Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 6 uppsatser.

Thioredoxin (Trx) and thioredoxin reductase (TrxR) along with an electron donor form a thioredoxin system. Such systems are widely distributed among the organisms belonging to the three domains of life. It is one of the major disulfide reducing systems, which provides electrons to several enzymes, such as ribonucleotide reductase, methionine sulfoxide reductase and glutathione peroxidase to name a few. It also plays an important role in combating oxidative stress and redox regulation of metabolism. Trx is a small redox protein, about 12 kDa in size, with an active site motif of Cys-X-X-Cys. The reduction of the disulfide in Trx is catalyzed by TrxR. Two types of thioredoxin reductases are known, namely NADPH thioredoxin reductase (NTR) with NADPH as the electron donor and ferredoxin thioredxoin reductase (FTR) which depends on reduced ferredoxin as electron donor. Although NTR is widely distributed in the three domains of life, it is absent in some archaea, whereas FTRs are mostly found in plants, photosynthetic eukaryotes, cyanobacteria, and some archaea. The thioredoxin system has been well studied in plants, mammals, and a few bacteria, but not much is known about the archaeal thioredoxin system. Our laboratory has been studying the thioredoxin systems of methanogenic archaea, and a major focus has been on Methanocaldococcus jannaschii, a deeply rooted archaeon that has two Trxs and one TrxR. My thesis research concerns the thioredoxin system of the late evolving members of the group which are exposed to oxygen more frequently than the deeply rooted members of the group, and have several Trxs and TrxRs. Methanosarcina mazei is one such organism, whose thioredoxin system is composed of one NTR, two FTRs, and five Trx homologs. Characterization of the components of a thioredoxin system sets the basis to further explore its function. I have expressed in Escherichia coli and purified the five Trxs and three TrxRs of M. mazei. I have shown the disulfide reductase activities in MM_Trx1 and MM_Trx5 by their ability to reduce insulin with DTT as the electron donor, and that in MM_Trx3 through the reduction of DTNB by this protein with NADPH as the electron donor, and in the presence of NTR as the enzyme. MM_Trx3 was found to be the only M. mazei thioredoxin to accept electrons through the NTR, and to form a complete Trx - NTR system. The Trx - FTR systems are well studied in plants, and such a system is yet to be defined in archaea. I have proposed a mechanism of action for one of the FTRs. FTR2 harbors a rubredoxin domain, and this unit is the only rubredoxin in this organism. Superoxide reductase, an enzyme that reduces superoxide radical to hydrogen peroxide without forming oxygen, utilizes rubredoxin as the direct electron source and this enzyme is found in certain anaerobes, including Methanosarcina species. Thus, it is possible that FTR2 provides electrons via a Trx to the superoxide reductase of M. mazei. This activity will define FTR2 as a tool in combating oxidative stress in M. mazei. In my thesis research I have laid a foundation to understand a complex thioredoxin system of M. mazei, to find the role of each Trx and TrxR, and to explore their involvement in oxidative stress and redox regulation. / Master of Science

Selenium is a required trace element which was originally discovered by the Swedish chemist Jons Jacob Berzelius in 1817. It was initially believed to be a toxin as it was identified as being the cause of hoof maladies and excessive hair loss in horses that feed upon plants with high selenium content. It wasn’t until 1957 that the potential contributions of selenium to physiology were first demonstrated. Selenium is now known to play a critical role in the maintenance of human health. Interestingly, unlike other trace metals/semi-metals, selenium is directly incorporated into proteins in the form of the amino acid selenocysteine (Sec) in a very complicated and energetically costly fashion. Though rare, being found in only 25 human proteins, Sec proteins are involved in numerous vital biological processes including maintenance of redox homeostasis and anti-oxidant defense. Even though Sec is essential, the reason that Sec replaces its structural analog cysteine (Cys) in only 25 proteins is not widely agreed upon. A previous model suggests that the replacement of Cys with Sec provides enzymes with a type of catalytic advantage. The presence of Cys-containing orthologs of mammalian Sec-enzymes in other eukaryotes argues against this model. A newer model to explain the use of Sec is that the gain of function imparted to an enzyme by replacing Cys with Sec is the ability of Sec to impart chemical reversibility. Building on previous results from our lab demonstrating the ability of Sec to confer proteins with the ability to resist over oxidation we have elucidated the mechanism by which Sec containing thioredoxin reductase (TrxR) resists over oxidation. The ability of Sec-TrxR to resist oxidative inactivation is due to the greater electrophilicity of Sec relative to Cys. This allows for quicker resolution and prevents over oxidation. Based on these findings we also investigate the utility of the alkylating agent dimedone to probe the oxidation state of Sec. Interestingly, it was discovered that dimedone will react with seleneninic acid with the resulting adduct being labile. Additonally it was discovered that dimedone will also react with seleninic acid, resulting in the formation of a dimedone dimer. These results call into question the usefulness of dimedone in deteremining the oxidation state of Sec. Finally, we provide evidence that Sec-TrxR enzymes are able to catalyze single electron reductions. This is most likely due to the formation of a stable Sec radical intermediate. As a whole this project provides support for the theory that Sec was selected for due to its ability to convey chemical reversiablity to proteins.

