The generation of knockout mutants of Escherichia coli is central to investigating gene function.... more The generation of knockout mutants of Escherichia coli is central to investigating gene function. A classical method for inactivating genes in vivo is transposon mutagenesis. 1-3 However, the method has certain requirements, such as mapping of the site of transposon insertion, which can make it quite time consuming. A newer transposon mutagenesis methodology based on a hyper-reactive Tn5 transposition system 4 facilitates efficient mutagenesis of genes in vitro as well as in vivo. 5
Transgenic lines of the potato cultivar White Lady expressing the trehalose-6-phosphate synthase ... more Transgenic lines of the potato cultivar White Lady expressing the trehalose-6-phosphate synthase (TPS1) gene of yeast exhibit improved drought tolerance, but grow slower and have a lower carbon fixation rate and stomatal density than the wild-type. To understand the molecular basis of this phenomenon, we have compared the transcriptomes of wild-type and TPS1-transgenic plants using the POCI microarray containing 42,034 potato unigene probes. We show that 74 and 25 genes were up-, and down-regulated, respectively, in the mature source leaves of TPS1-transgenic plants when compared with the wild-type. The differentially regulated genes were assigned into 16 functional groups. All of the seven genes, which were assigned into carbon fixation and metabolism group, were up-regulated, while about 42% of the assigned genes are involved in transcriptional and post-transcriptional regulation. Expression of genes encoding a 14-3-3 regulatory protein, and four transcription factors were down-regulated in the TPS1-transgenic leaves. To verify the microarray results, we used RNA gel blot analysis to examine the expression of eight genes and found that the RNA gel blot and microarray data correlated in each case. Using the putative Arabidopsis orthologs of the assigned potato sequences we have identified putative transcription binding sites in the promoter region of the differentially regulated genes, and putative proteinprotein interactions involving some of the up-and down-regulated genes. We have also demonstrated that starch content is lower, while malate, inositol and maltose contents are higher in the TPS1-transgenic than in the wild-type leaves. Our results suggest that a complex regulatory network, involving transcription factors and other regulatory proteins, underpins the phenotypic alterations we have observed previously in potato when expressing the TPS1 gene of yeast.
Background: Mangalicas are fatty type local/rare pig breeds with an increasing presence in the ni... more Background: Mangalicas are fatty type local/rare pig breeds with an increasing presence in the niche pork market in Hungary and in other countries. To explore their genetic resources, we have analysed data from next-generation sequencing of an individual male from each of three Mangalica breeds along with a local male Duroc pig. Structural variations, such as SNPs, INDELs and CNVs, were identified and particular genes with SNP variations were analysed with special emphasis on functions related to fat metabolism in pigs.
Vitamin C, B5, and B6 contents of potato tubers were measured by gas chromatography-mass spectrom... more Vitamin C, B5, and B6 contents of potato tubers were measured by gas chromatography-mass spectrometry (GC-MS). Two diploid potato populations derived from two different crosses were analyzed. Plants were grown at two locations under different environmental conditions. Measurements of vitamin content of tubers showed high variation in both populations. A weak correlation was found between the vitamin concentrations of tubers harvested at the two locations. Correlations between the mean values of vitamins in field-grown tubers and in different tissues derived from in vitro or greenhouse-grown potato plants were also determined. A very high correlation between the vitamin content of field-and greenhouse-grown tubers, and between field-grown tubers and sink leaves of greenhouse-grown plants was detected. This finding can facilitate breeding by preselection of individuals with improved vitamin content under greenhouse conditions at their early developmental stage.
The NocR protein of Ag,robacterium tumefa-cienswa1 found to regulate expression of the divergentl... more The NocR protein of Ag,robacterium tumefa-cienswa1 found to regulate expression of the divergently transcribed nocB and nocR genes of the pTiT3TTopiline catabolism (noc) region. Experiments using the firefly luciferase {Iuc) gene as reporter demonstrited that NocR represses and activates transcription from the nocB promoter in the absence and presence of nopaline, respectively. NocR also negatively autoregulaies its own synthesis irrespective of the presence of nopaline. Regulation of expression of both nocB and nocRls mediated by binding of the NocR protein to the nocR promoter. A 12 bp symmetrical seqluence, which lies 3 bp downstream of the -10 hexamer of the nocRpromotei, was confirmed to be essential for bindine of the NocR protein. Functional localization of the ricB and nocR promoters verified that they do not overlap at all, and that the interrupted dyad, at which NocR binds, is I37 bp upstream of the regulatednocB promoter. The in vivo and in vitro results described here and those published previously suggest that a novel type ofregulatory 6ssfuanis11, which may involve changes in DNA topology, controls gene expression in t[e noc operon of pTiT37.
