Matrix Biology 19Ž. 2000 671᎐682

Revised genomic structure of the human MAGP1 and identification of alternate transcripts in human and mouse tissues

Fernando Segadea,U, Thomas J. Broekelmanna, Richard A. Pierceb, Robert P. Mechama

aDepartment of Cell Biology and Physiology, Washington Uni¨ersity School of Medicine, St. Louis, MO 63110, USA bDepartment of Internal Medicine, Washington Uni¨ersity School of Medicine, St. Louis, MO 63110, USA

Received 21 June 2000; received in revised form 15 August 2000; accepted 17 August 2000

Abstract

The human MAGP1 Ž.or MFAP2 and mouse Magp1 code for the microfibril-associated glycoprotein-1 Ž.MAGP-1 , an extracellular matrix of microfibrillar structures. We report a revised 5Ј genomic structure including the use of a single start site that gives rise to a 32-bp 5Ј exon spanning a segment of the previously described exon B. No evidence of heterogeneous 5Ј ends from the use of alternative promoters was found in human tissues and cell lines. We located the genetic marker D1S170 to a position 3 kb downstream of the polyadenylation site. Large-scale comparison of the human and mouse genes revealed conservation of sequence outside the coding exons. Although the 5Ј flanking regions were found to be divergent certain cis-elements for transcription factors are conserved, including Sp1, AP-2, AP-4, NF-␬B, and c-ETS motifs. We identified a total of five splice variants in addition to the canonical MAGP1ArMagp1A form. These transcripts are species-specific and are generated by different processing mechanisms. The alternate forms MAGP1AЈ, MAGP1B, and MAGP1C are expressed in human tissues; and the two variants Magp1AЈЈ and Magp1D were found only in mouse. The alternatively spliced forms show restricted patterns of expression relative to the canonical isoform. ᮊ 2000 Elsevier Science B.V.rInternational Society of Matrix Biology. All rights reserved.

Keywords: MAGP1; Gene structure; Microfibrils; Alternate transcripts

Abbreviations: EST, expressed sequence tag; PIP, percent identity plot; RACE, rapid amplification of cDNA ends; tsp, transcription start point U Corresponding author. Tel.: q1-314-362-2212; fax: q1-314-362-2252. E-mail address: [email protected]Ž. F. Segade .

0945-053Xr00r$ - see front matter ᮊ 2000 Elsevier Science B.V.rInternational Society of Matrix Biology. All rights reserved. PII: S 0 9 4 5 - 0 5 3 XŽ. 0 0 0 0 1 1 5 - 3 672 F. Segade et al. rMatrix Biology 19() 2000 671᎐682

1. Introduction sites for either 5Ј exon and, therefore, their exact lengths were not establishedŽ. Faraco et al., 1995 . Microfibrils are 10᎐12 nm filaments found in most Our current research on the MAGP-1 gene expres- tissues, particularly elastic tissues, bone, and cartilage sion required a detailed sequence comparison between Ž.Mecham and Davis, 1994 . Microfibrils are complex human and mouse loci to search for motifs, mostly in structures formed by the assembly of a large number the flanking sequences and introns, whose high con- of , including, fibrillins-1 and -2, latent trans- servation across species are suggestive of a role in the forming growth factor ␤1-binding protein-2Ž. LTBP-2 , transcriptional regulation of the gene. We undertook and microfibril-associated glycoproteins-1Ž. MAGP-1 the characterization and complete sequencing of more and -2Ž Gibson et al., 1986, 1996; Mecham and Davis, than 10 kb of the mouse Magp1 locus for transcriptio- 1994.Ž . MAGP-1 is a small protein 31 kDa for the nal regulation studies. The availability of extensive mature secreted form. , with two distinctive domains: databases, both of expressed cDNAs and of extremely the amino-terminal half of the molecule is highly long contigs of genomic human sequence allowed us acidic, enriched in proline, and contains a clustering to compare the human and mouse loci in order to find of glutamine residues, whereas the carboxy-terminal out the conserved motifs probably related to gene portion contains all 13 cysteine residues and has an regulation and to clarify the role that differential overall net positive charge. The precise role that promoter usage could play in the regulation of the MAGP-1 plays in microfibril assembly has not been human MAGP1. By precise mapping of the 5Ј end of totally clarified, although there is evidence that it may the MAGP1 mRNA and amplification analysis of hu- serve to facilitate binding onto the microfibril man tissue cDNA, we establish that a segment of the Ž.Brown-Augsburger et al., 1996 . Disulfide bonding exon previously designated as exon B is the only 5Ј between cysteines apparently mediate the binding of non-coding exon in normal human tissue. We also MAGP-1 to the microfibril, since the presence of report the complete structure and sequence of the reducing agents is a strict requirement for the extrac- mouse Magp1 gene as well as the identification of tion of MAGP-1 from tissuesŽ. Gibson et al., 1991 . splice variants of the human and mouse genes that The protein undergoes post-translational removal of code for non-secreted forms of the protein. its signal peptide, sulfation of several tyrosines, and O-linked glycosylation. In mouse development, MAGP-1 mRNA is widely expressed, mainly in mes- 2. Experimental procedures enchymal and connective tissue cells where it is easily detected as early as day 8.5 of developmentŽ Chen et 2.1. Sources of DNA clones al., 1993. . In vascular development, MAGP-1 is ex- pressed by all vascular cells that make elastin and EST cDNA clones from the IMAGE consortium co-localizes to extracellular elastic fibers. Ž.Lennon et al., 1996 were obtained from Genome The mammalian genes for MAGP-1 have been Systems Inc.Ž. St. Louis, MO, USA . PAC clone 37C10 isolated in humanŽ.Ž Faraco et al., 1995 , mouse Chen containing the entire human MAGP1 gene sequence et al., 1993.Ž , and bovine Bashir et al., 1994 . . In from male blood library RPCI-1 was purchased from humans, MAGP-1 is encoded by a single copy gene the Roswell Park Cancer InstituteŽ Buffalo, NY, Ž.MAGP1 or MFAP2 located in 1p36.1- USA. . Human kidney genomic DNA was obtained p35Ž. Faraco et al., 1995 , a region with conserved from ClontechŽ. Palo Alto, CA, USA . synteny to mouse chromosome 4, where the ortholo- gous Magp1 gene mapsŽ. Chen et al., 1993 . MAGP1 2.2. Isolation and characterization of mouse Magp1 maps within the congenital muscular dystrophy with genomic clones rigid spine syndrome locus Ž.RSMD1 and has been proposed as a candidate geneŽ Moghadaszadeh et al., An EcoRIrNotI fragment corresponding to posi- 1998. . The human, mouse, and bovine genes are di- tions 16᎐1020 of the mouse Magp1 cDNAŽ Chen et vided into eight coding exons whose splice sites, co- al., 1993; GenBank Accession No. L23769. was ob- don splice phase, and protein coding sequence are tained by digestion of the IMAGE consortium clone highly conserved. Whereas a 32-bp 5Ј untranslated AA268095 and used as a probe. The probe was radi- exon is present in mouse, two alternatively used 5Ј olabeled by the random-priming method and used to untranslated exonsŽ. named A and B located 2.0 and screen approximately 1=106 plaques from a mouse 2.8 kb upstream of exon 2 have been described in the strain 129rSvJ genomic library constructed in the humanŽ. Faraco et al., 1995 . A complex regulation of ␭FIX II vectorŽ. Stratagene, La Jolla, CA, USA . MAGP1 through the generation of Plaques giving a positive signal were picked and puri- multiple transcripts was then envisioned. Neverthe- fied by two subsequent rounds of hybridization. Phage less, the precise location of the transcription initiation DNAs were analyzed by restriction endonuclease F. Segade et al. rMatrix Biology 19() 2000 671᎐682 673 mapping and DNA sequencing. Four independent electrophoretic separation of 15 ␮g of total RNA in Magp1 genomic clones were isolated. Clone 1.2% agarose-formaldehyde denaturing gels and visu- ␭Magp1.43 containing an insert of approximately 14 alized by ethidium bromide staining. kb was selected for the complete sequencing of the Magp1 transcription unit and flanking regions. The 2.5. Primer extension analysis Magp1 sequence was assembled from subcloned re- striction fragments, exon-to-exon PCR amplification The origin of transcription of the human MAGP1 products, or directly from the ␭Magp1.43 DNA. Final mRNA was established by primer extension analysis, assembly included sequence obtained from 129rSvJ performed according to published procedures genomic clones kindly provided by Dr J. Bonadio Ž.Sambrook et al., 1989 , using 5 ␮g of total RNA as a Ž.University of Michigan . Sequence data have been template and 1 pmol of end-labeled antisense primer deposited with the GenBank Data Library under Ac- OHMS-PEŽ. Table 1 . The primer and RNA were cession No. AF268473. preheated at 70ЊC for 10 min and then annealed at 37ЊC for 10 min. Following extension at 42ЊC for 30 2.3. DNA sequencing min in the presence of 200 units of Superscript II reverse transcriptaseŽ GibcoBRL, Gaithersburg, MD, Five hundred to 10 000 ng of template DNA was USA. , the reaction products were subjected to elec- sequenced using ABI Big Dye terminator chemistry trophoresis through a 6% polyacrylamider7 M urea Ž.PE Applied Biosystems, Foster City, CA, USA . Re- sequencing gel. A sequence ladder served as a molec- actions were purified by phenol᎐chloroformŽ 1:1 ular weight marker. volrvol. extraction and precipitation with 9 vols of n-butanolŽ. Tillett and Neilan, 1999 , dried in a speed- 2.6. Rapid amplification of MAGP1 cDNA 5Ј end( 5Ј vac evaporator, resuspended in loading buffer, and RACE) run on ABI sequencers at the Washington University Protein and Nucleic Acid Chemistry Laboratory DNA 5Ј RACEŽ. Frohman et al., 1988 was used to char- sequencing facility. Sequences were extracted and ma- acterize the 5Ј end of the human MAGP1 mRNA and nipulated with ABI software. was performed with the GibcoBRL 5Ј RACE kit. Total MG63 RNAŽ. 1 ␮g was reverse-transcribed 2.4. RNA isolation using 2.5 pmol of oligonucleotide OHMS-1Ž. Table 1 as a primer and 200 units of Superscript II reverse Total RNA was isolated from MG63 human transcriptase for 50 min at 50ЊC. After hydrolysis of osteosarcomaŽ. ATCC CRL-1427 or A2058 human the RNA template, purification and homopolymeric melanoma cellsŽ. ATCC CRL-11147 using the isothio- tailing of the cDNA were carried out following the cyanateracid phenol methodŽ Chomczynski and Sac- manufacturer’s indications. A first round of amplifi- chi, 1987. . The integrity of the RNA was checked by cation was performed using Ready-to-Go PCR beads

