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Structure of the Human Type Lv Collagen COLAA5 Gene*

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0 1994 by The American Society for Biochemistry and Molecular Biology, Inc Printed in U S A .

Structure of the Human Type

Lv

Collagen COLAA5 Gene*

(Received for publication, July 20, 1993, and in revised form, September 24, 1993)

Jing Zhou, Anu Leinonen, and Karl Tryggvasonj

From the Biocenter and Department of Biochemistry, University of Oulu, SF-90570 Oulu, Finland The complete exon size and distribution pattern of the

human aS(IV) collagen gene COZ.4A5 has been deter- mined. Seventeen genomic A phage clones, eight of which have been described previously (Zhou, J., Hos- tikka, S. L., Chow, L. T., and Tryggvason, K. (1991) Ge- nomic~ 9,1-9), spanning about 160 kilobases of DNA con- tained 140 kilobases of the gene itself. The clones covered the entire gene with the exception of exons 2

and 37 and their flanking regions so that the exact gene size could not be determined. The sequences of these two exons were, however, determined from polymerase chain reaction products. The C O W gene has a struc- ture highly homologous with that of COIAAl which en- codes the al(IV) chain. However, the COLA45 gene con- tains 51 exons, or one less than COL4Al. The exon size pattern of the genes are similar, with 41 exons having identical sizes. All the exons were assigned to distinct EcoRI restriction fragments. The results may be useful for characterization of mutations in COLA45 in patients with X chromosome-linked Alport syndrome.

Basement membranes are ubiquitous thin sheet-like extra- cellular structures located beneath epithelia and endothelia and they surround muscle, nerve, and other tissues of mesen- chymal origin. The basement membranes play a role in cell adhesion and differentiation, as well as in tissue regeneration. They also have an important function in the size-selective siev- ing of macromolecules in renal glomeruli. Basement mem- branes are affected in a number of genetic and acquired dis- eases. This is well exemplified by the X chromosome-linked Alport syndrome (hereditary nephritis) which is characterized by abnormal glomerular basement membrane structure and hematuria resulting from mutations in the gene for the a5 chain of type

IV

collagen (1-3).

Type

IV

collagen, which forms the structural meshwork of all basement membranes, is a triple-helical molecule composed of three a chains (4). To date, six genetically distinct a chains, al(IV), a2(IV), a3(IV), a4(IV), a5(IV), and a6(IV), have been identified (4-8). These 170-185-kDa homologous polypeptides contain collagenous Gly-X-Y-repeat sequences frequently inter- rupted by noncollagenous sequences. Each a chain contains a large globular noncollagenous domain at the carboxyl-terminal end. The al(IV) and a2(IV) chains are the major forms which are presumably present in all basement membranes, the mo-

*

This work was supported in part by grants from the Sigrid Juselius Foundation, The Academy of Finland, and Finland’s Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This 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($ U04470 to U04520.

$ To whom correspondence should be addressed: Biocenter and De- partment of Biochemistry, University of Oulu, SF-90570 Oulu, Finland.

Tel.: 358-81-553-1150; Fax: 358-81-553-1151.

lecular ratio being 2:l. The three other minor chains have a more restricted tissue distribution, but it is not known if they are present in homo- or heterotrimer molecules.

The six human type

IV

collagen genes have been shown to be located painvise on three different chromosomes. The COL4Al

and COL4A2 genes for the al(IV) and a2(IV) chains, respec- tively, have been localized to chromosome 13 (9, 10). They have a unique arrangement in that they are located head-to-head and are transcribed from opposite DNA strands from a common promoter (11, 12). Similar arrangement was recently demon- strated for the COL4A5 and COL4A6 genes on the X chromo- some (8). The COL4A3 and COL4A4 genes for the human

a3(IV) and a4(IV) chains, respectively, are also co-located as a

pair in proximity to each other on chromosome 2 (13, 141, but the structure or promoter arrangement of these genes has not been elucidated. The human COL4Al gene is over 100 kbl in size and contains 52 exons (15). The exon-intron structure of

COL4A2 has only been partially determined. Sequencing of the

six most 3’ end exons and five most 5’ end exons of COL4A2 has demonstrated that this gene has diverged extensively from

COL4A1, but it shares homology with the COL4A6 gene (8,161. Our previous sequencing of the first exon from from the 5’ end and the 19 most 3’ end exons of the human COL4A5 gene (8,171 has shown that it is evolutionarily closely related with

COL4Al. Thus, all 18 most 3’ end exons of COL4A5 have sizes identical to the corresponding ones in COL4Al. Additionally, the exon-intron boundaries of the 19th most 3‘ end exon of

COL4A5 match perfectly with those of the corresponding exon of COL4A1, although it is 3 bp shorter due to the deletion of one codon (17). The unique arrangement of closely located pairs of type

IV

collagen genes on chromosomes 2, 13, and X indicates that the genes have evolved by duplication of an ancestor gene on one chromosome, after which the gene pair was duplicated to other chromosomes.

The COL4A5 gene has turned out to be of considerable in- terest because it has been shown to be mutated in patients with

X chromosome-linked Alport syndrome (1-3). This is a progres- sive, heterogeneous kidney disease characterized by hematuria and end stage renal failure in males and it is frequently ac- companied by deafness and eye lesions. Over 30 mutations, both large gene rearrangements and point mutation, have now been reported in the gene in Alport patients (1,2, 18-29). The clinical manifestations and pathological findings, which are quite strictly restricted to the kidney, can be explained by the fact that the a5(IV) collagen chain is located in the kidney solely in the glomerular basement membrane. The a5(IV) chain must be essential for the normal structure and function of the glomerular basement membrane, since different mutations, in- cluding complete deletion of the COL4A5 gene lead to the

Al-

port phenotype (27). The entire cDNA-derived amino acid se- quence of the 1,685-residue polypeptide has recently been determined (20).

