Biochemical and genetic properties of HPRT Cape Town

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dc.contributor.advisorHarley, Eric Hen_ZA
dc.contributor.authorGalloon, Terryen_ZA
dc.date.accessioned2017-12-13T14:12:39Z
dc.date.available2017-12-13T14:12:39Z
dc.date.issued1987en_ZA
dc.description.abstractAn unusual partial HPRT deficient mutant, HPRT Cape Town was observed to have a low activity in erythrocyte lysates at high concentrations of the purine substrates, hypoxanthine and guanine. This substrate inhibition was not observed with the substrate PPRP. The low activity was not associated with changes in the Km or Vmax for any of the substrates (Steyn and Harley, 1984). The kinetics of the proband's enzyme was studied in lymphoblast extracts. The characteristic substrate inhibition was observed which showed that this phenomenon was not confined to erythrocytes but was a more generalized phenomenon. This result implies that the decreased HPRT activity observed in the proband is due to substrate inhibition by the purine bases. The HPRT enzyme is coded for by a gene which is located on the X chromosome (Pai et al., 1980). The proband's daughter was therefore studied in order to determine the cause of the mutation. It was not known whether the substrate inhibition was the result of a mutation in the gene coding for the enzyme, a mutation which results in altered post-translational modification or the absence or alteration of factors influencing normal HPRT kinetics. The daughter's transformed lymphoblasts exhibited growth patterns in selective media that resembled those of her father. The daughter's enzyme prepared from lymphoblast extracts exhibited the characteristic substrate inhibition. These results suggest that this cell line results from the selection of a clone or clones which have suppressed the function of the X chromosome carrying the maternal and presumably normal HPRT allele. The daughter's enzyme prepared from erythrocyte lysates exhibited intermediate enzyme activity between that of the proband and a normal control. This result suggests that the daughter is an obligate heterozygote and that the defect is due to a mutation in the HPRT gene itself. The defect was studied at the gene level. No difference was observed in the banding patterns of the proband's DNA and control DNA which were digested with various restriction enzymes and hybridized to ³²p-labelled HPRT cDNA. The size of the HPRT mRNA of the proband was the same as the control. These results imply that there is no major gene alteration; this is expected since the proband only has a partial deficiency of the enzyme. The HPRT cDNA was subcloned into a riboprobe vector, pGEM-3. The T7 promoter was used to transcribe antisense RNA strands which were then hybridized to the proband's RNA and control RNA. No difference was observed in the size of the protected fragment. This result does not exclude the possibility of a point mutation as the cause of the defect in HPRT Cape Town.en_ZA
dc.identifier.apacitationGalloon, T. (1987). <i>Biochemical and genetic properties of HPRT Cape Town</i>. (Thesis). University of Cape Town ,Faculty of Health Sciences ,Division of Chemical Pathology. Retrieved from http://hdl.handle.net/11427/26591en_ZA
dc.identifier.chicagocitationGalloon, Terry. <i>"Biochemical and genetic properties of HPRT Cape Town."</i> Thesis., University of Cape Town ,Faculty of Health Sciences ,Division of Chemical Pathology, 1987. http://hdl.handle.net/11427/26591en_ZA
dc.identifier.citationGalloon, T. 1987. Biochemical and genetic properties of HPRT Cape Town. University of Cape Town.en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Galloon, Terry AB - An unusual partial HPRT deficient mutant, HPRT Cape Town was observed to have a low activity in erythrocyte lysates at high concentrations of the purine substrates, hypoxanthine and guanine. This substrate inhibition was not observed with the substrate PPRP. The low activity was not associated with changes in the Km or Vmax for any of the substrates (Steyn and Harley, 1984). The kinetics of the proband's enzyme was studied in lymphoblast extracts. The characteristic substrate inhibition was observed which showed that this phenomenon was not confined to erythrocytes but was a more generalized phenomenon. This result implies that the decreased HPRT activity observed in the proband is due to substrate inhibition by the purine bases. The HPRT enzyme is coded for by a gene which is located on the X chromosome (Pai et al., 1980). The proband's daughter was therefore studied in order to determine the cause of the mutation. It was not known whether the substrate inhibition was the result of a mutation in the gene coding for the enzyme, a mutation which results in altered post-translational modification or the absence or alteration of factors influencing normal HPRT kinetics. The daughter's transformed lymphoblasts exhibited growth patterns in selective media that resembled those of her father. The daughter's enzyme prepared from lymphoblast extracts exhibited the characteristic substrate inhibition. These results suggest that this cell line results from the selection of a clone or clones which have suppressed the function of the X chromosome carrying the maternal and presumably normal HPRT allele. The daughter's enzyme prepared from erythrocyte lysates exhibited intermediate enzyme activity between that of the proband and a normal control. This result suggests that the daughter is an obligate heterozygote and that the defect is due to a mutation in the HPRT gene itself. The defect was studied at the gene level. No difference was observed in the banding patterns of the proband's DNA and control DNA which were digested with various restriction enzymes and hybridized to ³²p-labelled HPRT cDNA. The size of the HPRT mRNA of the proband was the same as the control. These results imply that there is no major gene alteration; this is expected since the proband only has a partial deficiency of the enzyme. The HPRT cDNA was subcloned into a riboprobe vector, pGEM-3. The T7 promoter was used to transcribe antisense RNA strands which were then hybridized to the proband's RNA and control RNA. No difference was observed in the size of the protected fragment. This result does not exclude the possibility of a point mutation as the cause of the defect in HPRT Cape Town. DA - 1987 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 1987 T1 - Biochemical and genetic properties of HPRT Cape Town TI - Biochemical and genetic properties of HPRT Cape Town UR - http://hdl.handle.net/11427/26591 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/26591
dc.identifier.vancouvercitationGalloon T. Biochemical and genetic properties of HPRT Cape Town. [Thesis]. University of Cape Town ,Faculty of Health Sciences ,Division of Chemical Pathology, 1987 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/26591en_ZA
dc.language.isoengen_ZA
dc.publisher.departmentDivision of Chemical Pathologyen_ZA
dc.publisher.facultyFaculty of Health Sciencesen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.subject.otherEnzymes - Analysisen_ZA
dc.subject.otherLesch-Nyhan syndromeen_ZA
dc.titleBiochemical and genetic properties of HPRT Cape Townen_ZA
dc.typeMaster Thesis
dc.type.qualificationlevelMasters
dc.type.qualificationnameMSc (Med)en_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceThesisen_ZA
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