GENOMICS 45, 304–312 (1997)
ARTICLE NO. GE974921

Structure of the Human Type XIX Collagen (COL19A1) Gene, Which
Suggests It Has Arisen from an Ancestor Gene of the FACIT Family
Mohammed Khaleduzzaman, Hideaki Sumiyoshi, Yasuyoshi Ueki, Kazuhito Inoguchi,
Yoshifumi Ninomiya, and Hidekatsu Yoshioka1
Department of Molecular Biology and Biochemistry, Okayama University Medical School, Okayama 700, Japan
Received April 30, 1997; accepted July 23, 1997

Type XIX collagen is a newly discovered member of
the FACIT (fibril-associated collagens with interrupted triple helices) group of extracellular matrix
proteins. Based on the primary structure, type XIX collagen is thought to act as a cross-bridge between fibrils
and other extracellular matrix molecules. Here we describe the complete exon/intron organization of
COL19A1 and show that it contains 51 exons, spanningmore than 250 kb of genomic DNA. The comparison ofexon structures of COL19A1 and other FACIT family
genes revealed several similarities among these genes.
The structure of exons encoding the noncollagenous(NC) 1–collagenous (COL) 1–NC 2–COL 2–NC 3–COL
3–NC 4 domain of the a1(XIX) chain is similar to thatof the NC 1–COL 1–NC 2–COL 3–NC 3 domain of thea2(IX) chain except for the NC 3 domain of a1(XIX).
The exons encoding the COL 5–NC 6 domain of a1(XIX)
are also similar to those of the COL 3–NC 4 domainof a1(IX) chain. Previously, COL19A1 was mapped tohuman chromosome 6q12–q14, where COL9A1 is also
located. Likewise, the present work shows that themouse Col19a1 gene is located on mouse chromosome
1, region A3, where Col9a1 has also been mapped.
Taken together, the data suggest that COL19A1 and
COL9A1 (Col19a1 and Col9a1) were duplicated fromthe same ancestor gene of the FACIT family. Three CA
repeat markers with high heterozygosity were foundin COL19A1. These markers may be useful for linkage
analysis of age-related inheritable diseases involvedin eyes and/or brain. q 1997 Academic Press

To date, 19 distinct types of collagen molecule, encoded by more than 30 genes, have been identified in
Sequence data from this article have been deposited with the
DDBJ/EMBL/GenBank Data Libraries under Accession Nos.
AB004584 to AB004634 and AB004688. The symbols COL19A1 and
Col19a1 represent the human and the mouse a1(XIX) collagen gene,
respectively.
1
To whom correspondence should be addressed at the Department of Molecular Biology and Biochemistry, Okayama University
Medical School 2-5-1, Shikata-cho, Okayama 700, Japan. Telephone: /81-86-235-7128. Fax: /81-86-222-7768. E-mail: mbb@
ccews2.cc.okayama-u.ac.jp.

0888-7543/97 $25.00
Copyright q 1997 by Academic Press
All rights of reproduction in any form reserved.

