THE JOURNAL OF BIOLOGICAL CHEMISTRY© 1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 272, No. 27, Issue of July 4, pp. 17104 –17111, 1997
Printed in U.S.A.

Ubiquitous Expression of the a1(XIX) Collagen Gene (Col19a1)
during Mouse Embryogenesis Becomes Restricted to a
Few Tissues in the Adult Organism*(Received for publication, November 26, 1996, and in revised form, February 21, 1997)

Hideaki Sumiyoshi, Kazuhito Inoguchi, Mohammed Khaleduzzaman,
Yoshifumi Ninomiya, and Hidekatsu Yoshioka‡
From the Department of Molecular Biology and Biochemistry, Okayama University Medical School, Okayama 700, Japan

Type XIX collagen is a poorly characterized member ofthe fibril-associated collagens with an interrupted triple
helices (FACIT) class of collagen molecules. As a firststep toward elucidating its function, we have isolated
full size cDNA clones from the mouse a1(XIX) collagengene (Col19a1) and established its pattern of expression
in the developing embryo and adult organism. Col19a1
transcripts can be detected as early as 11 days of gestation and in all embryonic tissues, except the liver, of an
18-day postcoitum mouse. In contrast, only a few adulttissues, brain, eye, and testis, seem to accumulate
Col19a1 mRNA. Col19a1 transcripts are at least 10 times
more abundant in adult than fetal brain and significantly less in adult than fetal muscle and skin. Consistent with the RNA data, polyclonal antibodies for a1(XIX)
collagen reacted with a 150-kDa protein in the neutral
salt extraction of adult mouse brain tissues. We therefore propose that type XIX collagen plays a distinct rolefrom the other FACIT molecules, particularly in the assembly of embryonic matrices and in the maintenance ofspecific adult tissues.

Collagenous networks play a critical role in the morphogenesis of the embryo and in the maintenance of tissue architecture of adult tissues. The 19 genetically distinct types of collagen molecules are expressed in developmental and tissuespecific manners (1– 4). The collagen superfamily can beclassified into several subgroups according to the structuralfeatures of its members, the supramolecular aggregates thatthey form, and the structure of the gene that the proteins are
encoded. One of the most interesting and less characterized
collagen subgroups is the one of the so-called FACIT1 (fibrilassociated collagens with interrupted triple helices) which includes types IX, XII, XIV, XVI, and XIX collagen. Available
evidence suggests that FACIT may play an important role inproviding tissue-specific molecular links between collagen* The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted
to the GenBankTM/EBI Data Bank with accession number(s)AB000636.
‡ Present address and to whom correspondence and reprints should
be addressed: Dept. of Molecular Biology and Biochemistry, Okayama
University Medical School, 2-5-1 Shikata-cho, Okayama 700, Japan.
Tel.: 81-86-235-7128; Fax: 81-86-222-7768; E-mail: mbb@ccews2.
cc.okayama-u.ac.jp.
1
The abbreviations used are: FACIT, fibril-associated collagens withinterrupted triple helices; bp, base pairs; kb, kilobases; d.p.c., days
postcoitum; RT-PCR, reverse transcription-polymerase chain reaction;
COL, collagenous; NC, noncollagenous; MBP, maltose binding protein;
PAGE, polyacrylamide gel electrophoresis.

fibrils and other extracellular matrix aggregates (5, 6). With
the exception of types IX and XII, virtually nothing is known
about the expression pattern of the other FACITs. Without thisinformation, no function has yet been postulated for types XIV,
XVI, and XIX collagen.
Type XIX collagen was originally discovered through cDNA
cloning of RNA transcripts from the human rhabdomyosarcoma cell line RD (CCL 136) (7). The predicted polypeptide wasfound to contain 1,142 amino acid residues with a 23-residue
signal peptide followed by the five collagenous (COL) domains,interspersed and flanked with the six non-collagenous (NC)domains (3, 4, 8). The coding region of the a1(XIX) mRNA issmall compared with the length of the entire transcript (10.4kb), due to the presence of more than 5-kb long 39-untranslated
region. Additionally, an unusual number of splicing events
appeared to occur in the RD cell line. More interestingly, thea1(XIX) collagen gene (COL19A1) was located to human chromosome 6q12-q14, syntenic to the a1(IX) collagen (COL9A1)
and a1(XII) collagen genes (COL12A1) (7, 9). Type IX collagenis found in tissues containing type II collagen, such as hyaline
cartilage and the vitreous body of the eye. On the other hand,type XII collagen is found in dense connective tissues, such as
tendons and ligaments, where type I is the major collagenous
component. Aside from the rhabdomyosarcoma cell line, there
is currently no information about the tissue distribution of type
XIX collagen and, consequently, about its possible function.
The present study was designed to fill this gap of knowledge
and to generate suitable reagents to eventually establish type
XIX collagen function using the gene targeting approach inmouse embryonic stem cells. Accordingly, we isolated overlapping cDNA clones coding for the entire mouse a1(XIX) chain;
we examined the pattern of mRNA accumulation in the tissuesof both the developing and adult mice; and we identified thea1(XIX) protein in adult brain tissues.
MATERIALS AND METHODS

