Carlo Bruschi - Academia.edu (original) (raw)
Papers by Carlo Bruschi
Molecular Genetics and Genomics, 2015
Chromosome translocation is a major genomic event for a cell, affecting almost every of its life ... more Chromosome translocation is a major genomic event for a cell, affecting almost every of its life aspects ranging from metabolism, organelle maintenance and homeostasis to gene maintenance and expression. By using the bridge-induced translocation system, we defined the effects of induced chromosome translocation on the chronological life span (CLS) of yeast with particular interest to the oxidative stress condition. The results demonstrate that every translocant strain has a different CLS, but all have a high increase in reactive oxygen species and in lipid peroxides levels at the end of the life span. This could be due to the very unique and strong deregulation of the oxidative stress network. Furthermore, the loss of the translocated chromosome occurs at the end of the life span and is locus dependent. Additionally, the RDH54 gene may play a role in the correct segregation of the translocant chromosome, since in its absence there is an increase in loss of the bridge-induced translocated chromosome.
Experimental systems exist, which are able to generate only specific reciprocal translocations be... more Experimental systems exist, which are able to generate only specific reciprocal translocations between engineered chromosomal loci of yeast and Drosophila but not in wild-type cells. Here we report the successfull induction of chromosome translocations in the yeast Saccharomyces cerevisiae by targeted DNA integration of the KANR selectable marker flanked by two DNA sequences homologous to two different chromosomes. Using this Bridge-Induced Translocation (BIT) system, from 2 to 4% of all integrants, depending on the length of the homologous DNA ends, showed targeted non-reciprocal translocations between chromosomes V-VIII and VIII-XV in two wild-type strains. The complex fate of the chromosomal fragments generated indicated the involvement of multiple repair pathways in a two-step integration dynamics. The possibility of inducing chromosomal translocations between any two desired genetic loci in a eukaryotic model system will be instrumental for their molecular characterization, as ...
Adaptation, which is often used as a synonym for evolution is usually due to a genetic sweep that... more Adaptation, which is often used as a synonym for evolution is usually due to a genetic sweep that gives an organism the selective advantage to survive in a harsh environment. This genetic sweep can come either from different point mutations that occur inside a single genetic element or from a wide and deep genomic rearrangement such as a chromosome translocation. The latter has been widely studied in many of its aspects. In fact it has been demonstrated that it affects global gene expression by de-regulating over a thousand genes [1], it is able to induce multiple aneuploidies [2] and that it may lead to loss of heterozygosity (LOH)[3]. Chromosome translocation can be induced by using a simple but effective method named Bridge Induced Translocation (BIT) [4] that allows to specifically target a translocation anywhere in the yeast genome. Combining the BIT method with the Chronological Life Span assay [5] we wanted to analyse how a yeast cell, after it underwent a BIT translocation, ...
Food Technology and Biotechnology
CDC6 is an essential gene of Saccharomyces cerevisiae involved in the initiation of DNA replicati... more CDC6 is an essential gene of Saccharomyces cerevisiae involved in the initiation of DNA replication. Interacting with ORC complex of proteins, Cdc6 protein has a pivotal role in loading Mcm proteins on origins of replication. Although much evidence about its func-tion in S phase of the cell cycle is available, only a few data indicate a regulatory function of this protein in G2/M transition phase of the cell cycle. By synchronisation with a micro-tubule-destabilising drug nocodazole and Fluorescence Activating Cell Sorting (FACS) analysis it was possible to provide evidence that CDC6 is responsible for a proper se-quence of genetic events and accurate chromosome segregation during mitosis.
Gene
Site-specific recombination systems from bacteriophage and yeasts are becoming precious tools for... more Site-specific recombination systems from bacteriophage and yeasts are becoming precious tools for manipulating DNA both in vitro and in living organisms. In this work we describe the isolation of yeast Saccharomyces cerevisiae segregants which have lost the highly stable 2 μm DNA plasmid, exploiting the site-specific recombination system of 2 μm itself. We efficiently isolated [ciro] segregants from two haploid yeast strains and also a diploid. Moreover, the effect of mutations in the core region of the FRT (Flp Recognition Target) sequence was investigated in vivo, studying the result of the recombination event between several mutated and wild-type FRT sequences. From our result it seems that the identity between the core regions of two FRT sites is necessary but not sufficient, indicating that the core sequence itself has a relevant function in the recombination mechanism in vivo.
