The structure of the rat aggrecan gene and preliminary characterization of its promoter.

K J Doege,K Garrison,S N Coulter,Y Yamada

doi:10.1016/s0021-9258(19)62035-1

K J Doege, K Garrison + Show 2 more

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https://doi.org/10.1016/s0021-9258(19)62035-1

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Abstract

Aggrecan is a major structural component of cartilage extracellular matrix and a specific gene product of differentiated chondrocytes. cDNA clones have been used to isolate rat aggrecan genomic clones from phage and cosmid libraries, producing over 80 kilobases (kb) of overlapping DNA containing the complete rat aggrecan gene, including 12 kb of 5'- and 8 kb of 3'-flanking DNA. DNA sequencing shows 18 exons, most of which encode structural or functional modules; exceptions are domains G1-B and G2-B, which are split into two exons and the G3 lectin domain, which is encoded by three exons. There is one expressed epidermal growth factor-like exon and in addition a non-expressed "pseudo-exon" encoding a heavily mutated epidermal growth factor-like domain. Intron sizes have been determined by restriction mapping and inter-exon polymerase chain reaction; a 30-kb intron separates exons 1 and 2. Exon 1 has been mapped by primer extension and S1 nuclease protection; it encodes 381 base pairs (bp) of 5'-untranslated sequence. There is a minor promoter which initiates transcription an additional 68 bp 5' of the major promoter start site. DNA sequence is reported for a 529-bp fragment encompassing exon 1, including 120 bp of 5'-flanking DNA comprising the promoter. This promoter is lacking the TATAA or CCAAT elements but has several putative binding sites for transcription factors. A 922-bp DNA fragment with 640-bp 5'-flanking DNA and 282-bp exon 1 sequence showed higher promoter activity in transfected chondrocytes than in fibroblasts, is completely inactive in the reverse orientation, and is strongly enhanceable in the forward direction by the SV40 enhancer.

Highlights

Aggrecan is a major structural component of cartilage from other aggregating proteoglycans in a tissue distribution extracellular matrix and a specific geneproduct of differentiated chondrocytes. cDNA clones have been used to isolate rat aggrecan genomic clones from phageand cosmid libraries, producing over 80 kilobases of overlapping DNA containing the complete rat aggrecan gene, including 12 kb of 5’-and 8 kb of 3”flanking DNA
This paper reports the first complete description of a gene from the aggrecan-like familyof proteoglycans
Therat aggrecan gene has been cloned from both phagaend cosmid libraries, yielding over80 kb of overlapping genomic clones

Summary

11 Present address

Biology Dept., Bucknell University, Lewisburg,PA provide attachment points for glycosaminoglycans and other carbohydrate chains; thesedomains are called keratan sulfate and chondroitin sulfate inaggrecan. Aggrecan is theonly family member with a third globular domain, G2, which contains additional link-related motifs B and B’; G2 is adjacent to G1, separated by an extended region called interglobular domain. The sample was heated at 90 "C for 10 min, made 77 lage matrix-deficient mouse (cmd)(30).The altered expression mM in potassium chloride, and slow cooled to 42 "C. Previous reports have included the sequence of five exons encodingthe chicken aggrecan G3 domain The pelleted nucleicacidswere dissolved in 80% formamide, 0.4 M PIPES, pH 6.4, 0.4 M NaCl, 1 mM EDTA, denatured for 10 minat 90 "C, and annealed for 3 hat 52-62 "C, according to GC content [34].The DNA-RNA hybrids were subjected to S1 nuclease digestion in 300 p1of 0.05 M ammonium acetate, pH 4.5, 0.25 M NaC1, 0.01 M zincchloride containing 6.25pg of denatured

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