From the Falk Cardiovascular Research Center, Stanford University School of Medicine, Stanford, California 94305-5246
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ABSTRACT |
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Apolipoprotein(a), (apo(a)), is the distinguishing protein portion of the lipoprotein(a) particle, elevated plasma levels of which are a major risk factor for cardiovascular disease. A search for enhancer elements that control the transcription of the apo(a) gene led to the identification of an upstream element that contains target binding sites for members of the Ets and Sp1 nuclear protein families. The enhancer element functions in either orientation to confer a greater than 10-fold increase in the activity of the apo(a) minimal promoter in cultured hepatocyte cells. Unexpectedly, the enhancer element is located within a LINE retrotransposon element, suggesting that LINE elements may function as mobile regulatory elements to control the expression of nearby genes.
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INTRODUCTION |
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Elevated levels of lipoprotein(a)
(Lp(a))1 are a major risk
factor for atherosclerosis, myocardial infarction, and cerebrovascular stroke (see Refs. 1 and 2 and references therein). Lp(a) is a
lipoprotein consisting of low density lipoprotein to which is
covalently linked a unique protein, apolipoprotein(a) (apo(a)) (3, 4).
Apo(a) is a large, highly polymorphic glycoprotein with sequence
homology to plasminogen. Depending on individual alleles, it contains
from 12 to 50 copies of a domain which resembles kringle four of
plasminogen, plus a single kringle five-like domain, and an inactive
protease domain (5, 6). When plasminogen is activated to plasmin, it
can cleave fibrin and several other protein substrates. By competing
with plasminogen for fibrin binding through its apo(a) component, Lp(a)
can reduce the conversion of plasminogen to the active plasmin and
inhibit clot lysis (7-9). By a similar mechanism, Lp(a) can reduce
plasmin-dependent activation of latent transforming growth
factor-, resulting in smooth muscle cell activation, lipid
accumulation, and inflammatory damage to the endothelium (10, 11).
Transgenic mice expressing the human apo(a) gene are more susceptible
to the development of fatty streak-type lesions when fed a high fat
diet, and they exhibit prolonged fibrinolysis (12, 13).
The concentration of Lp(a) in human plasma varies about 1000-fold in
the population and is inherited in a Mendelian co-dominant fashion.
Roughly 97% of this variation maps to the locus of the apo(a) gene
(14-16). Hence, the regulation of apo(a) gene expression is
significant in controlling pathological plasma levels of Lp(a). About
one half of the large variation of apo(a) plasma concentration is due
to transcription efficiency, and one half to the post-transcriptional effects of size polymorphism (16). Since isoforms of apo(a) with
greater numbers of kringle domains transit through the cell less
rapidly and efficiently, there is a rough inverse correlation between
size of apo(a) isoforms and plasma concentration (17, 18). However,
even within alleles matched for isoform size, there exists a greater
than 200-fold range of plasma concentration (19-22). Transcription
control is a likely cause of this variation. Numerous studies have
found a large variation in apo(a) mRNA quantity in humans and
primates, which corresponds with plasma concentration in most cases
(23-26). We have previously shown that the liver-specific expression
of the apo(a) gene is partly mediated by the apo(a) proximal promoter,
which is located from 98 to +130 relative to its transcription start
site and responds to the liver-enriched transcription factor HNF-1
(27). However, there are several indications that additional
transcriptional regulatory elements must contribute to the high levels
of apo(a) gene expression: activity of this core promoter is relatively
weak in in vitro transfection assays, it does not account
for the sex hormone responsiveness of the apo(a) gene, and it contains
far less sequence polymorphism than could account for the inherited
variation in gene expression, which is known to map to this locus. A
further indication that regulatory sequences exist further 5
to the
apo(a) gene comes from transgenic mouse experiments. Deletion of the
region 5
to the apo(a) promoter from a yeast artificial chromosome
severely compromises apo(a) expression in transgenic mice compared with a yeast artificial chromosome containing approximately 80 kb of upstream sequence.2
The apo(a) gene is part of a cluster of genes and pseudogenes on
chromosome six which share over 70% sequence identity (28, 29). The
apo(a) and the plasminogen genes are organized in a head to head
configuration separated by about 40 kb. We have systematically tested
this entire intergene region for additional apo(a) regulatory elements.
