Eur. J. Biochem. 248, 15-23 (1997) 0 FEBS 1997

The structural organisation of LAMA4, the gene encoding laminin a4 Allan RICHARDS ',*, Craig LUCCARINI ',* and F. Michael POPE '.2,3

' MRC Connective Tissue Genetics Group, Department of Pathology, University of Cambridge, Cambridge, UK * Strangeways Research Laboratory, Worts Causeway, Cambridge, UK

Department of Clinical Genetics, Addenbrookes Hospital, Cambridge, UK

(Received 16 April/l2 June 1997) - EJB 97 0528/2

We have determined the complete structural arrangement of LAMA4, the gene encoding the laminin a4 chain. Using both yeast artificial chromosome clones and total human genomic DNA and primers derived from the cDNA sequence, regions of the gene were amplified and sequenced to determine the splice donor and acceptor sites. The introns were sized by agarose gel electrophoresis of the PCR prod- ucts. The gene consisted of 39 exons spanning 122 kb. All of the splice sites conformed to the GTIAG rule, except intron 7 which possessed a GC dinucleotide at the donor splice site. The introdexon ratio was large at 17.8:1, mainly due to large introns at the 5' end of the gene. Regions at both the 5' and 3' end of the gene were subcloned from the yeast artificial chromosomes to enable untranscribed DNA to be sequenced. The gene represents the second of the laminin A gene family to be characterised and its structural organisation is similar to the equivalent regions of the LAMA2 gene.

Keywords: laminin ; gene structure.

The laminins are a family of connective tissue molecules and are the major non-collagenous component of basement mem- branes. Each laminin molecule is composed of three non-iden- tical chains which are each separate gene products [I-31. The original nomenclature of A, B1 and B2 chains has been altered [4] to al, pl and yl to accommodate the growing number of laminin genes being isolated, which now form the LAMA, LAMB and OZMC gene families. In addition each trimer has an Arabic numeral, i.e. alplyl is laminin 1. To date five a [5-91 three p 110-121 and two y [13, 141 chains have been characterised in either or both human and rodents. These combine as heterotrim- ers to form at least 10 different laminin isoforms [15]. The a chains differ from both p and y by the possession of a large globular C-terminal domain, which consists of five repeating LG modules (laminin globular modules). N-terminal to this is an a- helical coiled-coil domain, or long arm (domain 1/11), possessed by all three chains, through which they associate and assemble. At the N-terminal end the chains separate, into three short arms, to form either a cruciform or a Y shaped structure, each of these are composed of multiple LE modules (epidermal growth factor like laminin modules) which differ in number between chains, and form domains 111 and V.

Globular regions termed domains IV and VI are also present in most but not all of the variants. In a1 and a2, domains I11 and IV have been duplicated and so these molecules contain regions known as IIIa, IVa, IIIb and IVb. Domain IV of both a and y chains share some similarity, but differ from the corre- sponding region in p chains [I]. The p chains also differ by

Correspondence to A. Richards, Dept. of Pathology, University of Cambridge, Cambridge, CB21QP, England

Fax: +44 1223 333 346. E-mail: arichard@hgmp.mrc.ac.uk Abbreviations. LE module, epidermal-growth-factor-like laminin

module; LG module, laminin globular module; UTR, untranslated re- gion; YAC, yeast artificial chromosome.

Note. The nucleotide sequence data published here have been sub- mitted to the EMBL sequence data bank and are available under acces- sion number Y14240.

possession of a small extra (a) domain within the long arm. The a3, a4, p3 and y2 chains lack various of these short arm domains.

The importance of laminins in forming a contact between cells and the extracellular matrix has recently been underlined by the discovery that LAMA2 is the locus of some forms of congenital muscular dystrophy [16]. In addition the genes en- coding the a3p3y2 subunits of laminin 5 are defective in cases of junctional epidermolysis bullosa, an inherited blistering disor- der of the skin, where adhesion between basal keratinocytes and their underlying basement membrane is disrupted [ 17 - 191. Using mice lacking the lamB2 gene, the p2 chain was shown to be important for both differentiation of the neuromuscular junc- tion 1201 and normal kidney function [21]. The role of laminin a4 is as yet unknown, but in the adult it is highly expressed in the heart and lung.