Table 1 Primers used in the present worka

Name Species Position Orientation Sequence

OHMS-PE Humanb 30᎐47Ž. AS 5Ј-TCTCATGGCAACAAAGAG-3Ј OHMS-1 Humanb 645᎐663Ž. AS 5Ј-AAGCACCAGGTCAGGTAGC-3Ј OHMS-2 Humanb 506᎐527Ž. AS 5Ј-CACGCCACATTTGGAGAACTTG-3Ј HK-2 Humanb 324᎐345Ž. AS 5Ј-GGTACTGTTCCTCACGGCAGTCAAG-3Ј OHMS-A Humanc 393᎐409Ž. S 5Ј-CAGTGAACGGAGTCACA-3Ј OHMS-B Humanc 1321᎐1337Ž. S 5Ј-ACTCGGAGCGGGCGGCG-3Ј OHMS-5 Humanb 30᎐47Ž. S 5Ј-GCCTCTTTGTTGCCATGA-3Ј HM-5 Humanb 39᎐56Ž. S 5Ј-TTAGAATTCGCCATGAGAGCTGCCTAC-3Ј HM-3 Humanb 573᎐591Ž. AS 5Ј-TTATCTAGAGCAGCTCCCACAGCTCCT-3Ј OMM-5 Moused 10᎐31Ž. S 5Ј-CGCACTAGAGCACAGCGCAGAC-3Ј OMM-UTR Moused 600᎐621Ž. AS 5Ј-CACAGCTCCTGGCACAGGAGGC-3Ј OMM-5Eco Moused 47᎐62Ž. S 5Ј-TTAGAATTCTCAACGCCACCACCG-3Ј OMM-5Bam Moused 45᎐62Ž. S 5Ј-TTAGGATCCTCAACGCCACCACCG-3Ј OMM-3Xba Moused 615᎐630Ž. AS 5Ј-TTATCTAGAGCAGCCCCCACAGCT-3Ј

aNotes. AS, antisense; S, sense. b Human MAGP1 cDNA Accession No. U19718. c Human MAGP1 exons A and B Accession No. U19719. d Mouse Magpl cDNA Accession No. L23769. 674 F. Segade et al. rMatrix Biology 19() 2000 671᎐682