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vertebrates (van der Rest and Garrone, 1991; Kivirikko, 1993; Inoguchi et al., 1995). The collagen superfamily can be classified into subgroups, based on theirstructures and functions. The FACIT (fibril-associatedcollagens with interrupted triple helices) family of collagens is one of those groups and includes types IX,
XII, XIV, XVI, and XIX collagen. FACIT collagens areassociated with the surface of collagen fibrils where
they maintain the organization and stability of the extracellular matrix. Members of the FACIT family sharesome common structural features, the functional significance of which is based mainly on the study of type
IX collagen (Shaw and Olsen, 1991). The structure ofthese molecules comprises several collagenous (COL)
domains divided and flanked at both ends by noncollagenous (NC) domains. Type IX collagen is the best characterized molecule in the members. The COL domains
and the central NC domains of this molecule interact
with type II collagen through covalent cross-links toform fibrils (Diab et al., 1996). The amino-terminal NC
domain has a potential of interacting with other extracellular components.
We (Yoshioka et al., 1992; Inoguchi et al., 1995) andothers (Myers et al., 1993, 1994) have recently reported
the identification of a new member of the FACIT family, named type XIX collagen, through the isolation of
overlapping cDNA clones from a human rhabdomyosarcoma cell line (RD, CCL136). The predicted humanpolypeptide contains 1142 amino acid residues that include a 23-residue signal peptide and five COL domains
divided and flanked by six NC domains. The size of thecoding region is unexpectedly small compared to theoverall length of the mRNA (10.4 kb) due to the presence of a very long 3* untranslated region (ú5 kb).
Interestingly, the corresponding COL19A1 gene was
localized to 6q12–q14, the same region where thea1(IX) collagen gene (COL9A1) (Yoshioka et al., 1992)
and the a1(XII) collagen gene (COL12A1) reside (Oh etal., 1992). Type IX collagen is found in tissues containing type II collagen such as hyaline cartilage andthe vitreous body of the eye. On the other hand, type
XII collagen is found in dense connective tissues suchas tendons and ligaments associated with type I collagen fibrils.

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It is well established that mutations in different
kinds of collagen molecules are the cause of numerous
connective tissue disorders. They include osteogenesis
imperfecta, Ehlers–Danlos syndrome (types VII, IV,
and I), some forms of chondrodysplasias, Alport syndrome, and epidermolysis bullosa (dystrophic form)(Kivirikko, 1993). Until recently, there was no known
mutation in a FACIT collagen. Muragaki et al. (1996)
reported a COL9A2 mutation in a family affected withmultiple epiphyseal dysplasia, symptoms of which are
stiffness and pain in large joints combined with shortstature and stubby fingers. Moreover, studies in micealso showed that type IX collagen plays a critical role
during the process of skeletal formation. Transgenic
mice with a truncated a1(IX) collagen chain were found
to express mild chondrodysplasia and progressive osteoarthritis (Nakata et al., 1993), and mice homozygous
for a null mutation of Col9a1 developed progressive
osteoarthritis-like changes in articular cartilage (Faessler et al., 1994).
As a necessary step toward elucidating a1(XIX) collagen function and pathology, here we describe the complete exon/intron organization of COL19A1. The study
demonstrates that this gene contains 51 exons spanning more than 250 kb of DNA. We have identified
three highly polymorphic markers within COL19A1.
Additionally, we have mapped the mouse Col19a1 locus
to chromosome 1. These data may facilitate the identification of hereditary abnormalities of COL19A1 (orCol19a1).
MATERIALS AND METHODS
Isolation and characterization of human genomic clones. A human genomic library (HL1111j; Clontech) was screened with cDNAs
encoding various portions of the human a1(XIX) collagen chain (Inoguchi et al., 1995). The screening was performed according to standard procedures (Sambrook et al., 1989). Positive clones were characterized by genomic mapping and by Southern blot hybridization withdifferent probes. Isolation of clones coding exons 2, 12, 15, 17, 43,
and 44 was performed using exon-specific probes made from HY 67
or KI 40 cDNA clone (Inoguchi et al., 1995) as templates by PCR(polymerase chain reaction) procedure. The nucleotide sequences ofprimers for generating exon-specific probes are exon 2, (forward) 5*ATCCGTGGCCGTTCACATGG-3* and (reverse) 5*-GTCCCTAACGGTCACGGAAG-3*; exon 12, (forward) 5*-GGTGAACAAGGAGAAAAAGG-3* and (reverse) 5*-ATTTTCTCCATTAAGGCCAG-3*; exon
15, (forward) 5*-GGTTCCCTGGGGATACAAGG-3* and (reverse) 5*CCTCTGACCCTCTTTTCCAG-3*; exon 17, (forward) 5*-GGACCTCCTGGAATACAAGG-3* and (reverse) 5*-CTTGTTATCCTTGTTATAAT-3*; and exon 43/44, (forward) 5*-GGAAAACCTGGTGCCCCAGG-3* and (reverse) 5*-TGGCTTTCCTGAGGGTCCTT-3*.
PCR was performed using 10 ng of cDNA template in a 50-ml
volume containing 2 units of Tth DNA polymerase (Toyobo, Osaka)under the following conditions: 947C for 1 min, 557C for 1 min, and707C for 1 min for 30 cycles. The PCR products were subcloned into
TA vectors (Invitrogen). The screening was performed under lowstringency conditions using exon-specific probe. Hybridization was
carried out at 507C overnight in a mixture containing 51 SSC (11SSC is 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. The
washing was performed at 507C with 61 SSC for 30 min.
Suitable restriction fragments of the genomic clones were subcloned into pBluescript II vector for further characterization. DNA

