title: Isolation and characterisation of a locus disrupted in a transgenic mouse mutant exhibiting sex-linked cleft palate
Creators: name: Laverty, Hugh Gerard
Publisher: name: University of Glasgow
Year: 1995
Subjects: subject: Genetics.

description: A unique transgenic mouse line displayed the phenotype of sex-linked cleft palate. As no other mouse line expressing this transgene demonstrated a similar phenotype, it is argued that the disorder is due to the disruption of the cellular locus. At the very least this mutation results in redirecting expression of the transgene in a unique fashion, alternatively, a gene(s) at the locus is critical to normal secondary palate development. Therefore 32.8Kb of cellular DNA of the cellular locus disrupted in this mutant has been cloned and characterised. This 32.8Kb of wild type DNA has been characterised at the level of conservation of sequence between species and expression. No potential gene encoding sequences were initially found. Subsequently, using the technique of exon-trapping several exons have been isolated from the wild type locus. When these exons were hybridised to Northern blots of embryonic and post-natal RNAs they revealed a 4.5Kb, a 0.8Kb and a 0.5Kb transcript, each expressed in the head and body during embryonic development. The 0.8Kb message was shown to be expressed post-natally in a tissue specific manner. Five individual exons have been isolated and sequenced. They range in size from 81bp to 446bp. One exon (exon p50, 446bp in size) was found to be composed of repetitive sequence but it did not contain an open reading frame (ORF) that spanned the exon. The other four exons (exon p51, p52, p56 and p57) were found to contain an ORF that spanned the exon. Database analysis with the sequence of the exons revealed that two of the exons (exon p56 and exon p57) showed no homology to nucleotide sequences in databases, and another (exon p52) exhibited a high level of similarity to CaM kinase II at both the nucleotide and amino acid level. Using RT-PCR, two of the exons (exon p52 and exon p57) were shown to be expressed at the RNA level during development in the wild type mouse. Exon p52 and exon p57 were also found to be spliced together. Cloning and sequencing of this exon p52-exon p57 RT-PCR product revealed that this RT-PCR product contained a single ORF that spanned the sequence of both exons spliced together. The amino acid sequence of the putative peptide encoded by this ORF revealed 47% identity to the calmodulin-binding domain of CaM kinase II. Analysis of this RT-PCR product in RNA derived from transgenically positive spleen revealed that several products are present which are absent in the wild type. These novel products hybridise with exon p52 and exon p57 sequences as well as the transgene sequences, indicating that the splicing of the RT-PCR product is aberrant in the transgenic mutant. A human form of heritable sex-linked cleft palate has been well documented. The phenotypes observed in the transgenic mutant and the cloning of the gene from the wild type locus may lead to the identification of the human gene involved in heritable sex-linked cleft palate, as well as provide a model for the analysis of the disorder.