We have used the polymerase chain reaction to isolate fragments of Tyl-copia group retrotransposo... more We have used the polymerase chain reaction to isolate fragments of Tyl-copia group retrotransposons from a wide variety of members of the higher plant kingdom. 56 out of 57 species tested generate an amplified fragment of the size expected for reverse transcriptase fragments of Tyl-copia group retrotransposons. Sequence analysis of subclones shows that the PCR fragments display varying degrees of sequence heterogeneity. Sequence heterogeneity therefore seems a general property of Tyl-copia group retrotransposons of higher plants, in contrast to the limited diversity seen in retrotransposons of Saccharomyces cerevisiae and Drosophila melanogaster. Phylogenetic analysis of all these sequences shows, with some significant exceptions, that the degree of sequence divergence in the retrotransposon populations between any pair of species is proportional to the evolutionary distance between those species. This implies that sequence divergence during vertical transmission of Tyl-copia group retrotransposons within plant lineages has been a major factor in the evolution of Tyl-copia group retrotransposons in higher plants. Additionally, we suggest that horizontal transmission of this transposon group between different species has also played a role in this process.
The genomic organisation and diversity of the Ty1-copia group retrotransposons has been investiga... more The genomic organisation and diversity of the Ty1-copia group retrotransposons has been investigated in several crop plants and their relatives from both dicotyledonous and monocotyledonous families, including potato ( Solanum tuberosum), faba beans ( Vicia faba), Vicia melanops, Vicia sativa, barley ( Hordeum vulgare), rye ( Secale cereale), and onion ( Allium cepa). Extreme heterogeneity in the sequence of the Ty1-copia retrotransposons from all these plants was revealed following sequence analysis of reverse transcriptase fragments. The estimated copy numbers of the Ty1-copia group retrotransposons for the genomes of S. tuberosum, L. esculentum, A. cepa, S. cereale, and V. faba is highly variable, ranging from a few hundred to approximately a million copies per genome. In situ hybridisation data from metaphase and prophase chromosomes of V. faba, S. cereale, and H. vulgare suggest that retrotransposon sequences are dispersed throughout the euchromatic regions of the genome but are almost undetectable in most heterochromatic regions. In contrast, similar data from metaphase chromosomes of A. cepa suggests that although retrotransposon sequences are dispersed throughout the euchromatic regions of the genome, they are predominantly concentrated in the terminal heterochromatin. These results are discussed in the context of the role played by the Ty1-copia group retrotransposons in the evolution of the plant genome. Lastly, the application of retrotransposon sequences as genetic markers for mapping genomes and for studying genetic biodiversity in plants is presented.
The Ty1-copia group of LTR retrotransposons has been studied extensively in yeast and Drosophila,... more The Ty1-copia group of LTR retrotransposons has been studied extensively in yeast and Drosophila, the organisms in which they were first discovered, and more recently in higher plant and vertebrate species. Their properties, such as copy number, sequence homogeneity, transcriptional and transpositional activity vary greatly between these different hosts. We will try to resolve these apparent discrepancies between these properties, explain any fundamental differences in the biology of the Ty1- copia group between hosts, and propose a general model for LTR retrotransposon evolution.
The terminal sequences of long-terminal repeat (LTR) retrotransposons are a source of powerful mo... more The terminal sequences of long-terminal repeat (LTR) retrotransposons are a source of powerful molecular markers for linkage mapping and biodiversity studies. The major factor limiting the widespread application of LTR retrotransposon-based molecular markers is the availability of new retrotransposon terminal sequences. We describe a PCR-based method for the rapid isolation of LTR sequences of Ty1-copia group retrotransposons from the genomic DNA of potentially any higher plant species. To demonstrate the utility of this technique, we have identi®ed a variety of new retrotransposon LTR sequences from pea, broad bean and Norway spruce. Primers speci®c for three pea LTRs have been used to reveal polymorphisms associated with the corresponding retrotransposons within the Pisum genus.
We have used the polymerase chain reaction to analyse Ty1-copia group retrotransposons of floweri... more We have used the polymerase chain reaction to analyse Ty1-copia group retrotransposons of flowering plants. All eight species studied contain reverse transcriptase fragments from Tyl-copia group retrotransposons. Sequence analysis of 31 subcloned fragments from potato reveals that each is different from the others, with predicted amino acid diversities between individual fragments varying between 5% and 75%. Such sequence heterogeneity within a single species contrasts strongly with the limited diversity seen in such retrotransposons in yeast and Drosophila. The fragments from the other seven plant species examined are also heterogeneous, both within and between species, showing that this is a general property of this transposon group in plants. Phylogenetic analysis of all these sequences reveals that many of them fall into subgroups which span species boundaries, such that the closest homologue of one sequence is often from a different species. We suggest that both vertical transmission of Ty1-copia group retrotransposons within plant lineages and horizontal transmission between different species have played roles in the evolution of Ty1-copia group retrotransposons in flowering plants.
cDNA clones of two genes (TUB8 and TUB13) which show a 25–30-fold increase in transcript in the s... more cDNA clones of two genes (TUB8 and TUB13) which show a 25–30-fold increase in transcript in the stolon tip during the early stages of tuberisation, have been isolated by differential screening. These genes are also expressed in leaves, stems and roots and the expression pattern in these organs changes on tuberisation. Southern analysis shows homologous sequences in the non-tuberising wild type potato species Solanum brevidens and in Lycopersicon esculentum (tomato). Sequence analysis reveals a high degree of similarity between the TUB13 cDNA, and a human S-adenosylmethionine decarboxylase gene. The predicted TUB8 peptide sequence shows several repeats of alanine, glutamate and proline which suggests a structural role for the encoded protein.
