From Birds to Drug-Resistant Cancer, a novel In situ Methodology to Explore Divergent Genome Evolution

Fluorescent hybridisatio nmethodologies have not changed in principles over the past 30 years, with the increase of computational sequencing technologies causing the replacement of in situ hybridisations. Fluorescence in situ hybridisation (FISH) is in need of a refresh to be a worthwhile tool in a modern day cytogenetic laboratory to overcome short comings of these new methods. The creation of the novel multilayer FISH protocol has effectively eliminated many negative aspects of classic FISH based experiments, such as a large reduction in cost and is no longer as limited by fluorophore availability. Here presented within this thesis is the creation of this methodology and application to a wide variety of cytogenetic hypothesises.

Key species from the Galliform order were investigated in order to detect previously missed intrachromosomal rearrangements within their macrochromosomes, a premise formerly overlooked. Rearrangements were found within chromosomes of the galliforme species used such as E.chinensis which displays a intrachromosomal inversion on the p-arm of chromosome 2. Furthermore, the creation of an interphase state folding prediction tool has been used to assess the arrangement of macrochromosomes during cellular growth stages within G.gallus. Here it is noted that there are particular arrangements identified which are similar across chromosomes studied.

The chicken lymphoma cell line DT40 is of great importance in B-cell receptor studies along with gene disruption experiments. Presented here is an updated karyotype for the cell line. Here shows contrasting and more in-depth evidence of aberrations to further develop our understanding of the genomic arrangement of this useful cell line.

The level of tumour heterogeneity in a cancer is a diagnostic tool allowing clinicians to comment on therapeutic choices and prognosis of the disease. Found to be dominant in recurrent cancers, cytotoxic resistant tumour cell populations may indeed exist within initial primary tumours at low frequency to be positively selected during chemotherapy. Within a neuroblastoma cell line,and cyto-toxic resistant derivatives lines,there has been identified a level of genomic heterogeneity which may give clues towards the generation of drug resistance mechanisms.