A small cluster of laminin genes (LAMB3, LAMCZ and LAMC2) are sited on chromosome 1 [14, 22, 231. The other two p genes, LAMB1 and LAMB2, are on chromosomes 7q22 and 31321.3 respectively [lo, 241. The genes LAMA2 and LAMA4 are paired at 6q22-23 and 6q21, respectively 16, 81, while LAMA1 and LAMA3 are located either side of the centromere of chromo- some 18, at 18~11 .3 and 18q11.2, respectively [7, 251. As yet only the murine a5 cDNA sequence has been described and the location of the human gene is unknown. While all of the struc- tures of p and y chain genes have been fully determined [26- 301 only the structure of LAMA2 of the LAMA gene family has been characterised [31]. The LAMB and LAMC gene families have diverged considerably from each other [27]. The LAMA2 gene again differs from those two gene families [31]. Here we have studied the LAMA4 gene to determine if it is structurally similar to LAMA2.

MATERIALS AND METHODS

Yeast artificial chromosome (YAC) library screening. Oli- gonucleotide primers derived from the cDNA sequence were

16 Richards et al. ( E M 1. Biochem. 248)

used to amplify genomic DNA at either end of the gene. The PCR products were sequenced to confirm that they were indeed part of the LAMA4 gene. Similar amplification reactions were then performed using cells from the I. C. I. YAC library [32] held at the human genome mapping project (Hinxton, UK).

Gene structure. Our strategy for determining the exon/in- tron organisation of the LAMA4 gene was to use oligonucleotide primers, derived from the cDNA sequence, to amplify genomic DNA and to detect introns by an increase in size of the PCR product compared with that of cDNA. The templates used were either total human genomic DNA or YAC clones. Once an in- tron-containing product was obtained, it was sequenced from either end until the sequence diverged from that of the cDNA sequence, i.e. into the intron. The donor and acceptor splice site sequences of the intron were determined and then the introns were sized by electrophoresis in agarose gels, along with known standards. Once an intron had been positioned, primers were designed from within determined adjacent exon sequences and used in conjunction with other primers (designed from the cDNA 200-500 bp up or downstream) to amplify adjacent in- trons. Occasionally, when adjacent introns were relatively small, several were contained within a single PCR product. In these cases, primers were then synthesised according to known cDNA sequence, adjacent to the detected introns, and used to sequence, from both ends, the complete exon between the previously posi- tioned introns and into the undetermined splice sites.

For many introns cloned by this strategy, standard Tuq DNA polymerase and amplification conditions were sufficient. For other, larger introns, the technique of long PCR [33] was used. Intron 2 proved difficult to amplify due to non-specific amplifi- cation of DNA from both total genomic DNA and the YAC clones. So this intron was positioned and splice sites determined by two different approaches. Firstly the 5' splice site was sub- cloned from YAC 21HE6. Secondly both the 5' and 3' splice sites were amplified by inverse PCR.

Genomic PCR. Standard amplifications were performed using either total human genomic DNA isolated from blood leu- kocytes or DNA isolated from YAC clones which contained re- gions of the LAMA4 gene. Typically 100-500 ng of DNA was used in reactions containing 20 mM Tris/HCl, pH 8.4, 50 mM KC1, 2.5 mM MgCI,, 200 pM of each dNTP and 25 pmol of two 24-base oligonucleotides derived from the cDNA sequence. After an initial denaturation at 95 "C, amplification was achieved by cycling through temperatures of 95 "C 1.5 min, 65 "C 1.5 min and 72°C 3 min. Amplification of DNA larger than 4 kb was achieved using Taq Plus DNA polymerase (Stratagene) under conditions recommended by the manufacturer. After denatura- tion at 94°C for 3 min, 40 cycles, which typically consisted of, 94°C 0.5 min and 68°C 10 min, were performed.

Cloning and sequencing. Amplified DNA products were made blunt ended with Klenow DNA polymerase and phosphor- ylated with T4 polynucleotide kinase. The DNA was then cloned into the Bluescript vector (Stratagene), transformed into compe- tent Escherichia coli (XL-1 Blue, Stratagene) and recombinant clones selected by blue/white screening. Recombinant white col- onies were secondary screened by amplification using whole bacterial cells in the PCR. This cloned DNA was used for double-stranded DNA sequencing, with primers derived from the vector cloning site sequence. DNA was first denatured with NaOH, precipitated and then sequenced with Sequenase version 11 (Amersham International).

