Palotie, Aarno, M.D., Ph.D.

Research Area: Migraine, genetics of complex traits

Research Interests:  Dr. Palotie’s laboratory has a special interest in the genetics of migraine. Migraine is a common neurological disorder showing clustering in families. Family and twin studies suggest a strong genetic component, especially in migraine which is accompanied by aura symptoms. The mode of inheritance is unclear but based on segregation analysis, a polygenic inheritance is most likely. With the benefits of the genetically isolated population of Finland, Dr. Palotie’s group aims to identify genetic loci contributing to the predisposition of migraine disease.

Dr. Palotie’s laboratory also has a tradition of visual physical mapping and development of high resolution fluorescence in situ hybridization (FISH) techniques used in positional cloning. Thus Dr. Palotie is managing the FISH and Tissue Array core unit, located on the forth floor of the Gonda (Goldschmied) Neuroscience & Genetics Research Center.

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Schiestl, Robert, Ph.D.

Research Area:  Molecular Mechanism of Carcinogenesis

Research Interests:  Our work centers mostly on basic mechanisms and genetic control of homologous and illegitimate recombination, events involved in carcinogenesis.

Effects of cancer predisposing mutations on the frequency of spontaneous and carcinogen induced mitotic recombination in vitro and in vivo, investigation of the biological effects of "nonmutagenic" carcinogens. Genetic instability and deletions are involved in carcinogenesis. We have previously constructed and/or used assays (DEL assays) that select for DNA deletion events in yeast (Schiestl et al. 1988, Schiestl 1989, Schiestl et al. 1989b,c, Schiestl and Reddy 1990, Carls and Schiestl 1994, Brennan et al. 1994), in human cells (Aubrecht et al. 1995) and in vivo in the mouse (Schiestl et al. 1994, Schiestl et al. 1997, Schiestl et al. 1997, Schiestl et al. 1998). DEL events in all three formats are inducible by a wide variety of proven carcinogens, including carcinogens that are negative in many other short-term tests. We have shown that many Salmonella assay negative carcinogens induce oxidative stress in yeast. (Brennan and Schiestl 1997, 1998). We are interested in determining the differential effects of "mutagenic versus nonmutagenic" carcinogens in the mouse using a variety of assays including gene expression profiling. The first model to study recombination in vivo is the pun mouse which has a 70 kb internal gene duplication disruption in the p coat color gene. Reversion to the wildtype sequence occurs by recombination between the two copies and is inducible by carcinogens (Schiestl et al. 1994, Schiestl et al. 1997). We have crossed the pun mutation into different DNA repair deficient backgrounds and found exciting results that p53 is involved in ionizing radiation induced recombination (Submitted) implying that it may be involved in the processing of double-strand breaks. As second model we have constructed a transgenic mouse that carries a duplication of exons 2 and 3 of the Hprt gene and we have already developed a sensitive histochemical staining method to identify deletion events in different tissues of the mouse. We will study the genetic control of such deletion events. p53, ERCC1, ATM, and SCID mutations cause the human diseases Li-Fraumeni syndrome, Xeroderma Pigmentosum, Ataxia Telangiectasia and severe combined immuno deficiency respectively. Furthermore we received mRAD52 and Ku80 mice. We are currently investigating effects of these mutations on spontaneous, carcinogen induced recombination events.

