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Goals: We want to understand the function of genomic insulators which permit the topological and functional separation of gene domains and the directed action of enhancers/locus control regions. These aims are pursued as a contribution to phase II of the DHGP and as an approach for identifying active elements for the design of novel integrating and episomal vector systems for the systematic modification of cells, tissues and transgenic animal models.

Background: Vertebrate genomes are subdivided by 20-50000 scaffold/matrix-attachment regions (S/MARs) which enable the independent regulation (transcription, replication) of the enclosed domains. S/MARs are a novel adition to the group of cis-acting genomic elements which are apparently devoid of a sequence consensus. A unifying feature is their propensity to adopt secondary structures which is supported by their stable base-unpairing under negative superhelical tension. This property is now exploited to predict the localization and activity of S/MARs by dedicated biomathematical anylyses (stress-induced duplex destabilization, SIDD). S/MARs have proven to be important landmarks enabling the localization of functional genes and enhancers in the framework of various eukaryotic genome projects. As a paradigm we analyze the structure and function of the human type 1 interferon gene cluster on 9p21/22, the 26 members of which are separated by S/MARs regions. We characterize the scope of biological activities for these and other genomic insulators.

    For all mammals type I Interferons occur in a cluster consisting of “early” interferon genes (IFN-beta and IFN-alpha2 in humans) and “late” genes, which depend on the prior induction of the early members. Whereas the induction mechanism for IFN-beta is known in considerable detail the same is clearly not true for the alpha interferons. Present studies address this problem by a combination of biomathematics and laboratory experiments (see below).

Our results and their significance: The active members of the human interferon gene cluster are regularly organized by strong S/MARs; pseudogenes have lost this propery. The interpretation of SIDD profiles covering the 400 kb region has been used as a guideline for the localization in vivo not only of S/MARs but also of other dominant structures such as DNAse I hypersensitive and topoismerase II cleavage sites. These combined data lead to a deeper understanding of expression characteristics as well as of genomic instablity and apoptotic cleavage phenomena. Since deletion events on 9p frequently lead to the simultaneous loss of one or several tumor suppressor genes (prototype p16INK4A), they form the molecular basis for cellular immortalization and a wide variety of cancers. While the murine IFN-p16INK4A locus on chromosome 4 is clearly set up in an analogous fashion, its sequence analysis is still to come. Upon completion it will permit the design of transgeneic mouse deletion mutants in which the involvement of various IFN-members in host-pathogen interactions can be studied.

S/MARs and their associated sequences have a number of properties which make them powerful tools for designing predictable gene expression systems. Site specific recombinase-based assays (excision and recombinase-mediated cassette exchange, RMCE) have been used to dissect transcriptional ´augmentation´ functions which manifest themselves after but not before integration, their insulator function and their activity in methylation-related suppression phenomena. A recent addition to the spectrum of activities is the support of ORI functions: we constructed the first example of an episomal vector  which replicates extrachromosomally for at least several hundred generations in the absence of selection pressure and without the support by a virally encoded protein. In this context, obviously, the S/MAR sequence is capable of recruiting the endogenous replication and segregation apparatus of the host cell enabling the design of entirely novel tools for gene therapeutic and biotechnological purposes. See <current thoughts>  

Patent   

  • J, Bode, J. Seibler and D. SchĂĽbeler (2005) Methods for marker-free repetitive DNA expression of cassette exchange in the genome of cells or parts of cells EP0939120, US20010032341, CA2263482

Applications

  • Baiker, A., Bode, J., Fetzer, C., Lipps, H. J. & Piechaczek, C. (Deutsche und Internationale Patentanmeldung 10078487 / 1999) Episomal replizierender Vektor zur permanenten Expression eines Transgens in Säugerzellen.
  • Baiker, A., Stehle, I., Bode, J. & Lipps, H. J. (Deutsche und Internationale Patentanmeldung 2003) Synthetischer, episomal replizierender und mitotisch stabiler Vektor als Expressionssystem fĂĽr Säugerzellen.Europäische Patentanmeldung 103 29 027.3-41.
     

Teaching and Further Interests

http://de.wikipedia.org/wiki/Benutzer:Juergen_Bode

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The Nuclear Matrix Group (Heraklion, Crete 1999)

Dean Jackson/Steve Krawetz/Stefan Stamm/Frank Fackelmayer/ Amos Simon
Elena Matthia/Jordanka Zlatanova
Teni Boulikas/Paola Caiafa/Juergen Bode

bode.juergen@mh-hannover.de
juergenbode@gmx.de

Semester