YAC subcloning. To subclone DNA containing the 5' end of intron 2, total DNA from cells containing YAC 21HE6 was first digested with HindIII and Southern blotted. This was probed with labelled DNA amplified from exon 2. The probe hybridized to a band of around 4 kb. The HindIII digestion was

15 kb I

1 2

I

3

I I I

4

I

5 6 7 8 I I . I I 1

9 10 11 12 m I I I I I I .

13 14

I

15 16 17 18 19 1 . I I I 1 1 I w I

20 21 22 23 24 25 26 27 28 29 30 31 m i I I I . I . . .. I I I I I . .

32 33 34 35 36 37 38 I I I I m . I

H I I I I I

39

__1

Fig. 1. Organisation of LAMA4 The exodintron structure of LAMA4 is shown. Horizontal lines represent introns and vertical boxes exons which are numbered. Each line is equivalent to 15 kb.

then repeated and the fragments electrophoresed in an agarose gel, DNA of around 4 kb was excised from the gel, eluted, and cloned into Bluescript. Transformed cells were screened, with a labelled exon 2 probe, and positive clones isolated. These were then used to sequence both the 5' end of intron 2 and the 5' untranscribed region of the gene. The 3' end of the gene was similarly cloned using an exon 39 probe against DNA from YAC 35BF2. A 2.5-kb EcoRI fragment of this region was subcloned and partially sequenced.

Inverse PCR. The donor and acceptor splice sites of intron 2 were amplified in separate reactions by inverse PCR essen- tially as described by Silver [34]. Around 400 ng YAC 21HE6 was digested with either HindIII or EcoRI. After phenollchloro- form extraction and ethanol precipitation, the DNA was resus- pended at a concentration of 10 ng/pl and circularised with T4 ligase in 20 mM Tris/HCl, pH 7.6, 5 mM MgCI,, 5 mM dithio- threitol, and 1 mM ATP at 14°C for 16 h. After ethanol precipi- tation the circularised DNA was used in separate PCR using Taq Plus and either primers within exons 2 or 3, such that elongation proceeded in a direction to amplify the DNA 5' and 3' of each exon.

RESULTS

YAC isolation. A YAC library was PCR screened with primers which amplified regions at the 5' and 3' end of the LAMA4 gene. Clone 21HE6 contained the 5' end, whilst clone 35BF2 con- tained the 3' end of the gene. Clone 35BF2 was previously shown to be chimaeric, and we had previously used subclones constructed from this YAC to localise LAMA4 to 6q21 [8]. Fluo- rescent in situ hybridisation analysis was not performed with 21HE6 and so it is not known if this clone is also chimaeric, however, neither clone contained the complete gene. Further- more both clones lacked the central part of the gene, since PCRs using primers from this region successfully amplified total hu- man genomic DNA but not DNA from either YAC.

Richards et al. ( E m J. Biochem. 248) 17

Table 1. Splice-site sequences. The size of each exon is shown along with the splice-site sequences of each intron. The 5’ and 3’ splice sequences are in bold type. Introns are in lower case and exons in upper case. The phase of each intron is also indicated. Phase 0 introns are situated between complete codons, while phase 1 and 2 introns interrupt a codon after the first and second base respectively. The position within the amino acid sequence is shown under the DNA sequence.

