Estudos anteriores realizados no Laboratório de Fisiologia Vegetal do IBUSP com Guzmania monostachia demonstraram que quando essas plantas são submetidas ao déficit hídrico ocorre a indução do CAM, com maior expressão desse metabolismo na porção foliar apical. Para outra espécie (Vriesea gigantea), foi verificada a maior atividade da enzima nitrato redutase (NR) na porção basal durante o período diurno. Em uma bromélia terrestre (Ananas comosus) foi observada a sinalização por citocininas tanto na indução da expressão gênica, quanto na ativação da NR. Outros laboratórios evidenciaram que plantas de Mesembryanthemum crystallinum induzidas ao CAM apresentaram uma provável regulação negativa da fosfoenolpiruvato carboxilase (PEPC) por citocininas. Em decorrência desses conhecimentos acumulados, surgiram novos questionamentos: haveria variações diuturnas da atividade das enzimas PEPC e NR nas diferentes porções das folhas de G. monostachia induzidas ao CAM? A maior disponibilidade de esqueletos carbônicos à noite (acúmulo de acidez) influenciaria positivamente a atividade da NR, deslocando seu pico de atividade para o período noturno? As variações dos teores endógenos de citocininas acompanhariam as possíveis mudanças da atividade da PEPC e da NR, indicando, assim, a participação dessa classe hormonal na regulação dessas enzimas? O presente trabalho teve por objetivo principal investigar uma possível regulação da atividade das enzimas PEPC e NR por citocininas em folhas destacadas da bromélia epífita com tanque, Guzmania monostachia (Bromeliaceae) induzidas ao CAM. Foi esperado com esta pesquisa aprofundar os estudos sobre a inter-relação entre o comportamento fotossintético, a capacidade de assimilação de nitrogênio e a possível regulação das atividades da PEPC e da NR por citocininas endógenas. Análises de acidez titulável, ácidos orgânicos, amido endógeno e da atividade da enzima malato desidrogenase (MDH) foram realizadas, confirmando a indução do CAM nas folhas isoladas de G. monostachia mantidas em polietilenoglicol (PEG) a uma concentração de 30%. O uso desse composto foi eficiente na redução do conteúdo relativo de água e na imposição da deficiência hídrica foliar. Além disso, pôde-se verificar a maior expressão do CAM na porção apical das folhas mantidas em PEG 30%, quando comparada à porção basal. Análises da atividade da PEPC e da NR permitiram verificar a separação espacial dessas enzimas. A primeira apresentou maior atividade no ápice foliar, enquanto a segunda mostrou a maior atividade na porção basal. Apesar disso, não foi observada a separação temporal dessas enzimas, uma vez que ambas apresentaram picos de atividade noturna. A maior atividade da NR durante o período escuro (01 hora) foi verificada nas folhas-controle ou sob deficiência hídrica. Esse resultado sugere que outros fatores, diferentes do metabolismo CAM, influenciaram para a ocorrência da maior atividade dessa enzima durante o período noturno. Os resultados obtidos ainda sugerem que as citocininas possivelmente atuaram como um regulador negativo para a atividade da PEPC durante o dia, uma vez que os maiores níveis endógenos desse hormônio foram observados durante esse período, enquanto a maior atividade dessa enzima foi verificada durante a noite, quando os teores de Z+iP decaíram significativamente. A aplicação de Z ou iP resultou também num decréscimo da atividade dessa enzima. Por outro lado, as citocininas atuaram como um provável regulador positivo para a atividade da NR, uma vez que a maior atividade noturna dessa enzima foi antecedida em 3 ou 6 horas pelos maiores níveis endógenos de citocininas na porção basal das folhas mantidas em água ou PEG 30%, respectivamente. A aplicação de citocininas-livres aumentou significativamente a atividade da NR na base das folhas destacadas