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sequencing was carried out with the Sanger dideoxynucleotide chain
termination method (Sambrook et al., 1989). The ends of subcloned
fragments were sequenced with universal forward and reverse primers for pBluescript II vector. In addition, the deletion fragments generated with mung bean nuclease deletion kit (Takara, Inc., Ootsu,
Japan) or specific primers for insert fragments were useful for thesequence for the internal portions of the fragments. The sequencesamples were prepared using the AmpliTaq cycle sequencing kit(Perkin–Elmer, Inc.) following the manufacturer’s protocol. The
samples were applied to a 373S automatic sequencer (Perkin–Elmer,
Inc.). All reported sequences were confirmed by sequencing of bothstrands.
Allele detection using microsatellites. CA repeat sequences were
found in the three genomic DNA fragments mentioned above. To
identify family by polymorphism, primers for PCR were designed at
the flanking regions of the CA repeats (Table 1). PCR was carried out
according to previously described procedures (Oohashi et al., 1995).
Genomic DNAs were isolated from 25 male and 25 female unrelated
Japanese individuals using standard procedures (Sambrook et al.,1989).
Chromosomal mapping of Col19a1. A mouse genomic clone, gME
1, containing exons 5 and 6 of Col19a1 (H. Sumiyoshi et al., unpublished data) was used for chromosomal mapping of Col19a1. This
clone was isolated from the mouse genomic library (a gift from Dr.
F. Ramirez, Mount Sinai School of Medicine, New York, NY) using
a cDNA clone, ME 1, encoding a portion of the NC 6 domain of themouse a1(XIX) collagen chain as a probe (Sumiyoshi et al., 1997).
The DNA was purified using the Qiagen plasmid kit (Qiagen, Inc.,
Chatsworth, CA).
Mouse chromosomes were prepared according to the published procedure (Feng et al., 1994). Briefly, lymphocytes were isolated frommouse spleen and cultured at 377C in RPMI 1640 medium supplemented with 15% fetal calf serum, 3 mg/ml concanavalin A, 10 mg/
ml lipopolysaccharide, and 5 1 1005 M mercaptoethanol. After 44 h,
the cultured lymphocytes were treated with 0.18 mg/ml bromodeoxyuridine for an additional 14 h. The synchronized cells were washedand recultured at 377C for 4 h in a-minimal essential medium withthymidine (2.5 mg/ml). Chromosome slides were made by the conventional method used for human chromosome preparation (hypotonic
treatment, fixation, and air drying). The genomic probe was biotinylated with dATP using the BioNick labeling kit (Gibco BRL) at 157C
for 1 h (Heng et al., 1992).
The procedure for fluorescence in situ hybridization (FISH) detection was performed according to the previously published protocol(Heng et al., 1992; Heng and Tsui, 1993). Briefly, slides were baked
at 557C for 1 h. After RNase A treatment, the slides were denatured
in 70% formamide in 21 SSC for 2 min at 707C followed by dehydration with ethanol. Probes were denatured at 757C for 5 min in a
hybridization mix consisting of 50% formamide and 10% dextran
sulfate and mouse cot I DNA and prehybridized for 15 min at 377C.
Probes were loaded on the denatured slides. After overnight hybridization, slides were washed and signal was detected as well as amplified using published method (Heng et al., 1992). FISH signals and4,6-diamino-2-phenylindole (DAPI) banding pattern were recorded
separately by taking photographs, and the assignment of the FISH
mapping data with chromosomal bands was achieved by superimposing FISH signals with DAPI-banded chromosomes (Heng and Tsui,
1993).