The abbreviations used are: kb, kilobaseb); bp, base pairb); PCR, polymerase chain reaction.

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Human COL4A5 Collagen Gene

6609

5' II 11 I1 I II I I Y I II 1 u r n II I I n I II I I n I I I 1 n m 3' I 1 I I I I I I I 1 2 5 10 15 20 25 I l l 30 35 37 40 45 50 51 I I LA224 LA33

-"

LA210 LA25 PCR-22 L A B HPl00 LA26 Fa F7 E M H P 1 LA3 h a 2 FM13 M L 5 " Fz MG2 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 kb

FIG. 1. Structure of the human type IV collagen a6 chain gene. lbp, location of exons is shown by vertical bars and introns by a horizontal

line. The exons are numbered from the 5' end of the gene. Intron sequences of unknown size that are not contained in the genomic clones are indicated by circles. Middle, alignment of nine A phage clones characterized in this study together with eight clones identified previously (17). The

2.9-kb genomic segment PCR22 was generated by PCR amplification (see text). Bottom, scale in kilobases.

Detailed characterization of the COUA5 gene is essential for

studies on the nature of mutations in Alport syndrome. We

have previously reported the exon-intron structure of the 3' end

of the gene spanning the 19 most 3' end exons, as well as the

sequence of the first exon from the 5' end (8,171. In the present

study we have determined the sequences of the remaining ex- ons, thus providing the complete exon pattern of the gene

which contains 51 exons.

MATERIALS AND METHODS

Zsolation and Characterization of Genomic Clones--Two human ge-

nomic libraries: a human X chromosome-specific library (ATCC 57750)

and a human placenta genomic library (Clontech HLlOGJ), both gener- ated by partial Sau3A digestion, were screened with 3ZP-labeled cDNA inserts coding for human u5(IV) chain (7, 20) according to standard procedures (30). The isolated genomic clones were characterized by restriction mapping and by hybridization with different cDNA frag- ments or sequence-specific oligonucleotide probes. Suitable restriction fragments were subcloned into pUC or pBluescript vectors for further

restriction mapping and DNA sequencing carried out with the Sanger

dideoxynucleotide chain termination method (31).

Preparation of DNA Clones Containing Exon 22-Exon 22 was not present in the isolated genomic clones. In order to obtain a gene seg- ment containing this exon, the missing genomic fragment between the

clones LA26 and HPlOO was PCR-amplified (32) using oliogonucleotide

primers containing sequences from the 3' and 5' ends of the clones, respectively. The primers were synthesized in an Applied Biosystems DNA synthesizer. The primers were designed to contain EcoRI restric- tion sites at the 5' end for subsequent cloning of the amplified fragment into the pBluescript vector. The PCR reactions were carried out using Ampli-Taq DNA polymerase (Promega) under conditions recommended

by the manufacturer, and 800 ng of human lymphocyte DNA was used

as template in each reaction.

RESULTS

Isolation of Genomic Clones-We have previously described

the isolation of eight genomic clones containing the 3' end of

the human COMA5 gene containing the 19 most 3' end exons,

except exon 15 from the 3' end which was cloned and sequenced

using the polymerase chain reaction (17). In the present study nine new, mostly overlapping A phage clones covering the cen-

tral and 5' end of the gene were isolated (Fig. 1). One of those

clones, LA226, was shown to contain the first exon of COLAA5,

the intergene region, and the first two exons of COMA6 (8).

Restriction enzyme mapping and DNA sequencing analysis (see

below) demonstrated that the new clones contained the coding

sequences for exons 1, 3-21, and 23-32 (counted from the 5'

end). Extensive screening of the genomic libraries did not yield

clones containing exons 2 or 22. In order to obtain genomic

DNA containing exon 22, we PCR-amplified the gene segment

TABLE I

Comparison of exon sizes (bp) in the human COL4A5 and COL4Al genes

Exon numbers in parentheses represent exon number of COL4Al where it differs from that in COL4A5. Exons which differ in size be- tween genes are underlined.

Exon COUA5 C O U A l Exon EOUA5 C O U A l

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 (20) 20 (21) 21 (22) 22 (23) 23 (24) 24 (25) 25 (26) 202

+

81 60 90 45 45 63 54 27 81 63 36 42 93 54 57 45 54 42 133 174 84 93 71 192 169 - - - - - - - - 129

+

84 60 90 45 45 63 54 27 84 63 36 42 87 27 51 45 54 42 85 36 165 96 84 71 192 169 - - - - - - - - 26 (27) 27 (28) 28 (29) 29 (30) 30 (31) 32 (33) 31 (32) 33 (34) 34 (35) 35 (36) 36 (37) 37 (38) 38 (39) 39 (40) 40 (41) 41 (42) 42 (43) 43 (44) 44 (45) 45 (46) 46 (47) 47 (48) 48 (49) 49 (50) 50 (51) 51 (52) 93 93 105 105 98 98 151 151 114 114 168 168 90 150 90 - 153 99 - 99 ~~ 90 90 140 140 127 127 81 81 99 99 51 51 186 186 134 134 73 73 72 72 129 129 99 99 213 213 178 178 115 115 173 173 ~ 1245

1383

lacking between the clones HPlOO and LA26 using primer se-

quences obtained from the 3'- and 5'-end of the two clones,

respectively. This resulted in the amplification of a 2.9-kb frag-

ment, PCR22, containing exon 22. The same approach was not

successful for the exon 2 containing segment which was miss-

ing between clones LA226 and LA29.

Alignment of presently isolated clones, together with the

eight clones previously isolated, is shown in Fig. 1. The clones

span 10 kb of the 5'-flanking region, 140 kb of the structural

gene, and 9.5 kb of the 3"flanking region. The clones do not

contain exons 2 or 37 and, therefore, the sizes of introns 1,2,36,

and 37 could not be determined. The present data demonstrate

that the COMA5 gene is at least 140 kb in size.