Ž.Amersham Pharmacia, Piscataway, NJ, USA with 5 2.8. Software ␮l of dŽ. C -tailed cDNA as a template, the MAGP1- specific primer OHMS-2Ž. Table 1 , and the kit- Primary sequence input, assembly, basic alignment, provided Abridged Anchor Primer. After an initial conceptual translation, and restriction site prediction denaturation step at 94ЊC for 2 min, 25 cycles of were performed with the LASERGENE package of denaturation at 94ЊC for 30 s, annealing at 55ЊC for programsŽ. DNASTAR, Madison, WI, USA . The 30 s, and extension at 72ЊC for 1 min were carried Gapped-BLAST programŽ. Altschul et al., 1997 was out. The amplification products were then diluted used for the searches of the GenBank non-redundant 1:100 and 5 ␮l was used for a second round of PCR in and Expressed Sequence TagŽ. EST databases at the the same conditions as before but using the provided National Center for Biotechnology Information. Abridged Universal Amplification Primer and HK-2 Repetitive elements in the genomic sequences were as a specific nested primerŽ. Table 1 . RACE products identified and masked with the RepeatMasker pro- were gel-purified and sequenced in their entirety. gramŽ Smit and Green; at http:rrftp.genome. washington.edurRMrrepeatmasker.html. . The re- sulting sequence was aligned using the PIPMaker 2.7. Amplification of sure-RACE and rapid-scan cDNA programŽ Schwartz, et al., 2000; at http:rr panels globin.cse.psu.edurpipmakerr. and visualized as a plot in which regions between successive gaps were converted into segments of percent identity relative to Panels containing tissue-specific cDNAs generated positions in the human sequence, with the resulting by oligoŽ. dT -primer reverse transcription of human data then drawn as a percent identity plotŽ. PIP . q Ž.Ž.Ž.Sure-RACE or mouse Rapid-Scan poly A RNAs Segments with an identity of 50% or greater were were obtained from OriGene Technologies Inc. plotted, so regions that match poorly appear blank. Ž.Rockville, MD, USA . For the amplification of hu- Gaps within an alignment appear as discontinuities man MAGP1 cDNAs with alternate 5Ј ends, the between adjacent horizontal lines. The evaluation of sure-RACE cDNAs were dissolved in 25 ␮lof10mM evolutionary distances between the coding sequences Tris, pH 8.0, 1 mM EDTA, according to the manufac- of the human and mouse genes was carried out with turer’s recommendations. Five microliters Ž.␮lof SNAP softwareŽ SynonymousrNon-synonymous Anal- cDNA were used as templates in 25-␮l Ready-to-Go ysis Program. at http:rrhiv-web.lanl.gov. The pro- PCR reactions, with 10 pmol of the common anti- gram calculates synonymous and non-synonymous sense primer HK-2 and the appropriate 5Ј-end speci- substitution rates on a set of codon-aligned nu- fic primers OHMS-A or OHMS-BŽ. Table 1 . After an cleotide sequences, based on the method of Nei and initial denaturation step of 2 min at 94ЊC, the PCR GojoboriŽ. 1986 . Briefly, the number of synonymous proceeded for 30 cycles with 30 s each at 94, 55, and and non-synonymous codon changes are counted, as 72ЊC. The amplification of human MAGP1 splice vari- well as the number of potential synonymous and ᎐ ants from the Sure-RACE panel was carried out with non-synonymous changes. A Jukes Cantor correction 10 pmol each of the sense primer OHMS-5 and the is then applied to account for multiple hits to reach antisense OHMS-2Ž. Table 1 for 20 cycles with 1 min the final ds and dn expressed in Table 3. If the each at 94, 57, and 72ЊC. For the identification of proportion of substitution reaches 0.75, the correction mouse splice variants, the Rapid-Scan cDNAs were is not applied and a NA value is obtained. The Ј dissolved in 25 ␮l of amplification mix containing 10 analysis of the 5 flanking regions of the genes to identify putative transcription factor binding sites was pmol each of the primers OMM-5 and OMM-UTR done with the MatInspector version 2.2 program Ž.Table 1 , 0.2 mM each of the four dNTPs, and 0.5 Ž.Quandt et al., 1995 on the TRANSFAC Version 3.5 units of Taq DNA polymeraseŽ Roche Molecular databaseŽ. http:rrtransfac.gbf-braunschweig.de . Pre- Biochemicals, Indianapolis, IN, USA. . After an initial diction of protein localization sites in cells was done denaturation step of 3 min at 94ЊC, 25 cycles of with the PSORT II program at http:rr denaturation at 94ЊC for 1 min, annealing at 57ЊC for psort.nibb.ac.jp. 1 min, and extension at 72ЊC for 1 min were per- formed. The human MAGP1 or mouse Magp1 ampli- fication products were detected by Southern blotting using as probes the radioactively-labelled inserts in 3. Results and discussion the EST clones H27217 or AA268095, respectively. The bands identified as positive were sliced from gels 3.1. Characterization of the genomic clones run in parallel, purified through QiaExtract columns Ž.Qiagen Inc., Valencia, CA, USA , and sequenced. The structures of MAGP1 and Magp1 genes were F. Segade et al. rMatrix Biology 19() 2000 671᎐682 675 determined from human and mouse genomic clones, We could not locate any 5Ј flanking genes in respectively. To obtain Magp1 genomic sequences, ␭MAGP1.43 but the search of the mouse EST 1 000 000 clones from a mouse library were probed database with the 10-kb contig identified four ESTs with a full length 1.0-kb mouse Magp1 cDNA result- ŽAccession Nos. AI430252, AW125482, AI510075, and ing in four independent positive clones. Restriction AA052659. , with homology to the 3Ј end of the con- endonuclease digestion and Southern hybridization tig. The complete sequencing of EST clones AA052659 indicated that clones ␭ Magp1.43, ␭ Magp1.5, and AI430252 established that they code for the mouse ␭Magp1.6, and ␭Magp1.7 partially overlapped and ortholog of KIAA0445. However, in mouse the size of contained Magp1 gene sequences. Comparison the intergenic region is just 663 bp. Such a close between the hybridization patterns of mouse genomic proximity of the MAGP1rMagp1and KIAA0445 genes DNA and the isolated phage clones digested with the makes it possible to envision a coordinated mecha- appropriate restriction enzymes indicated that the nism for their expression. Our BLAST searches found 14-kb insert in clone ␭Magp1.43 comprised the entire that the D1S170 microsatellite marker relative to Magp1 gene. The analysis also verified that Magp1is which MAGP1 was originally mapped is contained in present as a single copy in the mouse genomeŽ data the 37C10 sequence. The 449-bp D1S170 sequence not shown. . The ␭Magp1.43 clone was selected for Ž.Accession No. AJ009946 is situated approximately 3 further analysis and sequencing. A 10.5-kb sequence kb downstream from MAGP1 within the KIAA0445 contig derived from ␭Magp1.43 was assembled, in- transcription unitŽ. positions 186096᎐186546 in 37C10 cluding the Magp1 transcribed region, 3.7 kb up- Ž.Fig. 1a . stream of the transcription initiation site, and 1.2 kb downstream of the polyadenylation signal. The se- 3.3. Large-scale sequence comparison quence has been deposited in GenBank with acces- sion number AF268473. A search of the GenBank The human and mouse genomic sequences were database using the Gapped-BLASTN comparison aligned and subjected to comparative analysis. A pair- programŽ. Altschul et al., 1997 with the human wise comparison found that a region with high se- MAGP1 cDNA sequenceŽ Accession No. U19718, ex- quence similarity to the mouse ␭MAGP1.43 contig ons 2-9; Faraco et al., 1995. matched to a 216-kb covers a 13.5-kb segment of 37C10 between positions genomic sequenceŽ. Accession No. AL049569 from 171 and 184 kb. A graphical representation was ob- PAC clone 37C10Ž Sanger Centre Human Chromo- tained using a percent identity plotŽ. PIP in which the some 1 Mapping Group. . Hybridization analysis and percent identityŽ. from 50 to 100% in each gap-free PCR amplification of DNA from human kidney or aligning segment is plotted using the coordinates of from clone 37C10 confirmed that the PAC contains the human sequenceŽ. Schwartz et al., 2000 . Four the entire MAGP1 gene and it is an accurate repre- gap-free sections were found in the alignment, namely, sentation of the genomic region surrounding the gene human 326᎐855 and mouse 587᎐1100 at 60% identity; Ž.not shown . human 4873᎐11152 and mouse 3632᎐9076 at 50% identity, within the MAGP1rMagp1 transcription unit; 3.2. Neighboring genes and markers human 12677᎐12755 and mouse 9723᎐9806 at 75% identity; and human 13117᎐13335 and mouse The availability of more than 200 kb of human 10072᎐10277 at 70% identity, corresponding to the 3Ј sequence from 37C10 allowed a long-range analysis to UTR of the KIAA0445rKiaa0045 geneŽ. Fig. 1b . The identify genes located in the neighborhood of MAGP1. majority of the coding exons could be identified by After masking the sequence to exclude repetitive ele- visual inspection of the PIP, being the longest ments from the comparisons, a BLAST search of the stretches with high similarity. Remarkably, very short non-redundant GenBank and the human EST sequences that are situated mostly within intronic databases located the transcription units of four pre- regions are even better conserved that most coding viously identified genes. Their relative sizes, accession segments. It is tempting to speculate that they repre- numbers, and orientation relative to MAGP1 are pre- sent putative regulatory regions such as binding sites sented in Fig. 1a. The closest gene in the 5Ј vicinity of for specific transcription factors. It is also noticeable MAGP1, situated 5.3 kb upstream from MAGP1, that no obvious sequence conservation is found in the matches the partial sequence AJ009947 and codes for 5Ј flanking regions of the genes where a number of a putative ATPase protein in a sense orientation repetitive sequences are present. A close analysis of relative to MAGP1. In the 3Ј flanking region and in the unaligned section of MAGP1 immediately up- an antisense orientation we located the 6.5 kb stream of the CpG island with the RepeatMasker KIAA0445 mRNAŽ Seki et al., 1997; Accession No. program detected a large number of inserted retro- AB007914. , whose 3Ј UTR ends approximately 1.5 kb transposed elements, including total or partial SINE downstream from the MAGP1 polyadenylation signal. sequencesŽ. 5 Alu and 1 MIR elements , two partial 676 F. Segade et al. rMatrix Biology 19() 2000 671᎐682