S-adenosylmethionine decarboxylase (SAMDC) is involved in the biosynthesis of the polyamines, spe... more S-adenosylmethionine decarboxylase (SAMDC) is involved in the biosynthesis of the polyamines, spermidine and spermine. Recently, we reported the isolation of a putative cDNA clone of the SAMDC clone of potato (Plant Mol Biol 20; 641–651). In order to confirm that the potato genes does encode SAMDC, a complementation experiment with a yeast strain that possesses a null mutation in the SAMDC gene was performed. The yeast strain contains a deletion-insertion mutation in the SAMDC gene and has an absolute requirement for the addition of exogenous spermidine for growth. When the full-length potato cDNA was expressed in the mutant yeast strain there was no longer a requirement for exogenous spermidine. Immunoblotting experiments suggest that the potato SAMDC gene product has an apparent molecular mass of 39 kDa. Expression of the SAMDC gene was high in the young and actively dividing tissues and low in the mature and non-dividing tissues of both vegetative and reproductive organs. Additionally, isolation and characterisation of the corresponding genomic clone is reported. The gene has one intron in its 5′-untranslated sequence but otherwise the transcribed portion is identical to the cDNA clone.
A molecular approach has been used to study the role of polyamines in plant development by manipu... more A molecular approach has been used to study the role of polyamines in plant development by manipulating the expression of the S-adenosylmethionine decarboxylase (SAMDC) gene. SAMDC is a key enzyme involved in the biosynthesis of the polyamines spermidine and spermine and is also known to influence the rate of biosynthesis of ethylene. Previously, a cDNA clone of the SAMDC gene of potato has been isolated and characterized. This cDNA clone has been used to make antisense and sense SAMDC constructs under the control of the 35S CaMV or tetracycline-inducible promoters. Agrobacterium-mediated transformation has been used to produce transgenic potato plants with the engineered antisense and sense SAMDC genes in order to downregulate or overexpress the SAMDC transcript, respectively. Decreases or increases in the level of SAMDC transcript in the antisense and sense transgenic plants were observed, respectively. Antisense transgenic plants which expressed the engineered SAMDC gene constitutively under the control of the 35S CaMV promoter with a duplicated enhancer region showed a range of stunted phenotypes with highly branched stems, short internodes, small leaves and inhibited root growth. The abnormal characteristics of the antisense plants correlated with the altered levels of SAMDC transcript, SAMDC activity, polyamine content and rate of ethylene evolution. Attempts to produce sense transgenic plants with the 35S SAMDC sense construct were unsuccessful indicating that a constitutive overexpression of the engineered SAMDC is lethal to the plants. When antisense and sense SAMDC transgenes were expressed under the control of a tetracycline-inducible promoter a number of transgenic plants were produced. In these antisense and sense plants, significant changes in the level of SAMDC transcript, SAMDC activity and free polyamine content were observed on tetracycline-induction of detached leaves.
Polyamines are low molecular mass polycations found in all living organisms. In plants, they have... more Polyamines are low molecular mass polycations found in all living organisms. In plants, they have been implicated in a wide range of biological processes, including growth, development and stress responses. Recently, several key genes of the polyamine biosynthesis pathways have been cloned and shown to be developmentally regulated. Experiments in which the synthesis of polyamines has been perturbed have clearly shown that these compounds are essential for normal growth and development. These findings have been further supported by recent genetic analyses of polyamine synthesis mutants of Arabidopsis. The research has revealed a much more detailed level of understanding than hitherto, opening up new avenues of investigation for the future.
We have used the polymerase chain reaction to isolate fragments of Tyl-copia group retrotransposo... more We have used the polymerase chain reaction to isolate fragments of Tyl-copia group retrotransposons from a wide variety of members of the higher plant kingdom. 56 out of 57 species tested generate an amplified fragment of the size expected for reverse transcriptase fragments of Tyl-copia group retrotransposons. Sequence analysis of subclones shows that the PCR fragments display varying degrees of sequence heterogeneity. Sequence heterogeneity therefore seems a general property of Tyl-copia group retrotransposons of higher plants, in contrast to the limited diversity seen in retrotransposons of Saccharomyces cerevisiae and Drosophila melanogaster. Phylogenetic analysis of all these sequences shows, with some significant exceptions, that the degree of sequence divergence in the retrotransposon populations between any pair of species is proportional to the evolutionary distance between those species. This implies that sequence divergence during vertical transmission of Tyl-copia group retrotransposons within plant lineages has been a major factor in the evolution of Tyl-copia group retrotransposons in higher plants. Additionally, we suggest that horizontal transmission of this transposon group between different species has also played a role in this process.