mantidas em água ou PEG 30% / Prior studies undertaken in the Laboratory of Plant Physiology on IBUSP with Guzmania monostachia have shown that during water shortage, CAM induction occurs with greater expression in the apical portion of the leaf. In the case of another species (Vriesea gigantean), more intense nitrate reductase (NR) enzyme activity was observed in the basal portion during the daytime. In a certain terrestrial bromeliad (Ananas comosus), signaling by cytokinins, both in the induction of gene expression as well as NR activation, was observed. According to other laboratories, the cytokinins seem to play a negative regulation of phosphoenolpyruvate carboxylase (PEPC) in CAM induced Mesembryanthemum crystallinum plants. As a result of accumulated knowledge, new questions have arisen, such as: Are there daily variations in PEPC and NR enzymes activity in the different portions of CAM induced leaves of G. monostachia? Would the more pronounced nocturnal availability of carbon skeletons (accumulation of acidity) positively influence NR activity, with consequential displacement of its peak of activity to this period? Would variations in endogenous cytokinins concentration accompany possible changes in PEPC and NR activity, thereby indicating the participation of this hormonal class in their regulation? The main aim in the present study was to investigate the possible regulation of PEPC and NR activity by cytokinins in detached CAM-induced leaves of the epiphyte tank bromeliad Guzmania monostachia (Bromeliaceae). The expectations with this research were to study more deeply the inter-relationship between photosynthetic behavior, the capacity for nitrogen assimilation and the possible regulation of PEPC and NR activity by endogenous cytokinins. Analyses of titratable acidity, organic acids, endogenous starch and malate dehydrogenase (MDH) enzyme activity confirmed CAM induction in isolated leaves of G. monostachia kept in polyethylene glycol (PEG) at a concentration of 30%. The use of this compound was efficient in reducing relative water content and imposing leaf water deficiency. Furthermore, compared to the basal portion, greater CAM expression could be observed in the apical portion of leaves kept in PEG 30%. Analyses of PEPC and NR activity allowed detecting their mutual spatial separation, seeing that, in the first greater activity was concentrated in the leaf apex, while in the second this was more pronounced in the basal portion. Even so, no temporal separation could be observed, since peak of activity for both occurred at night. The peak of nocturnal NR activity (1 hour) was observed in control leaves or those undergoing water deficiency, thereby implying that factors, other than CAM metabolism, exerted an influence on the occurrence of more intense activity of this enzyme at this time. Furthermore, there were indications that cytokinins possibly act as a negative regulator of PEPC activity during the daytime, when the highest endogenous levels of this hormone were observed, whereas it was apparent that the most intense activity of this enzyme actually occurred at night, when Z+iP rates decreased significantly. Z or iP application also induced a decrease in the activity of this enzyme. On the other hand, the cytokinins acted as a positive regulator of NR activity, since the nocturnal peak of activity of this enzyme was preceded by 3 or 6 hours by higher endogenous levels of cytokinins in the basal portion of leaves maintained in water or PEG 30%, respectively. The application of free cytokinins induced a significant increase in NR activity in the base of detached leaves kept in water or PEG 30%

Thesis (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains ix, 175 p. : ill. Includes abstract. Includes bibliographical references.

