Key words: Human molecular genetics, somatic-cell genetics, instability of simple sequence repeats
The general interest of my lab is in genetic instability in the development and progression of cancer. We are using a tissue culture system to study mutations that result in changes in the lengths of microsatellite sequences in mammalian cells. Microsatellites are tandem repeats of short sequences (usually from one to five base pairs per repeat unit, with tract lengths up to about 30 units); they are abundant and widely dispersed throughout mammalian and other eucaryotic genomes. There is extensive length polymorphism at most human microsatellite loci. Microsatellite instability has been shown to be a hallmark of certain cancers, including those that occur in families with hereditary nonpolyposis colorectal cancer (HNPCC) and a subset of related sporadic cancers. The hypermutability of microsatellites in these cancers results in at least some families from a defect in DNA mismatch repair
We have developed a plasmid vector into which various microsatellite sequences can be cloned. The cloning site is located within a fusion gene, composed of the Herpes simplex virus thymidine kinase (tk) gene fused to the 5' end of a bacterial neomycin-resistance (neo) gene. The cloning site is located near the 3' end of the tk gene, such that inserts affect the reading frame of the neo gene. Inserts that position the gene out-of-frame and consist of tandem repeats that are not multiples of three bases are being studied. Plasmids containing microsatellites are introduced by transfection into mammalian cells, where they integrate into the cellular genome, and clones resistant to the neomycin analogue G418 are selected. Increases and decreases in specific numbers of repeating units restore the reading frame of the neo gene, leading to drug-resistance.
We are using these microsatellites as markers of genetic instability in neoplasia. We are asking questions about the rates and kinds of mutations that occur in neoplastic cells with and without mismatch repair defects and about genes other than those involved in mismatch repair that may affect the stability of these sequences. The effects of microsatellite length and sequence composition on mutation rates and the nature of these mutations are being studied. A similar system yeast is being used to learn about the effects of the position in the genome on microsatellite mutations.