The genomic organisation and diversity of the Ty1-copia group retrotransposons has been investiga... more The genomic organisation and diversity of the Ty1-copia group retrotransposons has been investigated in several crop plants and their relatives from both dicotyledonous and monocotyledonous families, including potato ( Solanum tuberosum), faba beans ( Vicia faba), Vicia melanops, Vicia sativa, barley ( Hordeum vulgare), rye ( Secale cereale), and onion ( Allium cepa). Extreme heterogeneity in the sequence of the Ty1-copia retrotransposons from all these plants was revealed following sequence analysis of reverse transcriptase fragments. The estimated copy numbers of the Ty1-copia group retrotransposons for the genomes of S. tuberosum, L. esculentum, A. cepa, S. cereale, and V. faba is highly variable, ranging from a few hundred to approximately a million copies per genome. In situ hybridisation data from metaphase and prophase chromosomes of V. faba, S. cereale, and H. vulgare suggest that retrotransposon sequences are dispersed throughout the euchromatic regions of the genome but are almost undetectable in most heterochromatic regions. In contrast, similar data from metaphase chromosomes of A. cepa suggests that although retrotransposon sequences are dispersed throughout the euchromatic regions of the genome, they are predominantly concentrated in the terminal heterochromatin. These results are discussed in the context of the role played by the Ty1-copia group retrotransposons in the evolution of the plant genome. Lastly, the application of retrotransposon sequences as genetic markers for mapping genomes and for studying genetic biodiversity in plants is presented.
The Ty1-copia group of LTR retrotransposons has been studied extensively in yeast and Drosophila,... more The Ty1-copia group of LTR retrotransposons has been studied extensively in yeast and Drosophila, the organisms in which they were first discovered, and more recently in higher plant and vertebrate species. Their properties, such as copy number, sequence homogeneity, transcriptional and transpositional activity vary greatly between these different hosts. We will try to resolve these apparent discrepancies between these properties, explain any fundamental differences in the biology of the Ty1- copia group between hosts, and propose a general model for LTR retrotransposon evolution.
The terminal sequences of long-terminal repeat (LTR) retrotransposons are a source of powerful mo... more The terminal sequences of long-terminal repeat (LTR) retrotransposons are a source of powerful molecular markers for linkage mapping and biodiversity studies. The major factor limiting the widespread application of LTR retrotransposon-based molecular markers is the availability of new retrotransposon terminal sequences. We describe a PCR-based method for the rapid isolation of LTR sequences of Ty1-copia group retrotransposons from the genomic DNA of potentially any higher plant species. To demonstrate the utility of this technique, we have identi®ed a variety of new retrotransposon LTR sequences from pea, broad bean and Norway spruce. Primers speci®c for three pea LTRs have been used to reveal polymorphisms associated with the corresponding retrotransposons within the Pisum genus.
We have used the polymerase chain reaction to analyse Ty1-copia group retrotransposons of floweri... more We have used the polymerase chain reaction to analyse Ty1-copia group retrotransposons of flowering plants. All eight species studied contain reverse transcriptase fragments from Tyl-copia group retrotransposons. Sequence analysis of 31 subcloned fragments from potato reveals that each is different from the others, with predicted amino acid diversities between individual fragments varying between 5% and 75%. Such sequence heterogeneity within a single species contrasts strongly with the limited diversity seen in such retrotransposons in yeast and Drosophila. The fragments from the other seven plant species examined are also heterogeneous, both within and between species, showing that this is a general property of this transposon group in plants. Phylogenetic analysis of all these sequences reveals that many of them fall into subgroups which span species boundaries, such that the closest homologue of one sequence is often from a different species. We suggest that both vertical transmission of Ty1-copia group retrotransposons within plant lineages and horizontal transmission between different species have played roles in the evolution of Ty1-copia group retrotransposons in flowering plants.
cDNA clones of two genes (TUB8 and TUB13) which show a 25–30-fold increase in transcript in the s... more cDNA clones of two genes (TUB8 and TUB13) which show a 25–30-fold increase in transcript in the stolon tip during the early stages of tuberisation, have been isolated by differential screening. These genes are also expressed in leaves, stems and roots and the expression pattern in these organs changes on tuberisation. Southern analysis shows homologous sequences in the non-tuberising wild type potato species Solanum brevidens and in Lycopersicon esculentum (tomato). Sequence analysis reveals a high degree of similarity between the TUB13 cDNA, and a human S-adenosylmethionine decarboxylase gene. The predicted TUB8 peptide sequence shows several repeats of alanine, glutamate and proline which suggests a structural role for the encoded protein.
S-adenosylmethionine decarboxylase (SAMDC) is involved in the biosynthesis of the polyamines, spe... more S-adenosylmethionine decarboxylase (SAMDC) is involved in the biosynthesis of the polyamines, spermidine and spermine. Recently, we reported the isolation of a putative cDNA clone of the SAMDC clone of potato (Plant Mol Biol 20; 641–651). In order to confirm that the potato genes does encode SAMDC, a complementation experiment with a yeast strain that possesses a null mutation in the SAMDC gene was performed. The yeast strain contains a deletion-insertion mutation in the SAMDC gene and has an absolute requirement for the addition of exogenous spermidine for growth. When the full-length potato cDNA was expressed in the mutant yeast strain there was no longer a requirement for exogenous spermidine. Immunoblotting experiments suggest that the potato SAMDC gene product has an apparent molecular mass of 39 kDa. Expression of the SAMDC gene was high in the young and actively dividing tissues and low in the mature and non-dividing tissues of both vegetative and reproductive organs. Additionally, isolation and characterisation of the corresponding genomic clone is reported. The gene has one intron in its 5′-untranslated sequence but otherwise the transcribed portion is identical to the cDNA clone.