Fig. 1. Long-range organization of the greater human and mouse MAGP1rMagp1 regions.Ž. a Schematic overview of the location of genes identified in both the human and mouse genomic sequences. Arrows indicate the direction of transcription. The available human sequence spans all five genes as well as extensive amounts of flanking DNA whereas the generated mouse sequence ends in the last intron of the Kiaa0445 gene on the downstream side. From left: genes for PAD-H19 peptidyl arginine deiminase type IIŽ. Accession No. AB030176.1 , succinate dehydrogenase iron-protein subunitŽ.Ž sdhB Accession No. U17248 . , a putative ATPase Ž Accession No. AJ009947 . , MAGP1, and gene for KIAA0445Ž. Accession No. AB007914 . The position of the human microsatellite marker D1S170 is indicated below the KIAA0445 locusŽ.Ž.Ž. positions 186096᎐186546 in 37C10 . b PIP Schwartz et al., 2000 for human and mouse genomic sequences. Features in the PIP: tall black rectangle, exon; tall white rectangle, simple repeat; black pointed box, L2-type repeat; dark gray pointed box, LTR repeat; black triangle, MIR-type repeat; light gray triangles, other SINEŽ. Alu -type repeat; short white rectangle, regions where CpGrGpCG0.60; short dark gray rectangle, regions where CpGrGpCG0.75.Ž. c Domain structure of the MAGP1rMagp1 mRNA and encoded domains: SP, signal peptide; QRD, Gln-rich acidic domain; CRD, Cys-rich domains. Schematic depiction of the exons involved in the generation of the MAGP1rMagp1 splice variants. Stars indicate the use of cryptic splice sites within exons.Ž. d Nucleotide sequences of the splice junctions involved in the generation of human MAGP1AЈ and mouse Magp1AЈЈ variants. The exons are indicated by uppercase and the introns by lowercase letters. 5Ј and 3Ј splice sites in the alternate transcripts are in bold.