Isolation and Characterization of cDNA Clones—Poly(A)1 RNA was
isolated from 18 d.p.c. mouse whole embryo tissue according to thestandard protocol (10) and was used as a template for cDNA synthesis.
Double-stranded cDNA was synthesized using random primers andthen inserted into the lgt10 vector using a commercial kit (Amersham
Corp.). Three distinct mouse embryonic cDNA libraries were screened
under low stringency conditions using probes specific for the human
a1(XIX) collagen (4, 7). Hybridizations carried out at 55 °C overnight ina mixture containing 5 3 SSC (1 3 SSC; 0.15 M NaCl, 0.015 M sodium
citrate, pH 6.8), 1% N-laurylsarcosine, 50 mg/ml salmon sperm DNA,and 32P-labeled probes after prehybridization which is the same solution without probes for 1 h. Isolation and purification of the positive
clones were as described previously (7). Sequencing was carried out
according to the dideoxy chain termination methods using an ABI 373S
automatic sequencer. After sequencing validation of positive clones, a
0.6-kb gap in the a1(XIX) coding sequence was resolved by amplifying
an aliquot of reverse-transcribed embryonic cDNA using the polymerase chain technique (RT-PCR). Twenty microliters of reverse transcrip-

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This paper is available on line at http://www.jbc.org

Mouse a1(XIX) Collagen Gene Expression

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FIG. 1. The domain structure of the mouse a1(XIX) collagen chain deduced from nucleotide sequences of cDNA clones and their
partial restriction map. Five overlapping cDNAs, ME1, ME2, ME12, ME14, and ME23, were isolated from 18 d.p.c. mouse whole embryo library.
A clone, ME PCR, was generated from RNA of 18 d.p.c. mouse whole embryo using RT-PCR method. The alignment of these cDNAs was determinedby restriction enzyme digestions and DNA sequencing. Amino acid sequence deduced from the cDNA sequence allowed the prediction of the domain
structure of the mouse a1(XIX) chain (top of the figure). Numbers of the amino acid residues in individual domains of COL1–5 and NC1– 6 areshown in parentheses. Locations of the 16 cysteinyl residues are indicated by closed circles. Positions of the nine imperfections of the Gly-X-Y
repeated structure are shown by short bars above the COL domains. The signal peptide of 23 amino acid residues is indicated by a thicker bar at
the amino terminus of the chain. Locations of 59 and 39 mouse probes used for the Northern blotting analysis (in Fig. 6) are indicated by thin bars
with the numbers 1 and 2 in parentheses.
tion reaction mixture (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2,
10 mM dithiothreitol) containing 2 mg of total RNA from 18 d.p.c. mouseembryo, 0.25 mM dNTP, 2 units of RNasin (Toyobo, Osaka), 400 ng ofrandom hexamer, and 100 units of Moloney murine leukemia virus
reverse transcriptase (Life Technologies, Inc.) was incubated at 37 °C
for 1 h, heated to 70 °C for 10 min, and quick-chilled on ice, and diluted
with 80 ml of water. To get appopriate RCR products, the nested PCR
was performed. First PCR reaction was performed using 2 ml of cDNA
solution in 20 ml of mixture containing 0.8 units of Tth DNA polymerase(Toyobo) using primer 1 (forward, 59-GGAACTTAAAGACACATGCC-39,
Fig. 2) and primer 2 (reverse, 59-ATGTCTCCCAAGAGAGGAAG-39,
Fig. 2), under the following condition: 94 °C for 1 min, 60 °C for 1 min,and 70 °C for 3 min for 35 cycles. Five microliters of the first PCR
amplification product was used for the second PCR reaction with primer
3 (forward, 59-CCTCAATGGACAAGATGGTT-39, Fig. 2) and primer 4(reverse, 59-TTCTACCAGGGATTCCTACAC-39, Fig. 2) under same condition. PCR products were subcloned in TA vectors (Invitrogen). The
sequence was confirmed with both strands of two clones.
Northern Blot and RT-PCR Analysis—For Northern blotting analysis, approximately 3 or 5 mg of poly(A)1 RNA was electrophoresed in
0.8% agarose gel under denaturing conditions, blotted onto Hybond N
nylon filter (Amersham Corp.), and to hybridized species-specific probes(10). Sources of RNA included 18 d.p.c. mouse whole embryo, 5-week-old
mouse brain, human rhabdomyosarcoma cell line (RD), human glioma
cell line (U251MG), human infant skin fibroblast culture cells, andhuman adult skin fibroblast culture cells. The following mouse tissues
and cultured cells were instead used for RT-PCR amplification: 11, 12,
and 14 d.p.c. mouse whole embryo; different tissues from 18 d.p.c.
mouse embryo and 5-week-old mouse; the human rhabdomyosarcoma
cell lines RD, A204 and KYM1; the human glioma cell lines U251MG,
U373MG, and A172; the human neuroblastoma cell lines TGW and
NB1; and skin fibroblasts from mouse embryo and mouse adult andhuman infant and human adult. Single strand cDNA was synthesized
as mentioned above. PCR was performed with 35 cycles for a1(XIX) or
30 cycles for b actin using 0.8 units of Tth DNA polymerase at 94 °C for
1 min, at 60 °C for 2 min, and at 70 °C for 3 min. The nucleotide