A molecular approach has been used to study the role of polyamines in plant development by manipu... more A molecular approach has been used to study the role of polyamines in plant development by manipulating the expression of the S-adenosylmethionine decarboxylase (SAMDC) gene. SAMDC is a key enzyme involved in the biosynthesis of the polyamines spermidine and spermine and is also known to influence the rate of biosynthesis of ethylene. Previously, a cDNA clone of the SAMDC gene of potato has been isolated and characterized. This cDNA clone has been used to make antisense and sense SAMDC constructs under the control of the 35S CaMV or tetracycline-inducible promoters. Agrobacterium-mediated transformation has been used to produce transgenic potato plants with the engineered antisense and sense SAMDC genes in order to downregulate or overexpress the SAMDC transcript, respectively. Decreases or increases in the level of SAMDC transcript in the antisense and sense transgenic plants were observed, respectively. Antisense transgenic plants which expressed the engineered SAMDC gene constitutively under the control of the 35S CaMV promoter with a duplicated enhancer region showed a range of stunted phenotypes with highly branched stems, short internodes, small leaves and inhibited root growth. The abnormal characteristics of the antisense plants correlated with the altered levels of SAMDC transcript, SAMDC activity, polyamine content and rate of ethylene evolution. Attempts to produce sense transgenic plants with the 35S SAMDC sense construct were unsuccessful indicating that a constitutive overexpression of the engineered SAMDC is lethal to the plants. When antisense and sense SAMDC transgenes were expressed under the control of a tetracycline-inducible promoter a number of transgenic plants were produced. In these antisense and sense plants, significant changes in the level of SAMDC transcript, SAMDC activity and free polyamine content were observed on tetracycline-induction of detached leaves.
Polyamines are low molecular mass polycations found in all living organisms. In plants, they have... more Polyamines are low molecular mass polycations found in all living organisms. In plants, they have been implicated in a wide range of biological processes, including growth, development and stress responses. Recently, several key genes of the polyamine biosynthesis pathways have been cloned and shown to be developmentally regulated. Experiments in which the synthesis of polyamines has been perturbed have clearly shown that these compounds are essential for normal growth and development. These findings have been further supported by recent genetic analyses of polyamine synthesis mutants of Arabidopsis. The research has revealed a much more detailed level of understanding than hitherto, opening up new avenues of investigation for the future.
We have used the polymerase chain reaction to isolate fragments of Tyl-copia group retrotransposo... more We have used the polymerase chain reaction to isolate fragments of Tyl-copia group retrotransposons from a wide variety of members of the higher plant kingdom. 56 out of 57 species tested generate an amplified fragment of the size expected for reverse transcriptase fragments of Tyl-copia group retrotransposons. Sequence analysis of subclones shows that the PCR fragments display varying degrees of sequence heterogeneity. Sequence heterogeneity therefore seems a general property of Tyl-copia group retrotransposons of higher plants, in contrast to the limited diversity seen in retrotransposons of Saccharomyces cerevisiae and Drosophila melanogaster. Phylogenetic analysis of all these sequences shows, with some significant exceptions, that the degree of sequence divergence in the retrotransposon populations between any pair of species is proportional to the evolutionary distance between those species. This implies that sequence divergence during vertical transmission of Tyl-copia group retrotransposons within plant lineages has been a major factor in the evolution of Tyl-copia group retrotransposons in higher plants. Additionally, we suggest that horizontal transmission of this transposon group between different species has also played a role in this process.
The genomic organisation and diversity of the Ty1-copia group retrotransposons has been investiga... more The genomic organisation and diversity of the Ty1-copia group retrotransposons has been investigated in several crop plants and their relatives from both dicotyledonous and monocotyledonous families, including potato ( Solanum tuberosum), faba beans ( Vicia faba), Vicia melanops, Vicia sativa, barley ( Hordeum vulgare), rye ( Secale cereale), and onion ( Allium cepa). Extreme heterogeneity in the sequence of the Ty1-copia retrotransposons from all these plants was revealed following sequence analysis of reverse transcriptase fragments. The estimated copy numbers of the Ty1-copia group retrotransposons for the genomes of S. tuberosum, L. esculentum, A. cepa, S. cereale, and V. faba is highly variable, ranging from a few hundred to approximately a million copies per genome. In situ hybridisation data from metaphase and prophase chromosomes of V. faba, S. cereale, and H. vulgare suggest that retrotransposon sequences are dispersed throughout the euchromatic regions of the genome but are almost undetectable in most heterochromatic regions. In contrast, similar data from metaphase chromosomes of A. cepa suggests that although retrotransposon sequences are dispersed throughout the euchromatic regions of the genome, they are predominantly concentrated in the terminal heterochromatin. These results are discussed in the context of the role played by the Ty1-copia group retrotransposons in the evolution of the plant genome. Lastly, the application of retrotransposon sequences as genetic markers for mapping genomes and for studying genetic biodiversity in plants is presented.