Estudos anteriores realizados no Laboratório de Fisiologia Vegetal do IBUSP com Guzmania monostachia demonstraram que quando essas plantas são submetidas ao déficit hídrico ocorre a indução do CAM, com maior expressão desse metabolismo na porção foliar apical. Para outra espécie (Vriesea gigantea), foi verificada a maior atividade da enzima nitrato redutase (NR) na porção basal durante o período diurno. Em uma bromélia terrestre (Ananas comosus) foi observada a sinalização por citocininas tanto na indução da expressão gênica, quanto na ativação da NR. Outros laboratórios evidenciaram que plantas de Mesembryanthemum crystallinum induzidas ao CAM apresentaram uma provável regulação negativa da fosfoenolpiruvato carboxilase (PEPC) por citocininas. Em decorrência desses conhecimentos acumulados, surgiram novos questionamentos: haveria variações diuturnas da atividade das enzimas PEPC e NR nas diferentes porções das folhas de G. monostachia induzidas ao CAM? A maior disponibilidade de esqueletos carbônicos à noite (acúmulo de acidez) influenciaria positivamente a atividade da NR, deslocando seu pico de atividade para o período noturno? As variações dos teores endógenos de citocininas acompanhariam as possíveis mudanças da atividade da PEPC e da NR, indicando, assim, a participação dessa classe hormonal na regulação dessas enzimas? O presente trabalho teve por objetivo principal investigar uma possível regulação da atividade das enzimas PEPC e NR por citocininas em folhas destacadas da bromélia epífita com tanque, Guzmania monostachia (Bromeliaceae) induzidas ao CAM. Foi esperado com esta pesquisa aprofundar os estudos sobre a inter-relação entre o comportamento fotossintético, a capacidade de assimilação de nitrogênio e a possível regulação das atividades da PEPC e da NR por citocininas endógenas. Análises de acidez titulável, ácidos orgânicos, amido endógeno e da atividade da enzima malato desidrogenase (MDH) foram realizadas, confirmando a indução do CAM nas folhas isoladas de G. monostachia mantidas em polietilenoglicol (PEG) a uma concentração de 30%. O uso desse composto foi eficiente na redução do conteúdo relativo de água e na imposição da deficiência hídrica foliar. Além disso, pôde-se verificar a maior expressão do CAM na porção apical das folhas mantidas em PEG 30%, quando comparada à porção basal. Análises da atividade da PEPC e da NR permitiram verificar a separação espacial dessas enzimas. A primeira apresentou maior atividade no ápice foliar, enquanto a segunda mostrou a maior atividade na porção basal. Apesar disso, não foi observada a separação temporal dessas enzimas, uma vez que ambas apresentaram picos de atividade noturna. A maior atividade da NR durante o período escuro (01 hora) foi verificada nas folhas-controle ou sob deficiência hídrica. Esse resultado sugere que outros fatores, diferentes do metabolismo CAM, influenciaram para a ocorrência da maior atividade dessa enzima durante o período noturno. Os resultados obtidos ainda sugerem que as citocininas possivelmente atuaram como um regulador negativo para a atividade da PEPC durante o dia, uma vez que os maiores níveis endógenos desse hormônio foram observados durante esse período, enquanto a maior atividade dessa enzima foi verificada durante a noite, quando os teores de Z+iP decaíram significativamente. A aplicação de Z ou iP resultou também num decréscimo da atividade dessa enzima. Por outro lado, as citocininas atuaram como um provável regulador positivo para a atividade da NR, uma vez que a maior atividade noturna dessa enzima foi antecedida em 3 ou 6 horas pelos maiores níveis endógenos de citocininas na porção basal das folhas mantidas em água ou PEG 30%, respectivamente. A aplicação de citocininas-livres aumentou significativamente a atividade da NR na base das folhas destacadas mantidas em água ou PEG 30% / Prior studies undertaken in the Laboratory of Plant Physiology on IBUSP with Guzmania monostachia have shown that during water shortage, CAM induction occurs with greater expression in the apical portion of the leaf. In the case of another species (Vriesea gigantean), more intense nitrate reductase (NR) enzyme activity was observed in the basal portion during the daytime. In a certain terrestrial bromeliad (Ananas comosus), signaling by cytokinins, both in the induction of gene expression as well as NR activation, was observed. According to other laboratories, the cytokinins seem to play a negative regulation of phosphoenolpyruvate carboxylase (PEPC) in CAM induced Mesembryanthemum crystallinum plants. As a result of accumulated knowledge, new questions have