Evan E. Eichler[l], Susan Hoffman and Harvey Mohrenweiser.
Lawrence Livermore National Labs, Livermore, CA 94550.
Zinc finger (ZNF) genes represent one of the largest and most diverse family of genes found in the human genome. These genes encode transcriptional regulators which are believed to play critical roles in cellular and developmental differentiation processes. DNA binding of the encoded proteins is typically mediated by a zinc finger motif which consists either of two cysteines and two histidines (Kruppel family or C[2]/H[2] type) or four cysteines alone (steroid receptor or C[2]/C[2] type). It has been estimated that there are between 300-700 loci in the human genome which show homology to the C[2]/H[2]-type zinc finger motif. Despite their abundance, the function of the vast majority of these genes is not known. Chromosome 19 appears to be particularly enriched for zinc finger genes such that one third of all ZNF loci are distributed within three potential clusters corresponding to cytogenetic band locations l9pl2, pl3.2 and q34.
We are currently working to map and characterize all ZNF genes in the l9pl2 cluster between STS markers D19S269 and D19S450. Our strategy has been to develop a highly integrated physical map of this region using FISH, STS markers and conversion of overlapping YAC contigs to a cosmid clone map of this interval. Eight overlapping YAC clones have been identified in the l9pl2 region, creating a contig of approximately 2 megabases. YAC DNA was purified and used to screen a flow-sorted chromosome 19 arrayed cosmid library. A total of 200 cosmids were identified and assembled into contigs based on fluorescent fingerprinting methods. The location of cosmid contigs was confirmed using FISH and cosmids were anchored using a STS marker screening strategy. Sixteen cosmid contigs have been constructed comprising approximately 1.4 MB of this 2MB interval. In addition a detailed EcoR I enzyme restriction map has been developed for approximately 650 kb of this region. As a first attempt of interdigitating potential ZNF genes in this region, cosmids were screened with a degenerative 27-mer oligonucleotide probe to the conserved "H/C" link between zinc finger motifs. This analysis identified a minimum of 12 potential C[2]/H[2] ZNF genes in this region. Hybridization and sequence analysis has confirmed the identity, thus far, of one functional zinc finger gene (ZNF 85).
Once the physical map is completed, cDNAs will be identified and assigned to an EcoR 1 restriction enzyme map of this cosmid contig. The development of a high-resolution transcription map, followed by the determination of expression profiles of various ZNF cDNAs should provide valuable insight into the functional properties of active ZNF genes and their potential role in development and differentiation. Furthermore, the elucidation of the organization of this cluster by novel Alu-typing strategies as well as subfamily assignment will allow us to test models of gene duplication and may suggest other mechanisms of gene evolution for l9pl2 and other ZNF clusters in the human genome.
[l] DOE Human Genome Distinguished Postdoctoral Fellow