Exon Size Pattern-Restriction fragments of the genomic

clones were subjected to subcloning and sequencing in order to

determine the exon sizes. For sequencing, either universal prim-

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1 Exon 1 a g g g g q g a a g g a a g a g t a g c t c c t t c t t c t t c t t c t t t t t t t t t t c t t c c a c t c t t a a ~ a aqcttctttctcttcacccaagcctcactgtccctctccggctctagctctctc~tata aaccctcaagattatgtcaattggttagagccagc~ggaatttcgtgcgggtgctgaag gagctqcgggagccggagaaga ATGAAACTGCGTGGAGTCAGCCTGGCTGCCGGCTTG (283 bp) M K L R G V S L A A G L 1 TTCTTACTGGCCCTGAGTCTTTGGGGGCAGCCTGCAGAGGCTGCG gtaagtccttcctc F L L A L S L W G Q P A E A A CCCtCC 284 Exon 2

...

GCTTGCTATGGGTGTTCTCCAGGATCMGTGTGACTGC (60 bp) 28 A C Y G C S P G S K C D C AGTGGCATWGGGGAAAAG

...

S G I K G E K Exon 3 t c a t t c t c a t t t a a t t g c a g GGAGAGAGAGGGTTTCCAGGTTTGGAAGGACACCCAGGA 344 (90 bp) G E R G F P G L E G H P G TTGCCTGGATTTCCAGGTCCAGAAGGGCCTCCGGGGCCTCGGGGACAAAAG 48 g t a t g t a t L P G F P G P E G P P G P R G Q K catgttgccaac 434 (45 bp) Exon 4 a a a a a t a t t g c a t t t t t c a g GGTGATGATGGAATTCCAGGGCCACCAGGACCWGGA 18 G D D G I P G P P G P K G ATCAGA g t a a g t a g t a t t t t c t c t a t I R 419 Exon 5 g a t t t t a t t t c t t c t t a t a g GGTCCTCCTGGACTTCCTGGTTTCCAGGGACACCAGGT (45 bp) G P P G L P G F P G T P G 93 CTTCCT g t a a g t a g c a t t t c a c t t t t L P Exon 6 t q t t a t g t c q c t t t t c a a a g GGAATGCCAGGCCACGATGGGGCCCCAGGACCTWGGT 524 (63 bp) G M P G H D G A P G P Q G 108 ATTCCCGGATGCAATGGAACCAAG gtgagatccatccatttaat I P G C N G T K 587 (54 bp) Exon 7 t t a t t t t t a a c t c c t t c t a g GGAGMCGTGGATTTCCAGGCAGTCCCGGTTTTCCTGGT 129 G E R G F P G S P G F P G TTACAGGGTCCTCCA gtaagttataaaatttggqa L Q G P P

Exon 8 c c t t t t c t t t t t a a t a a t a g GGACCCCCTGGGATCCCAGGTATGAAG 641 gtaagcatctc

(27 bp) G P P G I P G M K 147 attctgggg 668 Exon 9 c c a t t g a t g g c t t c t t t t a g GGTGAACCAGGTAGTATAATTATGTCATCACTGCCAGGA (81 bp) G E P G S I I M S S L P G CCAAAGGGTAATCCAGGATATCCAGGTCCTCCTGGAATACAA 156 gtaagtatccagtgatt P K G N P G Y P G P P G I Q t t c I4 9 (63 bp) Exon 10 c t t t a c t c a c t t t a t a a c a q GGCCTACCTGGTCCCACTGGTATACCAGGGCCAATTGGT 183 CCCCCAGGACCACCAGGTTTGATG g t a a g c t g t g t t g t t t a a t t G L P G P T G I P G P I G P P G P P G L M 812 Exon 11 c t t t t g t c t t c t c t t c t t a g GGCCCTCCTGGTCCACCAGGACTTCCAGGACCTMG g t (36 bp) G P P G P P G L P G P K 204 a a t t t t c t t t t t c t t t a t 848

Exon 12 atgqaaacttctctctccag GGGAATATGGGCTTMTTTCCAGGGACCCAAAGGTGAA

(42 bp) G N H G L N F Q G P K G E AAA gtgagtaaagaaagagagct 216 K Exon 13 t t a t t t t a t c t t g c a a a c a g GGTGAGCMGGTCTTCAGGGCCCACCTGGGCCACCTGGG 890 (93 bp) G E Q G L Q G P P G P P G CAGATCAGTGAACAGAAAAGACCAATTGATGTAGAGTTTCAGAAAGGAGATCAG 230 gtgag Q I S E Q K R P I D V E F Q K G D Q

Exon 14 tttcccctactactgcatag GGACTTCCTGGTGACCGAGGGCCTCCTGGACCTCCAGGG 983

(54 bp) ,c, G L P G D R G P P G P P G ATACGTGGTCCTCCA gtaagtacctaaagtgcttt _”. I R G P P Exon 15 a a c t a t t t t t a t g t g t a c a g GGTCCCCCAGGTGGTGAGAAAGGTGAGAAGGGTGAGCAA 1031 (57 bp) G P P G G E K G E K G E Q G G A G A G C C A G G W G A gtaagtgatgtaactgctaa 219 G E P G K R 1094 Exon 16 c a t t t c t t t g t a t c c t a t a g G G T A A A C C A G G C A A A G A T G C ~ G C C ~ C C M C C A G G A (45 bp) G K P G K D G E N G Q P G ATTCCT gtaagtagctaaggttcttt 298 I P ~ ~~~ 1139 (54 bp) Exon 17 c t a t c c t c t a t g t t t t a a a g GGTTTGCCTGGTGATCCTGGTTACCCTGGTGAACCCGGA 313 G L P G D P G Y P G E P G A G G G A T G G T G W G g t a a g a a t t t t a a t a c t t t g R D G E K