Chromosome translocation brings a huge array of changes to the cell, which spreads from genetic t... more Chromosome translocation brings a huge array of changes to the cell, which spreads from genetic to phenotypic modifications [1, 2]. In addition, we recently discovered that this genomic rearrangement has an influence on the life span of yeast and on its telomere's length. Using the BIT (bridge-induced translocation) system created in our lab [3], here we show that any chromosome translocation can either increase or decrease the chronological life span of cells. The data gathered show that the structural change of the two chromosomes involved in translocation can have an opposite effects on the chronological life span, either shortening or elongating it by 30%-50%. Furthermore, we were able to define that BIT not only influences life span but also telomere length, shortening the telomeres of most of our translocants. The connection between chronological aging and the molecular mechanism underlying programmed cell death makes us suppose that chromosome translocation also effects a...
Gene
Site-specific recombination systems from bacteriophage and yeasts are becoming precious tools for... more Site-specific recombination systems from bacteriophage and yeasts are becoming precious tools for manipulating DNA both in vitro and in living organisms. In this work we describe the isolation of yeast Saccharomyces cerevisiae segregants which have lost the highly stable 2 microm DNA plasmid, exploiting the site-specific recombination system of 2 microm itself. We efficiently isolated [cir0] segregants from two haploid yeast strains and also a diploid. Moreover, the effect of mutations in the core region of the FRT (Flp Recognition Target) sequence was investigated in vivo, studying the result of the recombination event between several mutated and wild-type FRT sequences. From our result it seems that the identity between the core regions of two FRT sites is necessary but not sufficient, indicating that the core sequence itself has a relevant function in the recombination mechanism in vivo.
Plasmid
Temperature-sensitive Saccharomyces cerevisiae cdc6 mutants, under restrictive conditions, show a... more Temperature-sensitive Saccharomyces cerevisiae cdc6 mutants, under restrictive conditions, show an increase in recombination frequency, as well as chromosome and circular minichromosome loss. The role of the essential CDC6 gene was tested in trans and in cis to study circular plasmid stability. It was possible to demonstrate that the product of the CDC6 gene, acting in trans, is important for centromeric, episomal, and also 2-microns plasmid maintenance, while the gene sequence itself has no effect in cis on the stability of the plasmids tested. A high percentage of phenotypic revertants for the cdc6 mutation loses 2 microns upon shifting to the restrictive temperature and, under semipermissive conditions, the endogenous plasmid becomes very unstable, favoring a more efficient curing procedure. A positive correlation between centromeric plasmid size and stability was demonstrated even for small circular plasmids.
Nanotoxicology, 2015
The use of cadmium sulphide quantum dots (CdS QDs) is increasing, particularly in the electronics... more The use of cadmium sulphide quantum dots (CdS QDs) is increasing, particularly in the electronics industry. Their size (1-10 nm in diameter) is, however, such that they can be taken up by living cells. Here, a bakers' yeast (Saccharomyces cerevisiae) deletion mutant collection has been exploited to provide a high-throughput means of revealing the genetic basis for tolerance/susceptibility to CdS QD exposure. The deletion of 112 genes, some associated with the abiotic stress response, some with various metabolic processes, some with mitochondrial organization, some with transport and some with DNA repair, reduced the level of tolerance to CdS QDs. A gene ontology analysis highlighted the role of oxidative stress in determining the cellular response. The transformation of sensitive mutants with centromeric plasmids harbouring DNA from a wild type strain restored the wild type growth phenotype when the complemented genes encoded either HSC82, DSK2 or ALD3. The use of these simple eukaryote knock-out mutants for functional toxicogenomic analysis will inform studies focusing on higher organisms.