LINE-2 elements and two solitary retroviral long Ž.ŽTAACCCn Meyne et al., 1990 . extends for more terminal repeatsŽ.Ž. LTR Fig. 1b . A similar analysis of than 800 bp in a region situated between 1.0 and 1.8 the unaligned Magp1 sequence found only three kb upstream of mouse exon 1. As shown in the PIP transposable elements: two partial SINE sequences the sequence for the human-specific exon A of Ž.B1 Alu-like elements and a solitary retroviral LTR, MAGP1 is located overlapping the 5Ј end of a retrovi- with no obvious conservation of position or sequence ral LTR and the 3Ј end of an adjacent partial LINE-2 with the human elements. However, a very large sequence in the reverse orientationŽ. Fig. 1b , whereas tandem repeat with the consensus sequence exon B occurs in an 800-bp CqG-rich section that is F. Segade et al. rMatrix Biology 19() 2000 671᎐682 677 consistent with the CpG island feature located in the vicinity of the 5Ј ends of active genesŽ Larsen et al., 1992. . There are examples of genes whose promoters or other regulatory regions, even 5Ј UTRs, have been provided by the insertion of transposable elements Ž.Britten, 1997 . The fact that exon A has only been identified in MAGP1 and not in its orthologs in mouse and bovine suggested a transcriptional mechanism specific for the human gene based on the differential use of two alternative 5Ј exonsŽ. Faraco et al., 1995 . In order to clarify in which circumstances the two putative types of transcripts are generated we decided to study the pattern of expression of exon A- and B-containing mRNAs in normal human tissuesŽ. Chen et al., 1993 . Since a survey of human tissues using Northern blot hybridization was precluded by the almost identical size of the putative exon A- and exon B-containing transcripts, we used instead a panel of commercially available human tissue cDNAs as a source of tem- plates for PCR analysis. To specifically amplify cD- NAs with their 5Ј ends derived from either exon A or exon BŽ. Accession No. U19719 , parallel reactions were carried out with the same antisense primer, HK-2Ž. exons 6᎐7, Table 1 , and the exon-specific primers OHMS-A or OHMS-BŽ. Table 1 . The ampli- fication products were analyzed by Southern hy- bridization using a radiolabelled human MAGP1 cDNA as a probe. Results are shown in Fig. 2a, where a 570 bp band corresponding to MAGP1 mRNA amplification is detected only in those samples that Fig. 2. Identification of the transcription initiation site in human were amplified with the exon B-specific OHMS-B Ј Ј MAGP1.Ž. a Analysis of the 5 ends of MAGP1 transcripts by primer. No amplification of cDNAs containing 5 se- RT-PCR of a multitissue human cDNA panel. Samples were ampli- quence derived from exon A was observed even when fied using oligonucleotides OHMS-A or OHMS-B as sense primers, the amplification was extended to 45 cyclesŽ data not and HK-2 as antisense primer. The amplified DNA fragments were shown. . Similar results were obtained when RNA fractionated by agarosew 1.5%Ž. wrvx gel electrophoresis, trans- from MG63 and A2058 cells, known to express ferred to nylon membranes, and hybridized with a human full length MAGP1 cDNA. A representative result of the amplification MAGP1, was used as a template for reverse transcrip- with the exon B-specific primer OHMS-B is shown where the tion-coupled PCRŽ.Ž. RT PCR see below . 570-bp MAGP1 band is indicated. Parallel experiments with exon A-specific OHMS-A primer were unsuccessfulŽ.Ž. not shown . b 3.4. Characterization of the 5Ј end of human MAGP1 Primer extension analysis. The primer extension products obtained using an antisense primerŽ. OHMS-PE specific to exon 2 were Ј resolved on a sequencing gel. Lane 1, sequencing ladder of unre- To investigate the size and structure of the 5 UTR lated DNA used for size determination; lane 2, yeast tRNA; lane 3, of the MAGP1 mRNA we first determined the tran- A2058 RNA; and lane 4, MG63 RNA. The arrow indicates the start scription start siteŽ. s Ž tsp . of the gene by primer site of the MAGP1 transcripts, located 74 nt upstream of the extension. Total RNA isolated from the MAGP1-ex- initiator ATG.Ž. c Nucleotide sequence of the 5Ј flanking region Ј Ј pressing A2058 human melanoma cells or MG63 hu- and 5 end of the human MAGP1 gene obtained by 5 RACE. The nucleotide sequence of exon 1 is shown in uppercase, and the man osteosarcoma cells was used as template for the preceding sequence is in lowercase. The sequence reported as exon extension of an end-labeled primerŽ OHMS-PE, Table BŽ. Chen et al., 1993 is in italics. The Inr element is underlined. 1. corresponding to positions 47᎐30 on the non-cod- The A at the initiator sequence is defined as position 1 of the ing strand of the Faraco et al.Ž. 1995 cDNA sequence. MAGP1 mRNA. The 5Ј splice donor site of exon 1 is doubly As shown in Fig. 2b, the estimated size of the ex- underlined. tended product is 60 nt. Since the 5Ј ends of the first exons were not accurately established, but were only extension bands in the range of 90᎐100 nt. The fact estimated to be at least 73 bp for exon A, and 58 bp that no other specific band arising from a minor for exon BŽ. Faraco et al., 1995 , we expected primer transcript was detected in a number of experiments 678 F. Segade et al. rMatrix Biology 19() 2000 671᎐682

Table 2 Exonrintron organization and sequence similarity of MAGP1rMagp1a

Region HumanŽ. bp Mouse Ž. bp % Simil. Region Human Ž. kb Mouse Ž. kb % Simil.

Exon 1Ž. UTR 32 41 ns Intron 1 2.3 2.1 54 Exon 2Ž. UTR 41 39 74 Exon 2Ž. coding 37 37 89 Intron 2 1.0 0.9 47 Exon 3 90 87 83 Intron 3 0.2 0.2 54 Exon 4 27 27 89 Intron 4 0.1 0.1 67 Exon 5 87 81 83 Intron 5 0.2 0.1 55 Exon 6 45 54 70 Intron 6 0.8 0.5 46 Exon 7 88 88 94 Intron 7 0.3 0.1 34 Exon 8 74 74 81 Intron 8 0.2 0.2 51 Exon 9Ž. coding 104 104 89 Exon 9Ž. UTR 410 388 60

