH2 terminus of the esterase D protein.

In the first approach, rabbit polyclonal antibodies wereraised against the esterase D protein for screening expressioncDNA libraries (16). Affinity-purified anti-esterase D IgGspecifically detected esterase D in immunoblotting analysiswith a sensitivity of 1 ng. Since esterase D is widelydistributed in human tissues, two Xgt11 expression librariesconstructed from human hepatoma mRNA (22) and fromhuman placenta mRNA (23), respectively, were immuno-screened with this IgG. A total of four clones were obtainedfrom these two libraries. Two were very small, containingonly 150- to 200-bp inserts, while the other two containedidentical 1.1-kilobase (kb) inserts (Fig. 2B). These twoidentical clones, named EL22-a and -b, were induced toexpress f8-galactosidase fusion protein as shown in Fig. 2A.Using the specific anti-esterase D IgG in immunoblotting, afusion protein of 145 kDa was detected (Fig. 2A). Since thewild-type ,B-galactosidase protein was 114 kDa, the remainingfragment of -31 kDa was presumed to be encoded by theinsert. This insert should therefore contain >90o of thesequence for esterase D protein, which is known to have amolecular mass of 34 kDa. The 145-kDa fusion protein wasalso detected by two different monoclonal antibodies againstesterase D protein in the immunoblot (data not shown),suggesting that the fusion protein contains different epitopesrecognized by these antibodies.

In the second cloning approach, oligonucleotide probescorresponding to the nucleotide sequences deduced from theamino acid sequences of the CNBr-cleaved esterase Dpeptides were chemically synthesized as shown in Fig. 1B.All these probes, except one 23-mer oligonucleotide mixturederived from peptide IV, cross-hybridized significantly withX vector or E. coli DNA under stringent hybridizationconditions. The 23-mer oligonucleotide was therefore usedfor subsequent screening. To test whether the EL22 clonecontained the esterase D gene, the 32P-labeled 23-mer oligo-

nucleotide mixture was used as the probe in Southern blottinganalysis. As shown in Fig. 2B, one 1.1-kb EcoRI-digestedDNA fragment hybridized specifically to this probe. Thesedata also suggest that the EL22 clone contains the esterase Dgene.DNA Sequence Analysis of EL22 cDNA Clone. The EL22

clone was further characterized by DNA sequence analysis.The 1.1-kb cDNA was subcloned into the M13 phage mpll atthe EcoRI site, and deletion mutants were constructedfollowing a described protocol (25). These subclones weresubjected to sequence analysis by the method of dideoxy-nucleotide chain termination. Approximately 95% of thedouble-stranded DNA was sequenced (Fig. 3). A long openreading frame encoding a protein of 31 kDa was found.Moreover, the three stretches of amino acid sequencespreviously obtained from CNBr-cleaved peptides were iden-tically matched to the deduced protein sequence (Fig. 3).With these data and the results described above, we concludethat the EL22 clone is the esterase D cDNA. Based on aminoacid sequences and the esterase D protein size, it is evidentthat 20-30 amino acids at the NH2 terminus of esterase D arenot found in the EL22 clone.

Size of Esterase D mRNA and of Esterase D Genome. RNAblotting analysis was performed to determine the mRNA sizeof the esterase D gene by using poly(A)-selected mRNA fromtwo cell lines, Y79 and LA-N-5, as described (18). A mRNAof =14.5 S (1.3-1.4 kb) was hybridized with the 32P-labeledEL22 clone (Fig. 4A). Southern genomic blotting analysisusing the same probe showed that the esterase D gene wasdistributed over 20-40 kbp in the human genome (Fig. 4B).This indicates the presence of large introns within this gene.We have subsequently confirmed this conclusion by charac-terizing the complete genomic esterase D clone (unpublisheddata).Chromosome Mapping. The esterase D gene has been

mapped to the chromosome 13q14.11 region by correlatingloss of the esterase D enzyme activity with known deletionson chromosome 13 of various mutant cells (3, 4). To deter-mine the location of the EL22 clone, several human mutantcell lines containing well-characterized deletions of chromo-some 13 were selected. DNA extracted from these mutantcells were subjected to Southern genomic blotting analysis byusing the EL22 clone as probe. As shown in Fig. 4C, a

I SAA TTC 666 6CA AAA A6C AAT CAS CAA TS AC A66 AAA ASA ATS SCA TT6 AA CA6 ATT TCC ACAACAAM6 T6C nT 666 66A T6 CA6 916lu Ph. Sly Ala Lys Sir Asn 61n 61n Leu Asp Arg Lys Arg Net Ala Lou Lys Gin Ile Ser Ser Asn Lys Cys Phi Sly Sly Lou 61n

