Civill, N.D.; (2000) Characterisation of a bagpipe homologue in Xenopus laevis. Doctoral thesis , University of London.

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Abstract

Mutations of Drosophila bagpipe, an NK3 class homeobox gene, result in failure of visceral mesoderm to differentiate into stomach tissue. Thus bagpipe, in association with other factors, is a good candidate for specification of visceral mesoderm in Drosophila. The cloning and characterisation of a Xenopus bagpipe homologue was therefore of great interest. This study describes the isolation of a full length Xenopus cDNA clone that on the basis of database analysis and sequence comparisons has been assigned as Xenopus bagpipe (XBap). Previous studies had revealed that the majority of homeoproteins recognise DNA sites with a 5'-TAAT-3' core but the NK class of homeoproteins had been shown to bind specifically to sites containing a 5'-CAAG-3' core. Experiments described here, however, show the XBap DNA binding site to be an even more divergent, 5'-TTAAGTGG-- TTAAGTGG-3'. A series of mutant oligonucleotides revealed that the `T' of the 5'- T_{1}A_{2}A_{3}G_{4}-3' core, as well as the presence of two such cores, are indeed essential for optimal XBap DNA binding. The murine NK3 class homeoproteins, Nkx-3.1 and Bapxl, are demonstrated to have the same requirements for optimal DNA binding as XBap. Drosophila Bagpipe, however, was found to have a less stringent requirement for a `T' at position one of the core, binding equally well to a 'C' in this position, but the presence of two such cores is still necessary for optimal DNA binding. Preliminary studies using site directed mutagenesis attempted to define the amino acids responsible for the differences. The effect of XBap on transcription was studied using a Xenopus oocyte assay and two cell transfection assays. XBap was not found to act as a transcriptional activator in any of these assays but evidence was obtained to suggest that a C-terminal truncation of XBap could act as a repressor of transcription.

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