sequences for the primers used in these reactions are as follows:a1(XIX), 59-AACTGCCAGCAGCAATGTTG-39 (forward) and 59CAATCTTCTGGATTACATCT-39 (reverse); b actin, 59-AAGAGAGGTATCCTGACCCT-39 (forward) and 59- TACATGGCTGGGGTGTTGAA-39 (reverse).
For competitive RT-PCR, a1(XIX) collagen and b actin cDNA clones
were modified using restriction enzymes to delete 51 bp in the former
and 61 bp in the latter. These modified clones were then used ascompetitors in RT-PCR amplifications of various mouse samples. To
determine the optimal condition, a series of RT-PCR reactions containing 2-fold serial dilutions of competitor (ranging from 1 pg to 1 fg/ml fora1(XIX), and from 50 pg to 1.6 pg/ml for b actin) were first carried out(11). PCR was then performed described above except that 5 more cycles
were added to each reaction. Aliquots of each amplification were electrophoresed in 2% or 2.5% agarose gels (Sigma) containing 0.5 mg/ml
ethidium bromide; gels were photographed with Polaroid film (Polaroid
type 667); photographs were quantitatively scanned using the NIH
image software.
Preparation of Type XIX Collagen Antibodies—A recombinanta1(XIX) NC1-COL1-NC2 peptide was made by expressing a cDNA
fragment subcloned into EcoRI and PstI sites of pMALc2 (New England
BioLabs Inc.) expression plasmid. Production of the MBP (maltose
binding protein)-a1(XIX)COL fusion proteins was induced by the addition of isopropyl-b-D-thiogalactopyranoside (final concentration of 0.3mM) to the bacterial cultures incubated at 37 °C for 2 h. The cells werelysed by lysozyme treatment, and the fusion proteins were purified byaffinity chromatography on an amylase column as describe by the
manufacturer (New England Biolabs Inc.). After incubation of the fusion protein with Xa-specific peptidase, the a1(XIX)COL recombinant
protein was separated on 12.5% SDS-PAGE gel and isolated in a gel
slice after Coomassie Brilliant Blue R (Merck, Darmstadt, Germany)
staining (12). The gel slices containing approximately 500 mg of protein
in total were washed with phosphate-buffered saline, cut into the small
pieces, emulsified with the equal volume of complete Freund’s adjuvant(Difco), and then injected subcutaneously into a rabbit. Booster injections (200 mg) were given with incomplete Freund’s adjuvant (Difco) 3