The nocR gene of Agrobacterium tumefaciensTi plasmid pTiT37 is the regulatory gene of the nopalin... more The nocR gene of Agrobacterium tumefaciensTi plasmid pTiT37 is the regulatory gene of the nopaline catabolism (noc) operon of pTiT37. We have cloned and sequenced nocR, which encodes a DNA-binding protein.
In many bacteria, the concentration of L-arginine is controlled by a transcriptional regulator, t... more In many bacteria, the concentration of L-arginine is controlled by a transcriptional regulator, the arginine repressor. In Bacillus subtilis this transcription factor is called AhrC and has roles in both the repression and activation of the genes involved in arginine metabolism. It interacts with 18 bp ARG boxes in the promoters of arginine biosynthetic and catabolic operons. AhrC is a hexamer and each subunit has two domains. The C-terminal domains form the core, mediating inter-subunit interactions and L-arginine binding, while the N-terminal domains contain a winged helix-turn-helix DNAbinding motif and are arranged around the periphery. Upon binding of the co-repressor L-arginine there is a ∼ 15°relative rotation between core Cterminal trimers. Here, we report the X-ray crystal structure of a dimer of the N-terminal domains of AhrC (NAhrC) in complex with an 18 bp DNA ARG box operator, refined to 2.85 Å resolution. Comparison of the N-terminal domains within this complex with those of the free domain reveals that the flexible β-wings of the DNA-binding motif in the free domain form a stable dimer interface in the protein-DNA complex, favouring correct orientation of the recognition helices. These are then positioned to insert into adjacent turns of the major groove of the ARG box, whilst the wings contact the minor groove. There are extensive contacts between the protein and the DNA phosphodiester backbone, as well as a number of direct hydrogen bonds between conserved amino acid side chains and bases. Combining this structure with other crystal structures of other AhrC components, we have constructed a model of the repression complex of AhrC at the B. subtilis biosynthetic argC operator and, along with transcriptome data, analysed the origins of sequence specificity and arginine activation.
The genetic relationship between 195 Mangalica and 79 non-Mangalica pigs was studied using mitoch... more The genetic relationship between 195 Mangalica and 79 non-Mangalica pigs was studied using mitochondrial D-loop SNP genotyping. Altogether, 35 polymorphic sites and 27 haplotypes were identified. Of the haplotypes, eight and 16 are Mangalica and non-Mangalica specific, respectively, while three contain both Mangalica and non-Mangalica individuals. Genetic distance values and phylogenetic analysis indicate that Mangalica individuals are very closely related, and five haplotypes represent approximately 92% of the Mangalica pigs involved in the study, thus determining the major maternal lineages. In contrast to previous microsatellite studies, individuals of Mangalica could not be distinguished as three separate breeds using mtDNA genotyping. Comparing modern and archaeological mtDNA sequences revealed that present day Mangalica is related to pigs that lived in the Carpathian basin where postulated ancestors of Mangalica also lived. This is the first DNA-based genetic evidence to support the described breeding history of Mangalica.
Background: Mangalica breeds are indigenous to Hungary and their breeding history dates back to a... more Background: Mangalica breeds are indigenous to Hungary and their breeding history dates back to about 200-250 years ago. They are fat-type pigs and have a rare curly hair phenotype. The aim of our study was to establish the relationships between these unique breeds and other European breeds. Results: Based on a core sequence of 382 bp present in 2713 mitochondrial D-loop sequences from pigs belonging to 38 local breeds from nine countries, five cosmopolitan breeds and wild boars from 14 countries, we identified 164 haplotypes. More than half of the 2713 sequences belonged to either four haplotypes characteristic of continental European breeds or two haplotypes characteristic of British/cosmopolitan breeds; each haplotype is present in more than 100 individuals. Most Mangalica individuals belonged either to one of these common continental European haplotypes or to two Mangalica-specific haplotypes that were absent in all other breeds. In addition, we identified the ancestral mitochondrial D-loop signature present in these 2713 sequences and found that~80% carried the European ancient signatures, ANC-Aside and ANC-Cside or their closely related signatures, while most of the remaining sequences carried a modern Asian signature, ANC-Easia. Mangalica individuals carried the ANC-Aside signature, but not the ANC-Cside or ANC-Easia signatures.
Background: The development of drought-tolerant, elite varieties of potato (Solanum tuberosum L.)... more Background: The development of drought-tolerant, elite varieties of potato (Solanum tuberosum L.) is a challenging task, which might be achieved by introducing transgenic lines into breeding. We previously demonstrated that strains of the White Lady potato cultivar that express the yeast trehalose-6-phosphate synthase (TPS1) gene exhibit improved drought tolerance.