91 AAA 6TT TTT 6A CAT SAC A6T 6TT 6M CTA MC T6C AAA AT6 AAA TTT SCT 6TC TAC TTA CCA CCA M6 6CA 6M ACA 66A MS T6C CCT 181Lys Val Phi 61u His Asp Ser Val 61u Leu Asn Cys Lys Nlet Lys Phe A!lVi l Tyr Ley ProP!roLys Ala Gl Thr 61y Lys Cys Pro

181 6CA t6T ATT 66C TCT CCA 66T TTA ACT T6C ACA SAG CCA MA TTT TAT CAT CM MT CT6 6TT ATC ATC MT CT6 CTT CA6 MC CAT tT6 273Ala Cys 1le Sly Ser Pro Sly Liu Thr Cys Thr 61u Pro Lys Phe Tyr His 61n Aso Leu Val Ile Ile Sir Lu Leu 61n Asn His Leu

271 TCT t6T T6T CAT T6C TCC ABA TAC AMC CCT CST 6C6 T6C HAT ATT AAA 66T MA 6AT MAS AMC T66 6AC ITT SCS ACT 66T CST 66A TTT 361Ser Cys Cys His Cys Ser Arg Tyr Ser Pro Arg Ala Cys Asn Ile Lys Sly 61u Asp 61u Ser Trp Asp Phe Ala Thr Sly Arg 61y Phi

361 TAT 6TT SAT 6CC ACT 6AA MAT CCT T66 AAA ACC AAC TAC AMA AT6 TAC TCT TAT BTC ACA MAB 6A6 CTT CCC CAA CTC ATA AAT 6CC AAT 456Tyr Val Asp Ala Thr 61u Asp Pro Trp Lys Thr Asn Tyr Arg 1!j Ixr...l!_Yl Thr 6u 61u Leu Pro ln Leo Ile Asn Ala Asn

451 TTT CCA 6T6 MAT CCC CAA A66 ATS TCT ATT TTT 66C CAC TCC AT6 66A 66T CAT 66A 6CT CT6 ATC T6T 6CT TT6 AAA AAT CCT 66A AAA 546Phe Pro Val Asp Pro 61n Arg Met Ser Ile Phe 61y His Ser Net_6ly_61Hiysi6lyAtaLeuIiCynA Leu..Lys . Pyo61y Lys

IV541 TAC AAA TCT UTS TCA 6CA TTT BCT CCA ATT T6C AAC CCT 6TA CTC T6T CCC T66 66C AAA AAA 6CC TTT A6T 66A TAT TT6 66A ACA SAT 633

Tyr Lys Ser Val Ser Ala Phe Ala Pro Ile Cys Asn Pro Yal Leu Cys Pro Trp Sly Lys Lys Ala Phe Ser Sly Tyr Leu 61y Thr Asp

631 CAA A6T AAA T66 AA6 6CT TAT SAT BCT ACC CAC CTT 6T6 AAA TCC TAT CCA 66A TCT CA6 CT6 SAC ATA CTA ATT MAT CAA 666 AAA BAT 72361n Ser Lys Trp Lys Ala Tyr Asp Ala Thr His Leu Val Lys Ser Tyr Pro 61y Ser SIn Leu Asp Ile Leu Ile Asp 61n 61y Lys Asp

721 SAC CA6 TTT CTT TTA SAT GSA CA6 TTA CTC CCT SAT AAC TTC ATA 6CT 6CC T6T ACA 6AA AS AAA ATC CCC 6TT 6TT TTT C6A TT6 CAA C16Asp 61n Phe Leu Leu Asp 61y 61n Leu Leu Pro Asp Asn Phe Ile Ala Ala Cys Thr S1u Lys Lys Ile Pro Val Val Phe Arg Leu 61n

11 AS 66T TAT BAT CAT A6C TAC TAC TTC ATT SCA ACC TTT ATT ACT BAC CAC ATC AMA CAT CAT 6CT AAA TAC CT6 AAT 6CA T6A AAA AAC 9166Su 61y Tyr Asp His Ser Tyr Tyr Phe Ile Ala Thr Phe Ile Thr Asp His Ile Arg His His Ala Lys Tyr ku Asn Ala ---

961 TCC AAA TAA 6AG AAT CTC TTC A66 ATT ATA AAA 6TT 6TA AAA T6C AAC T6T ATT 6CT GAB CAA AAA AAA AAA AAA TTC AAA ACA TT6 BAT 991

991 TTT AVA ST6 CTA AAA 6GB CTT TAT TCT ATA 6TT BAA TCA CCT CT6 AAT AAA 6AT ATA AAA CCT AAA AAA ACC C6A ATT C

FIG. 3. Nucleotide and de-duced amino acid sequences ofEL22 cDNA. The deduced aminoacid sequences (underlined) werecompletely identical to the peptidesequences shown in Fig. 1B. Thefirst three amino acids encodedfrom the EcoRI linker used forconstructing the cDNA librarymay not be derived from the au-thentic esterase D gene.