17106

Mouse a1(XIX) Collagen Gene Expression

FIG. 2

Mouse a1(XIX) Collagen Gene Expression

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FIG. 2—continued
weeks after the first injection. The titers of antisera were measured
with enzyme-linked immunosorbent assay (13).
Collagen Purification—All steps were carried out at 4 °C or on ice(14). Brains, dissected from 40 5-week-old mice, were homogenized in
500 ml of cold isotonic buffer (0.25 M sucrose, 10 mM HEPES, pH 7.0, 2
mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM N-ethylmaleimide), and then stirred for 60 min. The homogenate was centrifuged for

30 min at 15,000 rpm. The pellet was suspended in 500 ml of extraction
buffer (1 M NaCl, 50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM N-ethylmaleimide) and incubated for
48 h with stirring. The suspension was centrifuged at 35,000 rpm for 30
min, and the solubilized proteins were precipitated with 4 M NaCl of
final concentration. The precipitate was collected by centrifugation at
25,000 rpm for 30 min, and then the pellet was suspended in 0.5 M acetic

FIG. 2. Nucleotide sequence of cDNA (top) and deduced amino acid sequence (middle) of the mouse a1(XIX) collagen chain. The
amino acid sequence of the human (bottom) has been aligned with that of the mouse chain. The residues of the human differing from those of themouse only are shown. The collagenous domains (COL) are indicated by boxes, and those of the imperfections are indicated by small boxes in them.
The asterisks, the triangle, and open squares indicate the amino acid residues missing in the mouse and a stop codon, the putative signal peptide
cleavage site, and cysteine residues. The horizontal arrows show the positions of the primers; primers 1– 4 were used for generating cDNA clone,
ME PCR, primer 5 and 6 for RT-PCR analysis, and primer 7 and 8 for generating DNA fragment to be ligated to expression vector.

Mouse a1(XIX) Collagen Gene Expression

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TABLE I
Developmental expression of a1(XIX) collagen mRNAs determined by
RT-PCR analysis
The symbols used are: 11, strongly positive; 1, weakly positive; 6,
trace; 2, negative; and ND, not determined.

Muscle
Heart
Aorta
Skin
Intestine
Brain
Whole
Cerebrum
Cerebellum
Eye
Limb
Vertebrae
Tail
Joint
Sternum
Calvaria
Lung
KidneyLiver
SpleenThyroid
AdrenalTestis

18 d.p.c. embryo

5-Week-old adult

1
1
ND
1
1

2a
2
6
2a
2

1
ND
1
1
1
ND
ND
1
1
1
2
ND
ND
ND
ND

11
1
1
ND
ND
2
2
2
ND
6
6
2
2
2
2
1

a

The transcripts are usually not detectable in this condition. Compare to the results of competitive RT-PCR in Table III.

TABLE II
Expression of a1(XIX) collagen mRNAs in the cultured cells
determined by RT-PCR analysis
The symbols used are: 11, strongly positive; 1, weakly positive; 6,
trace; and 2, negative.
Rhabdomyosarcoma (human)
RD
A204
KYM1
Glioma (human)
U251MG
U373MG
A172
Neuroblastoma (human)
TGW
NB1
Fibroblast (human)
InfantAdult
Fibroblast (mouse)
EmbryoAdult
Lymphocyte (human)
EB virus transformed

11
2
2
11
2
2
6
2
1
2
1
6
2

acid. The insoluble material was removed by centrifugation at 35,000rpm for 30 min. The supernatant was dialyzed against 5 mM acetic acidand lyophilized. The sample was resolved with 200 ml of 50 mM acetic
acid and stored at 4 °C.
Western Blotting—Proteins were separated on 6.0% SDS-PAGE in
the presence or absence of 2-mercaptoethanol (12). An aliquot of thesample was also digested with bacterial collagenase before loading onto
the gel. The sample for digestion was first neutralized with 0.5 M NaOH
and then incubated with bacterial collagenase solution (250 units/ml
Wakojunyaku Form III; 50 mM Tris-HCl, pH 7.5, 0.2 M NaCl, 5 mM
CaCl2) containing 5.5 mM CaCl2 for 5 h at 37 °C. Samples were transferred onto polyvinylidene difluoride membranes electrophoretically(15), and the gel was stained with Coomassie Brilliant Blue R. The
transferred sample was allowed to react with anti-a1(XIX)COL antibodies, followed by incubation with the peroxidase-conjugated secondary antibodies against anti-rabbit IgG. The signals were detected byenhanced chemiluminescence Western blotting detection reagents (Amersham Corp.).