arisen, such as: Are there daily variations in PEPC and NR enzymes activity in the different portions of CAM induced leaves of G. monostachia? Would the more pronounced nocturnal availability of carbon skeletons (accumulation of acidity) positively influence NR activity, with consequential displacement of its peak of activity to this period? Would variations in endogenous cytokinins concentration accompany possible changes in PEPC and NR activity, thereby indicating the participation of this hormonal class in their regulation? The main aim in the present study was to investigate the possible regulation of PEPC and NR activity by cytokinins in detached CAM-induced leaves of the epiphyte tank bromeliad Guzmania monostachia (Bromeliaceae). The expectations with this research were to study more deeply the inter-relationship between photosynthetic behavior, the capacity for nitrogen assimilation and the possible regulation of PEPC and NR activity by endogenous cytokinins. Analyses of titratable acidity, organic acids, endogenous starch and malate dehydrogenase (MDH) enzyme activity confirmed CAM induction in isolated leaves of G. monostachia kept in polyethylene glycol (PEG) at a concentration of 30%. The use of this compound was efficient in reducing relative water content and imposing leaf water deficiency. Furthermore, compared to the basal portion, greater CAM expression could be observed in the apical portion of leaves kept in PEG 30%. Analyses of PEPC and NR activity allowed detecting their mutual spatial separation, seeing that, in the first greater activity was concentrated in the leaf apex, while in the second this was more pronounced in the basal portion. Even so, no temporal separation could be observed, since peak of activity for both occurred at night. The peak of nocturnal NR activity (1 hour) was observed in control leaves or those undergoing water deficiency, thereby implying that factors, other than CAM metabolism, exerted an influence on the occurrence of more intense activity of this enzyme at this time. Furthermore, there were indications that cytokinins possibly act as a negative regulator of PEPC activity during the daytime, when the highest endogenous levels of this hormone were observed, whereas it was apparent that the most intense activity of this enzyme actually occurred at night, when Z+iP rates decreased significantly. Z or iP application also induced a decrease in the activity of this enzyme. On the other hand, the cytokinins acted as a positive regulator of NR activity, since the nocturnal peak of activity of this enzyme was preceded by 3 or 6 hours by higher endogenous levels of cytokinins in the basal portion of leaves maintained in water or PEG 30%, respectively. The application of free cytokinins induced a significant increase in NR activity in the base of detached leaves kept in water or PEG 30%

No description available.

Chloroplasts of many species of hornworts (Anthocerotae) have a structure that resembles the pyrenoid of green algae but whether these two structures are homologous has not been determined. We utilized immunogold labelling on thin sections to determine the distribution of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the major protein of algal pyrenoids, in sixteen hornwort species with and without pyrenoids. Several species (Phaeoceros laevis, Anthoceros punctatus, A. formosae, A. laminiferus, Folioceros fuciformis, Folioceros sp., Dendroceros tubercularis, D. japonicus, D. validus, Notothylas orbicularis, N. temperata, and Spaerosporoceros adscendens) have uniplastidic (or primarily uniplastidic) cells with large prominent multiple pyrenoids. In all of these species, the labelling is found exclusively in the pyrenoid and, with the exception of the Folioceros, Dendroceros, and Notothylas species, the labelling is randomly distributed throughout the pyrenoid. In the exceptional species, the pyrenoids have prominent pyrenoglobuli or other inclusions that are unlabelled. In Megaceros flagellaris and M. longispirus, the cells are multiplastidic (with the exception of the apical cell and some epidermal cells) and the chloroplasts lack pyrenoids. Anthoceros fusiformis and Phaeoceros coriaceus have primarily uniplastidic cells but the chloroplasts lack pyrenoids; only an area of stroma in the center of the plastid devoid of starch, reminiscent of a pyrenoid, is found. In all of the species lacking pyrenoids, RuBisCo is found throughout the stroma, including the stromal spaces made by the so-called channel thylakoids. No preferential accumulation of RuBisCo is found in the pyrenoid-like region in A. fusiformis and P. coriaceus. These data indicate that 1) the hornwort pyrenoid is homologous to algal pyrenoids in the presence of RuBisCo; 2) that at least some of the RuBisCo in the pyrenoid must represent an active form of the enzyme; and 3) that, in the absence of pyrenoids, the RuBisCo is distributed throughout the stroma, as in higher plants.