| 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 1 to 5 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, which have been particularly valuable as markers for
genetic mapping. 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. Very little
is known about the function, if any, of microsatellites or
about how they arise and are maintained in the genome, but
the hypermutability 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. We are also using this
system to study rates of mutation in microsatellite length
and the nature of these mutations. The effects of microsatellite
length and sequence composition are being studied to determine
whether the extent of polymorphism observed in the human
population is a direct reflection of differences in mutation
rates of these sequences. |