aNote. ns, no similarity. Value is below the statistical significance threshold for BLASTN alignmentsŽ. Altschul et al., 1997 . even after overexposure of the X-ray film indicates only 5Ј exon in MAGP1 by similarity to Magp1 we that a single tsp lies no more than 70 bp upstream of hereafter refer to it as MAGP1 exon 1. the ATG initiation codon. This result suggests that the 5Ј UTR of human MAGP1, and hence, the 5Ј 3.5. Analysis of the 5Ј flanking region exon, is smaller than previously reported and prompted us to perform a 5Ј RACE procedureŽ Froh- Inspection of the 5Ј flanking sequence of MAGP1 man et al., 1988. to obtain the complete 5Ј UTR and Magp1 outside the inserted repeated elements sequence. OHMS-1Ž. Table 1 was used as a specific revealed that both human and mouse tsp are located primer to reverse-transcribe total RNA from A2058 within CpG islands, as defined by Larsen et al.Ž. 1992 and MG63 cells. The MAGP1-specific single-stranded wpercentage ofŽ. CqG above 50% and a ratio of cDNA was dŽ. C -tailed in its 5Ј end and subjected to observed vs. expected CpGs greater than 0.6x . Al- two consecutive PCRs using nested antisense internal though the CpG islands occur in orthologous regions, primers derived from the cDNA sequenceŽ. Table 2 . they differ substantially. Thus, the MAGP1 CpG is- The amplification products were purified and com- land extends from position ᎐238 relative to the tsp to pletely sequenced. Both cell lines gave rise to the q553Ž. 793 bp with a CpG ratio of 0.78; whereas the same 5Ј RACE sequence that included 32 nt derived CpG island in Magp1 is smaller in size, extending from exon BŽ. Fig. 2c . The perfect agreement between from position ᎐142 to q109Ž. 251 bp , and is less rich 5Ј RACE and primer extension data allowed us to in GqC, with a CpG ratio of 0.66. Several possible map unequivocally the main initiation site to an cis-acting regulatory sequences were identified within adenine residue located 74 bp upstream of the the aligned region to position ᎐289 and ᎐238 in ATGŽ. Fig. 2c , within a sequence that conforms to the MAGP1 and Magp1, respectively. Sequences upstream loose consensus initiator element Ž.Inr motif of the transcription start site lack TATA boxes, but r PyPyAq1 NŽ. T Aq3 PyPy that spans the transcription they do contain a sequence corresponding to the start site in TATA-less genesŽ. Smale, 1997 . A similar loose Inr consensusŽ. Smale, 1997 . In both cases, approach was followed to determine the 5Ј end of the areas upstream from the Inr contain multiple Sp1 mouse Magp1 mRNA. Using total lung RNA as a binding sitesŽ positions ᎐164, y75, y22, and ᎐9, in template, the 5Ј RACE longest products confirmed MAGP1, and positions ᎐124, y55, and ᎐11, in Magp1., that the mouse tsp corresponds to position 1 in the as is typical of TATA-less promoters. No downstream cDNA reported by Chen et al.Ž. 1993 , and conforms promoter elementŽ. DPE was found downstream of to the Inr consensusŽ. not shown . the main tspŽ. Burke and Kadonaga, 1997 . Among the Taken together, our PCR, 5Ј RACE, and primer inducible factors that are usually present further up- extension results do not support the notion of two stream from the core promoter, binding sites at alternate 5Ј exons in human MAGP1. We can only roughly conserved positions in both sequences were speculate on the mechanisms that originated the exon found for AP-2, AP-4, NF-␬B, and c-ETS transcrip- A-containing clones in the original report by Faraco tion factors. et al.Ž. 1995 , and although an artifactual origin of the clones cannot be dismissed, the use of certain pro- 3.6. Analysis of the transcribed regions moter-like sequences present in the inserted retropo- sons in response to specific situations can be envi- A comparison between the transcribed regions of sionedŽ. Li et al., 1999 . Since exon B is most likely the MAGP1, with exon 1 as defined above, and Magp1 F. Segade et al. rMatrix Biology 19() 2000 671᎐682 679 genes show that they have an identical genomic orga- Table 3 nization, consisting of nine exons. The minimum sizes Synonymous and non-synonymous substitution distances of MAGP1 of the genes are 6.1 and 5.3 kb for the human and Coding sequence ds dn dnrds mouse genes, respectively. The slightly larger size of the human introns account for much of the difference Exon 2 0.38 0.11 0.29 Ž.Table 2 . There is, however, no increase in the num- Exon 3 1.13 0.03 0.03 Exon 4 NA 0.04 NA ber or size of inserted repeat elements in the introns. Exon 5 0.12 0.05 0.42 Thus, the RepeatMasker program detected one inte- Exon 6 0.28 0.14 0.47 grated element contributing 420 bp in MAGP1 intron Exon 7 0.30 0.01 0.05 2, compared to two interspersed elements contribut- Exon 8 1.38 0.02 0.01 ing 522 bp to Magp1 introns 1 and 2. Nucleotide Exon 9 0.19 0.06 0.29 ORF 0.57 0.04 0.07 sequences of the exonrintron boundaries show that all introns are flanked by the consensus GTrAG splice site sequenceŽ. Breathnach and Chambon, 1981 . 5, 6, and 9; Table 3. , with higher dnrds values. While As previously reportedŽ Chen et al., 1993; Faraco et the latter are consistent with the positive selection for al., 1995. , two of the possible codon disruption phases evolutionary change, it is worth noting that among the are present in the MAGP1 or Magp1 splice junctions. exons with slow evolutionary rate, exon 3 contains the Thus, intron 7 disrupts a codon between the second segment of the signal peptide that includes the pro- and the third nucleotideŽ. phase II , while the remain- tease processing site, and exons 7 and 8 code for ing seven introns interrupt a codon between the first seven of the 13 Cys residues in MAGP-1. Remark- and the second nucleotideŽ.Ž. phase I Patthy, 1987 . ably, exons 7 and 8 are highly conserved in size and The sizes of exons are well conserved between the sequence not only in MAGP-1 from different species species, except exon 6, which is significantly shorter in but they are also very similar to two exons of the MAGP1 Ž.Table 2 . Pairwise alignment of the tran- Cys-rich domain of the closely related protein, scribed regions resulted in an overall similarity score MAGP-2Ž. Hatzinikolas and Gibson, 1998 . We can of 84% identity, but while the coding sequences yield speculate that the amino acid sequences encoded in similarity values ranging from 70 to 94%Ž. Table 2 , the more evolutionarily constrained exons form the both 3Ј and 5Ј UTRs seem to be less well conserved. most important domains to carry out the essential Thus, the aligned segments of the 3Ј UTRs are only functions of MAGP-1. 60% similar, close to the levels of similarity between orthologous introns. The 5Ј UTR shows even less 3.7. Characterization of MAGP1 splice ¨ariants conservation of sequence, since the 32 nt in the human and 41 nt in the mouse that are transcribed Using the MAGP1 gene sequence in 37C10 as the from exon 1 cannot be aligned at a significant level, query for BLASTN analysis, we searched the human and only the aligned UTRs coded by exon 2 are 74% subset of the EST database and obtained numerous similar. matches with ESTs from a variety of tissues. Three Similarity alone is not a precise indicator of the differing cDNA sequences were identified through selective forces that act on a sequence, therefore in the alignment to human MAGP1 cDNA. The most order to evaluate evolutionary distances between abundant ESTs correspond to the published sequence MAGP1 and Magp1 coding sequences we calculated for human MAGP1 Ž.Faraco et al., 1995 encoding the the ratio between the number of non-synonymous previously characterized secreted protein of 183 amino Ž.dn and synonymous Ž.ds base substitution. The acids. We designate this splice form that codes for the analysis of the entire open reading frame gave a ds canonical protein as MAGP1A. Two alternate forms value of 0.57, and a dn value of 0.04. Both rates are of MAGP1 cDNA were represented by two single not significantly different from the mean values of a ESTs which were sequenced in their entirety. Se- data set of mouse and human orthologous pairs Ždss quence analysis confirmed that the 1021-bp clone 0.468"0.169; dns0.090"0.102; Makalowski and H27217, isolated from a normal breast library, repre- Boguski, 1998. . The low dnrds ratio, 0.07, obtained sents a splice variant, named MAGP1B, that is gener- for the MAGP1 coding sequences, is indicative of ated by the skipping of exon 3Ž. Fig. 1c . In MAGP1 strong evolutionary constraints for amino acid re- the putative processing site for the removal of the placement. However, a similar analysis of the individ- signal peptide is encoded in exon 3Ž a Gln residue at ual coding exons found that the dnrds ratio is not position 18. . By skipping exon 3, the open reading uniform throughout the open reading frame, allowing frame in MAGP1B codes for a 153 amino acid pro- the separation of the coding exons into two classes tein that lacks the C-terminal end of the signal pep- according to their dnrds ratios: four exons with lower tide and the signal peptidase site. Computer analysis ratiosŽ.Ž exons 3, 4, 7, and 8 ; and four exons exons 2, with the PSORT program predicts an intracellular 680 F. Segade et al. rMatrix Biology 19() 2000 671᎐682 localization for MAGP1-B. The complete determina- conform to the acceptor consensus sequence tion of the sequence in the EST clone N40280Ž 1180 PyNŽ.Ž CrA AG Stephens and Schneider, 1992 . , mak- bp. revealed a third splice form, termed MAGP1C, ing it less preferred. It seems noteworthy that the 3Ј retaining intron 4 of MAGP1 Ž.Fig. 1c . It is possible acceptor region in exon 3 that is involved in the that MAGP1C represents an unprocessed transcript formation of MAGP1AЈ is also the splice site skipped that is not translated but, if translated, the insertion in the generation of the MAGP1B isoform, perhaps of 102 bp of intronic sequence would disrupt MAGP-1 indicative of the presence of ‘weak’ splicing signals with a premature stop codon resulting in a truncated whose regulatory relevance deserves further study. protein of 58 amino acids. While sequencing RT-PCR To verify the specificity and patterns of expression fragments obtained from MG63 cells with primers of the human splice variants, we conducted a PCR HM-5 and HM-3Ž. Table 1 for cloning purposes, we analysis by using the human Sure-RACE panel with identified a fourth form, designated MAGP1AЈ, primers OHMS-5 and OHMS-2Ž. Table 1 , followed by showing a 3 nt deletion between exons 2 and 3 of hybridization of the amplification products as de- MAGP1 Ž.Fig. 1d . Comparison with the 37C10 genomic scribed in Materials and Methods. The identity of the sequence revealed that in the processing of the hybridizing bands was ascertained by complete se- MAGP1 transcript to generate MAGP1A, the pre- quencing of the gel-purified bands obtained from at dicted splice acceptor site of intron 2 is cacag, whereas least two lanes. The panel employed has not been in the case of MAGP1AЈ an alternate site from exon normalized for cDNA concentration, therefore it can- 3 which is 3 bp downstream, is used insteadŽ. Fig. 1d . not be used to ascertain relative levels of expression This change in mRNA deletes an Ala residue at of MAGP1 forms among tissues. Nevertheless, the position 13 within the putative MAGP-1 signal pep- parameters used for the PCR assay allow an estima- tide. tion of the relative levels between amplification To investigate the frequency of the MAGP1AЈ al- products in the same lane. The PCR results clearly ternative spliced form, we made use of restriction demonstrate that the alternate transcript forms are fragment length polymorphism of amplified tran- present in normal tissue RNA and are not cloning scripts. The splicing of exons 2 and 3 originates a PstI artifacts from the libraries from which EST clones site that is lost in the products derived from were derivedŽ. Fig. 3a . MAGP1AЈ. We analyzed the recombinants and found We amplified MAGP1 products from a majority of that the MAGP1AЈ transcript constitutes approxi- the tissue cDNAs surveyed and found that the pat- mately 15% of the MAGP1 mRNA in MG63 cells. terns of expression of the MAGP1AŽ. 498 bp band The observed difference in abundance between and MAGP1BŽ. 408 bp variants were distinctive. Thus, MAGP1A and MAGP1AЈ may reflect the use of the MAGP1A is widely expressed and the most abundant agcag splice site in MAGP1AЈ that does not perfectly splice variant in most tissues, while MAGP1B is found