Abstract Sorghum (Sorghum bicolor L. Moench) is the world’s fifth most common cereal crop and provides an important source of staple food in the semi-arid tropics and feed in many other countries. The plant has the ability to grow and yield in hot and dry climates. However, sorghum grain is less digestible than the other major staple crops such as rice, wheat and maize. Therefore, the aim of this project is to improve the nutritional quality of sorghum grain by applying cutting-edge biotechnologies which involve the use of tissue culture and genetic transformation. Recently, Agrobacterium has been used by many researchers to introduce foreign genes into the sorghum genome. This method has some advantages compared to particle bombardment, however, one limitation is the regeneration of transgenic tissues. In this study successfully transformed sorghum using Agrobacterium and regenerated transgenic plants via an organogenic tissue culture system is reported. The results of transformation efficiency were achieved with co-cultivation after 48 hours. Regeneration of the sorghum transgenic plants was improved by using organogenic tissues. The GUS reporter gene and the Hpt and bar selectable markers were used. Southern blots and PCR were used to confirm transgene presence in the T0 and T1 generations. In this study, stable transgenic sorghum plants have been produced. The factors found to most influence Agrobacterium transformation were the type of organogenic tissue from different genotypes. The genotypes and the period of co-cultivation, as well as the selectable marker gene and selection strategy used. However, the transformation efficiency from this method was low (1.12%) compared with the previous efficiencies published for Agrobacterium-mediated sorghum transformation. Therefore, to improve the transformation efficiency for this method further work may need to be done. Thioredoxin genes were transformed into the sorghum genotype 296B by particle bombardment. In the first experiment no transgenics over-expressing trx and ntr were confirmed by Southern blot. In subsequent experiments, a limited number of transgenics of the T1 generation were confirmed and used for further analysis. A transgenic line with both trx & ntr was created by crossing a trx line and a ntr line. The 2 genes in this line were confirmed and showed different levels of expression by Real Time PCR. Also, the level of expression in the T2 hybrid plants was higher compared to the T1 parents. The grains from the transgenic lines were different in gelatinization, viscosity, pasting properties and in-vitro digestibility. The ntr line was confirmed to be more digestible than the other transgenic lines and a non-transgenic line. There was a significant increase of 11% (P=0.02) in digestibility of the sorghum ntr line over the non-transgenic. However, the transgenic sorghum seeds did not germinate after storage for more than 6 months. Differences in the morphology of the starch granules and protein matrix of the transgenic lines when compared to non-transgenic were observed with Scanning Electron microscopy. The difference was observed from the transition to the central zone. Pores appeared in the starch granules of the sorghum transgenic lines, but not in the non-transgenic. This may be directly related to the changes in gelatinization, viscosity, pasting and digestibility. To find regulatory sequences which can direct expression of transgenes in developing endosperm, the β-kafirin promoter was identified and cloned. Two constructs of varying length were made to test tissue specificity of the promoter, by replacing the Ubi promoter of the pUBIGUS vector. The GUS gene was used as the marker gene under the control of the amplified β-kafirin promoter. The result was determined on different explants of sorghum by transient expression via particle bombardment. The result shows the successful identification of the β-kafirin promoter region and its effect on transient expression levels. Agrobacterium transformation of sorghum organogenic tissue was developed. The digestibility of grain sorghum was improved by over-expressing the thioredoxin genes. In conclusion, the sorghum grain digestibility can be improved by transforming sorghum with thioredoxin genes, via Agrobacterium-mediated transformation. Further experimentation is required to identify regulatory sequences to optimise transgene expression in sorghum endosperm. In order to determine the reason behind the difficulties of seed germination, larger numbers of independent transgenic lines need to be generated and tested to determine whether over-expression of trx & ntr always has detrimental effects on seed longevity and germination.

