From the Departments of Pediatrics, Internal Medicine, and Genetics, Yale University School of Medicine, New Haven, Connecticut 06520-8021
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ABSTRACT |
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Ankyrin 1, an erythrocyte membrane protein that
links the underlying cytoskeleton to the plasma membrane, is also
expressed in brain and muscle. We cloned a truncated, muscle-specific
ankyrin 1 cDNA composed of novel 5 sequences and 3
sequences
previously identified in the last 3 exons of the human ankyrin 1 erythroid gene. Northern blot analysis revealed expression restricted
to cardiac and skeletal muscle tissues. Deduced amino acid sequence of
this muscle cDNA predicted a peptide of 155 amino acids in length
with a hydrophobic NH2 terminus. Cloning of the
corresponding chromosomal gene revealed that the ankyrin 1 muscle
transcript is composed of four exons spread over ~10 kilobase pairs
of DNA. Reverse transcriptase-polymerase chain reaction of skeletal
muscle cDNA identified multiple cDNA isoforms created by
alternative splicing. The ankyrin 1 muscle promoter was identified as a
(G + C)-rich promoter located >200 kilobase pairs from the ankyrin 1 erythroid promoter. An ankyrin 1 muscle promoter fragment directed high
level expression of a reporter gene in cultured C2C12 muscle cells, but
not in HeLa or K562 (erythroid) cells. DNA-protein interactions were
identified in vitro at a single Sp1 and two E box consensus
binding sites contained within the promoter. A MyoD cDNA expression
plasmid transactivated an ankyrin 1 muscle promoter fragment/reporter
gene plasmid in a dose-dependent fashion in both HeLa and
K562 cells. A polyclonal antibody raised to human ankyrin 1 muscle-specific sequences reacted with peptides of 28 and 30 kDa on
immunoblots of human skeletal muscle.
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INTRODUCTION |
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Erythrocyte ankyrin, ankyrin 1, is the prototype of a family of homologous proteins that are involved in the local segregation of integral membrane proteins within functional domains on the plasma membrane (1-4). This important cellular localization of membrane proteins may be provided by the relative affinities of the many different isoforms of ankyrin for target proteins. This specialization appears to have evolved through the tissue-specific, developmentally regulated expression of multiple protein isoforms. The molecular mechanisms by which ankyrin has acquired distinct isoforms with specialized function(s) are beginning to be revealed. The isoform diversity of ankyrin arises from both different gene products and from differential, alternative splicing of the same gene product (5-9). In humans, the cDNAs for three ankyrin proteins have been cloned and their gene products studied. These ankyrins share similar antigenic sites and domain structures, differing in a number of ways such as their cellular patterns of expression and their relative affinities of binding to spectrin and band 3. Ankyrin binding has been described for a variety of proteins including membrane skeleton proteins, ion transport proteins, and cell adhesion molecules (1, 4).
Ankyrin 1, first discovered in preparations of erythrocyte membranes, provides the principal linkage between the spectrin-actin based erythrocyte membrane skeleton and the plasma membrane (1, 10-12). The primary structure of human ankyrin 1, deduced from cDNA clones obtained from a reticulocyte cDNA library, encodes a mature protein of 1881 amino acids (7, 8). Ankyrin 1 has been identified in erythroid tissue, brain, and muscle (7, 8, 13-17). The major form of ankyrin 1, ~210 kDa, is composed of three domains, an 89-kDa NH2-terminal domain composed of 24 conserved repeats known as cdc 10/ankyrin repeats that contain the binding site for band 3; a 62-kDa domain that contains the binding sites for spectrin and vimentin; and a 55-kDa COOH-terminal regulatory domain (1, 2, 4). Complex patterns of alternative splicing have been identified in the region encoding the regulatory domain (5, 13, 14, 17). The precise role(s) of the regulatory domain is unknown, but it does appear to modulate spectrin and band 3 binding (18, 19). Defects of ankyrin 1 have been implicated in approximately half of all patients with hereditary spherocytosis (20, 21).
