From the Pacific Biomedical Research Center, University of Hawaii, Honolulu, Hawaii 96822
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ABSTRACT |
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We have reported in this paper the complete
cDNA sequence, gene structure, and tissue-specific expression of
LOXL2, a new amine oxidase and a member of an emerging family of human
lysyl oxidases. The predicted amino acid sequence, from several
overlapping cDNA clones isolated from placenta and spleen cDNA
libraries, shared extensive sequence homology with the conserved
copper-binding and catalytic domains of both lysyl oxidase (LOX) and
the lysyl oxidase-like (LOXL) protein. These conserved domains are
encoded by five consecutive exons within the LOX,
LOXL, and LOXL2 genes that also maintained
exon-intron structure conservation. In contrast, six exons encoding the
amino-terminal domains diverged both in sequence and structure. Exon 1 of the LOXL2 gene does not encode a signal sequence that is
present in LOX and LOXL, suggesting a different
processing and intracellular localization for this new protein.
Expression of the LOXL2 gene was detected in almost all
tissues with the highest steady state mRNA levels in the
reproductive tissues, placenta, uterus and prostate. In
situ hybridization identified placental syncytial and
cytotrophoblasts responsible for the synthesis of LOXL2
mRNA and demonstrated a spatial and temporal expression pattern
unique to the LOXL2 gene.
Lysyl oxidase is a copper-dependent amine oxidase that
belongs to a heterogeneous family of enzymes that oxidize primary amine substrates to reactive aldehydes. This enzyme family is subdivided into
two main classes on the basis of the chemical nature of the co-factors
associated with these amine oxidases. Flavine adenine dinucleotide is
the co-factor of monoamine oxidase and of an intracellular form of
polyamine oxidase. A second group of amine oxidases contain topaquinone, a modified tyrosine side chain utilized as a redox co-factor. Diamine oxidase, monoamine-metabolizing
semi-carbazide-sensitive amine oxidase, and lysyl oxidase belong to
this latter subfamily of amine oxidases (1-3).
Most of the studies on lysyl oxidase have focused on the specific
cross-linking activity and catalytic mechanism of action of this enzyme
on the extracellular matrix substrates, collagen and elastin. Lysyl
oxidase participates in the critical post-translational modification
essential to the biogenesis of connective tissue by deaminating the
side chains of lysine residues in these proteins, thus catalyzing the
covalent cross-linking of several fibrillar collagen types and the
formation of desmosine and isodesmosine cross-links in elastin (4,
5).
Recently, multiple novel biological functions have been attributed to
lysyl oxidase (6, 7) that have suggested that other intracellular and
intranuclear substrates may be involved in these multiple functions (8,
9). The range of these novel activities of lysyl oxidase cover a
spectrum of biological functions from developmental regulation (10) to
tumor suppression (7, 11-14) and cell growth control (15, 16). An
attractive hypothesis to explain how a single protein can fulfill these
different functions is that a family of several different lysyl
oxidases may exist that individually function to perform these roles.
Over the past few years, we and others have described two lysyl
oxidase-related proteins that fulfill all the requirements of being
functional copper-dependent, but genetically distinct, lysyl oxidases that could potentially serve as the basis for a family
of proteins present in a variety of cellular and tissue locations, each
with an unique function.
The first of these
LOX1-related proteins was
called LOXL or lysyl oxidase-like (17, 18). A comparison of the
cDNA sequence of LOXL and LOX confirmed a
significant homology within the carboxyl-terminal end of these
proteins. This homology included a striking conservation of the copper
binding site, the catalytic domain, and the carbonyl co-factor binding
site. This domain conservation was also reflected in conservation of
exon size and exon-intron boundaries in five of the seven exons in both
of the LOX and LOXL genes encoding these
conserved domains (18, 19). We have also mapped this LOXL
gene to chromosome 15q23 (20), and the LOX gene was
previously mapped to chromosome 5q23.3 (21, 22). We have, moreover,
localized the LOXL protein to sites of de novo fibrosis in
the liver and showed co-regulated expression of the LOXL
gene with the colIIIA1 gene and the LOX gene with
the gene encoding pro- We now report the detailed structural and expression analysis of a new
member of the lysyl oxidase gene family encoding a protein we have
referred to as LOXL2. The unique temporal and spatial tissue-specific
expression pattern in reproductive tissues and the possible
intracellular localization of LOXL2 indicate a role for LOXL2 that is
distinctly different from either LOX or LOXL.