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I ItI -t A R jt-

S "] urn'sFIG. 4. Blotting analysis of esterase D mRNA (A) and genomic DNA (B) as well as the localization of the EL22 clone on human chromosome

13q14 (C). The probe used in these experiments was 32P-labeled pUCEL22 DNA. (A) Poly(A)-selected mRNA of Y79 cells (lane a) and LA-N-5cells (lane b) was used for RNA blotting analysis. These two cell lines were chosen because they contained a sufficient amount of esterase Dprotein immunoprecipitable by the rabbit anti-esterase D antibodies. The esterase D mRNA size of these two cell lines is -14.5 S. However,the apparent size of LA-N-5 mRNA appeared slightly larger than Y79, which was probably caused by partial degradation. (B) High molecularweight genomic DNA from human peripheral blood lymphocyte (PBL) was digested with Pst I (lane a) and Sst I (lane b) and electrophoresedon a 0.8% agarose gel, followed by Southern blotting analysis. The combined size of the DNA fragments with positive hybridization was 20-40kb, indicating that there are large intron sequences in the esterase D genome. (C) DNA was extracted from (z) PBL (normal peripheral bloodlymphocytes; lane a); (il) 34-2-3 (Chinese hamster-human hybrid cells containing human chromosomes 13,12 and the short arm of 6; lane b);(iiM) GM1142 [A13(q14:q22)I (lane c); (iv) GM2718 [A13(q12:q14)] (lane d); (v) GM3887 [A13(q22:qtr)] (lane e); and (vi) Y79 (human retinoblastomacells; lane 0. After complete digestion with EcoRI, DNA fragments were analyzed by Southern genomic blotting. All the cells used here haveesterase D enzyme; however, GM1142 and GM2718 contained quantitatively less activity than the others, which is consistent with the presenceof a single copy of the esterase D gene as shown here. The same result has been replicated in three separate experiments.

Chinese hamster ovarian cell (CHO)-human fibroblast hy-brid cell line 34-2-3, containing two or three copies ofchromosome 13, one copy of chromosome 12, and one copyof chromosome 6p in its tetraploid CHO genome, showed ahybridization pattern (lane b) indistinguishable from that ofhuman peripheral lymphocyte DNA (lane a). The hybridiza-tion intensity observed in this cell line was between thatobserved in haploid cells, as described below, and thatobserved in diploid cells such as peripheral lymphocytes(lane a), suggesting that the esterase D gene is located inhuman chromosome 13. In contrast, DNA from CHO cellshybridized weakly with the EL22 clone and showed adifferent pattern from that of human DNA (data not shown).Furthermore, DNA obtained from two mutant cell lines,GM1142 and GM2718, with visible deletions at 13ql2-14 and13ql4-22, respectively, showed an identical hybridizationpattern with about one-half the intensity of that found inlymphocytes (lanes c and d). This suggests that they containthe haploid esterase D gene, which is apparently not locatedat the deletion junction. However, this reduced intensity wasnot found in the mutant cell line GM3887, which was deletedat 13q22-ter or in Y79 cells, which had no visible deletion inchromosome 13. To provide an internal control, the same blotwas hybridized with the v-myc gene to detect the 13.5-kbEcoRI-digested DNA fragment of the c-myc gene. With theexception of the 34-2-3 cell line, which did not contain the13.5-kb c-myc gene, the rest of the DNA samples showedessentially identical hybridization intensity at the 13.5-kb

c-myc gene (data not shown), demonstrating that the quan-titation was reliable. These results, therefore, indicate thatthe EL22 clone containing the esterase D gene is mapped tothe chromosome 13q14 region.

DISCUSSIONWe present here the molecular cloning of the human esteraseD gene and its sequence. Using this esterase D cDNA asprobe, we found that (i) the size of the esterase D mRNA is1.3-1.4 kb; (it) the gene is around 20-35 kb, indicating thepresence of large introns scattered over this genome; and (iii)the esterase D gene is indeed located at chromosome 13q14.Our unpublished results also indicated that the deducedamino acid ofthe esterase D gene was unique when comparedto 4000 well-characterized proteins.

