Dominique Swennen - Academia.edu (original) (raw)

Papers by Dominique Swennen

Research paper thumbnail of Structure of the Tilapia ( Oreochromis mossambicus ) Prolactin I Gene

DNA and Cell Biology, 1992

The tilapia (Oreochromis mossambicus) prolactin-I (PRL-I) gene has been cloned and sequenced. Its... more The tilapia (Oreochromis mossambicus) prolactin-I (PRL-I) gene has been cloned and sequenced. Its transcript (3,677 bases long) begins with a guanine and is organized in five exons and four introns like the other known prolactin genes. Analysis of the 1,555-bp 5'-flanking region suggests that pituitary-specific expression of the gene could be regulated through a trans-factor related to the mammalian pituitary-specific factor Pit-1. Two potential binding sites for such a factor were found in the first intron, suggesting a possible regulatory role for this region. Moreover, two potential Z-DNA regions are located at positions -837 to -812 and -246 to -179 from the transcription start site. These two regions could play an important role in the regulation of PRL gene expression.

Research paper thumbnail of Cloning of SEC61 homologues from Schizosaccharomyces pombe and Yarrowia lipolytica reveals the extent of functional conservation within this core component of the ER translocation machinery

Journal of Cell Science

The Sec61 protein is required for protein translocation across the ER membrane in both yeast and ... more The Sec61 protein is required for protein translocation across the ER membrane in both yeast and mammals and is found in close association with polypeptides during their membrane transit. In Saccharomyces cerevisiae Sec61p is essential for viability and the extent of sequence similarity between the yeast and mammalian proteins (55% sequence identity) suggests that the role of Sec61p in the translocation mechanism is likely to be conserved. In order to further our understanding of the structure and function of Sec61p we have cloned homologues from both Schizosaccharomyces pombe and Yarrowia lipolytica. The S. pombe gene comprises six exons encoding a 479 residue protein which we have immunolocalised to the endoplasmic reticulum. Sequence comparisons reveal that S. pombe Sec61p is 58.6% identical to that of S. cerevisiae. The deduced amino acid sequence of the Y. lipolytica protein shares 68.8% sequence identity with S. cerevisiae Sec61p. Gene disruption studies have shown that the SE...

Research paper thumbnail of Differential gene retention as an evolutionary mechanism to generate biodiversity and adaptation in yeasts

Scientific Reports, 2015

The evolutionary history of the characters underlying the adaptation of microorganisms to food an... more The evolutionary history of the characters underlying the adaptation of microorganisms to food and biotechnological uses is poorly understood. We undertook comparative genomics to investigate evolutionary relationships of the dairy yeast Geotrichum candidum within Saccharomycotina. Surprisingly, a remarkable proportion of genes showed discordant phylogenies, clustering with the filamentous fungus subphylum (Pezizomycotina), rather than the yeast subphylum (Saccharomycotina), of the Ascomycota. These genes appear not to be the result of Horizontal Gene Transfer (HGT), but to have been specifically retained by G. candidum after the filamentous fungiyeasts split concomitant with the yeasts' genome contraction. We refer to these genes as SRAGs (Specifically Retained Ancestral Genes), having been lost by all or nearly all other yeasts, and thus contributing to the phenotypic specificity of lineages. SRAG functions include lipases consistent with a role in cheese making and novel endoglucanases associated with degradation of plant material. Similar gene retention was observed in three other distantly related yeasts representative of this ecologically diverse subphylum. The phenomenon thus appears to be widespread in the Saccharomycotina and argues that, alongside neo-functionalization following gene duplication and HGT, specific gene retention must be recognized as an important mechanism for generation of biodiversity and adaptation in yeasts.

Research paper thumbnail of Overview of a surface-ripened cheese community functioning by meta-omics analyses

PloS one, 2015

Cheese ripening is a complex biochemical process driven by microbial communities composed of both... more Cheese ripening is a complex biochemical process driven by microbial communities composed of both eukaryotes and prokaryotes. Surface-ripened cheeses are widely consumed all over the world and are appreciated for their characteristic flavor. Microbial community composition has been studied for a long time on surface-ripened cheeses, but only limited knowledge has been acquired about its in situ metabolic activities. We applied metagenomic, metatranscriptomic and biochemical analyses to an experimental surface-ripened cheese composed of nine microbial species during four weeks of ripening. By combining all of the data, we were able to obtain an overview of the cheese maturation process and to better understand the metabolic activities of the different community members and their possible interactions. Furthermore, differential expression analysis was used to select a set of biomarker genes, providing a valuable tool that can be used to monitor the cheese-making process.