Isolation of LOXL2 cDNA Clones--
DNA sequence obtained
from Human Genome Sciences (Applied Biosystems) expressed sequence tag
clone HSSAE08RB provided information for the design of PCR primers, and
we have used these primers to screen a human spleen cDNA library
(cDNA Isolation Service, Life Technologies, Inc.). A human placenta
library (Stratagene, La Jolla, CA) was also screened using a
LOXL2-specific PCR-amplified cDNA probe. The primers
used to generate this probe were LOXL2-34 (5' CTG GTG CGA CTG AGA GGC
G 3') and LOXL2-35 (5' GAA GGC GTT GCT GGC GAA TC 3'). The probe was
random primer-labeled, and filters with phage plaque lifts were
hybridized overnight at 42 °C, followed by four washes for 30 min
each at room temperature in 2 × SSC, 0.1% SDS, and two washes
for 30 min at 50 °C in 0.2 × SSC and 0.1% SDS. Positive
phages were purified, and the inserts were excised and sequenced.
LOXL2 Genomic Sequencing--
Two overlapping PAC clones, 17459 and 17460 (Genome Systems, Inc., St Louis, MO), containing the
LOXL2 gene were used as templates for DNA sequencing using
the Thermo Sequenase radiolabeled terminator cycle sequencing kit
(Amersham Pharmacia Biotech). Oligonucleotides were designed based on
LOXL2 cDNA sequences and synthesized by Genosys Biotechnologies
Inc. (The Woodlands, TX) and Life Technologies, Inc. Sequencing
reactions were performed using 32.5 fm PAC DNA in a reaction mixture
containing the following reagents: 2 µM each dATP, dGTP,
dTTP, dCTP, 8 units of Thermo Sequenase, 26 mM Tris-HCl, pH
9.5, 6.5 mM MgCl2, 0.45 µCi of
[33P]ddGTP, ddATP, ddTTP, and ddCTP. Each reaction was
terminated, and aliquots were loaded on a 6% denaturing polyacrylamide
gel. Following electrophoresis, gels were exposed for 2 days at
Intron-Exon Size Determination--
Exon sizes were determined
by genomic DNA sequencing from PAC clones 17459 and 17460. Intron sizes
were determined using sets of primers that corresponded to 5' and 3'
end sequences of previously identified exons of the LOXL2
cDNA and DNA from PAC clone 17459 and PAC clone 17460. Polymerase
chain reactions were performed using the GeneAmp kit (Perkin-Elmer)
with the following parameters: initial denaturation at 94 °C for 3 min was followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at primer-specific temperatures (between 52 and 66 °C)
for 30 s, and extension at 72 °C for 1 min with a final
extension at 72 °C for 7 min. PCR reactions were performed using 80 ng of DNA in 25 µl of reaction mixture containing final
concentrations of the following reagents: PCR buffer from Perkin-Elmer
containing 1.5 mM MgCl2, 0.2 mM
each of dGTP, dATP, dTTP, dCTP, and 0.625 unit of Taq
polymerase. Amplified products were electrophoresed on 1% agarose
gels, and size-separated DNA fragments were visualized by ethidium
bromide staining.