We have used the polymerase chain reaction to isolate fragments of Tyl-copia group retrotransposo... more We have used the polymerase chain reaction to isolate fragments of Tyl-copia group retrotransposons from a wide variety of members of the higher plant kingdom. 56 out of 57 species tested generate an amplified fragment of the size expected for reverse transcriptase fragments of Tyl-copia group retrotransposons. Sequence analysis of subclones shows that the PCR fragments display varying degrees of sequence heterogeneity. Sequence heterogeneity therefore seems a general property of Tyl-copia group retrotransposons of higher plants, in contrast to the limited diversity seen in retrotransposons of Saccharomyces cerevisiae and Drosophila melanogaster. Phylogenetic analysis of all these sequences shows, with some significant exceptions, that the degree of sequence divergence in the retrotransposon populations between any pair of species is proportional to the evolutionary distance between those species. This implies that sequence divergence during vertical transmission of Tyl-copia group retrotransposons within plant lineages has been a major factor in the evolution of Tyl-copia group retrotransposons in higher plants. Additionally, we suggest that horizontal transmission of this transposon group between different species has also played a role in this process.
The genomic organisation and diversity of the Ty1-copia group retrotransposons has been investiga... more The genomic organisation and diversity of the Ty1-copia group retrotransposons has been investigated in several crop plants and their relatives from both dicotyledonous and monocotyledonous families, including potato ( Solanum tuberosum), faba beans ( Vicia faba), Vicia melanops, Vicia sativa, barley ( Hordeum vulgare), rye ( Secale cereale), and onion ( Allium cepa). Extreme heterogeneity in the sequence of the Ty1-copia retrotransposons from all these plants was revealed following sequence analysis of reverse transcriptase fragments. The estimated copy numbers of the Ty1-copia group retrotransposons for the genomes of S. tuberosum, L. esculentum, A. cepa, S. cereale, and V. faba is highly variable, ranging from a few hundred to approximately a million copies per genome. In situ hybridisation data from metaphase and prophase chromosomes of V. faba, S. cereale, and H. vulgare suggest that retrotransposon sequences are dispersed throughout the euchromatic regions of the genome but are almost undetectable in most heterochromatic regions. In contrast, similar data from metaphase chromosomes of A. cepa suggests that although retrotransposon sequences are dispersed throughout the euchromatic regions of the genome, they are predominantly concentrated in the terminal heterochromatin. These results are discussed in the context of the role played by the Ty1-copia group retrotransposons in the evolution of the plant genome. Lastly, the application of retrotransposon sequences as genetic markers for mapping genomes and for studying genetic biodiversity in plants is presented.
The Ty1-copia group of LTR retrotransposons has been studied extensively in yeast and Drosophila,... more The Ty1-copia group of LTR retrotransposons has been studied extensively in yeast and Drosophila, the organisms in which they were first discovered, and more recently in higher plant and vertebrate species. Their properties, such as copy number, sequence homogeneity, transcriptional and transpositional activity vary greatly between these different hosts. We will try to resolve these apparent discrepancies between these properties, explain any fundamental differences in the biology of the Ty1- copia group between hosts, and propose a general model for LTR retrotransposon evolution.
The terminal sequences of long-terminal repeat (LTR) retrotransposons are a source of powerful mo... more The terminal sequences of long-terminal repeat (LTR) retrotransposons are a source of powerful molecular markers for linkage mapping and biodiversity studies. The major factor limiting the widespread application of LTR retrotransposon-based molecular markers is the availability of new retrotransposon terminal sequences. We describe a PCR-based method for the rapid isolation of LTR sequences of Ty1-copia group retrotransposons from the genomic DNA of potentially any higher plant species. To demonstrate the utility of this technique, we have identi®ed a variety of new retrotransposon LTR sequences from pea, broad bean and Norway spruce. Primers speci®c for three pea LTRs have been used to reveal polymorphisms associated with the corresponding retrotransposons within the Pisum genus.
We have used the polymerase chain reaction to analyse Ty1-copia group retrotransposons of floweri... more We have used the polymerase chain reaction to analyse Ty1-copia group retrotransposons of flowering plants. All eight species studied contain reverse transcriptase fragments from Tyl-copia group retrotransposons. Sequence analysis of 31 subcloned fragments from potato reveals that each is different from the others, with predicted amino acid diversities between individual fragments varying between 5% and 75%. Such sequence heterogeneity within a single species contrasts strongly with the limited diversity seen in such retrotransposons in yeast and Drosophila. The fragments from the other seven plant species examined are also heterogeneous, both within and between species, showing that this is a general property of this transposon group in plants. Phylogenetic analysis of all these sequences reveals that many of them fall into subgroups which span species boundaries, such that the closest homologue of one sequence is often from a different species. We suggest that both vertical transmission of Ty1-copia group retrotransposons within plant lineages and horizontal transmission between different species have played roles in the evolution of Ty1-copia group retrotransposons in flowering plants.
cDNA clones of two genes (TUB8 and TUB13) which show a 25–30-fold increase in transcript in the s... more cDNA clones of two genes (TUB8 and TUB13) which show a 25–30-fold increase in transcript in the stolon tip during the early stages of tuberisation, have been isolated by differential screening. These genes are also expressed in leaves, stems and roots and the expression pattern in these organs changes on tuberisation. Southern analysis shows homologous sequences in the non-tuberising wild type potato species Solanum brevidens and in Lycopersicon esculentum (tomato). Sequence analysis reveals a high degree of similarity between the TUB13 cDNA, and a human S-adenosylmethionine decarboxylase gene. The predicted TUB8 peptide sequence shows several repeats of alanine, glutamate and proline which suggests a structural role for the encoded protein.