Current Genetics
We have employed the analysis of spontaneous forward mutations that confer the ability to utilize... more We have employed the analysis of spontaneous forward mutations that confer the ability to utilize L--aminoadipate as a nitrogen source (-Aa+) to discern the events that contribute to mitotic segregation of spontaneous recessive mutations by diploid cells. -Aa- diploid cells yield -Aa+ mutants at a rate of 7.83.610-9. As in haploid strains, approximately 97% (30/31) of -Aa+ mutants are spontaneous lys2-x recessive mutations. -Aa+ mutants of diploid cells reflect mostly the fate of LYS2/lys2-x heterozygotes that arise by mutation within LYS2/LYS2 populations at a rate of 1.20.410-6. Mitotic recombination occurs in nonrandom association with forward mutation of LYS2 at a rate of 1.30.610-3. This mitotic recombination rate is tenfold higher than that of a control LYS2/lys2-1 diploid. Mitotic segregation within LYS2/lys2-x subpopulations yields primarily lys2-x/lys2-x diploids and a minority of lys2-x aneuploids. Fifteen percent of lys2-x/lys2-x diploids appear to have arisen by gene con...
BioTechniques, 2003
Sophisticated genome manipulation requires the possibility to modify any intergenic or intragenic... more Sophisticated genome manipulation requires the possibility to modify any intergenic or intragenic DNA sequence at will, without leaving large amounts of undesired vector DNA at the site of alteration. To this end, a series of vectors was developed from a previous gene knockout plasmid system to integrate nonselectable foreign DNA at any desired genomic location in yeast, with a minimum amount of residual plasmid DNA. These vectors have two mutated Flp recognition targets (FRT) sequences flanking the KanMX4 gene and multiple sites for subcloning the DNA fragment to be integrated. The selectable marker can be recycled by Flp site-specific excision between the identical FRTs, thereby allowing the integration of further DNA fragments. With this system, the NLS-tetR-GFP and DsRed genes were successfully integrated at the thr1 locus, and the RVB1 gene was tagged at the C-terminus with the V5-epitope-6-histidine tag. This plasmid system provides for a new molecular tool to integrate any DN...
Applied and environmental microbiology, 1998
Bacillus pumilus PS213 was found to be able to release acetate from acetylated xylan. The enzyme ... more Bacillus pumilus PS213 was found to be able to release acetate from acetylated xylan. The enzyme catalyzing this reaction has been purified to homogeneity and characterized. The enzyme was secreted, and its production was induced by corncob powder and xylan. Its molecular mass, as determined by gel filtration, is 190 kDa, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band of 40 kDa. The isoelectric point was found to be 4.8, and the enzyme activity was optimal at 55 degrees C and pH 8.0. The activity was inhibited by most of the metal ions, while no enhancement was observed. The Michaelis contant (Km) and Vmax for alpha-naphthyl acetate were 1.54 mM and 360 micromol min-1 mg of protein-1, respectively.
Yeast (Chichester, England), Jan 30, 1995
We report the sequence of an 11.1 kb fragment located on the left arm of chromosome VII of Saccha... more We report the sequence of an 11.1 kb fragment located on the left arm of chromosome VII of Saccharomyces cerevisiae. By sequence analysis we have detected six open reading frames (ORFs) longer that 300 bp, which cover 87% of the entire sequence. ORF G1645 is 100% identical to the KEM1 gene, also identified as DST2, XRN1, SEP1 and RAR5, while G1648 is 100% identical to the NSP49 or NUP49 gene. ORF G1642 shares some identity with a hypothetical protein of Caenorhabditis elegans, while the other four ORFs show no significant homology to known proteins.
Plasmid, 2002
A comparison of the selective fitness of four 2-microm-based shuttle-plasmids carrying the yeast ... more A comparison of the selective fitness of four 2-microm-based shuttle-plasmids carrying the yeast genes HIS3, LEU2, TRP1, and URA3 was performed. The effect of each marker on long-term growth rate and plasmid maintenance was measured. In selective medium, the LEU2 and URA3 plasmids were maintained at the lowest and the highest levels, respectively, while the HIS3 and TRP1 plasmids were maintained at an intermediate level. In synthetic complete medium, plasmid loss rate was lower for the genes TRP1 and URA3 than for the other two markers, and a similar pattern was observed for cells growing in rich medium. These results were confirmed by competition experiments among transformants with different plasmids in complete and rich media, indicating a different degree of fitness for the markers used. A potential correlation of the energy cost of plasmid maintenance with the secondary DNA structure and the level of expression of the selective markers is also investigated.
Nature, 1997
Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4... more Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.