Primer Extension--
Primer extension was performed on 5 µg
of total RNA from human placenta. 32P-Labeled antisense
primers LOXL2-2 (5'-TAC CGA GCT TAG GCT TA-3') and LOXL2-18 5'-CAC GAA
CAG TGT CTA GAC GA-3') were annealed, and extension reactions were
performed for 50 min at 42 °C with 200 units of Superscript II
reverse transcriptase (Life Technologies, Inc.) and terminated for 15 min at 70 °C. These extension products were treated with RNase A for
30 min at 37 °C, extracted with phenol, precipitated, re-dissolved,
and subjected to electrophoresis through an 8% denaturing
polyacrylamide gel. A sequencing reaction performed on PAC clone 17460 using primers LOXL2-2 or LOXL2-18 was loaded on the same gel. Extension
reactions lacking either RNA or primers were used as controls.
Northern Blot Analysis--
Two-µg aliquots of size-separated
poly(A)+ RNA obtained from adult heart, brain, placenta,
lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus,
prostate, testis, uterus, small intestine, colon, and peripheral blood
leukocyte were present in the two Multiple Tissue Northern filters used
(CLONTECH). Each Multiple Tissue Northern blot was
prehybridized for 30 min at 68 °C in 7 ml of ExpressHyb
hybridization solution (CLONTECH). Hybridization, using 32P-labeled cDNAs prepared by random primer
labeling, was carried out in 5 ml of the same solution used for
prehybridization at 68 °C for 1 h. The filters were then washed
twice in 2 × SSC, 0.5%SDS at room temperature and twice in
0.1 × SSC, 0.1% SDS at 50 °C and exposed to Kodak Biomax film
for 12 h at In Situ Hybridization--
Placentas with attached fetal
membranes and decidua were obtained from a patient after elective
pregnancy termination at 14 weeks of gestation and from patient after
elective cesarean section at term from Kapiolani Medical Center
(Honololu, HI). Tissues were obtained with the approval of the
Institutional Review Board and the Human Experimentation Committee of
the University of Hawaii. Tissues were transferred to ice within 1 h of removal, and small pieces were cut from the villous placenta,
avoiding the chorionic and basal plates. Tissue samples were frozen in
liquid nitrogen, and sections were cut in a cryostat as described
previously (24). For in situ hybridizations,
35S-labeled LOXL2-specific oligomer that
corresponded to DNA sequences 2184-2244 bp of the LOXL2
cDNA and a complementary control oligomer were incubated with
pretreated and prehybridized tissue sections. The specificities of
these oligomer probes to the LOXL2 mRNA were confirmed
by Northern blot analysis. Following post-hybridization washes and
autoradiography, counterstained tissue sections were visualized by dark
and bright field microscopy.
LOXL2 cDNA Isolation and Sequence Analysis--
A search of
the Human Genome Sciences Inc. protein data base identified three
expressed sequence tag clones with homology to lysyl oxidase. DNA
sequence analysis of one of these clones, HSSAE08RB, demonstrated a
45% homology to lysyl oxidase (LOX) and 48% homology to
the lysyl oxidase-like (LOXL) cDNA. PCR-derived DNA
fragments prepared using this expressed sequence tag sequence information were used to identify 16 cDNA clones from a human spleen cDNA library and an additional 8 clones from a human
placenta library. DNA sequencing and alignment of these clones resulted in a full-length cDNA sequence encoding LOXL2. An open reading frame of 1911 bp was identified that clearly demonstrated sequence homology within this LOXL2 cDNA to both LOX
and LOXL cDNAs.