In this study, we describe an apo(a) transcription control region (ACR)
that is located 20 kb 5 to the apo(a) transcription start site. The
enhancer region contains an essential element responding to the members
of the Ets transcription factor family. In addition, the binding of Sp1
as well as several other transcription factors around the Ets site also
contributes to maximal enhancer activity. Unexpectedly, the apo(a)
enhancer region resides within a LINE element. LINE 1 (L1) elements are
long interspersed repeat elements that number from 50,000 to 100,000 in
the human genome. Although greater than 90% of them are truncated,
full-length L1s are approximately 6 kb in length and contain a
5
-untranslated region with internal transcription regulatory elements,
two open reading frames, and a 3
-untranslated region that terminates
in a poly(A) tail (30-32). This is the first report of an L1 element affecting the expression of a flanking gene. In contrast, the corresponding regions from 10 other randomly selected human L1 elements
were found to have reduced enhancer activity as well as one or more
nucleotide differences in the core Ets binding site sequence. Hence the
L1 element upstream of the apo(a) gene may represent a small subset of
these repeat elements that have the ability to alter the expression of
neighboring genes.
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MATERIALS AND METHODS |
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pGL3-1.4RI and pGL3a Plasmid Construction--
Plasmid
pGL3-1.4RI was constructed from pGLA1 (27), with subsequent
manipulation to create a unique EcoRI site and to remove unnecessary polylinker sequences. It contains human apo(a) genomic 5
sequences from an EcoRI site 1.4 kb upstream of the
transcription start site to an MscI site 130 bp downstream
of the start site, fused to the luciferase gene in pGL3-basic
(Promega). pGL3a is a similar luciferase reporter plasmid containing a
fragment of the 5
-genomic sequence used above. It contains the minimal
promoter of apo(a) from
98 to +130 (27) and derives from pGL3-1.4RI by removal of an EcoRI-PvuII fragment and
subsequent regeneration of the EcoRI site.
Bacterial Artificial Chromosome (BAC) Library Screening and
Subcloning--
A human genomic DNA library in the BAC plasmid (Genome
Systems, Inc.) was screened with probes representing the 5-flanking regions of apo(a) and plasminogen genes. One of the two positive clones
(pBAC2), containing an insert of ~150 kb, was confirmed to have the
entire intergenic region as well as portions of the coding regions of
both apo(a) and plasminogen genes by Southern blot analysis with apo(a)
and plasminogen cDNA probes.
Cell Culture and Transfection Assays--
Human hepatoma HepG2
cells and HeLa cells were maintained in Dulbecco's modified medium
supplemented with 10% fetal calf serum. 2 × 105
cells were seeded in 12-well cell culture plates 24 h prior to transfection by a calcium phosphate co-precipitation method according to the manufacturer's protocol (Promega) as we described previously (27). Transfections were done in triplicate. Briefly, 3 µg of pGL3-based luciferase expression plasmid and 1 µg of the control pSV--galactosidase (Promega) control plasmid, together with or without 1 and 3 µg of Ets-1 expression plasmid were added to each well covered with 2 ml of culture medium. 48 h after transfection, the cells were harvested and lysed, and luciferase and
-galactosidase assays were performed using the Dual Light Kit
(Tropix, Inc.) according to the protocol provided. To control for
transfection efficiency, results are reported as the ratio of
luciferase to
-galactosidase activity.
Deletion, Mutagenesis, and Diagnostic Fragments-- Fragments of genomic DNA in reporter plasmids were removed using available restriction sites. Specifically, construct nos. 3 and 6 (Fig. 2) were the result of removing a NheI fragment (one NheI site from the vector upstream of EcoRI, and the second one located at nucleotide 386 of L1, according to the L1 numbering system of Swergold (30) from either the negative or the positive orientation of the 8b fragment (construct nos. 2 and 1) in pGL3a. Construct nos. 4 and 5 are the internal deletion products of PstI or XhoI fragments, respectively, from construct no. 2. Construct nos. 7 and 8 were generated by cutting with PvuII and EcoRI either the negative or the positive orientation of the fragment 8b-containing plasmid. Construct nos. 9 and 10 were generated by inserting a PCR subfragment of 8b into the EcoRI site in pGL3a in both positive or negative orientations. This fragment was generated with PCR primers L1-3 (gatcgaattcGCTAGCACAGCAGTCTGA) and L1-4 (gtacgaattcAGCTGCGTGCTGGGAGA) to yield the enhancer-containing region corresponding to nucleotides 386-659 of L1 (lowercase letters denote added linker sequences).