Frontiers in Oncology, 2013
Yeast has been established as an efficient model system to study biological principles underpinni... more Yeast has been established as an efficient model system to study biological principles underpinning human health. In this review we focus on yeast models covering two aspects of cancer formation and progression (i) the activity of pyruvate kinase (PK), which recapitulates metabolic features of cancer cells, including the Warburg effect, and (ii) chromosome bridge-induced translocation (BIT) mimiking genome instability in cancer. Saccharomyces cerevisiae is an excellent model to study cancer cell metabolism, as exponentially growing yeast cells exhibit many metabolic similarities with rapidly proliferating cancer cells. The metabolic reconfiguration includes an increase in glucose uptake and fermentation, at the expense of respiration and oxidative phosphorylation (the Warburg effect), and involves a broad reconfiguration of nucleotide and amino acid metabolism. Both in yeast and humans, the regulation of this process seems to have a central player, PK, which is up-regulated in cancer, and to occur mostly on a post-transcriptional and posttranslational basis. Furthermore, BIT allows to generate selectable translocation-derived recombinants ("translocants"), between any two desired chromosomal locations, in wild-type yeast strains transformed with a linear DNA cassette carrying a selectable marker flanked by two DNA sequences homologous to different chromosomes. Using the BIT system, targeted non-reciprocal translocations in mitosis are easily inducible. An extensive collection of different yeast translocants exhibiting genome instability and aberrant phenotypes similar to cancer cells has been produced and subjected to analysis. In this review, we hence provide an overview upon two yeast cancer models, and extrapolate general principles for mimicking human disease mechanisms in yeast.
eLS, 2001
Yeast artificial chromosomes are shuttle-vectors that can be amplified and modified in bacteria a... more Yeast artificial chromosomes are shuttle-vectors that can be amplified and modified in bacteria and employed for the cloning of very large DNA inserts (up to 1-2 megabase pairs) in the yeast Saccharomyces cerevisiae.
A 15 kb DNA fragment from the Bacillus subtilis chromosome between citB and ppsC has been sequenc... more A 15 kb DNA fragment from the Bacillus subtilis chromosome between citB and ppsC has been sequenced, and new ORFs encoding putative enzymes involved in lipopolypeptide synthesis, which complete a partial operon previously reported, and a new set of enzymes responsible for lipid metabolism have been identified. From the analysis of DNA sequence homology of the fragment it was deduced that these new peptide synthetase genes are part of an operon for the biosynthesis of the fungicide fengycin.
Molecular Genetics and Genomics, 2015
Chromosome translocation is a major genomic event for a cell, affecting almost every of its life ... more Chromosome translocation is a major genomic event for a cell, affecting almost every of its life aspects ranging from metabolism, organelle maintenance and homeostasis to gene maintenance and expression. By using the bridge-induced translocation system, we defined the effects of induced chromosome translocation on the chronological life span (CLS) of yeast with particular interest to the oxidative stress condition. The results demonstrate that every translocant strain has a different CLS, but all have a high increase in reactive oxygen species and in lipid peroxides levels at the end of the life span. This could be due to the very unique and strong deregulation of the oxidative stress network. Furthermore, the loss of the translocated chromosome occurs at the end of the life span and is locus dependent. Additionally, the RDH54 gene may play a role in the correct segregation of the translocant chromosome, since in its absence there is an increase in loss of the bridge-induced translocated chromosome.
Experimental systems exist, which are able to generate only specific reciprocal translocations be... more Experimental systems exist, which are able to generate only specific reciprocal translocations between engineered chromosomal loci of yeast and Drosophila but not in wild-type cells. Here we report the successfull induction of chromosome translocations in the yeast Saccharomyces cerevisiae by targeted DNA integration of the KANR selectable marker flanked by two DNA sequences homologous to two different chromosomes. Using this Bridge-Induced Translocation (BIT) system, from 2 to 4% of all integrants, depending on the length of the homologous DNA ends, showed targeted non-reciprocal translocations between chromosomes V-VIII and VIII-XV in two wild-type strains. The complex fate of the chromosomal fragments generated indicated the involvement of multiple repair pathways in a two-step integration dynamics. The possibility of inducing chromosomal translocations between any two desired genetic loci in a eukaryotic model system will be instrumental for their molecular characterization, as ...