Initial studies in muscle immunolocalized ankyrin to the sarcolemma adjacent to the Z lines co-distributed with spectrin, as well as at the neuromuscular junction, and at the muscle triads (22-26). Studies performed in muscle cells suggested that ankyrin accumulation and assembly into the membrane was determined by a control mechanism operative at the posttranslational level, triggered near the time of cell fusion and onset of terminal differentiation (27). Northern blot analyses by Birkenmeier and colleagues using an erythroid ankyrin 1 cDNA probe encoding the regulatory domain identified multiple transcripts in murine skeletal muscle RNA (13, 28). These transcripts ranged in size from 1.6 to 3.5 kb, compared with the 7.5 and 9 kb ankyrin 1 transcripts observed on Northern blots of erythroid RNA (29, 30). Northern blot analysis of RNA from chicken myotubes using an ankyrin 1 cDNA fragment as probe also identified a small 3.6-kb transcript (31).
Isoform diversity in different muscle cell types is frequently determined by the presence of muscle type-specific isoforms (32, 33). These isoforms may be encoded by separate genes, may be generated by alternative splicing of a given gene, or may be controlled by specific regulatory elements in or around a given gene at different times. For example, numerous isoforms of spectrin with varying patterns of cellular localization and developmental expression have been identified in muscle cells (3, 34-36). These isoforms are the products of separate genes or alternative splicing of individual genes (3, 36, 37). Recent studies have identified two populations of ankyrin 1 in muscle cells (38). One population was identified at the sarcolemma using an antibody to the spectrin binding domain of ankyrin. This localization is similar to previous observations. A second population was identified at the M and Z lines using an antibody to sequences identified previously in neural isoforms of ankyrin 1, also present in muscle (38).
This report describes the cloning of a novel, truncated, muscle-specific ankyrin 1 isoform, characterization of its corresponding genomic structure, study of its pattern of expression, and identification of its promoter. Because of similarities detected on Western blotting, the isoform described here is likely to be the one detected at the sarcoplasmic reticulum, providing additional evidence that two populations of ankyrin 1 are present in muscle. These observations extend the molecular basis of ankyrin 1 isoform diversity to include the use of an alternate NH2 terminus and a tissue-specific alternate promoter.
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MATERIALS AND METHODS |
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RNA Preparation and Northern Blot Analyses--
Total RNA was
prepared from human skeletal muscle, or from the human tissue culture
cell lines RD (human rhabdomyosarcoma, embryonal, ATCC 136-CCL), K562
(chronic myelogenous leukemia in blast crisis with erythroid
characteristics, ATCC CCL 243), and HeLa (epithelioid carcinoma,
cervix, ATCC CCL 2) as described previously (39). Multiple-tissue
Northern blots containing 2 µg of poly(A)+ mRNA per
tissue were obtained from CLONTECH (Palo Alto, CA). A human -actin cDNA probe was used as a control for loading in Northern blot analyses (40).
cDNA and Genomic DNA Cloning--
An 838-bp cDNA
fragment was generated by 5 RACE and PCR using primers A and B (Table
I) with human skeletal muscle cDNA as template. This fragment,
which contains the entire coding region of the human ankyrin 1 muscle-specific cDNA (see below) was used as the hybridization
probe to screen a random- and oligo(dT)-primed human skeletal muscle
cDNA library in
gt11 (CLONTECH). A human ankyrin 1 cDNA fragment, pAnk15 (8), containing the 3
end of the
human ANK-1 muscle transcript was used as a hybridization probe to
screen a human genomic DNA library. The library is a Charon
4A bacteriophage library containing fragments of genomic DNA partially
digested with AluI and HaeIII with
EcoRI linkers added. For both library screens, selected
recombinants that hybridized to the screening probes were purified,
subcloned, and analyzed by standard techniques.
Rapid Amplification of cDNA Ends (RACE)1-- 5' RACE was performed as described (41, 42). 1 µg of human skeletal muscle RNA was reverse transcribed using primer C (see Table I). Single-stranded oligonucleotide ligation and PCR amplification were carried out using primer D and primers A and B, respectively. Amplification products were subcloned and sequenced.
