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


HEISS

Functional characterisation of genes in Xq28

Nina S. Heiss, Zdenek Sedlacek, Annemarie Poustka

Deutsches Krebsforschungszentrum (DKFZ), Department of Molecular Genome Analysis, Heidelberg, Germany

The systematic generation and integration of high-coverage physical and transcript maps in the chromosomal region Xq28 has facilitated the isolation of most genes in this region, as well as the identification of genes causally associated with diseases. We are currently analysing the developmental and tissue-specific expression of a selection of genes in Xq28 by RNA in situ hybridisation. Functional analyses include determination of the intracellular localisation of the corresponding proteins by using the enhanced green fluorescent protein (EGFP) as a tag, as well as generating antibodies and working on murine models in some cases. To complement the functional analyses on genes in Xq28 we are also carrying out evolutionary studies in this region. The DKC1 gene is responsible for causing the bone marrow failure syndrome dyskeratosis congenita (DKC) and is one example of a gene which we are characterising at the RNA expression and protein level. The DKC1 transcript is expressed ubiquitously and very early in murine embryological development. The protein dyskerin is highly conserved and appears to be involved in the pseudouridylation and cleavage of pre-rRNA. Dyskerin fused to the EGFP and expressed in mammalian cell lines localises to the nucleoplasm, the nucleoli, and the coiled bodies which are functionally associated with the nucleoli. Mutant constructs permitted a delineation of the sequences responsible for the nuclear targeting and indicated that mislocalisation of dyskerin is not a mechanism involved in the pathogenesis of DKC. Examples of genes in Xq28 being studied at the evolutionary level are rab GDI and XAP5. The autosomal paralogues of genes related to rab GDI and to XAP5 were isolated and mapped, and the amphioxus and fugu orthologues of the rab GDI genes were isolated. A comparison of the tissue-specific expression indicates that the various homologues have specialised functions.


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