Using a recently developed technique for comparison of selected parts of the genome (Lavrentieva et al., FEBS Lett. 1999, v. 443, 341) the genome rearrangements occurring in tumor cells in the vicinities of sites of integrations of Human Endogenous Retroviruses will be investigated. Such a problem to our knowledge has not been systematically investigated by now in particular due to absence of the adequate experimental technique allowing whole genome comparisons of RE abundance and position in normal and tumor cells.

The following goals for the current proposal are put forward:
(i) Identification of the differences in the distribution of HERVs and their LTRs between the genomes of normal cells and their tumor relatives by means of the "selected genomic differential display" developed recently by participant 3;
(ii) Isolation, sequencing and mapping of the genomic sequences flanking the newly inserted/translocated/deleted HERV/LTRs. The technique will also allow identification of losses of heterozygosity;
(iii) Identification of the genes situated in the vicinities of the newly inserted/translocated/deleted RE and investigation of the changes in their transcriptional status during tumor transformation;
(iv) Analyses of the various tumors and evaluation of the tumor-specificities of the observed HERV-centred rearrangements.

To achieve these goals all research will be divided in four main blocks.
1. The surgery tissue collection of different stages of germ cell tumors, both already available and newly prepared, will be used for DNA preparation by the research team from the Cancer Research Centre, Moscow. Similarly, DNA from a collection of tumors from the Karolinska Institute will be prepared by the team;
2. These samples will be accompanied by unaffected tissue material from the same individuals. Informed consent of the patients for participation in this study will be obtained.
2. The comparative differential display of the DNA from normal and tumor tissues and isolation of differences will be implemented by the Moscow team at Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry according to the technique developed by this team (Lavrentieva et al, FEBS Lett. 1999, v. 443, 341). The differences will be isolated, reamplified and cloned, and then transferred to the group in Marburg for sequencing and mapping;
3. Sequencing and mapping of the differences identified according task 2 will be implemented in Marburg involving bioinformatic search of the homologous sequences, use of radiation hybrids, contigs of cosmids from the area identified with the hybrids, FISH-hybridisation with the clones obtained in task 2;
4. The identification of the known and unknown genes situated within the regions involved in the rearrangement will be undertaken both in Moscow at the Shemyakin-Ovchinnikov Institute and in Marburg with the use of bioinformatic resources and by direct selection of the cDNAs from the cDNA libraries with the cosmids containing DNA from the rearranged genome region. The cDNAs selected will be sequenced and the sequences will be searched to find matches in the existing EST database. In future comparative analyses of the expression of the corresponding genes in normal and tumor cells will be implemented by means of Northern-blot hybridisation and RT-PCR.
Expected Results: The technique applied will allow detecting genome rearrangements occurring during tumor progression, including losses of heterozygosity. In case of success of the research project the involvement of the HERVs in development of the tumor cell genome instability and deregulation will be demonstrated. The tumor specificity of the rearrangements will be revealed. The results will help to obtain a deeper insight into mechanisms of tumor formation and could be of importance for diagnostic and therapeutic purposes.