Exon 18 tttacaattgcattgaacag GGCCAAMAGGTGACACTGGCCCACCTGGACCTCCTGGA 1193

(42 bp) G Q K G D T G P P G P P G CTT g t a a g t t t t t t t t t t t t a g t 33 1 L Exon 19 t t t t t t c t t t g g t a a t a a a g GTAATTCCTAGACCTGGGACTGGTATAACTATAGGAGA 1235 (133 bp) V I P R P G T G I T I G E AAAGGAAACATTGGGTTGCCTGGGTTGCCTGGAGAFAWxGGGAGCGAGGTTTCCTGGA K G N I G L P G L P G E K G E R G F P G 345 ATACAGGGTCCACCTGGCCTTCCTGGACCTCCAG gtaaat9agattgCattt.t I Q G P P G L P G P P 1368 Exon 20 g a t c a c t t t t t t g a a t c t t a g GGGCTGCAGTTATGGGTCCTCCTGGCCCTCCTGGATTT (174 bp) G A A V M G P P G P P G F CCTGGAGAAAGGGGTCAGAAAGGTGATGATGAAGGACCACCTGGAATTTC~TTCCTGGACCT P G E R G Q K G D E G P P G I S I P G P CCTGGACTTGACGGACAGCCTGGGGCTCCTCCTGGGCTTCCAGGGCCTCCTG~CCTGCTGGC P G L D G Q P G A P G L P G P P G P A G 389 CCTCACATTCCTCCTA g t a a g c t a t a t t t t t c t c c t P H I P P Exon 21 t c c t t t c t t t a t c t t a c t c a g GTGATGAGATATGTGAACCAGGCCCTCCAGGCCCCCCA 1542 (84 bp) S D E I C E P G P P G P P GGATCTCCAGGTGATAAAGGACTCCAAGGAGAACAAGGAGTGAAAG 447 gtttqatctccaa G S P G D K G L Q G E Q G V K a c a t a t t Exon 22 t g t t a t t a t g a t t t c a c t a g GTGACAAAGGTGACACTTGCTTCAACTGCATTGGAACT 1626 G D K G D T C F N C I G T (93 bp) 475 GGTATTTCAGGGCCTCCAGGTCMCCTGGTTTGCCAGGTCTCCCAGGTCCTCCAG gtaa G I S G P P G Q P G L P G L P G P P attatgcctcagggta Exon 23 g t t t g t g t g t g t g t g t g t t a g GATCTCTTGGTTTCCCTGGACAGMAGGGGAAMAGGA 1119 (71 bp) G S L G F P G Q K G E K G CAAGCTGGTGCMCTGGTCCCAAAGGATTACCA 506 g t a a g t t t t g a g t a t a t t a t Q A G A T G P K G L P

FIQ. 2. Nucleotide sequence of the SI exona of the human COlRAd collagen gene and their immediate flanking intron sequences. The flanking sequences of exons 2 and 37 have not been determined. Intron sequences and 5’ and 3’ end untranslated sequences are depicted by lower case letters and exon sequences by capital letters with the derived amino acid residues shown with the one-letter code below the second base above of a codon. The nucleotides for coding sequences and the first nucleotide of an exon and the numbers of amino acid residues are shown below the first residue encoded by an exon. The exons are amino acid residues are numbered according to Ref. 20. The nucleotide numbers are shown numbered from the 5’ end. The translation stop codon in exon 51 is indicated by an asterisk (*I.