Primer Extension Analyses-- The transcription start site of the muscle-specific ankyrin 1 cDNA isoform was determined using primer extension analysis. Primers E or F (see Table I) were used in primer extension reactions as described elsewhere (43). Templates in these reactions were 20 µg of total RNA from the human cell lines RD and HeLa, or 10 µg of tRNA.
Cell Culture-- The tissue culture cell lines C2C12 (murine muscle myoblast, ATCC 1772-CRL), RD, K562, and HeLa were used to study expression of the putative promoter of the muscle-specific isoform of the ankyrin 1 gene. C2C12, RD, and K562 cells were maintained in RPMI 1640 medium with 10% fetal calf serum. HeLa cells were maintained in Eagle's minimal essential medium with 10% fetal calf serum. C2C12 cells were maintained as myoblasts for all experiments described.
Preparation of Promoter-Reporter Plasmids--
Test plasmids
were prepared by inserting a 2.1-kb fragment of the 5-flanking ankyrin
1 muscle-specific genomic DNA upstream of the firefly luciferase
reporter gene in the plasmid pGL2B (Promega, Madison, WI). Serial
truncations of this 2.1-kb fragment in the pGL2B plasmid were
constructed using convenient restriction enzyme sites or PCR
amplification. Test plasmids were sequenced to exclude cloning or
PCR-generated artifacts.
Transient Transfections and Transactivation Assays--
All
plasmids tested were purified using Qiagen columns (Qiagen, Inc.,
Chatsworth, CA) and at least two preparations of each plasmid were
tested. 107 K562 cells were transfected by electroporation
with a single pulse of 300 V at 960 uF with 20 µg of test plasmid and
0.5 µg of pCMV, a mammalian reporter plasmid expressing
-galactosidase driven by the human cytomegalovirus immediate early
gene promoter (CLONTECH). 105 C2C12 or
HeLa cells were transfected with 2.0 µg of test plasmid and 0.25 µg
of the pCMV
plasmid by lipofection using 4 µl of LipofectAMINE
(Life Technologies, Inc.). Twenty-four hours after transfection, cells
were harvested and lysed, and the activity of both luciferase and
-galactosidase activity was determined in cell extracts. All assays
were performed in triplicate. Differences in transfection efficiency
were determined by co-transfection with the pCMV
plasmid. For
transactivation assays, K562 and HeLa cells were transfected using 5 and 1 µg of reporter plasmid, respectively, and varying amounts of a
MyoD cDNA expression plasmid, phMyoD (EMBL no. X56677), and
reporter gene activity were assayed as above.
Gel Mobility Shift Analyses-- Nuclear extracts were prepared from RD, C2C12, K562, and HeLa cells by hypotonic lysis, followed by high salt extraction of nuclei as described by Andrews and Faller (44). Binding reactions were carried out as described (45, 46). Competitor oligonucleotides were added at molar excesses of 10- or 100-fold. Resulting complexes were separated by electrophoresis through 6% polyacrylamide gels at 21 °C.
Immunoblot Analyses-- A rabbit-specific polyclonal antibody was raised to a synthetic peptide, ISPRVVRRRVFLKGN, conjugated to keyhole limpet hemocyanin and bovine serum albumin (Immuno-Dynamics, La Jolla, CA). The sequence of this peptide is contained in the novel, muscle-specific region of ankyrin 1. After 12 weeks, anti-peptide antisera was collected, then affinity purified on a column to which the synthetic peptide had been covalently linked. Human erythrocyte membranes and skeletal muscle homogenates were prepared as described previously (47, 48). These erythroid and muscle fractions were separated by SDS-polyacrylamide gel electrophoresis on a 4-20% gel and either stained with Coomassie Blue or transferred onto nitrocellulose and immunoblotted. Immunoblotting was performed as described elsewhere (49).