Conservation of the nucleotide and deduced amino acid sequence within
the carboxyl-terminal end of LOXL2, LOX, and LOXL included the
copper-binding domain (WEWHSCHQHYH) in LOX and LOXL and
WIWHDCHRHYH in LOXL2 with the four histidines that supply the nitrogen
ligands for the copper coordination complex specific for lysyl oxidase proteins (26). The active site in LOX (DIDCQWWIDITDVXPGNY) and in LOXL2
(DIDCQWVDITDVPPPGDY) contains, in each, a Tyr residue (Y) at the
COOH-terminal end, which is known to participate together with a Lys
residue in the formation of the quinone co-factor that is present in
these proteins. Ten cysteines characteristic of LOX and LOXL were
similarly conserved in LOXL2 (4). A growth factor and cytokine receptor
domain present in the LOX and LOXL proteins was also identified within
the LOXL2-derived amino acid sequence. This domain is
consistent with the consensus sequence C-X9-C-X-W-X25-32-C-X10-15
(where X is a defined number of any amino acids) that is a
proposed ligand-binding domain for a number of receptors for cytokines
and growth hormones (18, 27). Three repeats of the scavenger receptor
cysteine-rich domain were also present at amino acids 13-125,
149-250, and 258-368 in the derived LOXL2 sequence (25,
28).
The hydropathy profile of the LOXL2 protein was determined and compared
with that of the LOX and LOXL proteins using the MacMolly analysis
program. Unlike other lysyl oxidases, LOXL2 had no obvious evidence for
a hydrophobic signal sequence.
Three major transcription termination sites were noted within 3'-UTR
domains in the 20 LOXL2 cDNA clones that we have
analyzed. The first termination site was 690 bp 3' of the termination
codon, the second site was 740 bp, and the final transcription
termination site was 900 bp 3' of the termination codon. These
mRNAs all had 3'-UTRs differing slightly in size but were detected
by Northern analysis as a single mRNA species of 3.6 kb. A less
abundant mRNA species of 4.9 kb that was detected in Northern blots
of heart, liver, and pancreas RNA samples using LOXL2
cDNA as a probe raises the possibility that an additional, but less
frequently used, transcription termination signal may exist further 3'
of those we have already noted.
Structure of the LOXL2 Gene--
Two overlapping PAC clones, PAC
17459 and PAC 17460, that were isolated using LOXL2-specific
PCR primers, served as templates for sequencing the exon-intron
boundaries, complete exons, and to determine intron sizes using PCR and
extra long PCR reactions. Most exon-intron boundaries of the
LOXL2 gene show the consensus sequence (C/T)AG-exon-GT(A/G).
The sizes of the 11 exons of the LOXL2 gene ranged from 112 to 940 bp. Although the LOXL2 gene has 11 exons, five
consecutive exons (exons 6-10), which encode the copper-binding and
catalytic domains, revealed 84% sequence similarity, and exon sizes
were very similar to the corresponding exons of the LOX and
LOXL genes (18, 19). All the other exons in the
LOXL2 gene are divergent in both sequence and size.
The sizes of the introns were the following from intron 1 to intron 10, respectively, with the exception of intron 6 that could not be
amplified by these methods and is most probably larger in size: 2.0, 0.4, 0.53, 2.15, 2.7, 0.61, 1.22, 3.0, and 0.75 kb. The structure of
the LOXL2 gene and the exact sizes of each exon and intron
and a comparison with the sizes of the corresponding exons and introns
of the LOX and LOXL genes are presented in Fig. 1.
The LOXL2 Promoter--
An 830-bp region of the 5'-flanking
sequence of the human LOXL2 gene that included the first
exon and the first intron was analyzed using the DNA sequence analysis
program GCG (Genetics Computer Group, Madison, WI) and the TF sites
data file to identify potential transcription factor binding sites. A
large number of potential transcription factor binding sites were
detected within the 5'-flanking region, the first exon and the first
intron, including 10 AP-2 sites and two SP1 sites. The LOXL2
gene promoter domain contains no typical TATA or CAAT box sequences.
