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Revised Genomic Structure of the Human Magp1gene And Matrix Biology 19Ž. 2000 671᎐682 Revised genomic structure of the human MAGP1 gene and identi®cation 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 genes code for the micro®bril-associated glycoprotein-1 Ž.MAGP-1 , an extracellular matrix protein of micro®brillar structures. We report a revised 5Ј genomic structure including the use of a single transcription 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 ¯anking 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 identi®ed a total of ®ve splice variants in addition to the canonical MAGP1ArMagp1A form. These transcripts are species-speci®c 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; Micro®brils; Alternate transcripts Abbreviations: EST, expressed sequence tag; PIP, percent identity plot; RACE, rapid ampli®cation 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 . Micro®brils are 10᎐12 nm ®laments 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 . Micro®brils are complex human and mouse loci to search for motifs, mostly in structures formed by the assembly of a large number the ¯anking sequences and introns, whose high con- of proteins, including, ®brillins-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 micro®bril-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 ®nd 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 micro®bril assembly has not been human MAGP1. By precise mapping of the 5Ј end of totally clari®ed, although there is evidence that it may the MAGP1 mRNA and ampli®cation analysis of hu- serve to facilitate elastin binding onto the micro®bril man tissue cDNA, we establish that a segment of the Ž.Brown-Augsburger et al., 1996 . Disul®de 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 micro®bril, 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 identi®cation 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 ®bers. 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 chromosome 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 gene expression through the generation of Plaques giving a positive signal were picked and puri- multiple transcripts was then envisioned. Neverthe- ®ed 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 ¯anking regions. The 2.5. Primer extension analysis Magp1 sequence was assembled from subcloned re- striction fragments, exon-to-exon PCR ampli®cation 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 puri®ed 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 ampli®cation 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.
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