Computer Analyses-- Computer-assisted analyses of derived nucleotide and predicted amino acid sequences were performed utilizing the sequence analysis software package of the University of Wisconsin Genetics Computer Group (UW GCG; Madison, WI) (50) and the BLAST algorithm, National Center for Biotechnology Information (Bethesda, MD) (51).
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RESULTS |
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Identification of Novel Ankyrin 1 Sequences in
Muscle--
Northern blot analyses of human skeletal muscle RNA with
human ankyrin 1 erythroid cDNA probes encoding the repeat-domain (pAnk58), the spectrin-binding domain (pAnk37), or the regulatory domain (pAnk15) (8) yielded hybridization signals of 2.3 and 1.6 kb
only when the regulatory domain probe was used (see below). These
results are in contrast to Northern blot analyses of human erythroid
RNA using these probes where hybridization signals of 7.3 and 9.0 kb
are seen with all three domain-specific probes (7, 8). To identify the
molecular basis of these truncated transcripts, we performed 5 RACE
using oligonucleotide primers A (sense, linker) and B and C (antisense,
both in the 3
-untranslated region of the erythroid cDNA), with
total human skeletal muscle RNA as a template. This yielded a set of
cDNA products, the longest 838 bp in length. Nucleotide sequence
analysis of this product revealed a novel 5
end including
5
-untranslated sequences, an initiator methionine, and 219 bp of novel
sequence with an open reading frame. The 3
end of this RACE product
was composed of sequences previously identified in erythroid or neural
ankyrin 1 cDNA transcripts, including 309 bp of 3
in-frame
sequence.
Isolation and Analysis of Recombinant cDNA and Genomic DNA
Clones--
This 838-bp skeletal muscle RACE product was used as probe
to screen a human skeletal muscle cDNA library. Eight clones that hybridized to the screening probe were isolated after primary screening
of a human skeletal muscle cDNA library. Three clones were
purified, subcloned and sequenced (Fig.
1). The clones varied in size from 1096 to 2543 bp. All three clones contained 5-untranslated sequence, an
open reading frame of 528 bp (the same identified in the RACE product)
and 3
-untranslated sequences (Fig.
2A). Primer extension
predicted an additional 43 bp of upstream sequence from the end of
clone
2, the clone extending the most 5
of analyzed clones (not
shown). An additional 42 bp of upstream 5
untranslated sequence was
obtained by 5
RACE. Sequences obtained by RACE were verified by
comparison to corresponding genomic DNA sequences (see below). The
sequences around the transcription start site, CCA+1CTCA,
closely match transcription initiation recognition sequences,
YYA+1NWYY (52). Collectively, these data suggest that this
cDNA sequence is at or very near the 5
end of the ankyrin 1 muscle
cDNA.
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Expression of the Novel Ankyrin 1 Exon Is Restricted to Cardiac and Skeletal Muscle Tissue-- Northern blot analysis using the 838-bp ankyrin 1 muscle cDNA RACE product detected abundant mRNAs of 2.3 and 1.6 kb in cardiac and skeletal muscle tissues (Fig. 5). Signals of 3.7 and 7.0 kb were also detected, but in lesser amounts compared with 2.3 and 1.6 kb. These signals may represent ankyrin 1 muscle transcripts generated by alternative splicing, or, for the 7.0-kb signal, cross-hybridization with the erythroid ankyrin 1 isoform.
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Identification of the Ankyrin 1 Gene Muscle Promoter--
The
nucleotide sequence of the 5-flanking genomic DNA upstream of the
human ankyrin muscle cDNA transcription start site is shown in Fig.
6. Inspection of the sequences reveals
features characteristic of a muscle-specific gene promoter including
lack of consensus CCAAT sequences and a high G + C content (61%,
between nucleotides
245 and +18).