Research paper thumbnail of Growth and adaptation of microorganisms on the cheese surface

FEMS microbiology letters, 2015

Microbial communities living on cheese surfaces are composed of various bacteria, yeasts and mold... more Microbial communities living on cheese surfaces are composed of various bacteria, yeasts and molds that interact together, thus generating the typical sensory properties of a cheese. Physiological and genomic investigations have revealed important functions involved in the ability of microorganisms to establish themselves at the cheese surface. These functions include the ability to use the cheese's main energy sources, to acquire iron, to tolerate low pH at the beginning of ripening and to adapt to high salt concentrations and moisture levels. Horizontal gene transfer events involved in the adaptation to the cheese habitat have been described, both for bacteria and fungi. In the future, in situ microbial gene expression profiling and identification of genes that contribute to strain fitness by massive sequencing of transposon libraries will help us to better understand how cheese surface communities function.

Research paper thumbnail of Investigation of Geotrichum candidum gene expression during the ripening of Reblochon-type cheese by reverse transcription-quantitative PCR

International Journal of Food Microbiology, 2015

Cheese ripening involves the activity of various bacteria, yeasts or molds, which contribute to t... more Cheese ripening involves the activity of various bacteria, yeasts or molds, which contribute to the development of the typical color, flavor and texture of the final product. In situ measurements of gene expression are increasingly being used to improve our understanding of the microbial flora activity in cheeses. The objective of the present study was to investigate the physiology and metabolic activity of Geotrichum candidum during the ripening of Reblochon-type cheeses by quantifying mRNA transcripts at various ripening times. The expression of 80 genes involved in various functions could be quantified with a correct level of biological repeatability using a set of three stable reference genes. As ripening progresses, a decrease in expression was observed for genes involved in cell wall organization, translation, vesicular mediated transport, and in cytoskeleton constituents and ribosomal protein genes. There was also a decrease in the expression of mitochondrial F1F0 ATP synthase and plasma membrane H(+) ATPase genes. Some genes involved in the catabolism of lactate, acetate and ethanol were expressed to a greater extent at the beginning of ripening. During the second part of ripening, there was an increased expression of genes involved in the transport and catabolism of amino acids, which could be attributed to a change in the energy source. There was also an increase in the expression of genes involved in autophagy and of genes possibly involved in lifespan determination. Quantification of mRNA transcripts may also be used to produce bioindicators relevant for cheesemaking, for example when considering genes encoding enzymes involved in the catabolism of amino acids.

Research paper thumbnail of Secretion of active anti-Ras single-chain Fv antibody by the yeasts Yarrowia lipolytica and Kluyveromyces lactis

Microbiology (Reading, England), 2002

Yarrowia lipolytica and Kluyveromyces lactis secretion vectors were constructed and assessed for ... more Yarrowia lipolytica and Kluyveromyces lactis secretion vectors were constructed and assessed for the expression of heterologous proteins. An anti-Ras single-chain antibody fragment (scFv) coding sequence was fused in-frame to different pre- or prepro-regions, or downstream from a reporter secretory gene (Arxula adeninivorans glucoamylase), separated by a Kex2 protease (Kex2p)-like processing sequence. Both organisms are able to secrete soluble scFv, with yields depending on the nature of the expression cassette, up to levels ranging from 10 to 20 mg l(-1). N-terminal sequence analysis of the purified scFv showed that fusions are correctly processed to the mature scFv by a signal peptidase or a Kex2p-type endoprotease present in Y. lipolytica and K. lactis. The scFv protein also retains the capacity to bind to a glutathioneS-transferase (GST)-Harvey-Ras(Val12) fusion, indicating that the antibody is functional. These results indicate that the yeasts Y. lipolytica and K. lactis have p...