Mechanism of Persistent Genetic Instability Multiple genetic changes are required for the development of a malignant tumor cell and many environmentally-induced cancers show a delayed onset of more than 20 years following exposure. The frequency of such changes found in cancer cells is higher than can be explained through random mutation and it was proposed that a sub-population of cells develop a mutator phenotype. Such a persistent elevated level of genetic instability is also a major contributor to the progressive, multistage development of malignant disease. This phenotype, sometimes called delayed reproductive death, has indeed been observed in mammalian cells after treatment with ionizing radiation but the mechanism has not been defined. We have observed a similar genomic instability more than 50 cell divisions after exposure to ionizing radiation in yeast. These effects cannot be due to the initial damage because of their persistence over many generations. Mutations in a single gene leading to an elevated level of genetic instability also cannot account for these effects because they occur in up to 70% of the exposed cells. It is more likely that a difference in gene expression accounts for the high frequency of deletions (HFD) phenotype. We will investigate the mechanism of these delayed inheritable changes with conventional genetic tools as well as determining the gene expression profile (6200 genes) for yeast HFD cultures and control cultures to identify genes which may be involved in the maintenance or destabilization of genetic integrity. Finally, we will alter the expression of genes that are up or down regulated in HFD clones, and determine the effect of the altered gene expression on the initiation and/or inheritance of the HFD phenotype. This project should characterize the phenomenon of persistently elevated genetic instability, give insights into its mechanism and might also provide molecular targets for intervention to reverse the phenotype.

Interindividual Differences in DNA double-strand break repair efficiency We have also constructed plasmid model systems to determine the efficiency of cells for DNA double strand break repair by homologous versus illegitimate recombination. We are currently determining interindividual differences in DNA double strand break repair between cells from different control people and cells from 17 cancer patients that showed unexpected hypersensitivity to radiation treatment. In this project we are collaborating with Drs. Jack Little from our department, Tom Lindahl from the ICRF in London and Mark Meuth from Sheffield Univ. England. This project is also carried out in collaboration with Gene Therapeutic Inc. to determine polymorphisms in 18 genes involved in double strand break repair by high throughput DNA sequencing. We are also collaborating with Carl Barrett from the NIEHS to determine gene expression profiles of the different human cell lines in response to radiation exposure. This approach will hopefully link phenotypes in double-strand break repair with genotypes in DSB repair genes and may uncover new genetic risk factors for cancer.

Investigation of the mechanism, genetic control and inducibility of illegitimate recombination and restriction enzyme-mediated recombination in yeast and in mammalian cells. A system to study illegitimate integration of transformed DNA fragments in yeast has been developed (Schiestl and Petes 1991). In the presence of a restriction enzyme in the transformation mixture, the DNA fragments integrated into the respective genomic restriction sites by Restriction Enzyme-Mediated Integration (REMI) (Schiestl and Petes 1991). These original findings have, in the meantime, found widespread use for insertional mutagenesis and RFLP mapping in different organisms. In the absence of the restriction enzymes, the DNA fragments integrated by illegitimate integration falling into different classes. About 40% of integration events happen by microhomology mediated integration (Schiestl et al. 1993). Another 40% are mediated by topoisomerase I (Zhu and Schiestl 1996). Mutations of the yeast RAD50 gene reduce the frequency of illegitimate recombination 100 fold, but actually increase the frequency of homologous integration (Schiestl et al. 1993). We also found that restriction enzymes increase the frequency of integration of DNA fragments into human cells and that XRCC5 (Ku70) is involved in REMI (Submitted). We have achieved regulated expression of the human topoisomerase I in a yeast strain deleted for its own TOP1 gene. Most surprisingly, after overexpression of the human gene the frequency of illegitimate integration increased 20 fold and all additional events had target sites in the rDNA. Addition of a topoisomerase I inhibitor abolished this increase. We are currently developing a selective assay for illegitimate recombination (without the need for yeast transformation). With this assay we want to isolate mutants that increase (hyperrecombination) or decrease (hyporecombination) the frequency of IR and we also want to determine environmental factors that may alter the frequency of IR. Current results also show that ionizing radiation and other mutagens induce illegitimate integration. Thus we are able to map DSBs (e.g. meiotic, X-ray induced etc.) in yeast just by transforming a fragment, cloning and sequencing of the integration junctions and comparing those with the sequences in the database. This will yield the genomic distribution and sequence specificity of such illegitimate integration events that were attracted by genomic double-strand breaks.

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