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The Human Ankyrin 1 Gene Muscle Promoter Contains Binding Sites for Sp1 and MyoD-- Consensus sequences for a number of potential DNA-binding proteins, including Sp1, GATA-1, and two E boxes were present in the ankyrin 1 gene muscle promoter (Fig. 6). E boxes are binding sites for members of the MyoD family of basic helix-loop-helix transcription factors that are important in controlling muscle-specific gene expression. To determine if nuclear proteins could bind these sites in vitro, double-stranded oligonucleotides containing the corresponding ankyrin 1 muscle sequences (Sp1 G + H; E box left I + J; E box-right K + L; GATA M + N; Table I) or control sequences (Sp1 O + P (56, 57); E box Q + R (58); GATA-1 S + T (59)) were prepared and used in gel shift analyses.
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MyoD Transactivates the Human Muscle Ankyrin 1 Gene Promoter in Heterologous Cells-- None of the ankyrin 1 muscle promoter fragments directed expression of a reporter gene in K562 or HeLa cells, but the addition of MyoD by co-transfection conferred promoter activity to these fragments. Co-transfection of 1 µg of the ankyrin 1 minimal muscle promoter/reporter plasmid, p-184, and increasing amounts of a MyoD cDNA expression plasmid into HeLa cells resulted in increasing promoter activity with increasing amounts of MyoD plasmid (Fig. 10, top). Similar results were observed in co-transfection experiments in K562 cells (Fig. 10, bottom). The ability of MyoD to transcriptionally activate the ankyrin 1 muscle promoter in these cells which do not contain this muscle-specific factor, correlates with the inability of the ankyrin 1 muscle promoter to function in these cells.
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Immunoblotting-- Immunoblots of human erythrocyte membrane ghosts and skeletal muscle homogenates using the affinity-purified anti-peptide antibody 2401, raised against sequences unique to the ankyrin 1 muscle isoform, detected bands of 28 and 30 kDa in skeletal muscle (Fig. 11). Longer exposures revealed a band at 70 kDa in both skeletal muscle and erythrocyte membranes and a band at 210 kDa in erythrocyte membranes. A polyclonal antibody raised to ankyrin 2.1 from erythrocyte membranes (kindly supplied by Jon S. Morrow) detected bands of 205 and 210 kDa in erythrocyte membranes (Fig. 11). Longer exposure demonstrated a band of 210 kDa in skeletal muscle. The identity of the 70-kDa band detected in skeletal muscle homogenates and erythrocyte membranes is unknown, but it was highly reproducible and was identified in immunoblots of RD cells (not shown).
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Computer Analyses-- When compared with sequences present in available data bases, significant homology was demonstrated only between the novel human ankyrin 1 muscle-specific gene sequence and a corresponding murine sequence. The identity between the translated sequence of the human ankyrin 1 muscle isoform and the translated murine sequence was 91% with a similarity of 94%. Searching using only the highly charged 73 amino acid NH2 terminus also failed to reveal any significant homologies.
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DISCUSSION |
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The diversity of the numerous ankyrin family isoforms appears to be critical for specific cellular functions. Of the three ankyrin family proteins cloned, ankyrin 1 is considered to have the most limited pattern of expression, with expression restricted to erythroid, muscle and neural tissue. Despite these "limitations," the ankyrin 1 erythroid cDNA has at least 15 different transcripts generated by alternative splicing and/or alternative polyadenylation (5). The identification of a muscle-tissue-specific isoform with multiple transcripts generated by alternative splicing under the control of an alternate, tissue-specific promoter adds to this diversity. Interestingly, truncated isoforms of ankyrin 3, the ankyrin isoform with the widest pattern of tissue distribution, have been localized to the cytoplasm and Golgi apparatus of kidney and muscle cells as well as to the lysosomes of macrophages (6, 60-62). These truncated isoforms, however, lack only the NH2-terminal membrane-binding domain.
The regulation of truncated, tissue-specific isoforms of the ankyrin 1 gene by the use of an alternate promoter is similar to that observed in MCL1/3 or dystrophin gene transcripts (63-68). In dystrophin, five autonomous promoters direct the transcription of respective alternate first exons in a cell-specific and developmentally controlled manner (63). Two of these promoters direct the expression of transcripts encoding only the COOH terminus of dystrophin, utilizing exons 56-79 or exons 63-79, respectively, in a manner similar to muscle ankyrin 1, which utilizes exons 40-42. Remarkably, like ankyrin-1, the tissue-specific promoters of dystrophin may be remote (>100 kb) from each other.