Research paper thumbnail of pIN-III- ompA secretion vectors: modification of the ompA signal peptide sequence for easier insert cloning

Nucleic Acids Research, 1988

Research paper thumbnail of Genome evolution in yeasts

Research paper thumbnail of Production and purification of biologically active recombinant tilapia (Oreochromis niloticus) prolactins

Journal of Endocrinology, 1991

Recombinant expression vectors carrying tilapia prolactin-I or -II (tiPRL-I or tiPRL-II) cDNA wer... more Recombinant expression vectors carrying tilapia prolactin-I or -II (tiPRL-I or tiPRL-II) cDNA were constructed and the tiPRL-I and II proteins were produced in E. coli as inclusion bodies. These inclusion bodies were dissolved in 6 mol urea/l. Refolding of the proteins was followed by SDS-PAGE under non-reducing conditions so as to visualize the oxidized state of the molecules. Proteins tiPRL-I and tiPRL-II were purified by gel filtration and ion-exchange chromatography. The N-terminal sequence and bioactivities of both purified proteins were then analysed. Recombinant tiPRL-I and tiPRL-II induced a significant rise in plasma calcium levels as well as in mucocyte density in the abdominal skin epithelium. When tested on kidney membrane, both proteins exhibited potency in competing with 125I-labelled tiPRL-I for binding sites, but tiPRL-I seemed to be more potent than tiPRL-II in competing for these sites. The results obtained for the biological activities tested suggest that both recombinant prolactins were correctly refolded and had retained the full biological activity previously observed with the natural hormone preparations extracted from the animals.

Research paper thumbnail of Structure of the Tilapia ( Oreochromis mossambicus ) Prolactin I Gene

DNA and Cell Biology, 1992

The tilapia (Oreochromis mossambicus) prolactin-I (PRL-I) gene has been cloned and sequenced. Its... more The tilapia (Oreochromis mossambicus) prolactin-I (PRL-I) gene has been cloned and sequenced. Its transcript (3,677 bases long) begins with a guanine and is organized in five exons and four introns like the other known prolactin genes. Analysis of the 1,555-bp 5'-flanking region suggests that pituitary-specific expression of the gene could be regulated through a trans-factor related to the mammalian pituitary-specific factor Pit-1. Two potential binding sites for such a factor were found in the first intron, suggesting a possible regulatory role for this region. Moreover, two potential Z-DNA regions are located at positions -837 to -812 and -246 to -179 from the transcription start site. These two regions could play an important role in the regulation of PRL gene expression.

Research paper thumbnail of Tilapia Growth Hormone: Molecular Cloning of cDNA and Expression in Escherichia coli

DNA, 1989

A cDNA library was prepared from poly(A)+RNA extracted from tilapia Oreochromis niloticus anterio... more A cDNA library was prepared from poly(A)+RNA extracted from tilapia Oreochromis niloticus anterior pituitaries. The recombinant clones carrying the cDNA sequence of tilapia growth hormone (tiGH) were selected using a fragment of the trout growth hormone (tGH) cDNA as hybridization probe. The nucleotide sequence of the full-length tiGH cDNA was determined. This cDNA encodes a protein of 204 amino acids, including the putative signal peptide of 17 amino acids. Mature tiGH cDNA was inserted in an Escherichia coli expression vector which led to the production of tiGH protein with a yield estimated to be 20% of the total bacterial proteins.

Research paper thumbnail of Rainbow Trout Prolactin cDNA Cloning in Escherichia coli

DNA, 1989

We describe the isolation and characterization of a cDNA for trout prolactin (tPrl). An extensive... more We describe the isolation and characterization of a cDNA for trout prolactin (tPrl). An extensive analysis of tPrl recombinant clones by restriction analysis and sequencing revealed the presence of only one form of tPrl mRNA. The deduced protein sequence consists of 210 amino acids, including a signal peptide of 23 amino acids. The amino acid sequence of the mature protein is compared among teleosts and mammals, showing two domains of strong similarity that may be involved in biological activity.