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / The expression of pyruvate dehydrogenase kinase (PDK) 2 and 4 are increased in the fasted state to inactivate the pyruvate dehydrogenase complex (PDC) by phosphorylation to conserve substrates for glucose production. To assess the importance of PDK2 and PDK4 in regulation of the PDC to maintain glucose homeostasis, PDK2 knockout (KO), PDK4 KO, and PDK2/PDK4 double knockout (DKO) mice were generated. PDK2 deficiency caused higher PDC activity and lower blood glucose levels in the fed state while PDK4 deficiency caused similar effects in the fasting state. DKO intensified these effects in both states. PDK2 deficiency had no effect on glucose tolerance, PDK4 deficiency produced a modest effect, but DKO caused a marked improvement, lowered insulin levels, and increased insulin sensitivity. However, the DKO mice were more sensitive than wild-type mice to long term fasting, succumbing to hypoglycemia, ketoacidosis, and hypothermia. Stable isotope flux analysis indicated that hypoglycemia was due to a reduced rate of gluconeogenesis. We hypothesized that hyperglycemia would be prevented in DKO mice fed a high saturated fat diet for 30 weeks. As expected, DKO mice fed a high fat diet had improved glucose tolerance, decreased adiposity, and were euglycemic due to reduction in the rate of gluconeogenesis. Like chow fed DKO mice, high fat fed DKO mice were unusually sensitive to fasting because of ketoacidosis and hypothermia. PDK deficiency resulted in greater PDC activity which limited the availability of pyruvate for oxaloacetate synthesis. Low oxaloacetate resulted in overproduction of ketone bodies by the liver and inhibition of ketone body and fatty acid oxidation by peripheral tissues, culminating in ketoacidosis and hypothermia. Furthermore, when fed a ketogenic diet consisting of low carbohydrate and high fat, DKO mice also exhibited hypothermia, ketoacidosis, and hypoglycemia. The findings establish that PDK2 is more important in the fed state, PDK4 is more important in the fasted state, survival during long term fasting depends upon regulation of the PDC by both PDK2 and PDK4, and that the PDKs are important for the regulation of glucose and ketone body metabolism.

The perception is that inborn errors of metabolism (IEM) are rare, but the reality is that more than 600 lEMs are now recognized. The organic aciduria, 3-methylcrotonyl-CoA carboxylase (MCC) deficiency arises when 3-methylcrotonyl-Coenzyme A (CoA) carboxylase that participates in the fourth step of the leucine catabolism is defective. Tandem mass spectrometry (MS/MS) based screening programmes in North America, Europe and Australia, showed that MCC deficiency is the most frequent organic aciduria detected, with an average frequency of 1:50 000. Therefore MCC deficiency is considered an emerging disease in these regions. The incidence of MCC deficiency in the Republic of South Africa (RSA) is not yet known. However, one 48 year old male Caucasian individual (HGS) was diagnosed suffering from mild MCC deficiency, since elevated levels of 3-hydroxyisovaleric acid, 3- hydroxyisovalerylcarnitine, 3-methylcrotonylglycine was present in his urine. Several groups are currently working on various aspects of this emerging disease with the focus on the molecular characterisation of MCC deficiency. In the RSA no molecular based diagnostic method which complements MS/MS screening programmes have yet been implemented. Therefore, the aim of this study was to implement the necessary techniques for the molecular characterisation of MCC deficiency, the determination of the sequence of the open reading frame (ORF) of mccA and mccB subunits to determine which mutation(s) are present in the South African MCC deficient patient. For the implementation of the molecular characterisation, a two-pronged approached was used to characterize MCC of a MCC non-deficient individual (CFC). This approach included the reverse transcriptase polymerase chain reaction (RT-PCR) amplification of the ORFs of the associated genes [mccA (19 exons) and mccB (17 exons] and the PCR amplification of selected (genomic deoxyribonucleic acid (gDNA) regions (exons mccA8, mccA11 , mccB5, mccB6 and mccB5-intron 5-6 exon 6 (mccB5-6) which