Abstract Sorghum (Sorghum bicolor L. Moench) is the world’s fifth most common cereal crop and provides an important source of staple food in the semi-arid tropics and feed in many other countries. The plant has the ability to grow and yield in hot and dry climates. However, sorghum grain is less digestible than the other major staple crops such as rice, wheat and maize. Therefore, the aim of this project is to improve the nutritional quality of sorghum grain by applying cutting-edge biotechnologies which involve the use of tissue culture and genetic transformation. Recently, Agrobacterium has been used by many researchers to introduce foreign genes into the sorghum genome. This method has some advantages compared to particle bombardment, however, one limitation is the regeneration of transgenic tissues. In this study successfully transformed sorghum using Agrobacterium and regenerated transgenic plants via an organogenic tissue culture system is reported. The results of transformation efficiency were achieved with co-cultivation after 48 hours. Regeneration of the sorghum transgenic plants was improved by using organogenic tissues. The GUS reporter gene and the Hpt and bar selectable markers were used. Southern blots and PCR were used to confirm transgene presence in the T0 and T1 generations. In this study, stable transgenic sorghum plants have been produced. The factors found to most influence Agrobacterium transformation were the type of organogenic tissue from different genotypes. The genotypes and the period of co-cultivation, as well as the selectable marker gene and selection strategy used. However, the transformation efficiency from this method was low (1.12%) compared with the previous efficiencies published for Agrobacterium-mediated sorghum transformation. Therefore, to improve the transformation efficiency for this method further work may need to be done. Thioredoxin genes were transformed into the sorghum genotype 296B by particle bombardment. In the first experiment no transgenics over-expressing trx and ntr were confirmed by Southern blot. In subsequent experiments, a limited number of transgenics of the T1 generation were confirmed and used for further analysis. A transgenic line with both trx & ntr was created by crossing a trx line and a ntr line. The 2 genes in this line were confirmed and showed different levels of expression by Real Time PCR. Also, the level of expression in the T2 hybrid plants was higher compared to the T1 parents. The grains from the transgenic lines were different in gelatinization, viscosity, pasting properties and in-vitro digestibility. The ntr line was confirmed to be more digestible than the other transgenic lines and a non-transgenic line. There was a significant increase of 11% (P=0.02) in digestibility of the sorghum ntr line over the non-transgenic. However, the transgenic sorghum seeds did not germinate after storage for more than 6 months. Differences in the morphology of the starch granules and protein matrix of the transgenic lines when compared to non-transgenic were observed with Scanning Electron microscopy. The difference was observed from the transition to the central zone. Pores appeared in the starch granules of the sorghum transgenic lines, but not in the non-transgenic. This may be directly related to the changes in gelatinization, viscosity, pasting and digestibility. To find regulatory sequences which can direct expression of transgenes in developing endosperm, the β-kafirin promoter was identified and cloned. Two constructs of varying length were made to test tissue specificity of the promoter, by replacing the Ubi promoter of the pUBIGUS vector. The GUS gene was used as the marker gene under the control of the amplified β-kafirin promoter. The result was determined on different explants of sorghum by transient expression via particle bombardment. The result shows the successful identification of the β-kafirin promoter region and its effect on transient expression levels. Agrobacterium transformation of sorghum organogenic tissue was developed. The digestibility of grain sorghum was improved by over-expressing the thioredoxin genes. In conclusion, the sorghum grain digestibility can be improved by transforming sorghum with thioredoxin genes, via Agrobacterium-mediated transformation. Further experimentation is required to identify regulatory sequences to optimise transgene expression in sorghum endosperm. In order to determine the reason behind the difficulties of seed germination, larger numbers of independent transgenic lines need to be generated and tested to determine whether over-expression of trx & ntr always has detrimental effects on seed longevity and germination.