RESULTS AND DISCUSSION

Isolation of COL19A1 Clones
We previously isolated cDNA clones covering the entire human a1(XIX) collagen chain, as well as four genomic clones, termed KIG 3, KIG 12, KIG 23, and KIG
13 (see Fig. 1) (Inoguchi et al., 1995). These genomic
clones contained the 5* flanking region of COL19A1
and the first exon, as well as the 3* region containing

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TABLE 1DNA Sequence of CA Repeats, Flanking Primers, and Loci
Clone

Locus

CA strand primer

Repeat sequence

GT strand primer

MKG 1
MKG 5
MKG 64Intron 2
Intron 5
Intron 12

CAT GTA AAT TTG AGA ATC AG
CCT GTG TTA CTA ATA CAG AG
CTA CTA TTA CTA CTA GGG TC(CA)13(CA)12(CA)6CC(CA)3CC(CA)15

TCT AGA TTC AGA GCA ATG CC
ATG GGG CAT TCT GGT GAA TG
CTA GCT CCC TAA TGC CCT AG

Note. Primer sequences are written in the 5* to 3* direction.

five exons, which codes for a portion of the COL 1–
NC 1–COL 2 region of a1(XIX). To complete the exon/
intron organization of COL19A1, we isolated additional
genomic clones using cDNA clones HY 67, KI 40, andKI 50 and their subfragments as probes (Inoguchi etal., 1995). Restriction enzyme mapping and DNA sequencing analysis demonstrated that 11 positive clones
contained the coding sequences for exons 3–11, 13, 14,
16, 18–42, and 50–51. Despite extensive screening of
the genomic library, we were unable to isolated clones
containing exons 2, 12, 15, 17, 43, and 44. To obtaingenomic clones containing these exons, we made specific probes of 116, 54, 54, 63, and 108 bp in length forexons 2, 12, 15, 17, and 43/44, respectively. Using
rather lower stringency hybridization and washingconditions, we finally isolated 5 clones (MKG 81, MKG

64, MKG 93, MKG 73, and MKG 66) that contain theseexons. The alignment of the new and old genomic clones
is shown in Fig. 1 in relation to the exon/intron organization of COL19A1. The exons that were previously
designated exon /1 to /5 (Inoguchi et al., 1995) correspond to exons 45–49, respectively. Likewise, the genomic fragments 3.5 and 1.48 kb in length previously
analyzed by Myers et al. (1994) proved to contain the
3* end of exon 36, exons 37–39, and 5* end of exon 40.
Structure of COL19A1
Although there are several gaps between the individual genomic fragments we isolated, the data predictthat the COL19A1 gene is at least 250 kb in size andcontains 51 exons. This represents the first time the

FIG. 1. Structure of the human a1(XIX) gene, COL19A1, and relative location of genomic clones. The COL19A1 gene contains 51 exons,
which are indicated by black boxes in the middle. The gene spans at least 250 kb in size. Oval-shaped circles among introns show thatthose regions are not covered by the 20 clones isolated and shown. Asterisks in introns 2, 5, and 12 indicate the locations of the CA repeats.
Relative locations of 20 phage clones are shown by horizontal bars with their sizes in kilobases in parentheses underneath. Clones KIG 3,
KIG, 12, KIG 23, and KIG 13 (shown in italic) were previously described (Inoguchi et al., 1995). The closed box, horizontal bars, and open
boxes at the top indicate a signal peptide, noncollagenous (NC), and collagenous (COL) domains, respectively. Numbers in parentheses
show amino acid residues in the individual domains of the signal peptide, the COL 1 to 5, and the NC 1 to 6 domains.