Exon Size Intron splice-site sequences Phase

5’ donor 3’ acceptor

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

bP 142

336

102

125

81

215

75

152

111

112

168

194

117

149

142

91

117

180

CGTACTgtaagctccg UTR

GCCGAGgtacagtgtc A E 64

TGTGTGgtaagttggg c v 98

GGCCAAgtaagtccta

140

TGAAnGgtaagccaat

167

GTGCAGgtgcagtatc

239

CTACAGgcatggactg

264

CTCAAAgtacgcagga L K 314

GAAAAGgtacgtatgt

351

TCCAAGgtaaatatat

389

ACGAACgtaggtgtat

445

CATGAGgtacagcgga H E 509

ATAAnGgtaaggacgc I K 548

A N

E R

A V

T G

E K

Q E

E L

GAGCAGgtaatatcct S R

598

TATGATgtgagtattc

645

AGTCTAgtaaatatct

67 8

AGAGAGgtaagcatca

717

Y D

s s

R G

ATGCAGgtatcactta

777 A V

atttcctaagCTGCTG UTR

tctgttgcagAAATGC

67

tgtctcatagCACTGC

101

ccaattttagTTTTGC

142

tcctcttcagATGTGC

169

tttcccacagTGTGCA

241

tcacctccagGCTGTG

266

tcttctgcagACAAAA

317

ttatttgtagGAAAAT

354

ttcctttcagAGATCA

391

ttcttcccagTACTGA

447

cttcccgcagAAACAA

512

ttttctttagAATGCG

55 1

ttattaacagGAAGTT

600

tcctttgtagGCGGTG

648

tcttccacagGCAGTG

680

ctgtgtaaagGGGATG

719

ttaactgcagTAAGAA

779

K C

H C

F

C

C

C

T K

E N

I

L

K Q

N A

K

A V

S

D

R

0

0

2

2

1

1

0

0

1

1

0

0

2

0

1

1

1

18 Richards et al. ( E m J . Biochem. 248)

Table 1. (continued).

Exon Size Intron splice-site sequences Phase

5' donor 3' acceptor

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

bP 140

174

146

163

134

1 7 2

132

143

139

138

134

165

154

188

190

156

160

131

94

120

143 bp + 566 bp 3' UTR

A G C A A G g t a a c t g a t g S K 823 A A A A A C g t a a g c a c a t K N 88 1 T G A A A G g t a c a g t g a a

930 T T C A A G g t c a g c c a t a

984 T G C C C G g t a a g a g a c a

1029 A A T G G A g t g a g t a a a a N G 1086 C A T G A G g t a c a a a t c a H E 1130 A T C C A G g t a a g g c t t t

1178 T C A C T T g t a a g a a c c a S L 1224 T C A G G G g t a a g t g t t t S G 1270 C A C A A G g t a t a g c t c c

1315 T A C C A G g t a t a a t a t t

1370 A A A A A G g t a a a a g a t a

1421 T G C C A A g t a a a g t a a c

1484 C A T G A T g t a a g t t g a a H D 1547 G T T C A G g t a a g c c a g g V Q 1599 T T C T A G g t a a c a t t a g

1653 G G A C A G g t a g t g t c t g G Q 1696 T T A C A G g t g a g a a a a c

1728 T T C C A G g t a a g t g t t g

1768

E R

F K

A R

S R

T R

T R

K K

A K

L D

T V

P E

g t c a c t c c a g A T C C A A

826 g a t a t t t c a g G C C A A A

884 a t c t c a a t a g G G T G G G

932 t a t t g c a t a g C T C C C T

987 t c c a t t t c a g A G A T A A

1031 c t c t c c t c a g A G T A T G

1089

I Q

A K

V

L P

D

S M

t c t c t c c c a g A T C T C A

1133 t c t t t t c c a g G G C C C T

1180 t g g c a c a c a g A T A T C T

1227 t g c a t t t c a g T C A G A C

1273 t c t t t t g c a g A T A T G A

1317 a c a a a c a c a g G G T G G A

1372 a c t t t t a t a g G G A G G G

1424 c a t t t a c c a g A T C T C A

1486 c t t c a t c c a g G T G A T A

1450 c t t c c c t c a g A T T A A C

1 6 0 2 c t c t c c a c a g A T G A A T

1655 t c t t t t g c a g G T C A T A

1699 t g c a t t t c a g T T A T T A

1730 c t c t t t t c a g A A T C T C

1770

I S

A

I S

S D

Y

V

G G

S

V I

I N

E

V I

I

S

0

0

2

0

2

0

0

2

0

0

2

2

0

2

0

0

1

0

1

1

Richards et al. (Eur: L Biochern. 248)

