Two genes, COL4A3 and COL4A4 coding for the human α3(IV) and α4(IV) collagen chains are arranged head-to-head on chromosome 2q36 1

We have devised a simple and efficient cDNA cloning strategy that overcomes many of the difficulties encountered in obtaining full-length cDNA clones of low-abundance mRNAs. In essence, cDNAs are generated by using the DNA polymerase chain reaction technique to amplify copies of the region between a single point in the transcript and the 3' or 5' end. The minimum information required for this amplification is a single short stretch of sequence within the mRNA to be cloned. Since the cDNAs can be produced in one day, examined by Southern blotting the next, and readily cloned, large numbers of full-length cDNA clones of rare transcripts can be rapidly produced. Moreover, separation of amplified cDNAs by gel electrophoresis allows precise selection by size prior to cloning and thus facilitates the isolation of cDNAs representing variant mRNAs, such as those produced by alternative splicing or by the use of alternative promoters. The efficacy of this method was demonstrated by isolating cDNA clones of mRNA from int-2, a mouse gene that expresses four different transcripts at low abundance, the longest of which is approximately 2.9 kilobases. After less than 0.05% of the cDNAs produced had been screened, 29 independent int-2 clones were isolated. Sequence analysis demonstrated that the 3' and 5' ends of all four int-2 mRNAs were accurately represented by these clones.

The collagen superfamily of proteins now contains at least 19 proteins formally defined as collagens and an additional ten proteins that have collagen-like domains. The most abundant collagens form extracellular fibrils or network-like structures, but the others fulfill a variety of biological functions. Some of the eight highly specific post-translational enzymes involved in collagen biosynthesis have recently been cloned. Over 400 mutations in 6 different collagens cause a variety of human diseases that include osteogenesis imperfecta, chondrodysplasias, some forms of osteoporosis, some forms of osteoarthritis, and the renal disease known as the Alport syndrome. Many of the disease phenotypes have been produced in transgenic mice with mutated collagen genes. There has been increasing interest in the possibility that the unique post-translational enzymes involved in collagen biosynthesis offer attractive targets for specifically inhibiting excessive fibrotic reactions in a number of diseases. A number of experiments suggest it may be possible to inhibit collagen synthesis with oligo-nucleotides or antisense genes.

The collagen IV molecule is 400 nm long and consists of two alpha 1 (IV) and one alpha 2 (IV) chains. Their genes COL4A1 and COL4A2 form a transcription unit in which they are arranged head-to-head and connected by a short common promoter region. Recently, four additional genes have been discovered. They are similarly arranged in the pairs COL4A3-A4 and COL4A5-A6 and code for additional collagen IV isoforms. in the macromolecular network of collagen IV, which provides the scaffold for basement membranes, the molecules are attached and cross-linked via their like end-regions and they interact laterally with their triple-helical domains in a not yet fully understood manner. Collagen IV is involved in interactions with cells and possesses two specific recognition sites for the integrins alpha 1 beta 1 and alpha 2 beta 1.