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TYPE XIX COLLAGEN GENE

organization of a human FACIT collagen gene has been
determined. In fact, only the chick and mouse a2(IX)
genes have been completely characterized so far. The
mouse and chick a2(IX) genes span 16 and 10 kb, respectively, and contain 32 exons that are rather compact compared to other collagen genes (Ninomiya et al.,
1990; Perala et al., 1994). In contrast to these genes,
the chick a1(IX) gene, which was partially characterized with four genomic clones containing 16 exons at
the 5* end and 2 exons at the 3* end, was estimated at
approximately 100 kb in size (Ninomiya et al., 1990).
Like the fibrillar collagen genes whose sizes vary from
18 (COL1A1) (Chu et al., 1984) to 750 kb (COL5A1)(Takahara et al., 1995), the sizes of the genes belonging
to the FACIT family also vary considerably.
The sizes of exons and the exon/intron boundaries
are summarized in Fig. 2. The 5* gt and the 3* ag endsof the introns are all conserved. Like other collagengenes, the 9-bp rule is basically preserved in the exons
covering five COL domains of the chain. Exon sizes
vary from 27 (exon 40) to 171 bp (exon 47). Split codons
are found in exons 2–5 and exons 48–51, even thoughthe 3* end of exon 49, exon 50, and the 5* end of exon 51
encode a collagenous domain (COL 1 domain). Amongother FACIT genes, split codons have been found, inthe portions encoding the NC 4 domain of the chicka1(IX) gene and the NC 2–COL 1–NC 1 domain of
Col9a2, chick a1(IX), and chick a1(XII). Split glycine
codons are never seen in the exons encoding the central
collagenous domain of the fibrillar collagen genes, butthey are sometimes found in the exons encoding a collagenous domain with the imperfection(s) of Gly-X-Y
or a noncollagenous domain harboring a collagenous
domain. For instance, more than half of the exons encoding collagenous domains in type IV collagen achains start with the second nucleotide of the glycine
codon, which is conserved throughout evolution (Oohashi et al., 1995). Likewise, these split codons are also
conserved at the 5* and 3* ends in the FACIT family.
The first exon of the COL19A1 gene encodes only
5* untranslated sequence. Usually, the first exon incollagen genes includes the first Met residue. In a couple of collagen genes, including mouse and humana2(IV) (Buttice et al., 1990; Hostikka and Tryggvason,
1987) and chick and human a1(X) collagen genes (LuValle et al., 1988; Reichenberger et al., 1992), the firstexon encodes only the 5* untranslated sequence. Exon
2 contains part of the 5* untranslated region, the putative signal peptide, and 8 amino acids of the aminoterminal portion of the NC 6 domain. The large portionof the NC 6 domain is encoded by exons 3–8, whose
sizes vary from 75 (exon 3) to 276 bp (exon 6). Exon 6
is the largest among the coding exons. COL 5, with 144
amino acids, is encoded by exons 9–16 and the 5* partof exon 17. There is a discontinuity in the Gly-X-Y repeat at the end of exon 14 due to the Ala for Gly substitution. The NC 5 domain consists of 20 amino acids
and is encoded by the 3* end of exon 17 and the 5* endof exon 18. The COL 4 domain, with 224 amino acids,