LAMA2

19

1 5 10 15 20 25 30 35 40 45 50 55 60 64

G DOMAIN

LAMA4 20 25 30 35 39

--- LE LE LE

LYVGGLP

LG MODULE TERMINAL CYSTEINE

DGXWH

Fig. 2. Structural comparison of the LAMA2 and LAMA4 genes. The exon arrangement of LAMA2 and LAMA4 without introns is shown. Exons coding for various domains of each molecule are indicated by lines drawn between the two structures. Exons containing sequences for conserved motifs within them are shaded as indicated. The exons of LAMA4 (3-7) coding for LE modules are expanded and shown in greater detail under the complete gene structure. Regions encoding the three complete LE modules are indicated by a line drawn under these exons.

Gene structure. By amplifying various regions of the LAMA4 gene we were able to assemble the overall structure of the gene without the need for clones covering the entire gene. All introns, except intron 2, were amplified from either total genomic DNA or one of the YAC clones, partially sequenced and sized. The splice sites of intron 2 were obtained by subcloning a 4-kb HindIII fragment from YAC 21HE6 and by inverse PCR. Ampli- fication reactions showed that the 5‘ splice site and around 2.5 kb of intron 2 had been cloned from the YAC. Both the 5‘ and 3’ splice sites were cloned following inverse PCR. Total DNA from YAC clone 21HE6 was digested with HindIII or EcoRI in separate reactions, circularised, and amplified. As ex- pected the product formed using HindIII digestion and exon 2 primers was around 4 kb, identical in size to that directly sub- cloned from the YAC with an exon 2 probe. Both this 4-kb and a 2.5-kb EcoRI, exon 3 containing, amplified product were sub- cloned and their ends sequenced. Oligonucleotides, designed from within the 5‘ end of intron 2 , were used for upstream and downstream PCR, i.e. towards exon 2 and exon 3, respectively. The second of these reactions indicated the size of the remaining uncloned intron 2. From the two reactions the total size of intron 2 was estimated as 12.6 kb.

The full exonlintron structure of LAMA4 extended to over 122 kb (Fig. l), and consisted of 39 exons. The start of exon 1 was taken to be that previously determined by 5’ rapid amplifi- cation of cDNA ends [35]. The end of exon 39 was presumed to be the 3’ cDNA sequence as described by Iivanainen et al. [36]. Sequencing of genomic clones of this region confirmed it to be contiguous to the 3’ cDNA end that we have previously described [35] and this would be consistent with the mRNA size as detected by northern blotting. The exons ranged from 75 bp to over 700 bp (Table 1). Although most of the exons within the coding region were between 100-200 bp. The intron sizes var- ied considerably, from 455 bp to 12.6 kb. The splice-site se- quences of each intron conformed to the GT/AG rule except for intron 7, where the 5’ donor splice site was a GC dinucleotide. Introns occurred at both boundaries of the region of the gene coding for the IIIb derived region, which encoded the LE mod- ules. This domain was coded for by five complete exons, num- bers 3-7. The long arm (domains I and 11) was coded for by exons 8-19, with the end of the long arm and start of the G

domain (as previously determined by comparison with the other laminin chains, [35]) situated within exon 20. The five LG mod- ules were coded for by exons 20-23 (LG1 module), 24-26 (LG2), 27-32 (LG3), 32-35 (LG4) and 35-39 (LG5).

This structure was compared with that of LAMA2, recently described by Zhang et al. [31] which consisted of 64 exons ex- tending over 260 kb. Although the intron sizes varied consider- ably between the two genes, the arrangement of the exons was similar (Fig. 2). The short arm exons 3-7 of LAMA4 were de- rived from exons 15-19 of LAMA2 (see Discussion). The long arm and G domain of a2 and a4 were also coded for by a similar number of exons. Six of these (9134, 13/37, 17/41, 33/59, 34/60, 36/62 of LAMA4ILAMA2) were identical in size, so most exons varied somewhat, between the two genes (Table 2). A number of larger differences could be seen. Two of these involved the small exons, 43 and 52 of LAMA2 (6 bp and 12 bp, respective- ly). The LAMA4 gene did not contain such small exons and the equivalent regions showed some rearrangement. Exon 19 of LAMA4 was a little smaller (140 bp) than the corresponding re- gion of LAMA2 i.e. exons 43 and 44 (6 bp and 155 bp, respec- tively). The 12 bp exon of LAMA2 appeared to be derived from exon 27 (137 bp) of LAMA4 which was split into exons 52 and 53 in LAMA2 (12 bp and 121 bp, respectively). Conversely exon 63 of LAMA2 was split into exons 37 and 38 in LAMA4 (see Discussion). Apart from these areas of rearrangement, i.e. exons 43 and 52 of LAMA2 and 37 of LAMA4, 33 of the 36 other introns of LAMA4 displayed the same phase as the equivalent introns in LAMA2. The three which differed were introns 1 1 , 25 and 29 of D1MA4.

