Objectives-We have investigated the functional significance of conserved sequences within the 9p21.3 risk locus for coronary artery disease (CAD) and determined the relationship of 9p21.3 to expression of ANRIL and to whole genome gene expression. Methods and Results-We demonstrate that a conserved sequence within the 9p21.3 locus has enhancer activity and that the risk variant significantly increases reporter gene expression in primary aortic smooth muscle cells. Whole blood RNA expression of the short variants of ANRIL was increased by 2.2-fold whereas expression of the long ANRIL variant was decreased by 1.2-fold in healthy subjects homozygous for the risk allele. Expression levels of the long and short ANRIL variants were positively correlated with that of the cyclin-dependent kinase inhibitor, CDKN2B (p15) and TDGF1 (Cripto), respectively. Relevant to atherosclerosis, genome-wide expression profiling demonstrated upregulation of gene sets modulating cellular proliferation in carriers of the risk allele. Conclusion-These findings are consistent with the hypothesis that the 9p21.3 risk allele contains a functional enhancer, the activity of which is altered in carriers of the risk allele. 9p21.3 may promote atherosclerosis by regulating expression of ANRIL, which in turn is associated with altered expression of genes controlling cellular proliferation pathways. Key Words: coronary artery disease Ⅲ atherosclerosis Ⅲ 9p21.3 Ⅲ noncoding RNA Ⅲ ANRIL Ⅲ cellular proliferation T he 9p21.3 risk locus, identified in several genome-wide association studies for coronary artery disease (CAD), 1,2 spans 58 kb and encompasses multiple single nucleotide polymorphisms (SNPs) in tight linkage disequilibrium (LD). Approximately 25% of whites carry 2 copies of the risk allele and have a 1.5-fold increased risk for CAD. 2 The increased risk is independent of all known risk factors including plasma lipids, hypertension, diabetes, obesity, and markers of inflammation. This implies a novel biological pathway relevant to atherosclerosis.BLAST searches against the NCBI nucleotide sequence database and inspection of the UCSC Genome Browser revealed no annotated genes within the 58-kb interval. The localization of the risk locus to a region devoid of known protein coding genes implicates a novel gene or regulatory element that promotes atherosclerosis independently of established risk factors. It is notable that the 9p21 risk locus overlaps a newly annotated noncoding RNA (ncRNA), termed ANRIL, recently identified through a deletion analysis of a large French kindred with hereditary melanoma. 3 ANRIL spans 126.3 kb, overlaps at its 5Ј end with CDKN2B (p15), and consists of 20 exons subjected to alternative splicing. Full-length ANRIL (3834 bp), deposited under GenBank accession number DQ485353, encompasses the first 12 exons plus exons 14 to 20. At least 2 shorter variants of ANRIL that end with an alternative exon 13 have been reported, DQ485454 and EU741058. DQ485454 (2659 bp) comprises the first 12 exons and an alternative exon 13,...

The family 2a carbohydrate-binding module (CBM2a) of xylanase 10A from Cellulomonas fimi binds to the crystalline regions of cellulose. It does not share binding sites with the N-terminal family 4 binding module (CBM4-1) from the cellulase 9B from C.fimi, a module that binds strictly to soluble sugars and amorphous cellulose. The binding of CBM2a to crystalline matrices is mediated by several residues on the binding face, including three prominent, solvent-exposed tryptophan residues. Binding to crystalline cellulose was analyzed by making a series of conservative (phenylalanine and tyrosine) and non-conservative substitutions (alanine) of each solvent-exposed tryptophan (W17, W54 and W72). Other residues on the binding face with hydrogen bonding potential were substituted with alanine. Each tryptophan plays a different role in binding; a tryptophan is essential at position 54, a tyrosine or tryptophan at position 17 and any aromatic residue at position 72. Other residues on the binding face, with the exception of N15, are not essential determinants of binding affinity. Given the specificity of CBM2a, the structure of crystalline cellulose and the dynamic nature of the binding of CBM2a, we propose a model for the interaction between the polypeptide and the crystalline surface.

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