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Human

COL4A5 Collagen Gene

6611

Exon l4 t t t t t c c t t a c t c a t t t c a g GGCATTCCAGGAGCTCCAGGTGCTCCAGGCTTTCCTGGA 1790 (192 bp) G I P G A P G A P G F P G TCTAAAGGTGAACCTGGTGATATCCTCACTTTTCCAGGAATGAAGGGTGACAAAGGAGAG S K G E P G D I L T F P G H K G D K G E TTGGGTTCCCCTGGAGCTCCAGGGCTTCCTGGTTTACCTGGCACTCCTGGACAGGATGGA L G S P G A P G L P G L P G T P G Q D G 530 TTGCCAGGGCTTCCTGGCCCGAAAGGAGAGCCT q t t g a q t t g q t t t g a t a t t t t L P G L P G P K G E P 1982 Exon 25 t t a a a c t t t t c c c t t t t t a q GGTGGAATTACTTTTAAGGGTGAAAGAGGTCCCCCTGGG (169 bp) G G I T F K G E R G P P G AACC~GGTTTACCAGGCCTCCCAGGGAATATAGG~CTATGGGTCCCCCTGGTTTCGGC N P G L P G L P G N I G P H G P P G F G CCTCCAGGCCCAGTAGGTGAAAAAGGCATACAAGGTGTGGCAGGAAATCCAGGCCAGCCA P P G P V G E K G I Q G V A G N P G Q P 594 GGAATACCAG q t a a q t t t a c t q t q t t t t q G I P Exon 26 t q a t t t a c t c t t q c t t t c a q GTCCTAAAGGGGATCCAGGTCAGACTATAACCCAGCCG 2151 (93 bp) G P K G D P G Q T I T Q P GGGMGCCTGGCTTGCCTGGTMCCCAGGCAGAGATGGTGATGTAGGTCTTCCAG q t a t q t q a q q a a t t t a t t t c 650 G K P G L P G N P G R D G D V G L P 2244 Exon 21 a t a a c t q c t q t t t c t c c a t a q GTGACCCTGGACTTCCAGGGCMCCAGGCTTGCCAGGG (105 bp) C O , G D P G L P G Q P G L P G ATACCTGGTAGCAI\AGGAGAACCAGGTATCCCTGGMTTGGGCTTCCTGGACCACCTGGT CCCAAAG I P Gq t a t q t t q q a a t q q q t a q c a S K G E P G I P G I G L P G P P G P K "I_ Exon 28 a a a t g a c t t a t c a t t t t a c a g GCTTTCCTGGAATTCCAGGACCTCCAGGAGCACCTGGG (98 bp) 2349 G F P G I P G P P G A P G 716 ACACCTGGAAGAATTGGTCTAGAAGGCCCTCCTGGGCCCTCCTGGGCCACCCGGCTTTCCAGGACCAAAG T P G R I G L E G P P G P P G F P G P K q t c t g q q a c a t t t t t c t t t a 2447 Exon 29 t t q t c a t q t q t a t q c t c a a q GGTGAACCAGATTTGCATTACCTGGGCCACCTGGGCCA (151) G E P G F A L P G P P G P C C A G G A C T T C C A G G T T T C A A A G G A G C A C T T G G T C C A R M G P G L P G F K G A L G P K G D R G F P G CCTCCGGGTCCTCCAGGACGCACTGGCTTAGATGGGCTCCCTGGACCA&AAG gtatgqa 749 P P G P P G R T G L D G L P G P K q q c t q t c a c t g c Exon 30 a c t q t a t t t a t t t c t t a a a q GTGATGTTGGACCAAATGGACAACCTGGACCRATGGGA 2598 (114 bp) G D V G P N G Q P G P M G 799 CCTCCTGGGCTGCCAGGAATAGGTGTTCAGGGACCACCAGGACCACCAGGGATTCCTGGG P P G L P G I G V Q G P P G P P G I P G CCAATAGGTCAACCTG g t a a q a t t a q a g t a a a t q t q P I G Q P 2112 837 G L H G I P G E K G D P G Exon 31 a t t g a t a t t q t a t t a a c t a g GTTTACATGGAATACCAGGAGAGAAGGGGGATCCAGGA (168 bp) CCTCCTGGACTTGATGTTCCAGGACCCCCAGGTGAAAGAGGCAGTCCAGGGATCCCCGGA P P G L D V P G P P G E R G S P G I P G GCACCTGGTCCTATAGGACCTCCAGGATCACCAGGGCTTCCAGGMAAG~GGT~CTCT A P G P I G P P G S P G L P G K A G A S GGATTTCCAG q t a a t t t q t t t a a a q t t t t c G F P &On 32 C t t a t t q a t a t t c t t c a a a q GTACCAAAGGTWTGGGTATGATGGGACCTCCAGGC 2880 G T K G E H G M H G P P G (90 bp) 893 CCACCAGGACCTTTGGGAATTCCTGGCAGGAGTGGTGTACCTGGTCTTAAAG gtaataa P P G P L G I P G R S G V P G L K t c a a q q t t t g c t q Exon 33 t t c a c a c a c a t t q a t t t t a q GTGATGATGGCTTGCAGGGTCAGCCAGGACTTCCTGGC 2970 (150 bp) G D D G L Q G Q P G L P G 923 CCTACAGGAGAAAAAGGTAGTAAAGGAGAGCC'CGGCCTTCCAGGCCCTCCTGGACCAATG P T G E K G S K G E P G L P G P P G P H GATCCAAATCTTCTGGGCTCGGAGAGAAGGGGGAACCTGGCTTACCAG q t q a g t q D P N L L G S K G E K G E P G L P a a t q a a t t t a t t t Exon 34 t c t t c t a c t c a t t c t t q q a a q GTATACCTGGAGTTTCAGGGCCMGGTTATCAGGGT 3120 (99 bp) G I P G V S G P K G Y Q G TTGCCTGGAGACCCAGGGCAACCTGGACTGAGTGAGTGGACAACCTGGATTACCAGGACCACCA L P G D P G Q P G L S G Q P G L P G P P 973 G g t a a g t q t q a t a q q c c a t t t Exon 35 t t a c c a a t t t q a c c t t t c t a q GTCCCAAAGGTAACCCTGGTCTCCCTGGACAGCCAGGT 3219 (90 bp) G P K G N P G L P G Q P G CTTATAGGACCTCCTGGACTTAAAGGAACCATCGGTGATATGGGTTTTCCAG q t q a q t q 1006 L I G P P G L K G T I G D H G F P a t q a a a a t c t t c c 3309 G P Q G V E G P P G P S G (140 bp) Exon 36 t a t a t c a c a t a t t t t c a a c a g GGCCTCAGGGTGTGGMGGGCCTCCTGGACCTTCTGGA 1036 GTTCCTGGACRACCTGGCTCCCCAGGATTACCTGGACAGAAAGGCGACAAAGGTGATCCT V P G Q P G S P G L P G Q K G D K G D P GGTATTTCAAGCATTGGTCTTCCAGGTCTTCCTGGTCCAAAG q t a a t c t t t g g c a t a t a G I S S I G L P G L P G P K stt 3449 Exon 31 GGTGAGCCTGGTCTGCCTGGATACCCAGGGAACCCTGGTATCAAAGGTTCTGTGGGAGAT ( 1 2 7 b p ) G E P G L P G Y P G N P G I K G S V G D 1083 CCTGGTTTGCCCGGATTACCAGGAACCCCTGGAGCAAMGGTATTAGTGGACAACCAGGCCTTCCTGGA P G L P G L P G T P G A K G Q P G L P G TTCCCAG F P Exon 38 a a a t t q a g c t c t t t a c t c t a q GAACCCCAGGCCCTCCTGGACCAAMGGTATTAGTGGTATTAGTGGC 3516 G T P G P P G P K G I S G (81 bp) 1125 CCTCCTGGGAACCCCGGCCTTCCAGGAGAACCTGGTCCTGTAG qtaaqcatqaaaaata P P G N P G L P G E P G P V acaq Exon 39 c t q c t g t a c t c a a t t t t t t a q GTGGTGGAGGTCATCCTGGGCAACCAGGGCCTCCAGGC (99 bp) G G G G H P G Q P G P P G 3657 1152 GAAMAGGCAAACCCGGTCAAGATGGTATTCCTGGACCAGCTGGACAGAAGGGTGAACCA E K G K P G Q D G I P G P A G Q K G E P G q t q c t q t a q t t t t t c a t t t t 3756 G Q P G F G N P G P P G L Exon 40 t t t t q t t t t g t a c ' t c t q a c a q GTCAACCAGGCTTTGGAAACCCAGGACCCCCTGGACTT (51 bp) 1185 CCAGGACTTTCTG q t a a a c c t t a a t a a a a c a t q P G L S Exon 41 a a t t a t a c t t t a c t t t c a t a q GCCMAAGGGTGATGGAGGATTACCTGGGATTCCAGGA (186 bp) G Q K G D G G L P G I P G 3801 1202 AATCCTGGCCTTCCAGGTCCGGGCGAACCAGGCTTTCACGGTTTCCCTGGTGTG~G N P G L P G P K G E P G F H G F P G V Q GGTCCCCCAGGCCCTCCTGGTTCTCCGGGTCCAGCTCTGGAAGGACCTAAAGGCAACCCT G P P G P P G S P G P A L E G P K G N P GGGCCCCAAGGTCCTCCTGGGAGACCAG q t a t g t c c q t q a q t g g t a q q G P Q G P P G R P RG. !&continued