The 5’ region. The genomic clones which contained the 5’ splice site of intron 2 also extended upstream to over 800 bp into the untranscribed region of the gene (Fig. 3). This part of the gene and that of the first intron was sequenced and analysed for regu- latory sequences, by the computer programme SIGNAL SCAN [37]. The gene lacked a TATA box, but three A+T rich regions between - 198 and - 146 may fulfil this function. Just upstream from exon 1 were two CTC boxes, which have previously been found in some extracellular matrix protein genes [38, 391 to which a CTC factor binds. The CTC box may also be a binding site for SP1 [40], and there was also another SP1-binding site

20 Richards et al. (Em J. Biochem. 248)

Table 2. Comparison of exon and intron sizes of LAMA2 and LAMAI. The sizes of exons and introns of LAMA2 and LAMA4 are shown in bp. The size of four of the LAMA2 introns was not determined (n.d.). Exons identical in size in both genes are in bold type. Exons of LAMA4 are placed adjacent to the equivalent exons in LAMA2.

LAMA2 LAMA4

no. . exon size intron size no. exon size intron size

1 2 3 4 5 6 7 8 9

10 11 12 13 14

15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63

64

bP ~

112 171 113 243 270 118 179 100 161 141 174 102 212 112

114 128 87

212 107 181 137 237 144 180 189 134 118 135 125 87

194 143 99

112 163 211 117 164 139 103 117 183

6 155 144 134 160 125 163 145 139

12 121 177 148 178 169 113 190 156 154 131 223

119 + UTR

15 000 n. d. n. d. n. d.

750 5 700 3 800

600 5 000 2 200 7 000 2 000 1100 4 000

3 500 6 500 5 100 3 700

13 000 3 100

n. d. 1600 1100

150 1100 4 400 8 500 8 000 3 900 6 300

300 3 900 8 000 3 700 1 500 7 500 1100 1500 7 000 2 200

10 000 2 000 2 500 5 500 1 000 1900 3 700 3 900

650 700

1800 2 200 2 300 4 700 9 000

300 8 500

450 1900 2 200 4 900 1900 1400

1 2 3 4 5 6 7

8 9

10 11 12 13 14 15 16 17 18

19 20 21 22 23 24 25 26

27 28 29 30 31 32 33 34 35 36 37 38 39

bP ~

142 336 102 125 81

215 75

152 111 112 168 I94 117 149 142 97

117 180

140 174 146 163 134 172 132 143

139 138 134 165 154 188 190 156 160 131 94

120 143 + UTR

167 12670 10230

5 780 9 260 2 300

940

2 385 7 050 2955 2 680 7 540 6380 3 14.5

590 4 590 2375 3510

2 940 5 80 455 855 455

2 750 1480

955

540 1645

895 80.5

6 650 1600

940 1235 1745 1040

500 3 675

in close proximity, within the first exon. The significance of the first intron within the 5’ UTR (untranslated region) is not known, but it consisted of an A+G rich domain flanked by two smaller C+T rich domains. This may be a region important for tran- scriptional regulation of the gene. A sequence AGAGGG occurs five times within the intron (Fig. 3). This sequence also occurs within the polyomavims B enhancer [41]. The transcription factor PEA3 is also known to bind to A+G rich sequences [42]. Alternative splicing of the first intron of the LAMB2 gene was proposed to be involved in translational regulation of the gene, by forming a branch structure close to the initiator codon [30]. Using the computer programme FOLDRNA [43], analysis of LAMA4 RNA structure, either with or without the first intron, showed that the structure around the initiator codon was essen- tially unchanged by inclusion of the first intron (data not shown).