We have used DNA arrays to investigate the effects of knocking out the methionine repressor gene,... more We have used DNA arrays to investigate the effects of knocking out the methionine repressor gene, metJ, on the Escherichia coli transcriptome. We assayed the effects in the knockout strain of supplying wild-type or mutant MetJ repressors from an expression plasmid, thus establishing a rapid assay for in vivo effects of mutations characterized previously in vitro. Repression is largely restricted to known genes involved in the biosynthesis and uptake of methionine. However, we identified a number of additional genes that are significantly up-regulated in the absence of repressor. Sequence analysis of the 5' promoter regions of these genes identified plausible matches to met-box sequences for three of these, and subsequent electrophoretic mobility-shift assay analysis showed that for two such loci their repressor affinity is higher than or comparable with the known metB operator, suggesting that they are directly regulated. This can be rationalized for one of the loci, folE, by the metabolic role of its encoded enzyme; however, the links to the other regulated loci are unclear, suggesting both an extension to the known met regulon and additional complexity to the role of the repressor. The plasmid gene replacement system has been used to examine the importance of protein-protein co-operativity in operator saturation using the structurally characterized mutant repressor, Q44K. In vivo, there are detectable reductions in the levels of regulation observed, demonstrating the importance of balancing protein-protein and protein-DNA affinity.
The whole genome of Mangalica animals has been screened on the Illumina porcine chip giving the p... more The whole genome of Mangalica animals has been screened on the Illumina porcine chip giving the possibility (1) to replace the previously applied ten microsatellite markers by nine SNP loci to classify the Blond, Swallow-Belly and Red Mangalica individuals into three different breed groups (P>0.95); (2) to propose 54 SNP loci for parentage testing in Mangalica pigs where the exclusion probability is 0.999115 if one parent is known and the probability of identity is 1.54×10 -23 .
Background: Wheat is the leading source of vegetable protein in the human diet, and metabolites a... more Background: Wheat is the leading source of vegetable protein in the human diet, and metabolites are crucial for both plant development and human nutrition. The recent advances in metabolomics provided an opportunity to perform an untargeted metabolite analysis in this important crop.
The generation of knockout mutants of Escherichia coli is central to investigating gene function.... more The generation of knockout mutants of Escherichia coli is central to investigating gene function. A classical method for inactivating genes in vivo is transposon mutagenesis. 1-3 However, the method has certain requirements, such as mapping of the site of transposon insertion, which can make it quite time consuming. A newer transposon mutagenesis methodology based on a hyper-reactive Tn5 transposition system 4 facilitates efficient mutagenesis of genes in vitro as well as in vivo. 5
Transgenic lines of the potato cultivar White Lady expressing the trehalose-6-phosphate synthase ... more Transgenic lines of the potato cultivar White Lady expressing the trehalose-6-phosphate synthase (TPS1) gene of yeast exhibit improved drought tolerance, but grow slower and have a lower carbon fixation rate and stomatal density than the wild-type. To understand the molecular basis of this phenomenon, we have compared the transcriptomes of wild-type and TPS1-transgenic plants using the POCI microarray containing 42,034 potato unigene probes. We show that 74 and 25 genes were up-, and down-regulated, respectively, in the mature source leaves of TPS1-transgenic plants when compared with the wild-type. The differentially regulated genes were assigned into 16 functional groups. All of the seven genes, which were assigned into carbon fixation and metabolism group, were up-regulated, while about 42% of the assigned genes are involved in transcriptional and post-transcriptional regulation. Expression of genes encoding a 14-3-3 regulatory protein, and four transcription factors were down-regulated in the TPS1-transgenic leaves. To verify the microarray results, we used RNA gel blot analysis to examine the expression of eight genes and found that the RNA gel blot and microarray data correlated in each case. Using the putative Arabidopsis orthologs of the assigned potato sequences we have identified putative transcription binding sites in the promoter region of the differentially regulated genes, and putative proteinprotein interactions involving some of the up-and down-regulated genes. We have also demonstrated that starch content is lower, while malate, inositol and maltose contents are higher in the TPS1-transgenic than in the wild-type leaves. Our results suggest that a complex regulatory network, involving transcription factors and other regulatory proteins, underpins the phenotypic alterations we have observed previously in potato when expressing the TPS1 gene of yeast.
