Abstract
Collagen IV, the major structural component of basement membranes, is composed of two genetically distinct polypeptide chains, alpha 1(IV) and alpha 2(IV). We have isolated a 522-base-pair (bp) cDNA to the 5' portion of the murine alpha 1(IV) chain mRNA from a library constructed by specific primer extension of poly(A)+ RNA from differentiated F9 cells. This cDNA includes 141 bp of 5' untranslated sequence and encodes a signal peptide plus a portion of the amino-terminal cross-linking (7 S) domain. This cDNA clone was used to obtain the 5' portion of the murine alpha 1(IV) gene from which the nucleotide sequence of exons 1-6 was determined. Exon 1 (234 bp) codes for the 5' untranslated sequence, and the first 28 residues of the protein. The 5' untranslated sequence is highly conserved between the mouse and human species and has the potential to form three mutually exclusive stem-loop structures which may play a role in post-transcriptional regulation. Exons 2-6, which code for the 7 S domain, were found to be 60, 90, 45, and 63 bp in size. The exon structure for the helical portion of the 7 S domain is different from that of the major helical domain, suggesting that they evolved differently.
Highlights
Collagen IV, thmeajor structural component of based- istinctive structural domains
These clones spanned approximately40kb The amino acid sequence of the signal peptide and theNH2of contiguous DNA and contained at least 28 exons whose terminalportion of the 7 S domain agree verywell with positions were determined by DNA sequencing and R-loop residues 1-129 of the human sequence showing only 7 amino analysis
The other cysteine and lysine residues and used to constructa specific primer extension cDNA which may participate in intermolecular cross-links are library in the vector Xgtll with poly(A)+ RNA from differ- preserved
Summary
241 TCTGGCTGTGG~TGTGACTGTCATGGCGTGMGGGAC-GGGAG~GAGGCTTG SerGlyCy ~ GlyLy ~ C y ~ A ~ ~ C y ~ ~ i ~ G l y v ~ l L y ~ GlyGlnLy ~ G l y G 5l3u A ~ gGlyL ~ u with a cDNA encoding a portion of the murine orl(1V) chain [16]. The most 5’ EcoRI fragment from clone MIV-5 was used as a probe to screen a library constructed from partial AluI and Had digests of murine genomic DNA in Charon 4A, three recombinant phage. 625,000 recombinant phage were screened with each probe, three clones were plaque-purified, and their DNA was isolated by standard methods. Exons 2, 3, and 4 were sequenced on both strands in plasmids translation initiation proposed by Kozak [25]. This codon is followed by an open reading frame coding for 126 residues beginning with a signal peptide. Sequence of the 7 S domain of the human al(1V) chain [18]
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