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Transforming genes in human malignant melanoma.

Hughes, David C. (1989) Transforming genes in human malignant melanoma. Doctoral thesis, University of Surrey (United Kingdom)..

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Human cutaneous malignant melanoma progresses through well-defined stages, culminating in metastatic melanoma. The underlying genetic changes have not be n identified. The aim of this work was to identify activated transforming genes in human melanoma samples. Transfection assays have been used to identify potential transforming genes in melanoma samples, and transmissable transforming activity was demonstrated in several cases. A secondary transfectant obtained following transfection of DNA from the cell line NKI4 retained a single human repeat sequence-containing EcoRI fragment of approximately 16 kb. This fragment was molecularly cloned and analysis revealed the presence of human sequences corresponding to a novel human gene, which was designated MEL. Homologous sequences were also present in mouse DNA. The MEL gene was expressed in human melanoma cell lines and NIH 3T3 mouse fibroblasts, and cDNA clones were isolated from a normal human fibroblast cDNA library. The human MEL gene was localised to a region of chromosome 19 (p13. 2) implicated in some human malignancies. However the clone isolated from the secondary transfectant lacked transforming activity, and was not retained in either tertiary transformants or additional primary transformants. A related sequence was isolated from the cell line NKI4, which was capable of transforming NIH 3T3 cells. This sequence also detected a restriction fragment length polymorphism in human melanoma cell lines. To conclusively identify the transforming gene in NKI4, transfectant DNA was "tagged" with a selectable marker, G418 resistance (neo). The sequences adjacent to the "tag" on one side were cloned and analysed, from which it was deduced that the transforming gene must lie on the other side of the "tag". Melanoma cell lines and tumour biopsies were analysed for the presence of activated RAS genes, using a combination of biological (transfection) and biochemical (restriction enzyme polymorphisms, PCR and sequence specific oligonucleotide hybridisation) assays. Mutations were detected in all three RAS genes (K12, H12, N61), at all stages of melanoma development, including benign naevi.

Item Type: Thesis (Doctoral)
Divisions : Theses
Authors :
Hughes, David C.
Date : 1989
Contributors :
Additional Information : Thesis (Ph.D.)--University of Surrey (United Kingdom), 1989.
Depositing User : EPrints Services
Date Deposited : 22 Jun 2018 13:02
Last Modified : 06 Nov 2018 16:52

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