S-adenosylmethionine decarboxylase (SAMDC) is involved in the biosynthesis of the polyamines, spe... more S-adenosylmethionine decarboxylase (SAMDC) is involved in the biosynthesis of the polyamines, spermidine and spermine. Recently, we reported the isolation of a putative cDNA clone of the SAMDC clone of potato (Plant Mol Biol 20; 641–651). In order to confirm that the potato genes does encode SAMDC, a complementation experiment with a yeast strain that possesses a null mutation in the SAMDC gene was performed. The yeast strain contains a deletion-insertion mutation in the SAMDC gene and has an absolute requirement for the addition of exogenous spermidine for growth. When the full-length potato cDNA was expressed in the mutant yeast strain there was no longer a requirement for exogenous spermidine. Immunoblotting experiments suggest that the potato SAMDC gene product has an apparent molecular mass of 39 kDa. Expression of the SAMDC gene was high in the young and actively dividing tissues and low in the mature and non-dividing tissues of both vegetative and reproductive organs. Additionally, isolation and characterisation of the corresponding genomic clone is reported. The gene has one intron in its 5′-untranslated sequence but otherwise the transcribed portion is identical to the cDNA clone.
A molecular approach has been used to study the role of polyamines in plant development by manipu... more A molecular approach has been used to study the role of polyamines in plant development by manipulating the expression of the S-adenosylmethionine decarboxylase (SAMDC) gene. SAMDC is a key enzyme involved in the biosynthesis of the polyamines spermidine and spermine and is also known to influence the rate of biosynthesis of ethylene. Previously, a cDNA clone of the SAMDC gene of potato has been isolated and characterized. This cDNA clone has been used to make antisense and sense SAMDC constructs under the control of the 35S CaMV or tetracycline-inducible promoters. Agrobacterium-mediated transformation has been used to produce transgenic potato plants with the engineered antisense and sense SAMDC genes in order to downregulate or overexpress the SAMDC transcript, respectively. Decreases or increases in the level of SAMDC transcript in the antisense and sense transgenic plants were observed, respectively. Antisense transgenic plants which expressed the engineered SAMDC gene constitutively under the control of the 35S CaMV promoter with a duplicated enhancer region showed a range of stunted phenotypes with highly branched stems, short internodes, small leaves and inhibited root growth. The abnormal characteristics of the antisense plants correlated with the altered levels of SAMDC transcript, SAMDC activity, polyamine content and rate of ethylene evolution. Attempts to produce sense transgenic plants with the 35S SAMDC sense construct were unsuccessful indicating that a constitutive overexpression of the engineered SAMDC is lethal to the plants. When antisense and sense SAMDC transgenes were expressed under the control of a tetracycline-inducible promoter a number of transgenic plants were produced. In these antisense and sense plants, significant changes in the level of SAMDC transcript, SAMDC activity and free polyamine content were observed on tetracycline-induction of detached leaves.
Polyamines are low molecular mass polycations found in all living organisms. In plants, they have... more Polyamines are low molecular mass polycations found in all living organisms. In plants, they have been implicated in a wide range of biological processes, including growth, development and stress responses. Recently, several key genes of the polyamine biosynthesis pathways have been cloned and shown to be developmentally regulated. Experiments in which the synthesis of polyamines has been perturbed have clearly shown that these compounds are essential for normal growth and development. These findings have been further supported by recent genetic analyses of polyamine synthesis mutants of Arabidopsis. The research has revealed a much more detailed level of understanding than hitherto, opening up new avenues of investigation for the future.
We have used the polymerase chain reaction to isolate fragments of Tyl-copia group retrotransposo... more We have used the polymerase chain reaction to isolate fragments of Tyl-copia group retrotransposons from a wide variety of members of the higher plant kingdom. 56 out of 57 species tested generate an amplified fragment of the size expected for reverse transcriptase fragments of Tyl-copia group retrotransposons. Sequence analysis of subclones shows that the PCR fragments display varying degrees of sequence heterogeneity. Sequence heterogeneity therefore seems a general property of Tyl-copia group retrotransposons of higher plants, in contrast to the limited diversity seen in retrotransposons of Saccharomyces cerevisiae and Drosophila melanogaster. Phylogenetic analysis of all these sequences shows, with some significant exceptions, that the degree of sequence divergence in the retrotransposon populations between any pair of species is proportional to the evolutionary distance between those species. This implies that sequence divergence during vertical transmission of Tyl-copia group retrotransposons within plant lineages has been a major factor in the evolution of Tyl-copia group retrotransposons in higher plants. Additionally, we suggest that horizontal transmission of this transposon group between different species has also played a role in this process.