were used. The sizes of most exons and introns were determined determined from a PCR-amplified genomic fragment made us- from heteroduplex analyses between the cDNAand the genomic ing primers for the tentative exon 2.

clones. The sizes of exons (data not shown) were in good agree- The sizes of exons and introns of the COL4A5 gene are sum-

ment with those obtained from nucleotide sequencing, thus vali- marized in Table I, where they are compared with the corre- dating the size assessment of introns. The size of exon 2 was sponding ones in the COIAAl gene. The entire COL4A5 gene

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6612

Exon 42 catttgctgtggattattaaq GTCTACCAGGTCCAGAAGGTCCTCCAGGTCTCCCTGGA 3993

(1% bp) G L P G P E G P P G L P G MTGGAGGTATTAAAGGAGAGMGGGAAATCCAGGCCMCCTGGGCTACCTGGC~GCCT N G G I K G E K G N P G Q P G L P G L P 1264 GGTTTGMGGAGATCAAGGACCACCAGGACTCCAG gtaqqaaatggaagtaqata G L K G D Q G P P G L Q 4127

Exon 43 taatgattttatttattcag GGTAATCCTGGCCGGCCGGGTCTCAATGGAATGMGGA

(73 bp) G N P G R P G L N G M K G

1309

GATCCTGGTCTCCCTGGTGTTCCAGGATTCCCAG gtatttgaagggatttttgt D P G L P G V P G F P

Exon 44 ctgtatqtaccttctgtgcag GCATGAAAGGACCCAGTGGAGTACCTGGATCAGCTGGC 4200

(72 bp) G M K G P S G V P G S A G

CCTGAGGGGGAACCGGGACTTATTGGTCCTCCAG gtaagacttattcctgaaga 1333 P E G E P G L I G P P

4272

G P P G L P G P S G Q S I Exon 45 tttgtgtgttttgtctcatag GTCCTCCTGGATTACCTGGTCCTTCAGGACAGAGTATC (129 bp) 1357 ATAATTAAAGGAGATGCTGGTCCTCCAGGAATCCCTGGCCAGCCTGGGCTAAAGGGTCTA I I K G D A G P P G I P G Q P G L K G L CCAGGACCCCAAGGACCTCAAGGCTTACCAG gtaccaatgcagatcatctt P G P Q G P Q G L P 4401 G P T G P P G D P G R N G (99 bp)

Exon 46 tgcctcattcttttcctgtag GTCCAACTGGCCCTCCAGGAGATCCTGGACGCAATGGA CTCCCTGGCTTTGATGGTGCAGGAGGGCGCMGGAGACCCAGGTCTGCCAGGACAGCCA

L P G F D G A G G R K G D P G L P G Q P 1400

G gtaagacaagtaaaacatgc

Exon 47 ctgattatttcgtggaaatag GTACCCGTGGTTTGGATGGTCCCCCTGGTCCAGATGGA 4500 G T R G L D G P P G P D G (213 bp) 1433 TTGCAAGGTCCCCCAGGTCCCCCTGGMCCTCCTCTGTTGCACATGGATTTCTTATTACA L Q G P P G P P G T S S V A H G F L l T CGCCACAGCCAGACAACGGATGCACCACAATGCCCACAGGGAACACTTCAGGTCTATGAA R H S Q T T D A P Q C P Q G T L Q V Y E GGCTTTTCTCTCCTGTATGTACAAGGMTAUAGAGCCCACGGTCAAGACTTGG gtga G F S L L Y V Q G N K R A H G Q D L gataatcaatatctaa

Exon 4% tttctctccaaatctttctag GGACGGCTGGCAGCTGCCTTCGTCGCTTTAGTACCATG 4713

(178 bp) G T A G S C L R R F S T M 1504 CCTTTCATGTTCTGU\ACATCAATAATGTTTGCAACTTTGCTTCMGMTGACTATTCT P F M F C N I N N V C N F A S R N D Y S TACTGGCTCTCTACCCCAGAGCCCATGCCAATGAGCATGCAACCCCTAAAGGGCCAGAGC Y W L S T P E P M P N S M Q P L K G Q S ATCCAGCCATTCATTAGTCG gtaaggcattqatttagctg I Q P F I S R