There is a single AP-1 site (TGAATCA) at position Transcription Initiation Sites--
Transcription initiation sites
of the LOXL2 gene were determined by primer extension
analysis using total RNA from human placenta. The sizes of these
fragments identified two transcription initiation sites at positions
LOXL2 and the Lysyl Oxidase-related cDNA--
Recently, a
lysyl oxidase-related cDNA was reported (25). This cDNA
(WS9-14) contained a 3' sequence homologous to sequences within our
LOXL2 cDNA. This region of homology started from bp 780 of the WS9-14 sequence and was identical to LOXL2
cDNA sequence with the following differences: at bp 278 of the
LOXL2 sequence there is an A, while at the corresponding
position at bp 1131 of WS9-14 there is a T; at bp 433 a
G in LOXL2 and an A in WS9-14; at 1350 an A in
LOXL2 and a C in WS9-14; at 1631 a C in
LOXL2 and a G in WS9-14; at 1715 an additional C
in LOXL2; at 1779 a T in LOXL2 and a C in
WS9-14. Several of these changes result in amino acid
differences between LOXL2 and WS9-14. A number of additional sequence
differences in the 3'-UTR were found between WS9-14 and
LOXL2. The LOXL2 cDNA, however, has no
sequence homology whatsoever to the most 5' 780 bp of
WS9-14. The significance and origin of this 780-bp part of
the WS9-14 sequence is unclear. Our analysis of the
LOXL2 gene has confirmed that the cDNA sequence we have
identified is encoded within 11 exons. The most 5' of our cDNA is
found at the 5' end of exon 1 and primer extension, and promoter
analysis has demonstrated that transcription initiation begins within
this region. From these studies, it is clear that the most 5' sequence
of our LOXL2 cDNA is derived from the most 5' exonic
sequences of the LOXL2 gene.
The most 5' 780-bp sequences of the WS9-14 mRNA were
generated by two independent PCR extension and 5' rapid amplification of cDNA ends reactions, and the authors of this work (25) had not
confirmed these results by corresponding cDNA sequences. We were
unable to isolate any cDNA or genomic clones that would contain identical or homologous sequences to this domain of WS9-14.
Therefore the origin of the WS9-14 mRNA is unclear; it
is possible that this 5' part of the WS9-14 cDNA is a
PCR product unrelated to the rest of the mRNA. It is certainly
unlikely to be a transcript derived from the LOXL2 gene that
we have mapped to chromosome 8p21.3 (29).
Tissue-specific Expression of the LOXL2 Gene--
Northern blot
analysis of the LOXL2 mRNA detected a 3.6-kb band in all
tissues with the exception of blood leukocytes. An additional, much
less abundant mRNA of 4.9 kb was also detected in heart, liver, and
pancreas. The steady state levels of the LOXL2 mRNA were
quantitated in all tissues relative to LOXL2 Expression in the Placenta--
As placenta showed the
highest level of LOXL2 mRNA, we have further analyzed
placental tissues and fetal membranes to identify the cells in these
tissues that are responsible for the synthesis of the LOXL2
mRNA. Placental villi and fetal membrane sections from 14 weeks of
gestation and at term were used for in situ hybridizations using a LOXL2-specific 60-mer oligonucleotide probe. There
was no hybridization signal detected with this
LOXL2-specific oligonucleotide probe with any of the cells
in fetal membranes under these conditions. A weak signal was detected
at 14 weeks gestation in the placental villi that was significantly
increased in intensity in the villi of the term placenta (Fig.
4). Positive autoradiographic signals were associated with the syncytiotrophoblasts, which are clearly responsible for the synthesis of this abundant LOXL2 mRNA in
placental tissue.
Based on the significant sequence homology and similarity in gene
structure of the lysyl oxidases, it is likely that at least parts of
the lysyl oxidase genes share a common ancestor. There is a closer
evolutionary relationship between LOX and LOXL
than between these two lysyl oxidases and LOXL2. It is
likely that at least parts of the LOX, LOXL, and
LOXL2 genes share a common ancestor, as the exons encoding
the functional domains of the mature protein have remained closely
homologous both in sequence and in size. In contrast, exons encoding
the 5'- and 3'-untranslated regions and the amino-terminal domains of
these proteins have diverged significantly, not only in sequence but
also in gene structure.