Background: Mangalicas are fatty type local/rare pig breeds with an increasing presence in the ni... more Background: Mangalicas are fatty type local/rare pig breeds with an increasing presence in the niche pork market in Hungary and in other countries. To explore their genetic resources, we have analysed data from next-generation sequencing of an individual male from each of three Mangalica breeds along with a local male Duroc pig. Structural variations, such as SNPs, INDELs and CNVs, were identified and particular genes with SNP variations were analysed with special emphasis on functions related to fat metabolism in pigs.
Vitamin C, B5, and B6 contents of potato tubers were measured by gas chromatography-mass spectrom... more Vitamin C, B5, and B6 contents of potato tubers were measured by gas chromatography-mass spectrometry (GC-MS). Two diploid potato populations derived from two different crosses were analyzed. Plants were grown at two locations under different environmental conditions. Measurements of vitamin content of tubers showed high variation in both populations. A weak correlation was found between the vitamin concentrations of tubers harvested at the two locations. Correlations between the mean values of vitamins in field-grown tubers and in different tissues derived from in vitro or greenhouse-grown potato plants were also determined. A very high correlation between the vitamin content of field-and greenhouse-grown tubers, and between field-grown tubers and sink leaves of greenhouse-grown plants was detected. This finding can facilitate breeding by preselection of individuals with improved vitamin content under greenhouse conditions at their early developmental stage.
The NocR protein of Ag,robacterium tumefa-cienswa1 found to regulate expression of the divergentl... more The NocR protein of Ag,robacterium tumefa-cienswa1 found to regulate expression of the divergently transcribed nocB and nocR genes of the pTiT3TTopiline catabolism (noc) region. Experiments using the firefly luciferase {Iuc) gene as reporter demonstrited that NocR represses and activates transcription from the nocB promoter in the absence and presence of nopaline, respectively. NocR also negatively autoregulaies its own synthesis irrespective of the presence of nopaline. Regulation of expression of both nocB and nocRls mediated by binding of the NocR protein to the nocR promoter. A 12 bp symmetrical seqluence, which lies 3 bp downstream of the -10 hexamer of the nocRpromotei, was confirmed to be essential for bindine of the NocR protein. Functional localization of the ricB and nocR promoters verified that they do not overlap at all, and that the interrupted dyad, at which NocR binds, is I37 bp upstream of the regulatednocB promoter. The in vivo and in vitro results described here and those published previously suggest that a novel type ofregulatory 6ssfuanis11, which may involve changes in DNA topology, controls gene expression in t[e noc operon of pTiT37.
The nocR gene of Agrobacterium tumefaciensTi plasmid pTiT37 is the regulatory gene of the nopalin... more The nocR gene of Agrobacterium tumefaciensTi plasmid pTiT37 is the regulatory gene of the nopaline catabolism (noc) operon of pTiT37. We have cloned and sequenced nocR, which encodes a DNA-binding protein.
In many bacteria, the concentration of L-arginine is controlled by a transcriptional regulator, t... more In many bacteria, the concentration of L-arginine is controlled by a transcriptional regulator, the arginine repressor. In Bacillus subtilis this transcription factor is called AhrC and has roles in both the repression and activation of the genes involved in arginine metabolism. It interacts with 18 bp ARG boxes in the promoters of arginine biosynthetic and catabolic operons. AhrC is a hexamer and each subunit has two domains. The C-terminal domains form the core, mediating inter-subunit interactions and L-arginine binding, while the N-terminal domains contain a winged helix-turn-helix DNAbinding motif and are arranged around the periphery. Upon binding of the co-repressor L-arginine there is a ∼ 15°relative rotation between core Cterminal trimers. Here, we report the X-ray crystal structure of a dimer of the N-terminal domains of AhrC (NAhrC) in complex with an 18 bp DNA ARG box operator, refined to 2.85 Å resolution. Comparison of the N-terminal domains within this complex with those of the free domain reveals that the flexible β-wings of the DNA-binding motif in the free domain form a stable dimer interface in the protein-DNA complex, favouring correct orientation of the recognition helices. These are then positioned to insert into adjacent turns of the major groove of the ARG box, whilst the wings contact the minor groove. There are extensive contacts between the protein and the DNA phosphodiester backbone, as well as a number of direct hydrogen bonds between conserved amino acid side chains and bases. Combining this structure with other crystal structures of other AhrC components, we have constructed a model of the repression complex of AhrC at the B. subtilis biosynthetic argC operator and, along with transcriptome data, analysed the origins of sequence specificity and arginine activation.