DISCUSSION

The laminin genes can be subdivided into LAMA, LAMB and LAMC families. All of the known LAMB and LAMC gene struc- tures have been determined [26-301. Until now, of the five dif- ferent LAMA genes, only the structure of LAMA2 had been de- termined [31] which contains 64 exons and is over 260 kb in size. Here, we have described the complete structural organisa- tion of LAMA4 which is the second gene structure of the LAMA gene family to be characterised. It consists of 39 exons and spans over 122 kb.

The short arm of a4 is truncated and is composed mainly of LE modules coded for by exons 3-7. These are similar in size (102, 125, 81, 215 and 75 bp) and identical in phase to,exons 15-19 of LAMA2 (114, 128, 87, 212 and 107 bp) which code for the N-terminal end of domain IIIb. Those exons coding for the a2 IIIa domain are exons 26-30 of LAMA2 (134, 118, 135, 125 and 87 bp) which differ markedly in both size and phase from exons 3-7 of LAMA4. This confirms our previous obser- vation, based on protein sequence, that the a4 short arm is de- rived from domain IIIb of the longer a chain molecules. The long arm and G domain of both a2 and a4 are coded for by a similar number of exons with only a few rearrangements. Six of the exons of LAMA4ILAMA2 are identical in size, three being long arm exons and the others coding for parts of the G domain. The five LG modules which form the G domain all contain con- served protein motifs namely an LYVGGLP [5] and an FXGCI sequence [35], and each LG module is terminated with a cyste- ine residue ([S], accession no. S14458). Modules LG4 and LG5 also contain a motif DGXWH [35]. If one ignores the small 12-bp exon of LAMA2 then in both LAMA2 and LAMA4 the pattern of exons coding for these motifs is the same. In modules LG1, LG2, LG4 and LG5, the exon containing the sequence for LYVGGLP is followed by an exon containing the sequence for FXGCI and the cysteine residue which defines the end of the LG module. For module LG3 the arrangement is different to that described above, but the same in both LAMA2 and LAMA4. In this case the LYVGGLP encoding exon, also contains the se- quence for FXGCI (exons LAMA2 56lLAMA4 30). The LG mod- ule terminal cysteine is encoded not by the next exon, but two exons downstfeam from the LYVGGLP encoding exon (LAMA2 58lLAMA4 32). The fusion of exons 37 and 38 of LAMA4 into the single exon 63 of LAMA2 is demonstrated by the DGXWH motif of modules LG5. In LAMA2 both DGXWH and the LYVGGLP motif are encoded by exon 63, whereas in LAMA4 they are in separate exons, namely 37 and 38. While the organi- sation of the two genes are similar, the size of the introns has not been maintained and very little similarity exists. However

