The keratan sulfate-enriched region of bovine cartilage proteoglycan consists of a consecutively repeated hexapeptide motif

We have determined the sequence of a cDNA clone encoding the keratan sulfate-rich domain of the large aggregating cartilage proteoglycan core protein. The C-terminal portion of the deduced amino acid sequence is homologous to the chondroitin sulfate-rich region (domain CS1) of the rat chondrosarcoma proteoglycan, and the N-terminal portion is homologous to the second globular domain (G2) of the rat proteoglycan (Doege, K., Sasaki, M., Horigan, E., Hassell, J. R., and Yamada, Y. (1987) J. Biol. Chem. 262, 17757-17767). We could identify, inserted between these regions, a region absent in the rat proteoglycan. This domain corresponds to the keratan sulfate-enriched region of the bovine proteoglycan. It consists of a highly conserved hexapeptide motif consecutively repeated 23 times. Transfer blot analysis of genomic DNA indicated a single gene. The coding region for the keratan sulfate-enriched region was present both in human and bovine DNA, whereas the coding region for this domain appears to be absent in the rat genome. Transfer blot analysis of RNA showed that the keratan sulfate-rich region is present in proteoglycans from fetal as well as adult sources. Furthermore, RNA protection assays of RNA isolated from adult and fetal bovine articular cartilage showed that no alternative splicing occurs within this keratan sulfate-enriched region. These experiments show that the fetal bovine cartilage proteoglycan contains the keratan sulfate attachment domain, although it lacks the keratan sulfate side chains.     

Structure of the bovine elastin gene and S1 nuclease analysis of alternative splicing of elastin mRNA in the bovine nuchal ligament

Genomic clones encompassing all the translated sequences, the 3' untranslated sequence, and 1 kb flanking the ATG translation initiation codon of bovine tropoelastin have been obtained and characterized by restriction enzyme analysis and extensive DNA sequencing. These analyses demonstrated that functionally distinct hydrophobic and cross-linking domains of the protein are segregated into separate exons throughout the gene. The putative promoter region lacks a TATA box, has an extremely high G+C content, and contains several SP1 binding sites. Comprehensive S1 analyses using probes covering the entire mRNA and RNA isolated from the nuchal ligament of bovine fetuses of different ages, neonate calves, and adult cows demonstrated that while only a single exon is alternatively spliced at high frequency, many exons are alternatively spliced at limited, variable frequencies. The results also suggest that such limited splicing is increased in the adult tissue relative to fetal and neonate tissues.     

Molecular cloning, structural characterization and chromosomal localization of human lipoyltransferase gene.

Lipoyltransferase catalyzes the transfer of the lipoyl group from lipoyl-AMP to the lysine residue of the lipoate-dependent enzymes. We isolated human lipoyltransferase cDNA and genomic DNA. The cDNA insert contained a 1119-base pair open reading frame encoding a precursor peptide of 373 amino acids. Predicted amino acid sequence of the protein shares 88 and 31% identity with bovine lipoyltransferase and Escherichia coli lipoate-protein ligase A, respectively. Northern blot analyses of poly(A)+ RNA indicated a major species of about 1.5 kb. mRNA levels of lipoyltransferase were highest in skeletal muscle and heart, showing good correlation with those of dihydrolipoamide acyltransferase subunits of pyruvate, 2-oxoglutarate and branched-chain 2-oxo acid dehydrogenase complexes and H-protein of the glycine cleavage system which accept lipoic acid as a prosthetic group. The human lipoyltransferase gene is a single copy gene composed of four exons and three introns spanning approximately 8 kb of genomic DNA. Some alternatively spliced mRNA species were found by 5'-RACE analysis, and the most abundant species lacks the third exon. The human lipoyltransferase gene was localized to chromosome band 2q11.2 by fluorescence in situ hybridization.