The genetic relationship between 195 Mangalica and 79 non-Mangalica pigs was studied using mitoch... more The genetic relationship between 195 Mangalica and 79 non-Mangalica pigs was studied using mitochondrial D-loop SNP genotyping. Altogether, 35 polymorphic sites and 27 haplotypes were identified. Of the haplotypes, eight and 16 are Mangalica and non-Mangalica specific, respectively, while three contain both Mangalica and non-Mangalica individuals. Genetic distance values and phylogenetic analysis indicate that Mangalica individuals are very closely related, and five haplotypes represent approximately 92% of the Mangalica pigs involved in the study, thus determining the major maternal lineages. In contrast to previous microsatellite studies, individuals of Mangalica could not be distinguished as three separate breeds using mtDNA genotyping. Comparing modern and archaeological mtDNA sequences revealed that present day Mangalica is related to pigs that lived in the Carpathian basin where postulated ancestors of Mangalica also lived. This is the first DNA-based genetic evidence to support the described breeding history of Mangalica.
Background: Mangalica breeds are indigenous to Hungary and their breeding history dates back to a... more Background: Mangalica breeds are indigenous to Hungary and their breeding history dates back to about 200-250 years ago. They are fat-type pigs and have a rare curly hair phenotype. The aim of our study was to establish the relationships between these unique breeds and other European breeds. Results: Based on a core sequence of 382 bp present in 2713 mitochondrial D-loop sequences from pigs belonging to 38 local breeds from nine countries, five cosmopolitan breeds and wild boars from 14 countries, we identified 164 haplotypes. More than half of the 2713 sequences belonged to either four haplotypes characteristic of continental European breeds or two haplotypes characteristic of British/cosmopolitan breeds; each haplotype is present in more than 100 individuals. Most Mangalica individuals belonged either to one of these common continental European haplotypes or to two Mangalica-specific haplotypes that were absent in all other breeds. In addition, we identified the ancestral mitochondrial D-loop signature present in these 2713 sequences and found that~80% carried the European ancient signatures, ANC-Aside and ANC-Cside or their closely related signatures, while most of the remaining sequences carried a modern Asian signature, ANC-Easia. Mangalica individuals carried the ANC-Aside signature, but not the ANC-Cside or ANC-Easia signatures.
Background: The development of drought-tolerant, elite varieties of potato (Solanum tuberosum L.)... more Background: The development of drought-tolerant, elite varieties of potato (Solanum tuberosum L.) is a challenging task, which might be achieved by introducing transgenic lines into breeding. We previously demonstrated that strains of the White Lady potato cultivar that express the yeast trehalose-6-phosphate synthase (TPS1) gene exhibit improved drought tolerance.
We have used DNA arrays to investigate the effects of knocking out the methionine repressor gene,... more We have used DNA arrays to investigate the effects of knocking out the methionine repressor gene, metJ, on the Escherichia coli transcriptome. We assayed the effects in the knockout strain of supplying wild-type or mutant MetJ repressors from an expression plasmid, thus establishing a rapid assay for in vivo effects of mutations characterized previously in vitro. Repression is largely restricted to known genes involved in the biosynthesis and uptake of methionine. However, we identified a number of additional genes that are significantly up-regulated in the absence of repressor. Sequence analysis of the 5' promoter regions of these genes identified plausible matches to met-box sequences for three of these, and subsequent electrophoretic mobility-shift assay analysis showed that for two such loci their repressor affinity is higher than or comparable with the known metB operator, suggesting that they are directly regulated. This can be rationalized for one of the loci, folE, by the metabolic role of its encoded enzyme; however, the links to the other regulated loci are unclear, suggesting both an extension to the known met regulon and additional complexity to the role of the repressor. The plasmid gene replacement system has been used to examine the importance of protein-protein co-operativity in operator saturation using the structurally characterized mutant repressor, Q44K. In vivo, there are detectable reductions in the levels of regulation observed, demonstrating the importance of balancing protein-protein and protein-DNA affinity.
The whole genome of Mangalica animals has been screened on the Illumina porcine chip giving the p... more The whole genome of Mangalica animals has been screened on the Illumina porcine chip giving the possibility (1) to replace the previously applied ten microsatellite markers by nine SNP loci to classify the Blond, Swallow-Belly and Red Mangalica individuals into three different breed groups (P>0.95); (2) to propose 54 SNP loci for parentage testing in Mangalica pigs where the exclusion probability is 0.999115 if one parent is known and the probability of identity is 1.54×10 -23 .
Background: Wheat is the leading source of vegetable protein in the human diet, and metabolites a... more Background: Wheat is the leading source of vegetable protein in the human diet, and metabolites are crucial for both plant development and human nutrition. The recent advances in metabolomics provided an opportunity to perform an untargeted metabolite analysis in this important crop.
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Papers by Ferenc Marincs