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307

is the largest COL domain and is encoded by the 3*
end of exon 18 and exons 19–30. Exons 20, 25, and 27
contain one, two, and one interruption, respectively, of
the Gly-X-Y. The NC 4 domain, consisting of 31 aminoacids, is encoded by exon 31 and the 5* end of exon 32,
which contains two Cys codons. The three chains oftype IX collagen have a similar amino acid sequence(Cys-Pro-X-X-Cys-Pro-X) at the end of the NC 3 domain. These Cys residues could possibly form a disulfide interaction with other chains (van der Rest et al.,1985). A similar sequence (Cys-X-X-Cys) is in the same
region of a1(XIX) and in the NC4 domain of the a1(XVI)
chain (Pan et al., 1992). This sequence may participate
in interchain bridges. The COL 3 domain consists of
108 amino acids and contains an interruption of GlyX-Y. It is encoded by the 3* end of exon 32, exons 33–
37, and the 5* end of exon 38. The NC 3 domain (23amino acids) is encoded by the 3* end of exon 38 andthe 5* end of exon 39. The 45, 51, and 45-bp exons that
Myers et al. (1994) reported correspond to exons 37–
39. It should be noted that 1 nucleotide has to be added
to the sequence of the 3* end of intron 36 (Myers et al.,1994). This was confirmed by additional direct sequencing of PCR products generated from three different
sources of human genomic DNA as templates. The COL
2 domain (168 amino acids) is encoded by the 3* endof exon 39 and exons 40–47. Exon 40 is 27 bp and thesmallest of all exons, whereas exon 47 (171 bp) is thelargest exon that encodes collagenous domain. The NC
2 domain (44 amino acids) is encoded by exon 48 andthe 5* end of exon 49. The COL 1 domain is 70 aminoacids, the smallest collagenous domain, and contains
two interruptions. It is encoded by the 3* end of exon
49, exon 50, and 5* end of exon 51. Two imperfections,
namely Gly-X sequence in exon 50 and Gly-X-Y-X-Y
sequence in exon 51, are conserved among FACIT
genes. In exon 50, the nucleotide and the location of
the imperfection matched exactly with exon 31 of
Col9a2 and the second exon from the 3* end of the chicka1(IX) gene, showing 63 and 52% identity and 79 and
76% similarity, respectively, in amino acids. The NC 1
domain consists of 19 amino acids and is encoded by apart of exon 51. The Cys residue at the end of the COL
1 domain and another at position 5 of the NC 1 domain
are conserved among chains of the FACIT. Despite thissimilarity, Myers et al. (1994) suggested that these two
Cys residues in a1(XIX) chain are involved in an intrachain interaction, whereas those in the other FACIT
chains participate in interchain disulfide bonds. The
large portion of exon 51 encodes a long 3* untranslated
region 5.3 kb in size.
Evolutionary Similarities between COL19A1 andCollagen IX Genes
Although the overall sizes are different (more than
250 kb vs 16 kb), the exon structures of COL19A1 and
Col9a2 are similar. The structure of exons encoding
the NC 1–COL 1–NC 2–COL 2–NC 3–COL 3–NC 4

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FIG. 2. Exon/intron junctions of the COL19A1 and the sizes of the exons. The nucleotide sequences of exon/intron boundaries are shown
by uppercase letters for exons and lowercase letters for introns. The 5* gt and the 3* ag of introns are given in boldface. Nucleotide sequencesof exons are shown by a block of three letters for codon and continuous letters for untranslated region. The total numbers of nucleotidesin each exon are shown in the middle in parentheses. Underlined nucleotide c in intron 36 was missing in a previous report (Myers et al.,1994).

domain of a1(XIX) chain is similar to that of the NC
1–COL 1–NC 2–COL 2–NC 3 domain of a2(IX) chain.
This is based on the relative sizes of exons encoding
each domain, the positions of the exons encoding the
portion containing Cys residues, and the locations ofexons with split codons. Although the exon encoding
this portion of the chick a1(IX) gene is still unavailable,
we predict that it is probably similar to that of the
Col9a2/chick a2(IX) gene. However, there is an excep-

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tion in this region. No corresponding portion to the NC
3 domain of a1(XIX) was found in a2(IX) chain. Myerset al. (1994) pointed out that the NC 2 and the NC 4
domains of a1(XIX) chain are similar to those ofa1(XVI) chain, another FACIT member, but that the
NC 3 domains of both chains do not resemble eachother. The portion encoding the NC 3 domain of
COL19A1 may have evolved from a different origin. In
a similar region, chick a2(IX) and a3(IX) genes may