The LOXL2 mRNA was detected in almost all human tissues
analyzed with highest expression in several reproductive tissues, such
as placenta, prostate, and uterus. Although both LOX and LOXL genes are expressed in placenta, the LOX
gene shows highest expression in heart and lung, while the highest
LOXL mRNA levels were noted in heart, skeletal muscle,
and kidney (18). The results of in situ hybridizations
confirmed abundant LOXL2 mRNA levels in the placenta and
have identified the syncytiotrophoblasts as responsible for this high
expression of the LOXL2 gene.
These findings support an earlier description of a protein that had
been identified in the placenta with LOX activity but which was,
however, different from LOX in its amino acid composition (30).
Moreover, while LOX has been shown to be present in the amnion, one of
the components that makes up the fetal membranes (31), we did not
detect any LOXL2 mRNA in the fetal membranes using these
conditions for the in situ hybridization that readily detected its expression in the placenta. Clearly, therefore,
LOX and LOXL2 are expressed in very different
locations within reproductive tissues. The highest steady state level
of LOX mRNA was reported at 12 weeks of gestation, both in amnion
and placenta (31, 32). Thereafter, levels of LOX mRNA and
cross-linking activity declined dramatically to levels of only 5-10%
by 42 weeks (31). In contrast, we have observed increasing levels of
LOXL2 mRNA between 14 and 40 weeks gestation in
placenta, suggesting not only a different spatial but also a different
temporal activity of the LOXL2 gene.
There is an additional and quite significant difference between LOXL2
and all the other lysyl oxidases. Comparison of hydropathy profiles of
LOX, LOXL, and LOXL2 revealed that the LOXL2 protein, strikingly, had
no evidence for the hydrophobic signal sequence necessary for
extracellular transport that has been noted in all the other lysyl
oxidases. This result suggested that the LOXL2 protein may be processed
and transported through an intracellular pathway that is different from
the processing of the other members of the lysyl oxidase family (33).
This prediction is supported by our immunohistochemical observations
using a LOXL2-specific antibody that localized LOXL2 intra- as well as
extracellularly.2
In summary, we have identified LOXL2 as a new member of the lysyl
oxidase gene family. Although its precise processing and localization
are not known, based on the presence of the copper-binding and
catalytic domains, it is very likely that this protein will have
a catalytic function either extra- or intracellularly.
A possible intracellular localization of LOXL2 coincides with recent
reports of novel intracellular and intranuclear lysyl oxidases.
However, it was not clear from these studies how the 32-kDa active and
secreted form of lysyl oxidase could also have an intranuclear location
(8, 9). A LOX-dependent alteration of chromatin structure
has also been observed (34). This raises the possibility that LOX, or
more likely an intracellular LOXL2, may directly or indirectly exert
effects on nuclear components (5, 8). An intracellular form of LOX was
also found in association with fine, filamentous structure in the
cytoplasm of fibroblasts, consistent with this form of LOX being a
cytoskeletal protein (9). Based on the significant sequence homology
and the intracellular localization of LOXL2, it is possible that this
new protein is responsible for these reported intracellular and
intranuclear catalytic functions and for at least some of the
developmental and growth regulatory functions previously attributed to
lysyl oxidase (LOX) (10, 15).
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1(I) collagen. These results suggested
different functions for LOX and LOXL (23).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
80 °C using Kodak X-Omat film.
70 °C. The DNA fragments used for radiolabeling
were as follows: a 1.36-kb LOXL2 cDNA and a 2.0-kb
-actin cDNA provided with the Multiple Tissue Northern blots.