The genomic organisation and diversity of the Ty1-copia group retrotransposons has been investiga... more The genomic organisation and diversity of the Ty1-copia group retrotransposons has been investigated in several crop plants and their relatives from both dicotyledonous and monocotyledonous families, including potato ( Solanum tuberosum), faba beans ( Vicia faba), Vicia melanops, Vicia sativa, barley ( Hordeum vulgare), rye ( Secale cereale), and onion ( Allium cepa). Extreme heterogeneity in the sequence of the Ty1-copia retrotransposons from all these plants was revealed following sequence analysis of reverse transcriptase fragments. The estimated copy numbers of the Ty1-copia group retrotransposons for the genomes of S. tuberosum, L. esculentum, A. cepa, S. cereale, and V. faba is highly variable, ranging from a few hundred to approximately a million copies per genome. In situ hybridisation data from metaphase and prophase chromosomes of V. faba, S. cereale, and H. vulgare suggest that retrotransposon sequences are dispersed throughout the euchromatic regions of the genome but are almost undetectable in most heterochromatic regions. In contrast, similar data from metaphase chromosomes of A. cepa suggests that although retrotransposon sequences are dispersed throughout the euchromatic regions of the genome, they are predominantly concentrated in the terminal heterochromatin. These results are discussed in the context of the role played by the Ty1-copia group retrotransposons in the evolution of the plant genome. Lastly, the application of retrotransposon sequences as genetic markers for mapping genomes and for studying genetic biodiversity in plants is presented.
The Ty1-copia group of LTR retrotransposons has been studied extensively in yeast and Drosophila,... more The Ty1-copia group of LTR retrotransposons has been studied extensively in yeast and Drosophila, the organisms in which they were first discovered, and more recently in higher plant and vertebrate species. Their properties, such as copy number, sequence homogeneity, transcriptional and transpositional activity vary greatly between these different hosts. We will try to resolve these apparent discrepancies between these properties, explain any fundamental differences in the biology of the Ty1- copia group between hosts, and propose a general model for LTR retrotransposon evolution.
The terminal sequences of long-terminal repeat (LTR) retrotransposons are a source of powerful mo... more The terminal sequences of long-terminal repeat (LTR) retrotransposons are a source of powerful molecular markers for linkage mapping and biodiversity studies. The major factor limiting the widespread application of LTR retrotransposon-based molecular markers is the availability of new retrotransposon terminal sequences. We describe a PCR-based method for the rapid isolation of LTR sequences of Ty1-copia group retrotransposons from the genomic DNA of potentially any higher plant species. To demonstrate the utility of this technique, we have identi®ed a variety of new retrotransposon LTR sequences from pea, broad bean and Norway spruce. Primers speci®c for three pea LTRs have been used to reveal polymorphisms associated with the corresponding retrotransposons within the Pisum genus.
We have used the polymerase chain reaction to analyse Ty1-copia group retrotransposons of floweri... more We have used the polymerase chain reaction to analyse Ty1-copia group retrotransposons of flowering plants. All eight species studied contain reverse transcriptase fragments from Tyl-copia group retrotransposons. Sequence analysis of 31 subcloned fragments from potato reveals that each is different from the others, with predicted amino acid diversities between individual fragments varying between 5% and 75%. Such sequence heterogeneity within a single species contrasts strongly with the limited diversity seen in such retrotransposons in yeast and Drosophila. The fragments from the other seven plant species examined are also heterogeneous, both within and between species, showing that this is a general property of this transposon group in plants. Phylogenetic analysis of all these sequences reveals that many of them fall into subgroups which span species boundaries, such that the closest homologue of one sequence is often from a different species. We suggest that both vertical transmission of Ty1-copia group retrotransposons within plant lineages and horizontal transmission between different species have played roles in the evolution of Ty1-copia group retrotransposons in flowering plants.
cDNA clones of two genes (TUB8 and TUB13) which show a 25–30-fold increase in transcript in the s... more cDNA clones of two genes (TUB8 and TUB13) which show a 25–30-fold increase in transcript in the stolon tip during the early stages of tuberisation, have been isolated by differential screening. These genes are also expressed in leaves, stems and roots and the expression pattern in these organs changes on tuberisation. Southern analysis shows homologous sequences in the non-tuberising wild type potato species Solanum brevidens and in Lycopersicon esculentum (tomato). Sequence analysis reveals a high degree of similarity between the TUB13 cDNA, and a human S-adenosylmethionine decarboxylase gene. The predicted TUB8 peptide sequence shows several repeats of alanine, glutamate and proline which suggests a structural role for the encoded protein.
S-adenosylmethionine decarboxylase (SAMDC) is involved in the biosynthesis of the polyamines, spe... more S-adenosylmethionine decarboxylase (SAMDC) is involved in the biosynthesis of the polyamines, spermidine and spermine. Recently, we reported the isolation of a putative cDNA clone of the SAMDC clone of potato (Plant Mol Biol 20; 641–651). In order to confirm that the potato genes does encode SAMDC, a complementation experiment with a yeast strain that possesses a null mutation in the SAMDC gene was performed. The yeast strain contains a deletion-insertion mutation in the SAMDC gene and has an absolute requirement for the addition of exogenous spermidine for growth. When the full-length potato cDNA was expressed in the mutant yeast strain there was no longer a requirement for exogenous spermidine. Immunoblotting experiments suggest that the potato SAMDC gene product has an apparent molecular mass of 39 kDa. Expression of the SAMDC gene was high in the young and actively dividing tissues and low in the mature and non-dividing tissues of both vegetative and reproductive organs. Additionally, isolation and characterisation of the corresponding genomic clone is reported. The gene has one intron in its 5′-untranslated sequence but otherwise the transcribed portion is identical to the cDNA clone.