Exon 49 tctccttttcctttaccag ATGTGCAGTATGTGAAGCTCCAGCTGTGGTGATCGCAGTT (115 bp) 4891 1564 C A V C E A P A V V l A V CACAGTCAGACGATCCAGATTCCCCATTGTCCTCAGGGATGGGATTCTCTGTGGATTGGT H S Q T l Q I P H C P Q G W D S L W I G TATTCCTTCATGATG gtattttacactcttccttg Y S F M M 5006

Exon 50 tttttccttgtcttttatag CATACAAGTGCAGGGGCAGAAGGCTCAGGTCAAGCCCTA

(173 bp) H T S A G A E G S G Q A L GCCTCCCCTGGTTCCTGCTTGGAAGAGTTTCGTTCAGCTCCCTTCATCGAATGTCATGGG A S P G S C L E E F R S A P F I E C H G AGGGGTACCTGTMCTACTATGCCAACTCCTACAGCTTTTGGCTGGCAACTGTAGATGTG R G T C N Y Y A N S Y S F W L A T V D V 1602 TCAGACATGTTCAG gtaaagtgcttatagcttta S D M F S

Exon 51 cttatttcttatttcccag TAAACCTCAGTCAGAAACGCTGAAAGCAGGAGACTTGAGG

(1245 bp) K P Q S E T L K A G D L R 5179 1 6 6 n ACACGAATTAGCCGATGTCAAGTGTGCATGAAGAGGACATAA attttgaaqaattcctt "" T R I S R C Q V C M K R T ' 1 6 8 5 ttgtgttttaaaatgtgatactatatatatataaaatt~taggatgca~gtctcattg tccccaactttactactqctgccgtcaatggtgctactatatatgatcaagataacatgc tqactagtaaccatqaaqattcagatgtacctcaqcaatgcgccagagcaaagtctctat t a t t t t t c t a c t a a a g a a a t a a g g a a g t g a a t t t a c t t t t t g g g t c c a g a a t g a c t t t c t ccaagaattataaqatgaaaattatatattttgcccagttactaaaatggtacattaaaa attcaattaagagaaqagtcacattgagtaaaataaaagactgcagtttgtgggaagaat tatttttcacggtgctactaatcctgctqtatcccgggtttttaatataaaggtgttaag cttattttgctttgtaagtaaagaatgtgtatattgtgaacaqccttttagctcaaaatg ttgagtcatttacatatgacatagcatgaatcactctttacagaaaatgtaggaaaccct agaatacaqacagcaatattttatattcatgtttatcaaagtgagaggacttatattcct acatcaagttactactqagagtaaatttattttgagttttatcccgtaagttctgttttg attttttttaaaaaacaaacccttttagtcactttaatcagaattttaaatgttcatgtt acataccaaattataatatctaatggaqcaatttgtcttttgctatattctccaagatta tctcttaagaccatatgccccctgttttaatgtttcttacat~tgtttttact~tttc tgactggacaaaqttcttccaaacaattctgagaaacaaaaatacacacgcaqaattaac aattcttttccctgtgcttcttatgtaagaatcctcctgtggcctctgcttgtacagaac tgggaaacaacacttggttagtctcttttaagttacaaaaagccaattgatgtttcttat tctttttaaattttaaatattttgttataaatactcacaggataccttatttccctagct atcatctcctgacttaatgttttttaaacccaccaatataaatttaattaaagatatatg ttgtaaqgatggtctgttgtgtatctcttcaqcctgtgtggaaaaaacccctgctcattt acagatgattaccaatctgcacatcaacaagtatctcttcccagggaatcagtttctcca cgctttgatcactctgttagtagatagaaaacatatagtaatgtaaactttttccaatga agaaaactacctattggaagacatttccaagataataaatttcttgacaacattgtgtta atggctaaqaaaqgaaacaactggcttctattt "" FIG. 2"continued contains 51 exons or one less than the COIAAl gene. The exons

can now be numbered correctly from the 5' end. Consequently, the previously reported most 3' end exons 1-19 as counted from the 3' end should now be numbered 51-33, respectively. The complete sequence of exons 1-32 elucidated in this study, to- gether with the sequence of exons 33-51 as well as the derived amino acid sequences are illustrated in Fig. 2. With the excep- tion of exons 2 and 37,20 nucleotides of the sequences flanking each exon are also provided.

The exon sizes (5'- and 3"untranslated sequences not in- cluded) vary between 27 and 213 bp. Exon 1 contains 283 bp with 202 bp of a 5'-untranslated sequence and 81 bp of a trans- lated sequence. The transcription initiation site was verified by primer extension analysis. The translated sequence of exon 1 encodes solely the tentative 26-residue long signal peptide (19). Exon 2 encodes the 14 noncollagenous amino-terminal end and two Gly-X-Y triplets. Thus, exons 2-47 encode the collagenous domain, exon 47 being a junction exon encoding the carboxyl- terminal end of the collagenous domain and a part of the non-

collagenous domain.

Comparison of Exons in the COIAA5 and COIAAl Genes-

The complete exon size pattern of the COUA5 gene, as deter- mined in present and previous (17) studies, and that of

COIAAl ( X ) , as summarized in Table I, demonstrates a close evolutionary relationship of the two genes. Out of 51 exons in the COIAA5 gene, 42 have identical sizes as corresponding exons in COIAAl which has 52 exons. All the exons differing in size between the two genes code for parts of the collagenous domain. It appears that the 133-bp exon 19 in COL4A5 is a

fusion exon, as exons 19 and 20 in COLAAl are smaller and contain 85 and 36 bp, respectively. The distribution of exons starting with complete or split codons is similar in the COIAA5

and COIAAl genes. In COUA5 exons 3-18 start with complete codon glycine as compared with exons 2-18 in COL4Al. Addi- tionally, there are five other -Gly-X-Y- repeat coding exons in both genes that start with a complete glycine codon. In COIAA5

all the other exons encoding the collagenous domain start with the second base for glycine except exon 19 that starts with a

(6)

6613

TABLE I1 human gene for the nonfibrillar type XI11 collagen a1 chain has

Location of exons in EcoRZ fiagments of the human COLA45 gene hen shown to be Over 140 kb in size (36).