Richards et al. ( E m J. Biochem. 248) 21

-329

-279

-229

-179

-129

-79

-2 9

22

72

122

172

222

272

322

372

422

472

cagaaaaaac ccqagaaacc ttgctcaaga aagtgggcac cctgaccctc

tgccttcaga gatcatctac ctttggttcc tgccctggaa tcccttttcc

acccactttc ctgcaaacta gcgggggcca cataaaatat ctatgtaaat

atagataaga catccggtac cttgactaca taaataagag tggtgaggaa

gcgtcacaaa agctgaatat tcttctggag cccttggagg ggctccaaac

tgaga+=+agaccq cagggaaagg cggacctcag tgtctgaaaa

gccagcttag ag*=i cctgggagtA GAAGGTAA~U AGGGAGTGGT 0

GAOAATGAAT GTGAGAAGGA AGCCAGGACA GCGCAGTCCC CAGTCCCGAA

CGGCCAGGGA GAGGAGGTGG CCTAGCGC+T+TCA CCCCAATCCG

TCTGCCTTTT GATGCCGTAC Tgtaagctcc gtccatctgt aggttcttga

CAGCCACCTC GGGATACTGC ACACGGAGAG OAOOOAAAAT AAGCGAGGCA

CCGCCGCACC ACGCGGGAGA CCTACGGAGA CCCACAGCGC CCGAGCCCTG

GAAGAGCACT ACTGGATGTC AGCGGAGAAA TGGCTTTGAG CTCAGCCTGG

CGCTCGGTTC TGCCTCTGTG GCTCCTCTGG AGCGCTGCCT GCTCCCGCGC R S V L P L W L L W S A A C S R A

M A L S S A W

Fig. 3. 5’ gene sequence. The 5’ untranscribed region and that of exon 1, intron 1 and part of exon 2 are shown. The start of exon 1 is + 1 and is indicated with a circle below the sequence. Untranscribed DNA and introns are in lower case, exons are in upper case. The A+T rich regions between ~ 196 and - 146 are underlined. The CTC boxes (CCCTCCC) at - 16 and -73 are boxed and are on the complementary strand. The SP1 site in exon 1 is also boxed. The AGAGGG sequences within intron 1 are marked with asterisks. The DNA coding for the signal peptide has the amino acid sequence written under the DNA sequence.

both genes are large with particularly high intronlexon ratios, 17.8: 1 for LAMA4 and over 27: 1 for LAMA2.

Unlike all other laminin chains including /3 and y chains, laminin a4 does not contain either a domain IV or domain VI (laminin a3A also has neither, but the alternative LAMA3 tran- script, a3B has a domain IV). This suggests that these regims have been deleted from LAMA4. A similar situation exists in the LAMB3 gene, which also lacks a region coding for part of the short arm [28]. The boundary of the deleted region in LAMA4, between the short and the long arm, has an unusual splice site. The donor splice site of intron 7 has a GC dinucleotide instead of the normal GT sequence. Although rare, this splice sequence has previously been noted in several genes and in particular three other genes coding for extracellular matrix proteins [30, 44, 451. Its significance is not known. In the LAMB2 gene it was associated with alternative splicing, but we have no evidence of this in LAMA4.

As predicted by Zhang et al. [31] the structural arrangement of LAMA4 is similar to LAMA2 for those regions which the two molecules have in common. Thus all three laminin gene families have diverged considerably from each other, yet intra family members remain structurally similar. However the degree of similarity between LAMA2 and LAMA4 is somewhat less than between genes within the LAMB or LAMC families. For instance while 6 of 39 LAMA4 exons are identical in size to LAMAZ, 211 33 exons of LAMB2 and 8/23 exons of LAMB3 are identical to LAMBI. The LAMC2 gene has 16 of 23 exons identical in size to the LAMCI gene. However, while the exon sizes vary, the

phase of the exonlintron boundaries is very well conserved be- tween LAMA2 and LAMA4. Although the laminins are of a mod- ular structure, they are old molecules and therefore do not ex- hibit the repetitive phase 1 introns characteristic of modules which have evolved via exon shuffling [46]. However, alter- ations to the genes during evolution could potentially alter the reading frame. The deletion in LAMA4 effectively removes 1893 bp of corresponding LAMA2 cDNA sequence, or exactly 631 amino acids. Thus the reading frame is unchanged. Exactly how the LAMA gene family has evolved is unclear. The close proximity of LAMA4 and LAMA2 (6q21 and 6q22-23) suggests that these have arisen by duplication. However the a4 chain is more closely related to the a3 and a5 chains [9, 351, so LAMA4 may be derived from one of these genes, rather than as suggested solely by chromosomal location. In any case one might expect that the organisation of LAMA3 and LAMAS would be more sim- ilar to LAMA4 for those regions coding for the long arm and G domain than LAMA2. The Drosophila LAMA gene is the equiva- lent of the vertebrate LAMAS gene [9]. However its structure is considerably different from the human LAMA2 and LAMA4 genes. It contains only 15 exons within 14 kb [47]. This prob- ably reflects both gain of intronic sequences in the human genes and possibly loss of such regions from the Drosophila gene. It will probably also differ greatly from the vertebrate LAMAS gene. Comparison of all these gene structures, when known, may provide some clues as to how this gene family has evolved. Alteration of the laminin a4 gene in transgenic mice may also produce some evidence of its biological function.

22 Richards et al. ( E m J. Biockem. 248)

C. Luccarini was supported by a grant from the dystrophic epider- molysis bullosa research association (DEBRA) UK.

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