FIG. 3. Competitive PCR products run on the agarose gels. The
gel shown here contains RT-PCR products of one representative experiment using the muscle, skin, brain, and liver RNAs as templates. The
concentration of the gels is 2.0% for a1(XIX) and 2.5% for b actin. The
products from RNA of b actin are shown only from the experiment using
the skin. The panels on the left show the products from RNAs of
18 d.p.c. mouse embryo, and on the right from 5-week-adult mouse. The
RT-PCR products of the competitor (open arrowhead) has been designed
to be 51 bp (XIX collagen) and 61 bp (b actin) smaller than those of theendogenous product (closed arrowhead). On each gel, lanes left to rightcontain amplified DNA synthesized from a constant amount of tissue
cDNA and decreasing amounts of competitor DNAs. The DNA competitor in the reaction mix was as follows: a1(XIX) collagen competitor:lane A, 1 pg; lane B, 500 fg; lane C, 250 fg; lane D, 125 fg; lane E, 62.5
fg; lane F, 31.3 fg; lane G, 15.6 fg; lane H, 7.8 fg; lane I, 3.9 fg; lane J,
1.9 fg; and lane K, 1 fg; and b actin competitor: lane 1, 50 pg; lane 2, 25
pg; lane 3, 12.5 pg; lane 4, 6.3 pg; lane 5, 3.2 pg; and lane 6, 1.6 pg.
RESULTS AND DISCUSSION

Primary Structure of Mouse a1(XIX) Chain—To identify thebest source for a1(XIX) collagen, we initially performed RTPCR amplifications using RNA from a variety of embryonic andadult mouse tissues and a couple of sets of primers derived
from the human sequence. The most convincing results of thisinitial survey were obtained using embryonic tissues. Sequencing of these amplification products showed that the clones werethe mouse counterparts of the human COL19A1 gene. Based onthese data, we constructed and screened a cDNA library from18 d.p.c. mouse whole embryo. Five overlapping clones wereisolated and, upon sequencing, found to cover all but 0.6 kb of
the region coding for the mouse a1(XIX) chain; this last gap
was finally covered by RT-PCR amplification (Fig. 1).
The deduced amino acid sequence of the mouse a1(XIX)
chain shows 82% identity to the human counterpart with thehighest level of sequence conservation at the carboxyl termini
than the amino termini. All of the potentially important structural-functional features previously noted in the human
a1(XIX) chain are also conserved in the mouse polypeptide (Fig.
2) The major difference between the two mammalian polypeptides is the total number of 1,136 amino acids in mouse versus
1,142 amino acids in human. The differences are due to thedeletion of three amino acids in each NC6 and NC5 domain andthe substitution of cysteinyl residues for serinyl residues in thesignal peptide of the mouse chain. Additional structural differences include the NC5 and NC3 domains, which are shorter inthe mouse compared with the human chain, and the relativeMouse a1(XIX) Collagen Gene Expression
position of small imperfection in the COL5 domain. These few
differences notwithstanding, the primary structure of thea1(XIX) collagen chains is remarkably conserved between thetwo mammalian species.
Tissue Distribution of Col19a1—To define the pattern ofexpression of Col19a1,2 we surveyed tissues from various embryonic stages and 5-week-old mouse tissues. The transcripts
were readily detectable by RT-PCR analysis in the whole embryo at 11, 12, and 14 d.p.c. (data not shown). Positive tissuesof 18 d.p.c. embryo include limbs, vertebrae, heart, brain, tail,kidneys, calvaria, lung, muscle, skin, and intestine (Table I). A
very different pattern was observed in adult tissues where
Col19a1 expression was only seen in the cerebrum, cerebellum,
eyes, and testis, and perhaps in the aorta, lungs, and kidneys(Table I). Based on these data, we used the same approach toidentify cultured cells that accumulate COL19A1 (andCol19a1) transcripts. This second survey included humanrhabdomyosarcoma cell lines (RD, A204, and KYM1), humanglioma cell lines (U251MG, U373MG, and A172), human neuroblastoma cell lines (TGW and NB1), human normal skinfibroblasts (infant and adult), and mouse normal skin fibroblasts (18 d.p.c. embryo and adult) (Table II). Among malignant
cell lines, only RD (rhabdomyosarcoma) and U251MG (glioma)
showed significant COL19A1 mRNA accumulation. Detectableamounts were also noted in human neonatal fibroblasts, mouseembryo fibroblasts, and to a much lesser extent, in adultfibroblasts.
To examine whether Col19a1 gene expression may change
during development, we compared transcript levels in the muscle, skin, brain, and liver of 18 d.p.c. embryos and 5-week-old
mice using the quantitative technique of competitive RT-PCR(Fig. 3). Relative quantitation of amplification from the endogenous transcript and the competitor plasmid revealed that 1 mlof cDNA from muscle, skin, and brain of 18 d.p.c. embryo and5-week-old mouse contained 52.3, 7.1, 24.5, 0.7, 0.4, and 149.3
fg, respectively. When normalized to the b actin values, therelative amounts of Col19a1 transcripts were significantly
higher in embryonic muscle and skin than in the adult counterparts, whereas transcript levels in the brain were approximately 10 times more in adult animals than embryos(Table III).
To identify the protein in a tissue that expresses the a1(XIX)
mRNA, we utilized specific polyclonal antibodies (Fig. 4) in a
Western blotting analysis of mouse adult brain tissue. The
The symbol Col19a1 for the mouse a1(XIX) collagen gene is approved HGMW.
2