Abstract Sorghum (Sorghum bicolor L. Moench) is the world’s fifth most common cereal crop and provides an important source of staple food in the semi-arid tropics and feed in many other countries. The plant has the ability to grow and yield in hot and dry climates. However, sorghum grain is less digestible than the other major staple crops such as rice, wheat and maize. Therefore, the aim of this project is to improve the nutritional quality of sorghum grain by applying cutting-edge biotechnologies which involve the use of tissue culture and genetic transformation. Recently, Agrobacterium has been used by many researchers to introduce foreign genes into the sorghum genome. This method has some advantages compared to particle bombardment, however, one limitation is the regeneration of transgenic tissues. In this study successfully transformed sorghum using Agrobacterium and regenerated transgenic plants via an organogenic tissue culture system is reported. The results of transformation efficiency were achieved with co-cultivation after 48 hours. Regeneration of the sorghum transgenic plants was improved by using organogenic tissues. The GUS reporter gene and the Hpt and bar selectable markers were used. Southern blots and PCR were used to confirm transgene presence in the T0 and T1 generations. In this study, stable transgenic sorghum plants have been produced. The factors found to most influence Agrobacterium transformation were the type of organogenic tissue from different genotypes. The genotypes and the period of co-cultivation, as well as the selectable marker gene and selection strategy used. However, the transformation efficiency from this method was low (1.12%) compared with the previous efficiencies published for Agrobacterium-mediated sorghum transformation. Therefore, to improve the transformation efficiency for this method further work may need to be done. Thioredoxin genes were transformed into the sorghum genotype 296B by particle bombardment. In the first experiment no transgenics over-expressing trx and ntr were confirmed by Southern blot. In subsequent experiments, a limited number of transgenics of the T1 generation were confirmed and used for further analysis. A transgenic line with both trx & ntr was created by crossing a trx line and a ntr line. The 2 genes in this line were confirmed and showed different levels of expression by Real Time PCR. Also, the level of expression in the T2 hybrid plants was higher compared to the T1 parents. The grains from the transgenic lines were different in gelatinization, viscosity, pasting properties and in-vitro digestibility. The ntr line was confirmed to be more digestible than the other transgenic lines and a non-transgenic line. There was a significant increase of 11% (P=0.02) in digestibility of the sorghum ntr line over the non-transgenic. However, the transgenic sorghum seeds did not germinate after storage for more than 6 months. Differences in the morphology of the starch granules and protein matrix of the transgenic lines when compared to non-transgenic were observed with Scanning Electron microscopy. The difference was observed from the transition to the central zone. Pores appeared in the starch granules of the sorghum transgenic lines, but not in the non-transgenic. This may be directly related to the changes in gelatinization, viscosity, pasting and digestibility. To find regulatory sequences which can direct expression of transgenes in developing endosperm, the β-kafirin promoter was identified and cloned. Two constructs of varying length were made to test tissue specificity of the promoter, by replacing the Ubi promoter of the pUBIGUS vector. The GUS gene was used as the marker gene under the control of the amplified β-kafirin promoter. The result was determined on different explants of sorghum by transient expression via particle bombardment. The result shows the successful identification of the β-kafirin promoter region and its effect on transient expression levels. Agrobacterium transformation of sorghum organogenic tissue was developed. The digestibility of grain sorghum was improved by over-expressing the thioredoxin genes. In conclusion, the sorghum grain digestibility can be improved by transforming sorghum with thioredoxin genes, via Agrobacterium-mediated transformation. Further experimentation is required to identify regulatory sequences to optimise transgene expression in sorghum endosperm. In order to determine the reason behind the difficulties of seed germination, larger numbers of independent transgenic lines need to be generated and tested to determine whether over-expression of trx & ntr always has detrimental effects on seed longevity and germination.