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have been changed during evolution (Ninomiya et al.,
1990; Har-El et al., 1992). Exons 19–24 in the chicka2(IX) gene have split codons, which are not present
in Col9a2. Likewise, the chick a3(IX) chain has an interruption of Gly-X-Y in the COL 2 domain, which is
not present in chick a1(IX) and a2(IX) chains.
The structure of the exon encoding the COL 5 domainof a1(XIX) chain seems to be similar to that of the COL
3 domain of both a1(IX) and a2(IX) chains. Likewise,
the NC 6 domain of a1(XIX) resembles the NC 4 domain of a1(IX). The primary structure of the NC 6
domain of a1(XIX) chain is similar to that of the NC 4
domain of a1(IX) chain, the NC 3 domain of a1(XII)
chain, the NC 3 domain of a1(XIV) chain, and the NC
11 domain of a1(XVI) chain. It has been suggested thatthey may have a b-sheet structure and may be involved
in molecular recognition functions (Inoguchi et al.,
1995; Moradi-Ameli et al., 1994). In addition, this domain is also conserved in the minor fibrillar collagenchains, i.e., a1(V) and a1(XI), in which this domain
is called PARP (proline–arginine-rich peptide) or thethrombospondin N-terminal module (Brown andTimpl, 1995). The gene structure of the NC 6 is slightly
similar to that of PARP with regard to the existence ofexons encoding the portion containing conserved Cys
residues (Takahara et al., 1995). Probably, the portions
encoding these NC domains share a common origin.
The structure of the exon coding for the COL 4 and theNC 5 domains of a1(XIX) is less similar than any COL
and NC domain of a(IX). Additionally, the portion en-

coding the COL 4 domain seems to have been generated
by duplication of the 9-bp exon.
Allele Detection in COL19A1
CA repeat sequences were found in three of the genomic fragments. These CA repeats were located 1.9 kb
upstream of exon 3 (i.e., intron 2), 120 bp upstream ofexon 6 (i.e., intron 5), and 1.0 kb downstream of exon
12 (i.e., intron 12) (Fig. 1). These three microsatellites
of COL19A1 from 50 unrelated individuals showedpolymorphic variations in a Japanese population.
As shown in Table 2, the markers, COL19MK1,
COL19MK2, and COL19MK3, indicated six alleles
with 0.68, five alleles with 0.50, and six alleles with
0.40 heterozygosity. Expected heterozygosity and PIC(polymorphism information content) values (Botstein
et al., 1980) were 0.62 and 0.56, 0.42 and 0.39, and 0.45
and 0.43, respectively. The combined heterozygosity
was 0.82, indicating that these markers are useful for
linkage study of heritable connective tissue diseases.
Recently, mutations of COL9A2 in humans (Muragaki et al., 1996) and of Col9a1 in mice (Nakata etal., 1993; Faessler et al., 1994) have been reported.
Contrary to mutations in the type II (Kivirikko, 1993)
or XI collagens (Li et al., 1995), which are coordinately
expressed, the type IX collagen mutations were not lethal but showed progressive changes in the cartilageof the aging organism. This finding suggested that type
IX collagen contributes to the structural integrity ofcartilage and/or maintains cartilage homeostasis. The

TABLE 2
Informativeness of New Markers for COL19A1 Gene
Marker and allele
COL19MK1
A1
A2
A3
A4
A5
A6
COL19MK2
B1
B2
B3
B4
B5
COL19MK3
C1
C2
C3
C4
C5
C6

Size
(bp)

Allelefrequency a

112
110
108
102
100
96

0.07
0.04
0.04
0.32
0.52
0.01

204
202
198
196
194

0.03
0.13
0.03
0.06
0.75

204
202
200
198
196
194

0.01
0.08
0.73
0.08
0.07
0.03

Heterozygosity b

Expectedheterozygosity c

PIC d

0.68

0.62

0.56

0.50

0.42

0.39

0.40

0.45

0.43

Note. Mendelian inheritance: three markers show codominant inheritance (three families, nine meioses).
aStudied in 50 unrelated Japanese people.
b
Combined observed heterozygosity 0.82.
c
Expected heterozygosity was calculated by the formula 1 0 S(Pi)2, where Pi is the frequency of allele i.
d
PIC, polymorphism information content value (Botstein et al., 1980).

