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).
6337
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Proc. Natl. Acad. Sci. USA 83 (1986) 6339
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.
Biochemistry: Lee and Lee
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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.
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