Esterase D as the Starting Point for Cloning the RB Gene.Recent fine mapping data have shown that the esterase Dgene is located at chromosome 13q14:11 with no meioticrecombination observed with the RB gene (4, 27). Althoughthe exact kilobase-pair distance between these two genes isnot known, the maximal DNA content of a band such asq14:11, should not exceed 1000 kb on the average. Thisestimation is based on the following calculation:

3 x 106 kb (total human haploid genome DNA). 23 (chromosome number)

. 13 (number of bands in chromosome 13)- 9 (sub-band of the q14 region) = 1100 kb.

A at b

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However, the distance between the RB gene and the esteraseD gene may just be a few kilobases. Based on the lack ofesterase D activity in LA-RB 69 retinoblastoma cells, it waspreviously suggested that a submicroscopic deletion hadoccurred in the tumor cells resulting in the loss of both the RBand the esterase D genes (13). Our unpublished resultsindicated that there was no deletion in the coding sequencesof the esterase D gene in the tumor cells. However, someabnormality, perhaps in the regulatory region, must haveoccurred to cause a substantial reduction in the expression ofthe esterase D gene and diminution of the enzyme activity. Itis plausible to suggest that this abnormality would be likelyto interfere with RB gene expression leading to tumor-igenesis. Since the esterase D gene is known to be the closestmarker to the RB gene, it will serve as the starting point forcloning the RB gene by chromosomal walking. DNA frag-ments isolated from this process can then be used as probesto examine qualitative or quantitative differences in mRNAfrom fetal retinal cells and retinoblastoma cells. One wouldexpect to detect such differences because substantial evi-dence suggested somatic mutations occurred in the RB geneof tumor cells (10, 13, 14). The DNA fragments correspond-ing to the defective mRNA are the best candidate for the RBgene. Moreover, the availability of mutant cells with knowndeletions in 13ql3.1-14.11 and 13q14.11-q22, respectively (4,28), would enable one to determine the correct direction ofwalking toward the RB gene. Several important genetic locisuch as the bithorax gene of Drosophila (29) and themultidrug-resistant locus of hamster cells (30), have beendefined successfully by this method.

Esterase D as a Genetic Marker in Clinical Diagnosis. Theesterase D gene is closely linked to the RB gene and thegene(s) involved in Wilson disease. The polymorphic mark-ers of the esterase D gene can therefore be used to diagnoseretinoblastomas and Wilson disease through the method ofrestriction fragment length polymorphism (31). In addition, amethod for detecting single point mutations, as described byMyers et al. (32), may also be applied. The polymorphicnature of the esterase D protein has been used for thispurpose (3, 14, 15); however, an informative polymorphismwas seldom found because of the presence of only threeisoenzymes (EsD 1-1, EsD 2-1, and EsD 2-2). In contrast, thelarge size of the intron sequences in the esterase D genomeas described in this report should provide sufficient possi-bilities for finding more variety of polymorphic markers. Thedistinct characteristics of the esterase D gene and its adjacentDNA fragments will offer highly specific and accurate diag-nosis of these hereditary diseases.

Searching for Other Nonspecific Esterase Genes. In additionto the esterase D gene, there are at least eight separatestructural gene loci determining human esterase isozymeswith unknown physiological roles (2). These nonspecificesterases share similar enzymatic activities and may consti-tute a large gene family. Moreover, our recent resultsindicated that esterase D may have a role in detoxification(16). It is therefore of interest to examine whether othernonspecific esterases have similar functions. The presentesterase D clone, which is the first such esterase sequenced,could serve as a probe for future exploration of the remainingmembers of structurally related esterase genes.

We dedicate this work to the late Dr. P. Lampert, chairman of theDepartment of Pathology, whose interest and support were greatlyappreciated. We thank Drs. J. DeWet and J. Millan for providingtheir cDNA libraries; Dr. A. P. Chou and W. Wheatley for technicalassistance; Dr. R. Bookstein for computer analysis; Dr. H. S. U andDr. A. Hsueh for critical reading of the manuscript; and Mrs. V.

Othen for typing this manuscript. We are grateful to Dr. A. Hsueh forsponsoring the National Institutes of Health postdoctoral training-ship of E.Y.-H.P.L. and to Dr. G. Walter for his encouragement.This work was supported by research grants from the National EyeInstitute EY-05758, Academic Senate (University of California atSan Diego) and departmental funds to W.-H.L.

1. Hopkinson, D. A., Mestriner, M. A., Cortner, J. & Harris, H.(1973) Ann. Hum. Genet. 37, 119-137.

2. Coates, P. M., Mestriner, M. A. & Hopkinson, D. A. (1975) Ann.Hum. Genet. 39, 1-20.

3. Sparkes, R. S., Sparkes, M. E., Wilson, M. G., Towner, J. W.,Benedict, W., Murphree, A. L. & Yunis, J. J. (1980) Science 208,1042-1044.

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