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FIG. 3. FISH mapping. (a and b) The FISH signals on chromosome 1 and the same mitotic figure stained with DAPI to identify
chromosome 1, respectively. (c) A diagram of FISH mapping, in which each dot represents the signal detected on mouse chromosome 1.

Col19a1 is widely expressed during mouse embryogenesis, but restricted to a few adult tissues such as brain,eye, and testis (Sumiyoshi et al., 1997). We have previously located the COL19A1 to chromosome 6q12–
q14, close to where Stargadt disease type 3 maps aswell (Stone et al., 1994). This autosomal dominant disorder features central vision loss due to age-related
macular degeneration. In one family, multipoint linkage analysis resulted in a lod score of 6.2 in the interval
between markers D6S313 and D6S252. The gene expression and gene mapping data make COL19A1 astrong candidate gene for congenital degenerative diseases affecting eye and/or brain. Thus, it would be intriguing to analyze COL19A1 of affected persons in thisfamily.
Chromosomal Mapping of Col19a1
COL19A1 and COL9A1 reside on human chromosome 6q12–q14 (Yoshioka et al., 1992). The other
FACIT member, COL12A1, is also located on chromosome 6 (Oh et al., 1992). However, Col9a1 and Col12a1
are located on chromosomes 1 and 9 of mouse, respectively (DeBry and Seldin, 1996). To determine how
closely Col19a1 links to Col9a1 or Col12a1, chromosomal mapping of Col19a1 was performed.
A mouse genomic DNA fragment of 14 kb containing
exons 5 and 6 was used as a probe for FISH. Under
the conditions used, hybridization efficiency was 94%.
(Among 100 checked mitotic figures, 94 of them showedsignals on one pair of the chromosomes). Since the
DAPI banding was used to identify the specific chromosome, the assignment between signal from probe andmouse chromosome 1 was obtained. An example of the
mapping results is presented in Fig. 3. The detailed
position was further determined based on a summaryof 10 photos. According to the summary, this gene islocated on chromosome 1, region A3 (Fig. 3C).

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Among the human genes assigned to human chromosome 6, only two mouse counterparts, Bpag1 and
Col9a1, are localized to mouse chromosome 1, at positions 15.0 and 16.5 cM, respectively. Col19a1 was assigned to the same portion of mouse chromosome 1.
Col19a1 and Col9a1 might be duplicated from an ancestor gene on that chromosomal region. However, we
do not know yet whether these two genes are localizedwith head to head arrangement or head to tail arrangement. Among collagen genes, the former arrangement
is seen in COL4A1/COL4A2, COL4A3/COL4A4, and
COL4A5/COL4A6 on chromosomes 13, 2, and X, respectively (Oohashi et al., 1994), and the latter in
COL6A1/COL6A2 on chromosome 21 (Heiskanen etal., 1995). A search for mouse mutants with eye and/or brain abnormalities failed to identify candidate phenotypes for Col19a1 mutations. However, many mutations responsible for congenital degenerative eye and/or brain disease have yet to be located on the mouse
genome.
CONCLUSION

In conclusion, we have elucidated the genomic organization of COL19A1. From data on the primary structure of a1(XIX) collagen chain, the gene structure of
COL19A1 resembles that of type IX collagen genes.
These data suggest that this gene has probably arisen
from an ancestor gene of the FACIT family. The finestructures of COL19A1 and its polymorphic markers
as well as gene mapping of Col19a1 will facilitate analysis of hereditary diseases caused by the mutated genein human and mouse.
ACKNOWLEDGMENTS
The authors are very much indebted to Dr. F. Ramirez and his
graduate student, Ms. H. Zhang, with whom we started this project.

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TYPE XIX COLLAGEN GENE
We are also grateful to Dr. Henry Heng (SeeDNA Biotech, Inc.) for
his excellent mouse chromosome mapping technique and Dr. Ernst
Reichenberger (Harvard Medical School) for critical comments to themanuscript.

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