17109

antibodies were raised in rabbits against an a1(XIX) recombinant peptide. As shown Fig. 5C, the antibodies reacted withpeptides of 150 and 145 kDa in size from a fraction of neutral
salt extraction of mouse adult brain. Consistent with theiridentity, the 150- and 145-kDa bands were digested with bacterial collagenase (Fig. 5C, lane 2). Under nonreducing conditions, the reacting peptide migrated at about 400 kDa which
probably represents the trimeric form of type XIX collagen.
The above results suggest that type XIX collagen is an embryonic FACIT, since its expression is mostly confined to embryonic tissues and only a few adult organ systems. HighCol19a1 expression in a couple of tumor lines indirectly supports the idea of a mostly embryonic gene product. We postulate that the uniqueness of this FACIT molecule extends to thekind of supermolecular aggregate it is associated with. We notethat Col19a1 expression in the embryo does not follow theTABLE III
Mean ratios of mouse a1(XIX) collagen to b actin mRNA (31023)
Values are means 6 S.D. n 5 5 (p , 0.015). ND, not detectable.

Muscle
Skin
Brain
Liver

18 d.p.c. embryo

5-Week adult8.03 6 4.13
2.00 6 1.20
1.15 6 0.47
ND

0.14 6 0.10
0.15 6 0.19
13.57 6 8.08
ND

FIG. 4. Enzyme-linked immunosorbent assay using anti-type
XIX collagen polyclonal antibody. The antibody reacted with thea1(XIX)-MBP fusion protein (closed circles) but never cross-reactedwith total Escherichia coli proteins containing MBP (open squares).

FIG. 5. Protein analysis for the type XIX collagen using mouse adult brain extract. A, purified recombinant proteins were run on 12.5%
SDS-PAGE gel under a reducing condition and stained with Coomassie Brilliant Blue. Lane 1, MBP-COL fusion protein purified with amylose
affinity column; lane 2, digested fusion protein with factor Xa; lane 3, digested protein with bacterial collagenase. An arrow indicates COL peptide
in lane 2, which disappears in lane 3. B, gel electrophoresis of the extract prepared from mouse brain. The sample was separated on 6.0%
SDS-PAGE gel and stained with Coomassie Brilliant Blue. Lane 1, neutral 1 M NaCl extract from mouse adult brain tissues with b-mercaptoethanol; lane 2, the same sample of lane 1 treated with bacterial collagenase; lane 3, the same sample of lane 1 under nonreducing condition; lane
4, mouse type I collagen; lane 5, mouse type I collagen digested with bacterial collagenase. C, Western blot analysis for neutral 1 M NaCl extract
prepared from mouse adult brain tissues. Lanes 1–3 correspond with lanes 1–3 of B, respectively. Molecular mass is indicated on the left side of
the panels.