have been found to have mutations associated with MCC deficiency in Caucasians. The sequence analyses produced surprising results of the amplified ORFs (CFCmccA and CFCmccB) of the MCC non-deficient individual CFC. A non-synonymous single nucleotide polymorphism (SNP) (1391C→A, H464P) associated with MCC deficiency (Gallardo et al., 2001) was identified in the CFCmccA subunit. Another SNP (1368G→A, A456A) recently listed in GenBank was observed in the amplified CFCmccB ORF. No significant novel variations or described mutations were identified in the amplified genomic regions mccA8, mccA11 ,mccB5, mccB6 and mccB5-6. The implemented molecular approach was used to characterise MCC of our MCC deficient patient (HGS). The patient did not have any mutation in the four selected exons mccA8, mccA11, mccB5, mccB6 or the genomic region mccB5-6. The RT-PCR amplification of both ORFs (HGSmccA and HGSmccB) resulted in multiple amplicons. Gel extracted amplicons of the expected size were sequenced. Of the 36 exons, 34 exons were sequenced. This includes all 19 exons of HGSmccA and 15 of 17 exons of HGSmccB (exons 1-6 and exons 9-17). The non-synonymous SNP (1391C→A, H464P) detected in CFCmccA (MCC non-deficient individual), seems to be present in the HGSmccA subunit of the MCC deficient individual, HGS. The HGSmccB amplicons could not be entirely sequenced. However, the region exon 1-6 and 9-17 was sequenced but no described or novel mutations were identified. The lack of sequence data of region exon 7-8 led to an incomplete molecular characterisation of the MCC deficiency in HGS. In conclusion, the basic methods and techniques for the molecular characterisation of MCC deficient patients have been implemented locally. A few additional sequencing primers need to be designed to cover mccB7 and mccB8 as well as the entire coding and non-coding strands of each MCC gene (mccA and mccB). The primers for RT-PCR of both mccA and mccB need to be further refined to ensure better specificity. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2007.

Doctor of Philosophy / Department of Agronomy / Tesfaye T. Tesso / The lack of effective post-emergence weed control options is often highlighted as one of the major factors behind dwindling acreage under sorghum (Sorghum bicolor (L.) Moench) in the United States. The discovery of herbicide resistance sources in wild sorghum population and subsequent efforts to incorporate them into cultivated sorghum was received with much optimism to change weed management practices in sorghum. As the development of the technology advances, especially of the Acetolactate synthase (ALS) resistance, concerns over the temporary interveinal chlorosis and reduced seedling vigor in some of the resistant families became heightened. This thesis research is designed to shed light on the genetic basis of seedling chlorosis and assess its impacts on yield potential. The study has three parts; the first part is focused on identifying the genetic causes and plant mechanisms associated with the chlorotic phenotype. ALS herbicide resistant sister-lines expressing normal and chlorotic phenotypes were analyzed via RNA sequencing at four time points during seedling growth. The study identified several variants of genes coding chloroplast precursors and those that cause epigenetic modifications. Once confirmed, genetic markers can be developed to track these gene variants in the breeding population and eliminate segregates genetically prone to chlorosis/yellowing. The second part of the study focuses on assessing the effect of ALS resistance associated chlorosis on agronomic and nutritional parameters of sorghum inbred lines. A set of ALS resistant lines expressing different levels of the chlorotic phenotype were evaluated in replicated field trials and laboratory methods. Results showed that interveinal chlorosis delays flowering but does not have negative effect on yield and nutritional parameters with and without herbicide treatment. The last part addresses whether there is any yield drag that may be associated with herbicide resistance traits and foliar interveinal chlorosis. For this, we synthesized a large set (182) of hybrids from ALS resistant, ACCase resistant and regular (susceptible) seed and pollinator parents. The hybrids were then evaluated in three sets at multiple locations during the 2014 and 2015 crop seasons along with commercial checks. The results revealed that resistance to both herbicides do not cause any drag to grain yield. The traits also do not have any negative impact on grain and nutritional quality of resistant hybrids.