Abstract Sorghum (Sorghum bicolor L. Moench) is the world’s fifth most common cereal crop and provides an important source of staple food in the semi-arid tropics and feed in many other countries. The plant has the ability to grow and yield in hot and dry climates. However, sorghum grain is less digestible than the other major staple crops such as rice, wheat and maize. Therefore, the aim of this project is to improve the nutritional quality of sorghum grain by applying cutting-edge biotechnologies which involve the use of tissue culture and genetic transformation. Recently, Agrobacterium has been used by many researchers to introduce foreign genes into the sorghum genome. This method has some advantages compared to particle bombardment, however, one limitation is the regeneration of transgenic tissues. In this study successfully transformed sorghum using Agrobacterium and regenerated transgenic plants via an organogenic tissue culture system is reported. The results of transformation efficiency were achieved with co-cultivation after 48 hours. Regeneration of the sorghum transgenic plants was improved by using organogenic tissues. The GUS reporter gene and the Hpt and bar selectable markers were used. Southern blots and PCR were used to confirm transgene presence in the T0 and T1 generations. In this study, stable transgenic sorghum plants have been produced. The factors found to most influence Agrobacterium transformation were the type of organogenic tissue from different genotypes. The genotypes and the period of co-cultivation, as well as the selectable marker gene and selection strategy used. However, the transformation efficiency from this method was low (1.12%) compared with the previous efficiencies published for Agrobacterium-mediated sorghum transformation. Therefore, to improve the transformation efficiency for this method further work may need to be done. Thioredoxin genes were transformed into the sorghum genotype 296B by particle bombardment. In the first experiment no transgenics over-expressing trx and ntr were confirmed by Southern blot. In subsequent experiments, a limited number of transgenics of the T1 generation were confirmed and used for further analysis. A transgenic line with both trx & ntr was created by crossing a trx line and a ntr line. The 2 genes in this line were confirmed and showed different levels of expression by Real Time PCR. Also, the level of expression in the T2 hybrid plants was higher compared to the T1 parents. The grains from the transgenic lines were different in gelatinization, viscosity, pasting properties and in-vitro digestibility. The ntr line was confirmed to be more digestible than the other transgenic lines and a non-transgenic line. There was a significant increase of 11% (P=0.02) in digestibility of the sorghum ntr line over the non-transgenic. However, the transgenic sorghum seeds did not germinate after storage for more than 6 months. Differences in the morphology of the starch granules and protein matrix of the transgenic lines when compared to non-transgenic were observed with Scanning Electron microscopy. The difference was observed from the transition to the central zone. Pores appeared in the starch granules of the sorghum transgenic lines, but not in the non-transgenic. This may be directly related to the changes in gelatinization, viscosity, pasting and digestibility. To find regulatory sequences which can direct expression of transgenes in developing endosperm, the β-kafirin promoter was identified and cloned. Two constructs of varying length were made to test tissue specificity of the promoter, by replacing the Ubi promoter of the pUBIGUS vector. The GUS gene was used as the marker gene under the control of the amplified β-kafirin promoter. The result was determined on different explants of sorghum by transient expression via particle bombardment. The result shows the successful identification of the β-kafirin promoter region and its effect on transient expression levels. Agrobacterium transformation of sorghum organogenic tissue was developed. The digestibility of grain sorghum was improved by over-expressing the thioredoxin genes. In conclusion, the sorghum grain digestibility can be improved by transforming sorghum with thioredoxin genes, via Agrobacterium-mediated transformation. Further experimentation is required to identify regulatory sequences to optimise transgene expression in sorghum endosperm. In order to determine the reason behind the difficulties of seed germination, larger numbers of independent transgenic lines need to be generated and tested to determine whether over-expression of trx & ntr always has detrimental effects on seed longevity and germination.