Site-specific mutagenesis was carried out according to the mismatch PCR primer protocol (33) with oligonucleotides L1-3 and L1-4 as the outside primers. The internal primers contained the mutations described in Fig. 5A. The fragments were digested with EcoRI and subcloned into pGL3a and confirmed by sequencing prior to transfection assays. To test the enhancer containing fragment 8b with a heterologous promoter, it was ligated into the MluI and BglII sites of the pGL3-promoter vector (Promega) upstream of the SV40 early promoter.Footprint and Gel Shift Analysis--
Nuclear extracts from
HepG2 cells were prepared as described previously (27). Gel mobility
shift assay and DNase I footprint protection analysis protocols were
described previously (34, 35). The sense strand of oligonucleotide
probe (containing site A; underlined) used for gel mobility shift assay
is CAAAGCAGCCCGGAAGCTCGAAC. The oligonucleotides containing
mu-1, mu-2, and mu-3 used for the competition assays were the same as
site A except mutated at the putative Ets site as indicated in Fig.
3A. The DNase I footprint probes were generated from
construct nos. 9 and 10 (denoted ACR(+) and ACR(); plus and minus
strands from the positive or negative orientations of ACR) by first
end-labeling the MluI site with
-32P, and the
radiolabeled inserts were then released by digestion with
BglII. The sequence of the Sp1 oligonucleotide used in the footprint competition assay is aattcGCTGGGGGAGGGGCGCCg.
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RESULTS |
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Identification of an Apo(a) Gene Transcription Enhancer--
The
genetic locus of the apo(a) and plasminogen genes is shown in Fig.
1. The apo(a) and plasminogen genes are
organized in a head to head configuration with about a 40-kb intergenic
region separating them (Fig. 1). This region was isolated in a BAC
plasmid clone, designated pBAC2, which contains a 150-kb insert
including the whole intergenic region plus parts of the apo(a) and the
plasminogen genes (Fig. 1). As described under "Materials and
Methods," fragments encompassing the intergene region were ligated in
front of the apo(a) minimal promoter and luciferase reporter cDNA.
We previously described the core promoter region that spans nucleotides
98 to +130 relative to the transcription start site of the human apo(a) gene and responds to the liver-enriched transcription factor HNF-1
(27). Only two fragments produced activity significantly above
the parent reporter plasmid containing only the core apo(a) promoter.
One of these contains a previously described plasminogen enhancer
located 2 kb upstream of the plasminogen transcription start site (36).
It is not surprising to find that it enhances transcription in
vitro from both apo(a) and plasminogen core promoters, since the
two promoter regions contain about 90% sequence identity. The
physiological relevance of the plasminogen enhancer on apo(a) gene
expression remains to be evaluated. The most dramatic effector of
apo(a) transcription was a 1.8-kb SacI-KpnI
fragment that is located about 20 kb upstream of the apo(a) promoter
(Fig. 1, fragment 8b; Fig. 2,
construct nos. 1 and 2). It coincides with one of the four liver-specific DNase I hypersensitive sites, DH III, reported
by Magnaghi et al. (37). This 1.8-kb fragment is capable of
enhancing apo(a) transcription in an orientation-independent manner and
was therefore studied in detail.
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Minimal Enhancer Region Is Defined by Deletion Analysis--
To
locate the enhancer elements in this region, it was subjected to
deletion analysis. As shown in Fig. 2, construct no. 7, deletion of the
KpnI-PvuII fragment at the 5 end of the 1.8-kb SacI-KpnI fragment (orientation relative to the
apo(a) gene) resulted in a total loss of the enhancer activity,
indicating the presence of essential positive elements within this
region. By itself, this KpnI-PvuII fragment is
sufficient to confer an over 10-fold activation (Fig. 2, no.