Adaptation, which is often used as a synonym for evolution is usually due to a genetic sweep that... more Adaptation, which is often used as a synonym for evolution is usually due to a genetic sweep that gives an organism the selective advantage to survive in a harsh environment. This genetic sweep can come either from different point mutations that occur inside a single genetic element or from a wide and deep genomic rearrangement such as a chromosome translocation. The latter has been widely studied in many of its aspects. In fact it has been demonstrated that it affects global gene expression by de-regulating over a thousand genes [1], it is able to induce multiple aneuploidies [2] and that it may lead to loss of heterozygosity (LOH)[3]. Chromosome translocation can be induced by using a simple but effective method named Bridge Induced Translocation (BIT) [4] that allows to specifically target a translocation anywhere in the yeast genome. Combining the BIT method with the Chronological Life Span assay [5] we wanted to analyse how a yeast cell, after it underwent a BIT translocation, ...
Food Technology and Biotechnology
CDC6 is an essential gene of Saccharomyces cerevisiae involved in the initiation of DNA replicati... more CDC6 is an essential gene of Saccharomyces cerevisiae involved in the initiation of DNA replication. Interacting with ORC complex of proteins, Cdc6 protein has a pivotal role in loading Mcm proteins on origins of replication. Although much evidence about its func-tion in S phase of the cell cycle is available, only a few data indicate a regulatory function of this protein in G2/M transition phase of the cell cycle. By synchronisation with a micro-tubule-destabilising drug nocodazole and Fluorescence Activating Cell Sorting (FACS) analysis it was possible to provide evidence that CDC6 is responsible for a proper se-quence of genetic events and accurate chromosome segregation during mitosis.
Gene
Site-specific recombination systems from bacteriophage and yeasts are becoming precious tools for... more Site-specific recombination systems from bacteriophage and yeasts are becoming precious tools for manipulating DNA both in vitro and in living organisms. In this work we describe the isolation of yeast Saccharomyces cerevisiae segregants which have lost the highly stable 2 μm DNA plasmid, exploiting the site-specific recombination system of 2 μm itself. We efficiently isolated [ciro] segregants from two haploid yeast strains and also a diploid. Moreover, the effect of mutations in the core region of the FRT (Flp Recognition Target) sequence was investigated in vivo, studying the result of the recombination event between several mutated and wild-type FRT sequences. From our result it seems that the identity between the core regions of two FRT sites is necessary but not sufficient, indicating that the core sequence itself has a relevant function in the recombination mechanism in vivo.
Chromosome translocation brings a huge array of changes to the cell, which spreads from genetic t... more Chromosome translocation brings a huge array of changes to the cell, which spreads from genetic to phenotypic modifications [1, 2]. In addition, we recently discovered that this genomic rearrangement has an influence on the life span of yeast and on its telomere's length. Using the BIT (bridge-induced translocation) system created in our lab [3], here we show that any chromosome translocation can either increase or decrease the chronological life span of cells. The data gathered show that the structural change of the two chromosomes involved in translocation can have an opposite effects on the chronological life span, either shortening or elongating it by 30%-50%. Furthermore, we were able to define that BIT not only influences life span but also telomere length, shortening the telomeres of most of our translocants. The connection between chronological aging and the molecular mechanism underlying programmed cell death makes us suppose that chromosome translocation also effects a...
Gene
Site-specific recombination systems from bacteriophage and yeasts are becoming precious tools for... more Site-specific recombination systems from bacteriophage and yeasts are becoming precious tools for manipulating DNA both in vitro and in living organisms. In this work we describe the isolation of yeast Saccharomyces cerevisiae segregants which have lost the highly stable 2 microm DNA plasmid, exploiting the site-specific recombination system of 2 microm itself. We efficiently isolated [cir0] segregants from two haploid yeast strains and also a diploid. Moreover, the effect of mutations in the core region of the FRT (Flp Recognition Target) sequence was investigated in vivo, studying the result of the recombination event between several mutated and wild-type FRT sequences. From our result it seems that the identity between the core regions of two FRT sites is necessary but not sufficient, indicating that the core sequence itself has a relevant function in the recombination mechanism in vivo.
Plasmid
Temperature-sensitive Saccharomyces cerevisiae cdc6 mutants, under restrictive conditions, show a... more Temperature-sensitive Saccharomyces cerevisiae cdc6 mutants, under restrictive conditions, show an increase in recombination frequency, as well as chromosome and circular minichromosome loss. The role of the essential CDC6 gene was tested in trans and in cis to study circular plasmid stability. It was possible to demonstrate that the product of the CDC6 gene, acting in trans, is important for centromeric, episomal, and also 2-microns plasmid maintenance, while the gene sequence itself has no effect in cis on the stability of the plasmids tested. A high percentage of phenotypic revertants for the cdc6 mutation loses 2 microns upon shifting to the restrictive temperature and, under semipermissive conditions, the endogenous plasmid becomes very unstable, favoring a more efficient curing procedure. A positive correlation between centromeric plasmid size and stability was demonstrated even for small circular plasmids.