The perception is that inborn errors of metabolism (IEM) are rare, but the reality is that more than 600 lEMs are now recognized. The organic aciduria, 3-methylcrotonyl-CoA carboxylase (MCC) deficiency arises when 3-methylcrotonyl-Coenzyme A (CoA) carboxylase that participates in the fourth step of the leucine catabolism is defective. Tandem mass spectrometry (MS/MS) based screening programmes in North America, Europe and Australia, showed that MCC deficiency is the most frequent organic aciduria detected, with an average frequency of 1:50 000. Therefore MCC deficiency is considered an emerging disease in these regions. The incidence of MCC deficiency in the Republic of South Africa (RSA) is not yet known. However, one 48 year old male Caucasian individual (HGS) was diagnosed suffering from mild MCC deficiency, since elevated levels of 3-hydroxyisovaleric acid, 3- hydroxyisovalerylcarnitine, 3-methylcrotonylglycine was present in his urine. Several groups are currently working on various aspects of this emerging disease with the focus on the molecular characterisation of MCC deficiency. In the RSA no molecular based diagnostic method which complements MS/MS screening programmes have yet been implemented. Therefore, the aim of this study was to implement the necessary techniques for the molecular characterisation of MCC deficiency, the determination of the sequence of the open reading frame (ORF) of mccA and mccB subunits to determine which mutation(s) are present in the South African MCC deficient patient. For the implementation of the molecular characterisation, a two-pronged approached was used to characterize MCC of a MCC non-deficient individual (CFC). This approach included the reverse transcriptase polymerase chain reaction (RT-PCR) amplification of the ORFs of the associated genes [mccA (19 exons) and mccB (17 exons] and the PCR amplification of selected (genomic deoxyribonucleic acid (gDNA) regions (exons mccA8, mccA11 , mccB5, mccB6 and mccB5-intron 5-6 exon 6 (mccB5-6) which have been found to have mutations associated with MCC deficiency in Caucasians. The sequence analyses produced surprising results of the amplified ORFs (CFCmccA and CFCmccB) of the MCC non-deficient individual CFC. A non-synonymous single nucleotide polymorphism (SNP) (1391C→A, H464P) associated with MCC deficiency (Gallardo et al., 2001) was identified in the CFCmccA subunit. Another SNP (1368G→A, A456A) recently listed in GenBank was observed in the amplified CFCmccB ORF. No significant novel variations or described mutations were identified in the amplified genomic regions mccA8, mccA11 ,mccB5, mccB6 and mccB5-6. The implemented molecular approach was used to characterise MCC of our MCC deficient patient (HGS). The patient did not have any mutation in the four selected exons mccA8, mccA11, mccB5, mccB6 or the genomic region mccB5-6. The RT-PCR amplification of both ORFs (HGSmccA and HGSmccB) resulted in multiple amplicons. Gel extracted amplicons of the expected size were sequenced. Of the 36 exons, 34 exons were sequenced. This includes all 19 exons of HGSmccA and 15 of 17 exons of HGSmccB (exons 1-6 and exons 9-17). The non-synonymous SNP (1391C→A, H464P) detected in CFCmccA (MCC non-deficient individual), seems to be present in the HGSmccA subunit of the MCC deficient individual, HGS. The HGSmccB amplicons could not be entirely sequenced. However, the region exon 1-6 and 9-17 was sequenced but no described or novel mutations were identified. The lack of sequence data of region exon 7-8 led to an incomplete molecular characterisation of the MCC deficiency in HGS. In conclusion, the basic methods and techniques for the molecular characterisation of MCC deficient patients have been implemented locally. A few additional sequencing primers need to be designed to cover mccB7 and mccB8 as well as the entire coding and non-coding strands of each MCC gene (mccA and mccB). The primers for RT-PCR of both mccA and mccB need to be further refined to ensure better specificity. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2007.