8). A further truncation (no. 6) yields a fragment
without activity. We postulated that the functional enhancer domain
resides within the 274-bp NheI-PvuII fragment, which gives a greater than 10-fold activation when ligated in either
orientation 5
to the apo(a) core promoter and luciferase cDNA
(Fig. 2, nos. 9 and 10). We designated this the
apo(a) transcription control region (ACR). Deleting substantial amounts
of the 1.8-kb SacI-KpnI fragment downstream of
the ACR (construct nos. 4, 5, 8,
9, and 10) resulted in an increase of activity
compared with the entire fragment, due either to potential silencer
elements in these regions or to the nonspecific reduction in luciferase activity caused by increasing the size of the reporter plasmid.
ACR Is Part of a LINE Element--
DNA sequence analysis
surprisingly revealed that the ACR is located in the 5-untranslated
region of a highly repetitive L1 element. The ACR
(NheI-PvuII fragment) is located from nucleotide 386-659 relative to the transcription start site of the consensus L1
element reported by Swergold (30). This raised the question whether the
enhancer activity is specific to ACR or is a universal property of all
the L1s. To this end, primers L1-3 and L1-4, designed according to
consensus L1 sequences, were used to generate by PCR this
274-nucleotide region from 10 random human L1 elements. When subcloned
into the pGL3a expression plasmid, each of the random L1 fragments
produced from 20 to 50% of the luciferase activity shown by the ACR
construct in transfected HepG2 cells (data not shown). Sequence
analysis of the 10 randomly selected L1 fragments revealed several
scattered differences, consistent with the estimated 5% sequence
difference among human L1 elements. Only two sites (Fig. 5B,
sites A and B, shaded) were found to be consistently different between the ACR and the random L1s. The ACR
is the only one that contains a CCCGGAAG in its sequence within site A, while each of the random L1s has either CG,
CA, TG, or GG at this position. At
site B, the sequence of the ACR is a CA, but is
GG in random L1s. (Site A, but not site B, was shown to be
functionally active; see below.) Thus, the ACR may represent a
genetically different class of L1 element that can serve as a more
efficient enhancer of, at least, the apo(a) gene when located in its
proximity.
An Ets Element Is Critical for the Enhancer Activity of
ACR--
The sequence containing the site A, unique to the ACR,
resembles the consensus binding site for the Ets family of
transcription factors (38). Three mutations were created to test its
importance in enhancing expression from the apo(a) core promoter. As
shown in Fig. 3A, one, two,
and five nucleotide substitutions increasingly disrupted the activity
of the ACR. The effect of Ets-like proteins as trans-acting factors was
demonstrated by co-transfection of the ACR reporter plasmid ACR()
with an expression plasmid for the Ets-1 protein. The Ets-1 expression
plasmid increased ACR-driven luciferase expression in a
dose-dependent manner (Fig. 3B). However, Ets-1
protein fails to activate the ACR-driven expression to the same extent
when the Ets binding site is disrupted. The residual enhancing activity
produced by co-transfection with Ets-1 plasmid in this case may be the
result of other weak Ets binding sites present in the ACR.
Co-transfection of the Ets-1 plasmid had little effect on the activity
of the core promoter region contained in pGL3a. These data strongly
suggest that members of the Ets transcription factor family are capable
of enhancing apo(a) gene expression through site A in the ACR.
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Footprint Analysis Revealed Many Protein Protection
Sites--
DNase I footprint analysis of the ACR was carried out to
identify other DNA binding sites that may contribute to the ACR
enhancer activity. Nuclear extracts prepared from HepG2 cells protected eight regions of the 274-nucleotide ACR region from digestion with
DNase I (Figs. 4 and
5). Computer analysis of the ACR sequence revealed similarity to the consensus binding sites of transcription factors in most of the protected regions: AP-4 (footprint
I); Sp1 (footprints II, V, and
VI); CRE-BP (III); HNF-3 (III,
VII), Ets-1 (IV); and Oct-1 (VIII).
