POSTER NO: 224

Protein interaction profiles for genes from the telomeric end of the MHC Class III region

Jennifer I Semple, Stephanie E. Brown, Christopher M. Sanderson, R. Duncan Campbell
UK MRC HGMP Resource Centre, Hinxton, Cambridge, CB10 1SB

The Major Histocompatibility Complex (MHC) is located on the short arm of chromosome 6. A number of autoimmune diseases such as insulin dependent diabetes mellitus (IDDM), rheumatoid arthritis (RA) and ankylosing spondylitis (AS) are associated with the MHC region. There is evidence to indicate that in some cases this association cannot be wholly explained by association with Class I and Class II genes. The Class III region, which lies between the MHC Class I and Class II regions, spans ~730kb and contains ~62 genes. Aside from a few well characterised genes such as those encoding TNF, C4A and C4A complement proteins and heat shock proteins, most of the genes in this region have no known function, or at best, function is inferred through sequence homology.

In order to better interpret disease association data it is important to have a better understanding of the biochemical and cellular role of the proteins encoded by genes within these loci. Therefore, we have used the yeast two hybrid system to characterise the protein-protein interactions of genes from the telomeric end of the Class III region. We selected genes whose protein products were predicted to be expressed intracellularly, as these proteins are more likely to be amenable to yeast two hybrid analysis. In total, 11 genes were analysed: CLIC1, BAT5, CSNK2B, BAT4, BAT3, BAT2, AIF1 and its splice variant IRT1, NFKBIL1, ATP6G, BAT1, and a novel gene adjacent to BAT1. The full length open reading frame (ORF) of each gene was amplified by RT-PCR and cloned into the pGBDU vector in frame with the Gal4 DNA binding domain. Genes with particularly long ORFs - BAT3 (3.4kb) and BAT2 (6kb) - were cloned in 2-3 different fragments and screened separately. The baits were screened against a library of K562 cDNA fragments fused to the activation domain of Gal4. In addition to confirming previously described protein-protein interactions we also found a number of novel putative interactions.

High throughput yeast two hybrid screens have been carried out for proteins encoded by the S. cerevisiae and C. elegans genomes. This pilot study of genes from the MHC Class III region is, to our knowledge, the first report of an attempt to apply this approach to the human genome.