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A genomic interval of approximately 1-1.5 Mb centered at the MSR marker on 8p22 has emerged as a possible site for a tumor suppressor gene, based on high rates of allele loss and the presence of a homozygous deletion found in metastatic prostate cancer. The objective of this study was to prepare a bacterial contig of this interval, integrate the contig with radiation hybrid (RH) databases, and use these resources to identify transcription units that might represent the candidate tumor suppressor genes. Here we present a complete bacterial contig across the interval, which was assembled using 22 published and 17 newly originated STSs. The physical map provides twofold or greater coverage over much of the interval, including 17 BACs, 15 P1s, 2 cosmids, and 1 PAC clone. The position of the selected markers across the interval in relation to the other markers on the larger chromosomal scale was confirmed by RH mapping using the Stanford G3 RH panel. Transcribed units within the deletion region were identified by exon amplification, searching of the Human Transcript Map, placement of unmapped expressed sequence tags (ESTs) from the Radiation Hybrid Database (RHdb), and from other published sources, resulting in the isolation of six unique expressed sequences. The transcript map of the deletion interval now includes two known genes (MSR and N33) and six novel ESTs.

Bacterial artificial chromosome clones (BACs) are widely used at present in human genome physical mapping projects. To extend the utility of these clones for functional genomic studies, we have devised a method to modify BACs using Cre recombinase to introduce a gene cassette into the loxP sequence, which is present in the vector portion of the BAC clone. Cre-mediated integration is site specific and thus maintains the integrity of the genomic insert sequences, while eliminating the steps that are involved in restriction digest-based DNA cloning strategies. The success of this method depends on the use of a DNA construct, RETRObac, which contains the reporter marker green fluorescent protein (GFP) and the selectable marker neomycin phosphotransferase (neo), but does not contain a bacterial origin of replication. BAC clones have been modified successfully using this method and the genomic insert shows no signs of deletions or rearrangements. Transfection efficiencies of the modified BACs into human or murine cell lines ranged from 1% to 6%. After culture in media containing G418 for 3 weeks, approximately 0. 1% of cells previously sorted for GFP expression acquired stable antibiotic resistance. Introduction of a human BAC clone that contains genomic p53 sequences into murine NIH3T3 cells led to expression of human p53 mRNA as determined by RT-PCR, demonstrating that sequences contained on the BAC are expressed. We believe that GFP-neo modified BAC clones will be a valuable resource in efforts to study biological effects of known genes as well as in efforts to clone and analyze new genes and regulatory regions.