17110

Mouse a1(XIX) Collagen Gene Expression

FIG. 6. Northern blot analysis of the a1(XIX) mRNA. A, the mRNA samples isolated from human rhabdomyosarcoma cells RD (3 mg, lanes
1 and 2) and 18 d.p.c. mouse whole embryo (5 mg, lanes 3 and 4) were electrophoresed on 0.8% agarose gels, blotted onto nylon filters, and hybridized
with various probes. Each lane was hybridized with 59 EcoRI/PstI fragment of HY 67 that encodes a portion of NC 6 domain (4) (59 human probe(lane 1)), 39 HindIII fragment of KI 65 that encodes a portion of 39-untranslated region (4) (39 human probe (lane 2)), 59 EcoRI fragment of ME12
that encodes 59-untranslated region and a portion of NC 6 domain (indicated by (1) in Fig. 1) (59 mouse probe (lane 3)), and 39 EcoRI/PstI fragment
of ME14 that encodes a portion of 39-untranslated region (indicated by (2) in Fig. 1) (39 mouse probe (lane 4)). B, the mRNA samples isolated fromRD (3 mg, lane 1) was hybridized with 59 human probe, and those from mouse 18 d.p.c. whole embryo (5 mg, lane 2) and adult brain (5 mg, lane 3)
with 59 mouse probe. C, the mRNA samples isolated from RD (3 mg, lane 1), U251MG (human glioma, 5 mg, lane 2), human infant fibroblasts (5mg, lane 3), and human adult fibroblasts (5 mg, lane 4) were hybridized with 59 human probe.

pattern of either of the major fibrillar collagens (types I or II)
for it includes both cartilagenous and non-cartilagenous tissues. Even more intriguing is the expression of type XIX in theadult aorta, testis, and brain. The last organ, in particular,
raises provoking new ideas. For example, type XIX collagenmay be produced by glial cells to serve as a connecting bridge ofthe proteoglycan network or as an anchor for the surrounding
cells. Irrespective of the alternatives, the evidence strongly
supports the original prediction of Gordon and Olsen (16) forthe existence of FACIT molecules that may provide highly
specific properties to selected organ systems and/or at specificdevelopmental stages.
Alternative Splicing of the COL19A1 Transcript—We havepreviously reported an unusual number of alternatively spliced
COL19A1 products in the rhabdomyosarcoma cell line RD (4).
Here we examined whether or not alternatively spliced products appear to be tissue- and/or stage-specific. Northern analysis of mRNA from 18 d.p.c. whole embryo yielded a single bandwith 59 (Fig. 6A, lane 3) and 39 (lane 4) cDNA fragment asprobes. Transcripts from mouse adult brain also showed asingle band with more strong signal (Fig. 6B, lane 3) than thatfrom 18 d.p.c. mouse embryo (Fig. 6B, lane 2) when 59 probe
was used. Likewise, mRNA from infant skin fibroblasts showed
only one hybridizing species (Fig. 6C, lane 3). In contrast,
mRNA from glioma cell line (U251MG) yielded multiple bands(Fig. 6C, lane 2).
Alternative splicing is a widely used means to generate protein isoforms or even functionally distinct products from thesame transcript. Alternative splicing has been reported to occur for several different collagen types (17–24), although its
functional significance is understood only for a few of them. In
the case of the a1(XIX) chain, the results are apparently contradictory. Unlike two tumor lines, multiple transcripts havenot been seen in the present survey of normal tissues andcultured cells. We therefore conclude that the alternatively

spliced COL19A1 transcripts seen in the tumor cells are probably aberrant products of no physiological significance andwithout counterparts in the normal organism. It is however
unclear the reason for and the mechanism behind this strange
phenomenon in cancer cells.
Conclusion—In summary, this study has provided new andimportant information about the possible role of type XIX collagen. We believe the expression data are consistent with theidea that type XIX collagen is somehow involved in the assembly of specialized structures of the developing embryo and/or
the function of particular organ systems in the adult organism.
Irrespective of the hypothesis, it is safe to predict that type XIX
collagen will eventually prove to be unique among other FACIT
members. The information and reagents produced by the present study provide the means to address this question using thepowerful technique of gene targeting in the mouse.
Acknowledgments—We are indebted to Dr. F. Ramirez for the review
and critical comments of the manuscript. We also thank M. Kohmoto
and K. Frith for typing the manuscript.
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