The specific activity of all radiolabeled DNA fragments used in these
incubations was 5 × 109 dpm/µg.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Structure of the human LOXL2
gene. A comparison of the exon-intron structure of the
LOX, LOXL, and LOXL2 genes is
presented. Nonconserved exons are shown as gray rectangles,
conserved exons are shaded in black, and exons encoding
3'-UTR sequences are represented as hatched rectangles.
Sizes of exons are given in base pairs above each exon; intron sizes
are provided under "Results." Exon sizes were determined by genomic
DNA sequence analysis, and intron sizes were determined by PCR
analysis.
522. There are four
CAP/CRP-lac sites (ACACTTT) at positions
439 and
816, within the
first exon at +130 and within the first intron (AAAGTGT). There is a
single MRE site at
716 (TGCACAC), two malT sites (GGAGGA) at
400
and
55, a single GAGA site (AAGAGAG) at
716, one GR-uteroglobin (AGAACA) at
442, four GR-MT sites (TGTCCT) at
879,
560,
53, and
one within the first exon at +113. A zeste-white (CACTCA) at position
499 and a JCV (GGGTGGGG) at
856 were also present within this
promoter domain of the human LOXL2 gene. The DNA sequence of
the promoter region of the LOXL2 gene and the major
transcription factor binding site consensus sequences are shown in Fig.
2.
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Fig. 2.
The 5'-UTR region of the LOXL2
gene. The first nucleotide preceding the ATG codon is
numbered 1 and A is +1. Potential consensus regulatory and
transcription factor binding site sequences are indicated by
shaded boxes. Transcription initiation sites determined by
primer extension analysis are indicated by black dots.
95 and
100 and two additional but less abundant sites at
54 and
110.
-actin mRNA. The
expression of the LOXL2 mRNA was significantly higher in
placenta, prostate, uterus, and pancreas (ratios between 2 and 3)
compared with lower expression in brain, lung, skeletal muscle, thymus,
and kidney (ratios below 0.5). The results of these experiments are
presented in Fig. 3.
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Fig. 3.
Tissue-specific expression of the
LOXL2 gene. Multiple tissue Northern blots
containing 2 µg of poly(A)+ RNA from several human
tissues were hybridized to 32P-labeled LOXL2
cDNA probe and -actin probe (A and C). The
quantitation of the LOXL2 mRNA in each tissue is also
presented (B and D).
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Fig. 4.
In situ hybridization analysis of
LOXL2 mRNA distribution in term placental
tissue. Placental tissue sections were obtained and prepared for
in situ hybridization analysis as described previously (24).
An 35S-labeled LOXL2-specific oligomer was
incubated with pretreated and prehybridized placenta tissue
sections. Following post-hybridization washes and autoradiography,
counterstained tissue sections were visualized by dark field light
microscopy. A, a section of placental villi counterstained
with toluidine blue viewed by regular light microscopy,
demonstrating the syncytiotrophoblasts. B, a dark field
image of a consecutive section showing the placental tissue after
incubation with a radiolabeled LOXL2 oligomer. C, a dark
field image using an LOXL2 sense oligomer. D, a diagram
illustrating the structure and position of the syncytium in placental
villous tissue.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants CA766580 and HD24314 and supported in part by Grant RR03061 to the University of Hawaii under the Research Centers in Minority Institution Program.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) AF117949 (LOXL2 cDNA sequence) and AF117950 (LOXL2 gene promoter).
To whom correspondence should be addressed: Pacific Biomedical
Research Center, University of Hawaii, 1960 East-West Rd., Honolulu, HI 96822. Tel.: 808-956-9452; Fax: 808-956-9481;
kciszar{at}aol.com.
2 C. Jourdan-Le Saux, H. Tronecker, L. Bogic, G. D. Bryant-Greenwood, C. D. Boyd, and K. Csiszar, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: LOX, lysyl oxidase; LOXL, lysyl oxidase-like; PCR, polymerase chain reaction; kb, kilobase(s); bp, base pair(s); UTR, untranslated region; PAC, bacteriophage P1-derived vector.
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