Exon 32 contains an internal EcoRI site. The COMA5 gene contains 51 exons, or one less than the

EcoRI Clones isolated Clones isolated COLAAl collagen gene. AS reported previously (17), the trans- Exon f r a ( r t and in present previous Exon k a r t EcoRI and in present previous lated sequences of the five exons coding for the carboxyl-termi-

(17) studies (17) studies nal noncollagenous domain identical in length and the location 1.4 LA226 27 2.1 LA26 of introns are identical. Out of the 45 exons encoding the " col-

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 20 19 21 22 23 24 25 26 11.0 5.6 1.4

+

2.3 3.7 3.7 5.6 5.6 5.6 1.0 3.0 3.0 3.0 2.1 2.1 2.1 2.2 3.0 2.5 5.0 5.0 5.0 4.2 4.2 4.2 I n not isolated LA29 LA29 LA29 LA29 LA21 LA21 LA210 LA210 LA33 LA33 LA33 LA25 LA25 LA25 LA25 LA25 LA25 IF-100 IIP-100 LA26 LA26 LA26 T & o n 4.6 lAtl&o 28 4.0 29 4.0 30 8.8 31 4.3 32 4.3

+

0.5 33 34 0.5 35 1.5 36 2.8 37 2.8 38 4.5 39 5.5 40 5.5 41 5.5 42 1.2 43 8.0 1.2 44 45 8.0 46 8.0 47 2.3 48 2.3 49 6.5 50 6.5 51 6.5 1.4 LA26 LA26 LA3 LA3 ML2 ML2 ML2 ML2 ML2 F7 F7 F7 F7 FM13 FM13 FM13 FM13 MG-2 MG-2 MG-2 MG-2 MG-2 MG-2

complete codon for valine and exon 21 that starts with the second base for the aspartate codon. The situation is similar in

COLAAl, except that exon 22 starts with the second base of an asparagine codon.

Assignment of Exons in COLA45 to EcoRI Restriction Fragments-Since the COUA5 gene has been shown to be mutated in patients with X chromosome-linked Alport syn- drome, knowledge about the gene structure and exact location of exons in certain endonuclease restriction fragments is useful for thorough analysis of mutations, such as for the determina- tion of breakpoints of deletions, or for the characterization of other major gene rearrangements.

To

facilitate such analyses we determined in which EcoRI restriction fragments the exons are located (Table 11).

DISCUSSION

In the present study we have determined the entire exon pattern of the human type

IV

collagen a5 chain gene

(COLA45). Altogether, 17 A phage clones and one PCR ampli- fied gene segment spanning the exon 22 region covered 10 kb of the 5'-flanking region, 140 kb of the structural gene, and 9.5 kb of the 3"flanking region. The eight most 3' end clones have been reported earlier (17) and, additionally, characterization of the LA226 clone containing the 5' ends and intergenic region of

COL4A5 and COLAA6 have recently been reported (8). The clones were not completely overlapping and lacked regions con- taining exons 2 and 37. Therefore, the exact size of the gene could not be determined. We have previously shown that the closely related gene for the type

IV

collagen a1 chain is over 100 kb in size (151, and Cutting et al. (33) have estimated that the COL4A2 gene for the a2(IV) collagen chain is 100-160 kb.

Accordingly, it appears that, in general, the type

IV

collagen genes are considerably larger than, e.g. the genes for fibrillar collagens, such as the human al(1) gene which is 18 kb (34) and the chick a2(I) gene which is 39 kb (35), although the coding sequences of all these genes are of similar size order. Also, the

lagenous domain, 36 have identical sizes in the two genes. Both genes are similar in that a number of corresponding exons start with split codons. The highly related structure and head-to- head arrangement of the genes indicates that they evolved through duplication. This is supported by the fact that the translation products of the two genes, the a5(IV) and al(IV) collagen chains, have very homologous primary structure (20, 37). The reason for the COL4A5 gene having one exon less than

COLQAl appears to be a fusion and some other rearrangement of exons 19 and 20 in COIAAl.

The detailed structure of the COLQA5 gene determined in this study will be important for characterization of mutations in X-linked Alport syndrome and it will facilitate the develop- ment of diagnostic procedures for the disease. Alport syndrome is one major cause of end stage renal disease in males, with an estimated gene frequency of 1:5000 (38). The major manifesta- tions are loss of kidney function accompanied by hematuria and sensorineural deafness (38-40). Ultrastructural changes such as thinning and thickening and lamellation of the glomerular basement membrane led earlier to the hypothesis that type

IV

collagen, the main structural protein of the glomerular base- ment membrane, was defective in the disease (41, 42). The identification of the a5(IV) collagen chain (7, 43) and localiza- tion of its gene to the Xq22 (7) together with data showing highly specific expression of the gene in kidney glomeruli (7), made COLAA5 a strong candidate gene for Alport syndrome. This turned also out to be the case, and currently over 30 mutations have been reported in COUA5 in Alport syndrome (1,2,18-29). Thus far, no single mutations in COLAA6 causing Alport syndrome have been identified, but deletions spanning the 5' ends of the COL4A5 and COLAA6 cause a rare combi- nation of Alport syndrome associated with diffuse esophageal leiomyomatosis (8,251. The present assignment of exons in the

COLAA5 gene to distinct EcoRI restriction fragments will be useful for exact determination of breakpoints of deletions and for characterization of other rearrangements such as inver- sions, insertions, and duplications of DNA segments in Alport syndrome patients. Furthermore, the intron sequences adja- cent to all the exons, except for exons 2 and 37, will enable investigators to design primers for PCR amplification and se- quencing of exon regions when characterizing small mutations in exons of their immediate proximity.

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