Nanotoxicology, 2015
The use of cadmium sulphide quantum dots (CdS QDs) is increasing, particularly in the electronics... more The use of cadmium sulphide quantum dots (CdS QDs) is increasing, particularly in the electronics industry. Their size (1-10 nm in diameter) is, however, such that they can be taken up by living cells. Here, a bakers' yeast (Saccharomyces cerevisiae) deletion mutant collection has been exploited to provide a high-throughput means of revealing the genetic basis for tolerance/susceptibility to CdS QD exposure. The deletion of 112 genes, some associated with the abiotic stress response, some with various metabolic processes, some with mitochondrial organization, some with transport and some with DNA repair, reduced the level of tolerance to CdS QDs. A gene ontology analysis highlighted the role of oxidative stress in determining the cellular response. The transformation of sensitive mutants with centromeric plasmids harbouring DNA from a wild type strain restored the wild type growth phenotype when the complemented genes encoded either HSC82, DSK2 or ALD3. The use of these simple eukaryote knock-out mutants for functional toxicogenomic analysis will inform studies focusing on higher organisms.
Current Genetics
We have employed the analysis of spontaneous forward mutations that confer the ability to utilize... more We have employed the analysis of spontaneous forward mutations that confer the ability to utilize L--aminoadipate as a nitrogen source (-Aa+) to discern the events that contribute to mitotic segregation of spontaneous recessive mutations by diploid cells. -Aa- diploid cells yield -Aa+ mutants at a rate of 7.83.610-9. As in haploid strains, approximately 97% (30/31) of -Aa+ mutants are spontaneous lys2-x recessive mutations. -Aa+ mutants of diploid cells reflect mostly the fate of LYS2/lys2-x heterozygotes that arise by mutation within LYS2/LYS2 populations at a rate of 1.20.410-6. Mitotic recombination occurs in nonrandom association with forward mutation of LYS2 at a rate of 1.30.610-3. This mitotic recombination rate is tenfold higher than that of a control LYS2/lys2-1 diploid. Mitotic segregation within LYS2/lys2-x subpopulations yields primarily lys2-x/lys2-x diploids and a minority of lys2-x aneuploids. Fifteen percent of lys2-x/lys2-x diploids appear to have arisen by gene con...
BioTechniques, 2003
Sophisticated genome manipulation requires the possibility to modify any intergenic or intragenic... more Sophisticated genome manipulation requires the possibility to modify any intergenic or intragenic DNA sequence at will, without leaving large amounts of undesired vector DNA at the site of alteration. To this end, a series of vectors was developed from a previous gene knockout plasmid system to integrate nonselectable foreign DNA at any desired genomic location in yeast, with a minimum amount of residual plasmid DNA. These vectors have two mutated Flp recognition targets (FRT) sequences flanking the KanMX4 gene and multiple sites for subcloning the DNA fragment to be integrated. The selectable marker can be recycled by Flp site-specific excision between the identical FRTs, thereby allowing the integration of further DNA fragments. With this system, the NLS-tetR-GFP and DsRed genes were successfully integrated at the thr1 locus, and the RVB1 gene was tagged at the C-terminus with the V5-epitope-6-histidine tag. This plasmid system provides for a new molecular tool to integrate any DN...
Applied and environmental microbiology, 1998
Bacillus pumilus PS213 was found to be able to release acetate from acetylated xylan. The enzyme ... more Bacillus pumilus PS213 was found to be able to release acetate from acetylated xylan. The enzyme catalyzing this reaction has been purified to homogeneity and characterized. The enzyme was secreted, and its production was induced by corncob powder and xylan. Its molecular mass, as determined by gel filtration, is 190 kDa, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band of 40 kDa. The isoelectric point was found to be 4.8, and the enzyme activity was optimal at 55 degrees C and pH 8.0. The activity was inhibited by most of the metal ions, while no enhancement was observed. The Michaelis contant (Km) and Vmax for alpha-naphthyl acetate were 1.54 mM and 360 micromol min-1 mg of protein-1, respectively.