The functions of the two populations of ankyrin 1 in muscle are unknown. The co-localization of the 210-kDa ankyrin isoform with spectrin at the sarcomere suggests a role for ankyrin in providing a linkage between the membrane skeleton to the plasma membrane, as it does in the erythrocyte. The truncated ankyrin 1 isoform lacking the membrane and spectrin binding domains localized to the Z and M lines of internal myofibrils and was highly enriched in the sarcoplasmic reticululm (38). The hydrophobic NH2 terminus of the truncated ankyrin 1 isoform could insert into the sarcoplasmic reticulum membrane, with the COOH terminus serving as a ligand for myoplasmic proteins. The specificity of the truncated ankyrin 1 for different protein ligands could be provided by the isoforms generated by alternative splicing. The antibody used to immunolocalize the truncated muscle ankyrin isoform was raised to sequences shared by ankyrin 1 neural and muscle cDNA isoforms (5, 38). There were similarities detected on immunoblots of skeletal muscle using this antibody and our muscle-specific antibody 2401. Together, these data suggest that the isoform described here is likely to be the same one detected at the sarcoplasmic reticulum. The sequence of this isoform does not match any others in available data bases, suggesting that this may represent a novel class of proteins.
Defects in ankyrin 1 are the most common cause of typical hereditary
spherocytosis (HS) in humans. Interestingly, kindreds with HS and
co-segregating myopathic manifestations have been described, including
two brothers with HS, a movement disorder and myopathy (69), and a
three-generation Russian kindred with co-segregating HS and
hypertrophic cardiomyopathy (70). It is tempting to speculate that
these patients have a mutation in the very 3 end of the ankyrin 1 gene
in the region that is common to both erythroid and muscle ankyrin 1 transcripts or in critical tissue-specific control elements.
Different mutations or deletions of the dystrophin muscle promoter have
been described in patients with Becker muscular dystrophy and in
patients with severe cardiomyopathy, demonstrating that a mutation may
specifically affect either the cardiac or skeletal muscle expression of
a gene that is expressed in both cell types (71-74). It will be
important to identify the factors that control cardiac- and skeletal
muscle-specific expression of ankyrin 1, as this information may aid in
the identification of the defects in HS patients with co-segregating
skeletal muscle or cardiac myopathic symptoms. One potential regulatory
factor is GATA-4, a member of the GATA family of transcription factors
expressed in cardiac and foregut derivatives (75, 76). A potential GATA binding site is located in upstream 5-flanking genomic DNA of the
ankyrin 1 muscle promoter. GATA-4 appears to direct expression of
a number of muscle-associated genes primarily in cardiac muscle (72, 77-82).
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ACKNOWLEDGEMENTS |
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We thank C. Wong and Y. Wang for skilled technical assistance, C. Birkenmeier and J. Barker for helpful discussions and for communication of unpublished sequence information, Dr. Sonia Pearson-White for the MyoD cDNA expression plasmid, and Dr. Jon S. Morrow for the ankyrin 1 antibody.
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FOOTNOTES |
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* This work was supported in part by grants from the National Institutes of Health, the March of Dimes Birth Defects Foundation, and the American Heart Association-Connecticut Affiliate.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF005213, AF005214, and AF005215.
To whom all correspondence should be addressed: Dept. of Pediatrics,
Yale University School of Medicine, 333 Cedar St., P. O. Box 208064, New Haven, CT 06520-8064. Tel.: 203-737-2896; Fax: 203-785-5426;
E-mail: Patrick_Gallagher{at}QM.Yale.edu.
1 RACE, rapid amplification of cDNA ends; HS, hereditary spherocytosis; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase pair(s).
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REFERENCES |
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