Abstract Sorghum (Sorghum bicolor L. Moench) is the world’s fifth most common cereal crop and provides an important source of staple food in the semi-arid tropics and feed in many other countries. The plant has the ability to grow and yield in hot and dry climates. However, sorghum grain is less digestible than the other major staple crops such as rice, wheat and maize. Therefore, the aim of this project is to improve the nutritional quality of sorghum grain by applying cutting-edge biotechnologies which involve the use of tissue culture and genetic transformation. Recently, Agrobacterium has been used by many researchers to introduce foreign genes into the sorghum genome. This method has some advantages compared to particle bombardment, however, one limitation is the regeneration of transgenic tissues. In this study successfully transformed sorghum using Agrobacterium and regenerated transgenic plants via an organogenic tissue culture system is reported. The results of transformation efficiency were achieved with co-cultivation after 48 hours. Regeneration of the sorghum transgenic plants was improved by using organogenic tissues. The GUS reporter gene and the Hpt and bar selectable markers were used. Southern blots and PCR were used to confirm transgene presence in the T0 and T1 generations. In this study, stable transgenic sorghum plants have been produced. The factors found to most influence Agrobacterium transformation were the type of organogenic tissue from different genotypes. The genotypes and the period of co-cultivation, as well as the selectable marker gene and selection strategy used. However, the transformation efficiency from this method was low (1.12%) compared with the previous efficiencies published for Agrobacterium-mediated sorghum transformation. Therefore, to improve the transformation efficiency for this method further work may need to be done. Thioredoxin genes were transformed into the sorghum genotype 296B by particle bombardment. In the first experiment no transgenics over-expressing trx and ntr were confirmed by Southern blot. In subsequent experiments, a limited number of transgenics of the T1 generation were confirmed and used for further analysis. A transgenic line with both trx & ntr was created by crossing a trx line and a ntr line. The 2 genes in this line were confirmed and showed different levels of expression by Real Time PCR. Also, the level of expression in the T2 hybrid plants was higher compared to the T1 parents. The grains from the transgenic lines were different in gelatinization, viscosity, pasting properties and in-vitro digestibility. The ntr line was confirmed to be more digestible than the other transgenic lines and a non-transgenic line. There was a significant increase of 11% (P=0.02) in digestibility of the sorghum ntr line over the non-transgenic. However, the transgenic sorghum seeds did not germinate after storage for more than 6 months. Differences in the morphology of the starch granules and protein matrix of the transgenic lines when compared to non-transgenic were observed with Scanning Electron microscopy. The difference was observed from the transition to the central zone. Pores appeared in the starch granules of the sorghum transgenic lines, but not in the non-transgenic. This may be directly related to the changes in gelatinization, viscosity, pasting and digestibility. To find regulatory sequences which can direct expression of transgenes in developing endosperm, the β-kafirin promoter was identified and cloned. Two constructs of varying length were made to test tissue specificity of the promoter, by replacing the Ubi promoter of the pUBIGUS vector. The GUS gene was used as the marker gene under the control of the amplified β-kafirin promoter. The result was determined on different explants of sorghum by transient expression via particle bombardment. The result shows the successful identification of the β-kafirin promoter region and its effect on transient expression levels. Agrobacterium transformation of sorghum organogenic tissue was developed. The digestibility of grain sorghum was improved by over-expressing the thioredoxin genes. In conclusion, the sorghum grain digestibility can be improved by transforming sorghum with thioredoxin genes, via Agrobacterium-mediated transformation. Further experimentation is required to identify regulatory sequences to optimise transgene expression in sorghum endosperm. In order to determine the reason behind the difficulties of seed germination, larger numbers of independent transgenic lines need to be generated and tested to determine whether over-expression of trx & ntr always has detrimental effects on seed longevity and germination.

Diss. (sammanfattning) Stockholm : Karol inst., 2003. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.