Fig. 3. Expression pattern of the human MAGP1 and mouse Magp1 alternative transcripts.Ž. a RT-PCR with a human multiple tissue Ž cDNA panel. . PCR was performed with oligonucleotides located in exons 2 and 9 and, after agarose gel electrophoresis and transfer to nylon membranes, the amplification products were detected by hybridization using human MAGP1 cDNA as a probe. The sizes of the indicated bands are 498 bp Ž.MAGP1A and 408 bp Ž.MAGP1B . PBL, peripheral blood leukocytes. Ž. b RT-PCR with a mouse multiple tissue cDNA panel. PCR was performed with oligonucleotides located in exons 2 and 9, and the PCR products detected by hybridization with a mouse Magp1 cDNA probe. The PCR products with sizes of 610 bp Ž.Magp1A and 360 bp Ž.Magp1D are indicated. F. Segade et al. rMatrix Biology 19() 2000 671᎐682 681 exclusively in four samplesŽ spleen, liver, colon, and that we named Magp1D, and formed by the splicing peripheral leukocytes. . Among tissues in which we of exon 2 to exon 7Ž. Fig. 1c . As in the case of the detected coexpression of both MAGP1Aand human MAGP1B, the encoded 102 amino acid pro- MAGP1B, the relative levels of both forms was simi- tein lacks a signal peptide and is predicted by PSORT lar only in placenta. No amplification product derived to be located inside the cell. Unlike in humans, where from the MAGP1C splice variant can be seen in the the alternate form MAGP1B seems to be the major panel shown in Fig. 3a since the PCR conditions transcript in various tissues, the minor mouse Magp1D required for its detection were outside the established variant is always coexpressed with the Magp1A form, parameters for the accurate evaluation of the expres- suggesting a different regulatory mechanism. sion of the MAGP1A and MAGP1B forms. However, Because a minor splice form was found in the by extending the amplification to 45 cycles we ob- amplification band corresponding to the major human tained a 600-bp product from placenta that, upon MAGP1 transcript, we examined the results of the RT purification and sequencing, was found to derive from PCR of MEF RNA with primers OMM-5Eco and the MAGP1C variant. The additional bands present OMM-3Xba for mouse equivalents to the human in certain tissues, such as those visible in the testis MAGP1AЈ form. We identified a third spliced form of lane, were also purified and fully sequenced but they Magp1 that is generated by the use of a cryptic splice represent either truncation artifacts from oligonu- site present in a coding exon. This variant, named cleotide misprimingŽ. lower band , or an aberrantly Magp1AЈЈ, contains a deletion of 3 nt between exon 4 migrating MAGP1A productŽ. upper band . and exon 5 of Magp1 Ž.Fig. 1d . Comparison to the The characterization of splice variants in the hu- Magp1 genomic sequence found that instead of the man prompted us to investigate whether the mouse predicted splice site tacag, an alternate site in exon 5, Magp1 gene gives rise to more than the canonical agaag, is used. The 3-nt difference in coding sequence splice form. To this end we searched the mouse deletes a Glu at position 53. We established the ratio subset of the EST database for Magp1 cDNAs. A between Magp1A and Magp1AЈЈ in the PCR products large number of ESTs from independent libraries by cloning the 590 bp band into a plasmid vector and matching to Magp1 gene were found. However, these subsequent sequence of the recombinants. As in the ESTs either did not include the sequences involved in case of the human MAGP1AЈ variant, the use of a alternative splicing in the human gene or, if full- non-consensus splice acceptor by Magp1AЈЈ possibly length, they corresponded to the already character- explains its 25% frequency relative to Magp1A. These ized mouse Magp1 cDNAŽ. Chen et al., 1993 which, findings demonstrate that both human and mouse for similarity reasons, was designated Magp1A. genes are highly polymorphic, generating a total of six The absence of alternate forms in the mouse splice variants. However, the mechanisms underlaying database might reflect a lack of alternative splicing in this diversity of isoforms are apparently highly specific the mouse or, alternatively, that the minor forms are given that, except for the major transcript, no homol- present at a very low frequency. We proceeded to ogous variants were found in both species. survey a multitissue panel of presynthesized cDNA to amplify any possible variants present in mouse tissue at low levels, as we had done in human tissues. The Acknowledgements mouse Rapid-Scan cDNAs were amplified with primers complementary to exon 1Ž. OMM-5 and 3Ј We thank Dr Jeffrey Bonadio for his gift of Magp1 UTRŽ.Ž. OMM-UTR Table 1 , and the amplification clones. This work was supported by NIH Grants products were subsequently hybridized with a full HL53325, HL62295, and HL61006 to Dr Robert length mouse Magp1 cDNA probe. A representative Mecham. result of the Southern analysis of the multitissue panel can be seen in Fig. 3b. Two bands that hy- References bridized to the probe are visible in the figure: a 610 bp band present in almost every lane with the ex- Altschul, S.F., Madden, T.L., Schaffer,¨ A.A. et al., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database pected size for Magp1A form; and a second band ᎐ - Ž search programs. Nucleic Acids Res. 25, 3389 3402. 400 bp that was observed in five samples small Bashir, M.M., Abrams, W.R., Rosenbloom, J. et al., 1994. Micro- intestine, testis, adrenal gland, 12.5 day total embryo, fibril-associated glycoprotein: characterization of the bovine gene and involuting mammary gland. . The minor product and of the recombinantly expressed protein. Biochemistry 33, was purified from the gel region of every lane in 593᎐600. which it was detected, reamplified, and sequenced in Breathnach, R., Chambon, P., 1981. Organization and expression of eucaryotic split genes coding for proteins. Annu. Rev. Biochem. its entirety. Sequence analysis and comparison to the 50, 349᎐383. Magp1 gene sequence demonstrated that the 360 bp Britten, R.J., 1997. Mobile elements inserted in the distant past product corresponds to a novel alternate transcript have taken on important functions. Gene 205, 177᎐182. 682 F. Segade et al. rMatrix Biology 19() 2000 671᎐682