A molecular approach has been used to study the role of polyamines in plant development by manipu... more A molecular approach has been used to study the role of polyamines in plant development by manipulating the expression of the S-adenosylmethionine decarboxylase (SAMDC) gene. SAMDC is a key enzyme involved in the biosynthesis of the polyamines spermidine and spermine and is also known to influence the rate of biosynthesis of ethylene. Previously, a cDNA clone of the SAMDC gene of potato has been isolated and characterized. This cDNA clone has been used to make antisense and sense SAMDC constructs under the control of the 35S CaMV or tetracycline-inducible promoters. Agrobacterium-mediated transformation has been used to produce transgenic potato plants with the engineered antisense and sense SAMDC genes in order to downregulate or overexpress the SAMDC transcript, respectively. Decreases or increases in the level of SAMDC transcript in the antisense and sense transgenic plants were observed, respectively. Antisense transgenic plants which expressed the engineered SAMDC gene constitutively under the control of the 35S CaMV promoter with a duplicated enhancer region showed a range of stunted phenotypes with highly branched stems, short internodes, small leaves and inhibited root growth. The abnormal characteristics of the antisense plants correlated with the altered levels of SAMDC transcript, SAMDC activity, polyamine content and rate of ethylene evolution. Attempts to produce sense transgenic plants with the 35S SAMDC sense construct were unsuccessful indicating that a constitutive overexpression of the engineered SAMDC is lethal to the plants. When antisense and sense SAMDC transgenes were expressed under the control of a tetracycline-inducible promoter a number of transgenic plants were produced. In these antisense and sense plants, significant changes in the level of SAMDC transcript, SAMDC activity and free polyamine content were observed on tetracycline-induction of detached leaves.
Polyamines are low molecular mass polycations found in all living organisms. In plants, they have... more Polyamines are low molecular mass polycations found in all living organisms. In plants, they have been implicated in a wide range of biological processes, including growth, development and stress responses. Recently, several key genes of the polyamine biosynthesis pathways have been cloned and shown to be developmentally regulated. Experiments in which the synthesis of polyamines has been perturbed have clearly shown that these compounds are essential for normal growth and development. These findings have been further supported by recent genetic analyses of polyamine synthesis mutants of Arabidopsis. The research has revealed a much more detailed level of understanding than hitherto, opening up new avenues of investigation for the future.
We have used the polymerase chain reaction to isolate fragments of Tyl-copia group retrotransposo... more We have used the polymerase chain reaction to isolate fragments of Tyl-copia group retrotransposons from a wide variety of members of the higher plant kingdom. 56 out of 57 species tested generate an amplified fragment of the size expected for reverse transcriptase fragments of Tyl-copia group retrotransposons. Sequence analysis of subclones shows that the PCR fragments display varying degrees of sequence heterogeneity. Sequence heterogeneity therefore seems a general property of Tyl-copia group retrotransposons of higher plants, in contrast to the limited diversity seen in retrotransposons of Saccharomyces cerevisiae and Drosophila melanogaster. Phylogenetic analysis of all these sequences shows, with some significant exceptions, that the degree of sequence divergence in the retrotransposon populations between any pair of species is proportional to the evolutionary distance between those species. This implies that sequence divergence during vertical transmission of Tyl-copia group retrotransposons within plant lineages has been a major factor in the evolution of Tyl-copia group retrotransposons in higher plants. Additionally, we suggest that horizontal transmission of this transposon group between different species has also played a role in this process.
The genomic organisation and diversity of the Ty1-copia group retrotransposons has been investiga... more The genomic organisation and diversity of the Ty1-copia group retrotransposons has been investigated in several crop plants and their relatives from both dicotyledonous and monocotyledonous families, including potato ( Solanum tuberosum), faba beans ( Vicia faba), Vicia melanops, Vicia sativa, barley ( Hordeum vulgare), rye ( Secale cereale), and onion ( Allium cepa). Extreme heterogeneity in the sequence of the Ty1-copia retrotransposons from all these plants was revealed following sequence analysis of reverse transcriptase fragments. The estimated copy numbers of the Ty1-copia group retrotransposons for the genomes of S. tuberosum, L. esculentum, A. cepa, S. cereale, and V. faba is highly variable, ranging from a few hundred to approximately a million copies per genome. In situ hybridisation data from metaphase and prophase chromosomes of V. faba, S. cereale, and H. vulgare suggest that retrotransposon sequences are dispersed throughout the euchromatic regions of the genome but are almost undetectable in most heterochromatic regions. In contrast, similar data from metaphase chromosomes of A. cepa suggests that although retrotransposon sequences are dispersed throughout the euchromatic regions of the genome, they are predominantly concentrated in the terminal heterochromatin. These results are discussed in the context of the role played by the Ty1-copia group retrotransposons in the evolution of the plant genome. Lastly, the application of retrotransposon sequences as genetic markers for mapping genomes and for studying genetic biodiversity in plants is presented.
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Papers by Amar Kumar