Sukumaran, Madhu; (1997) Cloning and molecular characterisation of the murine 'c-ret' locus. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

To study the regulation of expression of the murine c-ret proto-oncogene, we have cloned and initiated the molecular characterisation of the c-ret locus. cDNA clones representing the long and short isoforms of the murine c-ret proto-oncogene have been cloned from a cDNA library synthesised from mRNA isolated from adult mouse salivary glands. Isolated cDNA clones have been used to screen genomic libraries to isolate cosmid clones representing the murine c-ret locus. Approximately 60kb of the mouse genome, representing the entire c-ret gene structure, have been cloned as five overlapping cosmid clones spanning 12 kb 5' of the gene locus. Primer extension, S1 nuclease protection and DNA sequences analysis have been used to identify the transcription initiation site and to characterise the promoter of the murine c-ret gene. Two different transcription initiation sites, 40 bps apart, which synthesises a major and a minor population of c-ret mRNA have been identified in NB2α cells. The major transcription initiation site of the locus have been localised to be 254 bps upstream of translation initiation site of the gene. DNA sequence analysis of the promoter of the gene show the murine c-ret locus, like the human c-ret gene, to lack the TATA initiation consensus motif and have high GC content. The G+C content (67%) and CG:GC ratio (0.95) over the promoter and 5'UTR of the murine c-ret locus (719 bps) implies the presence of a putative methylation-free CpG island within this segment of the genome. Functional significance (if any) for the presence of this putative CpG island within the c-ret promoter remains to be analyzed. Analysis of interspecies promoter DNA sequence conservation showed low (37%) overall sequence homology within the mouse and human c-ret promoters. Apart from the conservation of putative Sp1 binding GC box sequences at -40 and -110 bps, there were no other significant alignment and conservation of transcription factor binding consensus motifs within the two promoters. Promoter deletion analysis of 5' flanking sequences of the murine c-ret locus in transgenic mice suggest a locus organised as linked modules of independent regulatory domains. The distal 6kb of the 5' flanking sequences harbours the putative regulatory sequences of the locus which induce the early expression of the gene at the proximal end of the primitive streak and facial ganglion. The proximal 6 kb of 5' flanking sequences of the locus harbours the putative cis-regulatory elements which direct expression to the PNS, CNS and the urogenital system. The 5' flanking sequences of the locus recapitulate the complex and dynamic expression of the endogenous gene in the sensory branch of the PNS. In addition to these, the 5' flanking sequences of the locus can also direct expression to a specific subset of cells of the thyroid and parathyroid. However, the 5' flanking sequences of the locus do not possess the cis- elements which regulate the full spectrum of the endogenous gene expression: the elements which direct expression to the enteric and sympathetic nervous systems and the mesonephros seems to be present elsewhere in the locus. Further characterisation of the reminder of the locus has to be performed in order to identify and characterise the regulatory elements which regulate the full spatial expression profile of the endogenous gene. In addition to these studies, we have initiated genetic experiments using the established transgenic lines to study the ontogeny of the c-ret expressing cell-lineage in the null genetic background. Preliminary analysis indicate a reduction in Lac Z staining intensity in DRG isolated from mutant transgenic neonates compared to wild type transgenic neonates. Conversely, preliminary experiments indicate increased numbers of Lac Z staining c-cells in the thyroids of mutant transgenic neonates compared to the wild type transgenic thyroids. Further, more detailed quantitative analysis is required to substantiate these interesting preliminary findings. The detailed analysis of the possibility of plasticity, functional compensation or redundancy in c-ret function within the neural crest cell linage which establish the sensory nervous system and the c-cells of the thyroid, remains for further study.

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