Footprint IV co-localizes with site A, the previously identified
putative Ets binding site. A hallmark of Ets binding to the major
groove of DNA is a DNase I protection region in the sense strand and a
hypersensitive site on the antisense strand, similar to the pattern
seen in Fig. 4 (38).
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Mutagenesis of DNase I Protected Sites in the ACR-- To assess the effects of the other footprint binding sites within the ACR, we subjected the entire ACR to further deletion and mutagenesis analysis. Mutations were created in these sites and tested in transfection assays in HepG2 cells. Fig. 5A shows each of the DNase I-protected sites boxed and numbered from I to VIII with mutations shown underneath in lowercase letters for base substitutions or as a line denoting a deletion mutation. As shown in Fig. 5B, mutations of footprints II, III-2, and VI, as well as deletion of the first 50 nucleotides, which eliminates all of footprint I and most of footprint II, do not cause a significant reduction in luciferase expression, compared with those of the wild type ACR construct. Deletion of the region including footprints VII and VIII also had no significant effect (data not shown). (It is noteworthy that the mutation in footprint VI alters site B, which was earlier noted to be one of the two sites where the sequence of the ACR differed from the 10 random L1 examples.) In contrast, destruction of the binding sites for footprints III-1, III-3, IV, and V significantly reduce the enhancer activity. The identity of the transcription factors which bind to footprint III-1 and III-3 are unknown. Footprint IV represents the target recognition site for a member of the Ets family, while footprint V is a likely target of an Sp1-related protein.
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DISCUSSION |
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We report the screening of the 40 kb that lie between the 5 ends
of the human apo(a) and plasminogen genes for cis elements other than
the apo(a) core promoter that regulate the expression of the apo(a)
gene in an in vitro system. We have discovered an apo(a)
transcriptional control region (ACR), located midway between the apo(a)
and the plasminogen genes, that is able to confer a greater than
10-fold activation when linked in either orientation to the apo(a)
minimal promoter. Sequence analysis of the ACR revealed it to be part
of a LINE repeat element, whose members number up to 100,000 in the
human genome. Functional analysis of 10 randomly selected,
corresponding regions of other human L1 elements show that they are
less efficient than the ACR in enhancing the activity of the apo(a)
core promoter, and that they contain sequence differences in a site
corresponding to the consensus target of the Ets family of
transcription factors. We also inspected 60 additional L1 sequences available in GenBankTM, and found that they differ from the
ACR in the Ets target sequence. Mutagenesis showed this Ets binding
site to be indispensable for the apo(a) enhancer activity.
Co-transfection of an Ets-1 expression plasmid caused a
dose-dependent activation of ACR-driven expression, further
implicating the role of these trans-acting proteins. DNase I footprint
analysis and further site-directed mutagenesis indicate the involvement
of additional factors including Sp1 in the ACR transcription enhancer
activity and suggest likely interactions between several DNA binding
proteins in the ACR.
Previous data from our laboratory have shown that the apo(a) minimal
promoter is mediated by a liver-enriched transcription factor HNF-1
and can confer liver specific expression (27). In many cases, both
promoters and more distant enhancer elements control the tissue
specific expression of genes. In the case of apo(a), the ACR does not
appear to be liver-specific, suggesting that the apo(a) proximal
promoter is sufficient to determine tissue specificity of its
expression. (Liver represents the sole organ of significant apo(a)
production; much reduced levels of its mRNA have also been detected
in brain and in testes of rhesus monkeys (41), but upon liver
transplant, human subjects entirely convert their isoform of
circulating apo(a) from recipient to donor phenotype (42).) The ACR may
function only by increasing the expression level of the rather weak,
but tissue-specific apo(a) promoter. One example of an interplay
between liver-specific and nonspecific elements of an opposite type is
found in the apolipoprotein E gene, where tissue specificity is
determined by a hepatic control region that is located 19 kb from the
start of the apoE gene (43). A number of other genes are now known to
be controlled by remote transcription elements located thousands of
nucleotides from transcription start sites (43-46). The function of
the ACR must ultimately be evaluated in its normal genomic context
in vivo. We have begun to engineer transgenic mice with
mutagenized and normal regions of the human apo(a) locus in yeast
artificial chromosomes. Human subjects can also be screened for
polymorphisms or mutations in this region which can be shown to effect
apo(a) expression.