Yeast (Chichester, England), Jan 30, 1995
We report the sequence of an 11.1 kb fragment located on the left arm of chromosome VII of Saccha... more We report the sequence of an 11.1 kb fragment located on the left arm of chromosome VII of Saccharomyces cerevisiae. By sequence analysis we have detected six open reading frames (ORFs) longer that 300 bp, which cover 87% of the entire sequence. ORF G1645 is 100% identical to the KEM1 gene, also identified as DST2, XRN1, SEP1 and RAR5, while G1648 is 100% identical to the NSP49 or NUP49 gene. ORF G1642 shares some identity with a hypothetical protein of Caenorhabditis elegans, while the other four ORFs show no significant homology to known proteins.
Plasmid, 2002
A comparison of the selective fitness of four 2-microm-based shuttle-plasmids carrying the yeast ... more A comparison of the selective fitness of four 2-microm-based shuttle-plasmids carrying the yeast genes HIS3, LEU2, TRP1, and URA3 was performed. The effect of each marker on long-term growth rate and plasmid maintenance was measured. In selective medium, the LEU2 and URA3 plasmids were maintained at the lowest and the highest levels, respectively, while the HIS3 and TRP1 plasmids were maintained at an intermediate level. In synthetic complete medium, plasmid loss rate was lower for the genes TRP1 and URA3 than for the other two markers, and a similar pattern was observed for cells growing in rich medium. These results were confirmed by competition experiments among transformants with different plasmids in complete and rich media, indicating a different degree of fitness for the markers used. A potential correlation of the energy cost of plasmid maintenance with the secondary DNA structure and the level of expression of the selective markers is also investigated.
Nature, 1997
Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4... more Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.
Frontiers in Oncology, 2013
Yeast has been established as an efficient model system to study biological principles underpinni... more Yeast has been established as an efficient model system to study biological principles underpinning human health. In this review we focus on yeast models covering two aspects of cancer formation and progression (i) the activity of pyruvate kinase (PK), which recapitulates metabolic features of cancer cells, including the Warburg effect, and (ii) chromosome bridge-induced translocation (BIT) mimiking genome instability in cancer. Saccharomyces cerevisiae is an excellent model to study cancer cell metabolism, as exponentially growing yeast cells exhibit many metabolic similarities with rapidly proliferating cancer cells. The metabolic reconfiguration includes an increase in glucose uptake and fermentation, at the expense of respiration and oxidative phosphorylation (the Warburg effect), and involves a broad reconfiguration of nucleotide and amino acid metabolism. Both in yeast and humans, the regulation of this process seems to have a central player, PK, which is up-regulated in cancer, and to occur mostly on a post-transcriptional and posttranslational basis. Furthermore, BIT allows to generate selectable translocation-derived recombinants ("translocants"), between any two desired chromosomal locations, in wild-type yeast strains transformed with a linear DNA cassette carrying a selectable marker flanked by two DNA sequences homologous to different chromosomes. Using the BIT system, targeted non-reciprocal translocations in mitosis are easily inducible. An extensive collection of different yeast translocants exhibiting genome instability and aberrant phenotypes similar to cancer cells has been produced and subjected to analysis. In this review, we hence provide an overview upon two yeast cancer models, and extrapolate general principles for mimicking human disease mechanisms in yeast.
eLS, 2001
Yeast artificial chromosomes are shuttle-vectors that can be amplified and modified in bacteria a... more Yeast artificial chromosomes are shuttle-vectors that can be amplified and modified in bacteria and employed for the cloning of very large DNA inserts (up to 1-2 megabase pairs) in the yeast Saccharomyces cerevisiae.
A 15 kb DNA fragment from the Bacillus subtilis chromosome between citB and ppsC has been sequenc... more A 15 kb DNA fragment from the Bacillus subtilis chromosome between citB and ppsC has been sequenced, and new ORFs encoding putative enzymes involved in lipopolypeptide synthesis, which complete a partial operon previously reported, and a new set of enzymes responsible for lipid metabolism have been identified. From the analysis of DNA sequence homology of the fragment it was deduced that these new peptide synthetase genes are part of an operon for the biosynthesis of the fungicide fengycin.