Identification Of Candidate Genes

The HGP is rapidly and accurately generating genomic information that aids in the identification of unknown genes and disease loci. Once a disease-linked region is found, it can be scrutinized to identify target genes. Positional cloning is used to map the location of a human disease gene through marker analysis and to employ the mapped location on the chromosome to clone the gene and identify its function. Dong et al. (13) identified KAI1 through positional cloning by complementing a defect in rat prostatic tumor cell lines following introduction of human chromosome 11. We have employed a similar strategy to identify human liver tumor suppressor genes by introducing human chromosome 11 into rat liver epithelial tumor cell lines. It is possible that DNA from the introduced chromosome could be lost from microcell hybrid cell lines when the technique of microcell-mediated chromosome transfer is used. Therefore, extensive analysis of each hybrid cell line is required to confirm the presence of the transferred chromosome. In human-human hybrids, polymorphic markers are used to distinguish endogenous DNA from the introduced allele. Tumor suppressor genes can be localized if a correlation between suppression of the tumorigenic phenotype and the presence of specific chromosomal regions is observed (14-16). Through microsatellite polymerase chain reaction (PCR) analysis of specific chromosomal sequences, regions of chromosomal loss or retention can be distinguished rapidly. The application of this technology in human tumor models is hindered by the requirement for heterozygosity at the marker loci. There is no such limitation in rodent-human hybrids, where the introduced human allele can be easily distinguished from sequences in the recipient rat cell genome.

Studies in our laboratory suggest the presence of a human liver tumor suppressor gene in the p11.2-p12 region of human chromosome 11 (17-19). Introduction of human chromosome 11 into rat liver epithelial tumor cell lines via microcellmediated chromosome transfer produced microcell hybrid (MCH) cell lines that exhibit suppression of tumorigenicity in vivo (17). Because of the fragmentation of the chromosome when inserted into the donor cell lines, it was possible to define the region of the chromosome that conferred suppression on 11p11.2-p12 (17). Further molecular characterization of the MCH cell lines localized the putative liver tumor suppressor locus to a 950-kb region of 11p11.2.-p12 (18) and showed that this same locus suppresses the tumorigenic potential of other rat liver tumor cell lines (19). These studies demonstrated that a genetic locus (or loci) on human chromosome 11 could suppress the tumorigenic potential and alter the phenotypic properties of rat liver tumor cell lines. The information acquired in these previous studies made it possible to pursue advanced mapping of the 11p11.2-p12 region and placement of genes into the critical tumor suppressor region.

Human chromosome 11 contains several known or putative tumor suppressors whose deletion is critical to the pathogenesis of various malignancies. We have used a positional cloning strategy to identify candidate liver tumor suppressor genes from human chromosome region 11p11.2-p12 through investigation of a rat liver epithelial tumor cell line and derived MCH cell lines. We previously established a sequence tagged site (STS)-based map that defined the liver tumor suppressor locus to the D11S1361-D11S1357 interval of 11p11.2-p12 (18). We used EST and gene markers from the HGP to map the p11.2-p12 region of chromosome 11 that corresponded to the critical region of tumor suppression in our MCH cell lines. We integrated genomic maps from GeneMap'99 GB4 panel, NCBI Map Viewer, and the Human Genome Browser (University of California-Santa Cruz) to obtain the order of EST markers in the 11p11.2-p12 region. The superimposition of this EST-based map on our STS-based map resulted in the identification of candidate liver tumor suppressor genes from 11p11.2-p12, as well as increasing the number of informative genetic markers corresponding to this chromosomal region. From the 11p11.2-p12 region, 142 EST and gene markers were analyzed through PCR-based deletion mapping. Of these markers, 19 mapped to the critical region of tumor suppression in our MCH cell lines. Eleven of the 19 were expressed in the MCH cell lines, but not a tumor cell line, suggesting that 1 of these might be involved in liver tumor suppression in this model system.

Refinement of the number of candidate liver tumor suppressor genes was accomplished by screening MCH-derived tumor cell lines and additional MCH cell lines for the presence and expression of these candidate genes. Retention of EST markers in the DNA MCH-derived tumor cell lines and/or continued expression of these genes, would argue against their role as a tumor suppressor. These "functional" gene-expression-based analyses narrowed the number of candidate liver tumor suppressor genes to three. During our investigation of the 11p11.2-p12

chromosomal region, the HGP laboratories were in the process of sequencing the human genome. The accumulation of more and more sequencing data into the GenBank database resulted in the positioning of known genes to 11p11.2-p12. We used these genes to continue the mapping of 11p11.2-12. Thirty-nine known genes were localized to the p11.2-p12 region of human chromosome 11. Screening of these genes with the tumor cell line and the MCH cell lines resulted in the identification of three candidate genes (20). Investigations into the sequences of these candidates revealed that the three ESTs each shared 100% homology with three of the four newly identified genes, leaving the final number of candidate liver tumor suppressor genes at four (21).

Pregnancy Guide

Pregnancy Guide

A Beginner's Guide to Healthy Pregnancy. If you suspect, or know, that you are pregnant, we ho pe you have already visited your doctor. Presuming that you have confirmed your suspicions and that this is your first child, or that you wish to take better care of yourself d uring pregnancy than you did during your other pregnancies; you have come to the right place.

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