Beyond the Identification of Transcribed Sequences: Functional and Expression Analysis

9th Annual Workshop, October 28-31, 1999

Co-sponsored by the U.S. Department of Energy


THIESEN

Genomic sequences of KOX zinc finger gene clusters are used to determine (auto) regulatory networks of gene regulation

Dirk Koczan1, Christian Sina1, Peter Lorenz1, Tom Hearn2, Saleh Ibrahim1, Andreas Rump3, Andre Rosenthal3, Michael Jackson2, M.-F. Rousseau-Merck4,  I. Legrand4, and Hans-Juergen Thiesen1

1Institute of Immunology, University of Rostock, Rostock, Germany
2Human Molecular Genetics Unit, Univ Newcastle upon Tyne, United Kingdom
3IMB, Jena, Germany
4Institute Curie, Paris, France

The evolutionarily conserved KRAB domain is encoded in about one-third of all human Krueppel-type zinc finger genes. Initially, the KRAB domain of KOX1 has been identified to display the strongest repression activity  identified in mammalian organisms. To study regulatory networks of gene regulation, high density PAC zinc finger gene filters have been generated to map Krueppel-type cDNAs to the human genome. 364 PAC zinc finger clones initially identified by a mixture of KOX cDNAs (KOX3, 4, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 22 and 28) were mapped by FISH to individual chromosomes. In particular, a contig of PAC clones encoding human KRAB zinc finger genes has been generated  representing the zinc finger gene cluster on chromosome 10p11.2 encoding the genes of KOX19 (ZNF25), of KOX21 (ZNF37) and of KOX31 (ZNF33) extended by two novel ZNF genes designated ZNF248 and ZNF249 physically linked to ZNF25. To determine whether these genes are coregulated within their clusters Northern blots complemented by TaqMan analysis were performed. Interestingly, differential splice forms were detected for KOX19 and their distribution are currently quantitated by quantitative real time RT-PCR (TaqMan ) analysis. In addition, more than 300.000 bp describing the zinc finger gene cluster of chromosome 10p11.2 are currently instrumentalized  to determine DNA binding sites for KOX1 and KOX19 proteins by making use of our TDA selection system. Our ongoing analysis indicates the presence of autoregulatory networks within and between zinc finger gene clusters mediated by target sequences of repetitive nature.


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