Brown-Augsburger, P.B., Broekelmann, T., Rosenbloom, J., cal stresses increase mouse short interspersed elementŽ. SINE Mecham, R.P., 1996. Functional domains on elastin and MAGP RNA expression in vivo. Gene 239, 367᎐372. involved in elastic fiber assembly. Biochem. J. 318, 149᎐155. Makalowski, W., Boguski, M., 1998. Evolutionary parameters of the Burke, T.W., Kadonaga, J.T., 1997. The downstream core promoter transcribed mammalian genome: an analysis of 2820 orthologous element, DPE, is conserved from Drosophila to humans and is rodent and human sequences. Proc. Natl. Acad. Sci. USA 95, recognized by TAFII 60 of Drosophila. Genes Dev. 11, 9407᎐9412. 3020᎐3031. Mecham, R.P., Davis, E.C.@ 1994. ‘Elastic Fiber Structure and Chen, Y., Faraco, J., Yin, W., Germiller, J., Francke, U., Bonadio, Assembly’. In: P. D. Yurchenko, P.D. et al.Ž. Eds. , ‘‘Extracellular J., 1993. Structure, chromosomal localization, and expression Matrix Assembly and Structure’’, Academic Press, San Diego. pattern of the murine Magp gene. J. Biol. Chem. 268, pp. 281-314. ᎐ 27381 27389. Meyne, J., Baker, R.J., Hobart, H.H. et al., 1990. Distribution of Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA non-telomeric sites of theŽ. TTAGGG n telomeric sequence in ᎐ ᎐ isolation by acid guanidinium thiocyanate phenol chloroform vertebrate . Chromosoma 99, 3᎐10. ᎐ extraction. Anal. Biochem. 162, 156 159. Moghadaszadeh, B., Desguerre, I., Topaloglu, H. et al., 1998. Iden- Faraco, J., Bashir, M., Rosenbloom, J., Francke, U., 1995. Charac- tification of a new locus for a peculiar form of congenital terization of the human gene for microfibril-associated glycopro- muscular dystrophy with early rigidity of the spine on chromo- Ž. tein MFAP2 , assignment to chromosome 1p36.1-p35, and link- some 1p35-36. Am. J. Hum. Genet. 62, 1439᎐1445. ᎐ age to D1S170. Genomics 25, 630 637. Nei, M, Gojobori, T., 1986. Simple methods for estimating the Frohman, M.A., Dush, M.K., Martin, G.R., 1988. Rapid production numbers of synonymous and nonsynonymous nucleotide substi- of full-length cDNAs from rare transcripts: amplification using a tutions. Mol. Biol. Evol. 3, 418᎐426. single gene-specific oligonucleotide primer. Proc. Natl. Acad. Patthy, L., 1987. Intron-dependent evolution: preferred types of Sci. USA 85, 8998᎐9002. exons and introns. FEBS Lett. 214, 1᎐7. Gibson, M.A., Hughes, J.L., Fanning, J.C., Cleary, E.G., 1986. The Quandt, K., Frech, K., Karas, H., Wingender, E., Werner, T., 1995. major antigen of elastin-associated microfibrils is a 31-kDa gly- MatInd and MatInspector ᎏ new fast and versatile tools for coprotein. J. Biol. Chem. 261, 11429᎐11436. detection of consensus matches in nucleotide sequence data. Gibson, M.A., Sandberg, L.B., Grosso, L.E., Cleary, E.G., 1991. Nucleic Acids Res. 23, 4878᎐4884. Complementary DNA cloning establishes microfibril-associated glycoproteinŽ. MAGP to be a discrete component of the elastin- Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: associated microfibrils. J. Biol. Chem. 266, 7596᎐7601. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Gibson, M.A., Hatzinikolas, G., Kumaratilake, J.S., Sandberg, L.B., Cold Spring Harbor, NY. ᎏ Nicholl, J.K., Sutherland, G.R., Cleary, E.G., 1996. Further char- Schwartz, S., Zhang, Z., Frazer, K.A. et al., 2000. PipMaker a acterization of proteins associated with elastic fiber microfibrils web server for aligning two genomic DNA sequences. Genome ᎐ including the molecular cloning of MAGP-2. J. Biol. Chem. 271, Res. 10, 577 586. 1096᎐1103. Seki, N., Ohira, M., Nagase, T. et al., 1997. Characterization of Hatzinikolas, G., Gibson, M.A., 1998. The exon structure of the cDNA clones in size-fractionated cDNA libraries from human ᎐ human MAGP-2 gene. Similarity with the MAGP-1 gene is brain. DNA Res. 4, 345 349. confined to two exons encoding a cysteine-rich region. J. Biol. Smale, S.T., 1997. Transcription initiation from TATA-less pro- Chem. 273, 29309᎐29314. moters within eukaryotic protein-coding genes. Biochim. Bio- Larsen, F., Gundersen, G., Lopez, R., Prydz, H., 1992. CpG islands phys. Acta 1351, 73᎐88. as gene markers in the . Genomics 13, 1095᎐1107. Stephens, R.M., Schneider, T.D., 1992. Features of spliceosome Lennon, G., Auffray, C., Polymeropoulos, M., Soares, M.B., 1996. evolution and function inferred from an analysis of the informa- The I.M.A.G.E. Consortium: an integrated molecular analysis of tion at human splice sites. J. Mol. Biol. 228, 1124᎐1136. genomes and their expression. Genomics 33, 151᎐152. Tillett, D., Neilan, B.A., 1999. n-Butanol purification of dye termi- Li, T.H., Spearow, J., Rubin, C.M., Schmid, C.W., 1999. Physiologi- nator sequencing reactions. BioTechniques 26, 606᎐610.