Abstract
A cDNA for human microfibril-associated glycoprotein-2 (MAGP-2) was used to screen a human leukocyte genomic DNA library in EMBL-3 vector. One clone, clone H (10 kilobase pairs (kbp)), was isolated that contained most of the MAGP-2 gene. The remainder of the 3' end of the gene was obtained by direct polymerase chain reaction amplification of genomic DNA. The human MAGP-2 gene was found to be about 11 kbp in size and to contain 10 evenly distributed exons. The internal exons range in size from 30 base pairs (bp) to 88 bp with exons 4 and 6 the only exons of equal size (45 bp). All internal intron:exon junctions are defined by canonical splice donor and acceptor sites. Each junction has a 1/2 codon split with the exception of the exon 8/9 junction, which has a 2/1 split. The translation initiation codon is in exon 2, and the final exon contains 110 bp of coding sequence, including 2 cysteine codons. Primer extension experiments identified only one major transcription initiation site, 213 bases upstream of the ATG site. Rapid analysis of cDNA ends-polymerase chain reaction analysis of the 5' end of MAGP-2 mRNA from placenta confirmed this result and did not detect any alternative splicing of transcripts. The putative promoter region of the MAGP-2 gene was found to be AT-rich and it lacked a TATA box and other common regulatory elements. However the sequence surrounding the transcription start site CTCA(+1)TTCC was similar to the consensus CTCA(+1)NTCT (N is any nucleoside) for an initiator element found in terminal deoxynucleotidyltransferase and a number of other highly regulated genes. Comparison with the previously characterized human MAGP-1 gene showed that structural similarity was largely confined to the exact size, sequence, and junction alignment of the two penultimate exons which encode the first six of the seven cysteine residues that are precisely spaced in both proteins. The findings are consistent with the growing evidence that, although MAGP-1 and MAGP-2 are both intimately involved in the biology of fibrillin-containing microfibrils, the MAGPs are structurally, functionally, and developmentally diverse proteins which share one characteristic cysteine-rich motif.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF071547, U37283, and AF084918-AF084927
The findings are consistent with the growing evidence that, MAGP-1 and microfibril-associated glycoprotein-2 (MAGP-2) are both intimately involved in the biology of fibrillincontaining microfibrils, the MAGPs are structurally, functionally, and developmentally diverse proteins which share one characteristic cysteine-rich motif
The major structural components of the microfibrils are rod-like, 350-kDa glycoproteins named fibrillin 1 and 2 that appear to be arranged as parallel bundles of 4 – 8 molecules joined in series in a head to tail manner (6 –10)
Summary
A 318-bp digoxigenin-labeled fragment of human MAGP-2 cDNA was prepared from cDNA clone H61 [1] by PCR amplification and simultaneous incorporation of digoxigenin-11-dUTP (Boehringer Mannheim). The genomic DNA inserts were amplified using EMBL-3-specific primers and the Expand Long Template PCR system (Boehringer Mannheim) as per the manufacturer’s instructions. Direct sequencing (fmol kit, Promega) of these products was used to confirm the presence of exons from the MAGP-2 gene. Further sequencing of both DNA strands with appropriate primers was used to elucidate the sequences surrounding intron:exon junctions. The 3Ј region of the MAGP-2 gene was directly amplified and sequenced from human genomic DNA by PCR using a forward primer Gen F3, 5Ј-GGCCGGTTAAACAATGCATTCATCAG-3Ј (bases 509 –534), and reverse primer R10, 5Ј-AAGCTGGACATTGCAAAAGGATT-3Ј (bases 841– 863) from the 3Ј-untranslated region of the MAGP-2 cDNA
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