Based on functional criteria and on comparison to the sequence of other L1 elements, the most notable feature of the ACR is the presence of an Ets family target site. Evidence for its functional importance derives from footprint, gel shift, and mutagenesis data, and the effect of co-transfection with an Ets-1 expression plasmid. Ets is an oncogene that was first discovered in avian erythroleukemia virus to encode a transcription factor that is capable of activating erythrocyte-specific gene expression. It is now known that there is a family of Ets-like transcription factors with a variety of tissue distributions and target genes. For example, the thrombopoietin gene, which is predominantly expressed in liver, contains a functional Ets-binding site that is regulated by a ubiquitously expressed Ets-related transcription factor, E4TF1, in hepatocytes (47). Further studies will be needed to clarify which member(s) of the Ets family are responsible for binding to the ACR in hepatocytes. Ets proteins have been reported to act synergistically with other transcription factors, including Sp1, in the regulation of many viral and cellular genes (48, 49). Although an intact Ets binding site is essential for the enhancer activity of the ACR, our data indicate that binding sites for Sp1 as well as other transcription factors also contribute to the optimal function of this apo(a) gene enhancer.
The fact that the ACR resides within the 5-untranslated region of a
LINE sequence may have significant implications for the functions of L1
elements, as well as for the regulation of the apo(a) gene. The nearly
100,000 L1 long interspersed repeats comprise an estimated 5% of the
human genome. Roughly 10% of these represent full-length, 6-kb
elements, while only an estimated 30-60 are active elements that are
capable of transposition via reverse transcription of an RNA
intermediate (50). The LINE sequence within the apo(a) locus appears to
be full-length. However, it does not meet the criteria of a mobile L1
element, as it contains numerous stop codons in both consensus open
reading frames necessary for such activity. Nor is it a member of the
Ta subclass of L1 elements to which most of the mobile elements belong
(50, 51). Although it is unable to code for active mobility proteins,
our data indicate that this particular L1 element is capable of
interacting with DNA binding proteins and influencing the expression of
an adjacent gene.
It has been proposed that methylation of CpG dinucleotides in the 5
region of L1 sequences interferes with their expression (52).
Methylation at such sites may interfere with the interaction of
DNA-binding proteins such as Ets (53). Southern blotting with
methylation-sensitive and insensitive restriction enzymes and a probe
5
to the apo(a) gene L1 element indicate that the CpG dinucleotide in
the Ets binding site (site A) is unmethylated in human liver cells
(data not shown). This is consistent with an in vivo
interaction of an Ets family protein with this site.
There have been several reported cases in which de novo insertions of L1 sequences in either the germline or somatic cells have interrupted genes and caused human disease (54-58). To our knowledge, we describe the first example of a LINE element that acts as a cellular enhancer to stimulate the expression of a nearby gene. In a related example, a subclass of Alu DNA repeats has been found to function as an estrogen receptor-dependent enhancer (59). This raises the possibility that L1 elements may serve as potentially mobile gene control elements that could influence the evolution of gene expression in mammals, as well as help to control the expression of the potentially atherogenic apo(a) gene.
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FOOTNOTES |
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* This research was supported in part by National Institutes of Health Program Project Grant 48638 and post-doctoral fellowships from the National Institutes of Health, American Heart Association California Affiliate, and Swiss National Science Foundation.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.
To whom correspondence should be addressed: Falk Cardiovascular
Research Center, Stanford University School of Medicine, 300 Pasteur
Drive, Stanford CA 94305-5246. Tel.: 650-725-4494; Fax: 650-725-1599;
E-mail: richard.lawn{at}forsythe.stanford.edu.
1 The abbreviations used are: Lp(a), lipoprotein(a); apo(a), apolipoprotein(a); HNF, hepatocyte nuclear factor; kb, kilobase pair(s); bp, base pair(s); PCR, polymerase chain reaction; ACR, apo(a) gene transcription control region; L1, LINE element 1; BAC, bacterial artificial chromosome.
2 S. Hughes and E. Rubin, personal communication.
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REFERENCES |
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