Abstract
A novel type IV collagen, alpha 3(IV), has previously been isolated from a collagenase digest of bovine and human glomerular and lens basement membranes. The cloning and sequencing of a cDNA encoding the alpha 3(IV) chain is described here. Using the polymerase chain reaction, with primers derived from the known 27-residue bovine alpha 3(IV) amino acid sequence, a 68-base pair bovine genomic fragment (KEM68) which encodes the known peptide sequence, was synthesized. KEM68 was then used to screen a bovine lens cDNA library and a 1.5-kilobase partial cDNA clone obtained, encoding 471 residues of the bovine alpha 3(IV) chain: 238 residues from the triple helical collagenous domain and all 233 residues of the noncollagenous domain. The collagenous repeat sequence has three interruptions, coinciding with those in the alpha 1(IV) chain. The noncollagenous domain has 12 cysteine residues in identical positions to those of other type IV collagens and 71, 61, and 70% overall similarity with the human alpha 1(IV), alpha 2(IV), and alpha 5(IV) chains. The noncollagenous domain of alpha 3(IV) is of particular interest as it appears to be the component of glomerular basement membrane that reacts maximally with the Goodpasture antibody. Furthermore, such antigenicity is absent from collagenase digests of the glomerular basement membrane of some patients with Alport syndrome. The alpha 3(IV) cDNA clone described here now permits study of the molecular pathology of COL4A3 in Alport syndrome.
Highlights
Kilobase partial cDNA clone obtained, encoding 471 residues of the bovine a3(IV) chain: 238 residues from (9,lO)
The noncollagenous a3(IV) and a4(IV) are of particular interest as they have domain has 12 cysteine residues in identical positions been implicated in thepathogenesis of Goodpasture syndrome to those of other typeIV collagens and 71, 61,and 70% and Alport-type familial nephritis, two clinical syndromes overall similarity with thehuman a l ( I V ), a2(IV), and that affect GBM and cause functional kidney impairment a5(IV) chains
The noncollagenousdomain of a3(IV) is of particular interest as it appeartso be the componentof glomerular basement membrane that reacts maximally with the Goodpasture antibody. Such antigenicity is absent from collagenase digests of the glomerular basement membrane of some patients withAlport syndrome
Summary
Reactions to ensure that small producotsf -70 bp would be amplified under the PCR conditioncshosen. PCR Protocols-Standard PCR reactions were performed in a 50pl volume containing 10-20 ng of either bovinegenomic, human genomic, or human cDNA template, pmol of each oligonucleotide. With primers Fl-F4 and Rl-R4, annealing was for 30 s at 68 "C for the first cycle, at 66 "C for 30 s for the second, a t 64 "C for 30 s for the. Subcloning and Sequencing-The 68-bp product(KEM68)obtained using primers F4 and R3 and bovine genomic template was cloned into the EcoRV site of the phagemid pBluescript I1 (Stratagene). Reaction was performed containing 5 Pmol of Primers F4 and R3,50 ~ 9 a*nd ~ 9 a*re primers complementary to corresponding regions of pg Of pKEM68, lopMdATP,dGTP*dTTP* and 9.4 pM dCTP. A total of 3 X 10"clones Step 1: denature, 94 1min; anneal, 68 oc, 30 (2 cycles). The complete sequence was obtained usi1n7g-residue oligonucleotide primers designed from known sequencesionfstehret (Fig. 4)
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