Research paper thumbnail of Yarrowia lipolytica vesicle-mediated protein transport pathways

BMC Evolutionary Biology, 2007

Background: Protein secretion is a universal cellular process involving vesicles which bud and fu... more Background: Protein secretion is a universal cellular process involving vesicles which bud and fuse between organelles to bring proteins to their final destination. Vesicle budding is mediated by protein coats; vesicle targeting and fusion depend on Rab GTPase, tethering factors and SNARE complexes. The Génolevures II sequencing project made available entire genome sequences of four hemiascomycetous yeasts, Yarrowia lipolytica, Debaryomyces hansenii, Kluyveromyces lactis and Candida glabrata. Y. lipolytica is a dimorphic yeast and has good capacities to secrete proteins. The translocation of nascent protein through the endoplasmic reticulum membrane was well studied in Y. lipolytica and is largely co-translational as in the mammalian protein secretion pathway.

Research paper thumbnail of Folding Proteome of Yarrowia lipolytica Targeting with Uracil Permease Mutants

Journal of Proteome Research, 2010

The acquisition of the correct folding of membrane proteins is a crucial process that involves se... more The acquisition of the correct folding of membrane proteins is a crucial process that involves several steps from the recognition of nascent protein, its targeting to the endoplasmic reticulum membrane, its insertion, and its sorting to its final destination. Yarrowia lipolytica is a hemiascomycetous dimorphic yeast and an alternative eukaryotic yeast model with an efficient secretion pathway. To better understand the quality control of membrane proteins, we constructed a model system based on the uracil permease. Mutated forms of the permease were stabilized and retained in the cell and made the strains resistant to the 5-fluorouracil drug. To identify proteins involved in the quality control, we separated proteins extracted in nondenaturing conditions on blue native gels to keep proteins associated in complexes. Some gel fragments where the model protein was immunodetected were subjected to mass spectrometry analysis. The proteins identified gave a picture of the folding proteome, from the translocation across the endoplasmic reticulum membrane, the folding of the proteins, to the vesicle transport to Golgi or the degradation via the proteasome. For example, EMC complex, Gsf2p or Yet3p, chaperone membrane proteins of the endoplasmic reticulum were identified in the Y. lipolytica native proteome.

Research paper thumbnail of Structure of the Tilapia ( Oreochromis mossambicus ) Prolactin I Gene

DNA and Cell Biology, 1992

The tilapia (Oreochromis mossambicus) prolactin-I (PRL-I) gene has been cloned and sequenced. Its... more The tilapia (Oreochromis mossambicus) prolactin-I (PRL-I) gene has been cloned and sequenced. Its transcript (3,677 bases long) begins with a guanine and is organized in five exons and four introns like the other known prolactin genes. Analysis of the 1,555-bp 5'-flanking region suggests that pituitary-specific expression of the gene could be regulated through a trans-factor related to the mammalian pituitary-specific factor Pit-1. Two potential binding sites for such a factor were found in the first intron, suggesting a possible regulatory role for this region. Moreover, two potential Z-DNA regions are located at positions -837 to -812 and -246 to -179 from the transcription start site. These two regions could play an important role in the regulation of PRL gene expression.

Research paper thumbnail of Cloning of SEC61 homologues from Schizosaccharomyces pombe and Yarrowia lipolytica reveals the extent of functional conservation within this core component of the ER translocation machinery

Journal of Cell Science

The Sec61 protein is required for protein translocation across the ER membrane in both yeast and ... more The Sec61 protein is required for protein translocation across the ER membrane in both yeast and mammals and is found in close association with polypeptides during their membrane transit. In Saccharomyces cerevisiae Sec61p is essential for viability and the extent of sequence similarity between the yeast and mammalian proteins (55% sequence identity) suggests that the role of Sec61p in the translocation mechanism is likely to be conserved. In order to further our understanding of the structure and function of Sec61p we have cloned homologues from both Schizosaccharomyces pombe and Yarrowia lipolytica. The S. pombe gene comprises six exons encoding a 479 residue protein which we have immunolocalised to the endoplasmic reticulum. Sequence comparisons reveal that S. pombe Sec61p is 58.6% identical to that of S. cerevisiae. The deduced amino acid sequence of the Y. lipolytica protein shares 68.8% sequence identity with S. cerevisiae Sec61p. Gene disruption studies have shown that the SE...

Research paper thumbnail of Differential gene retention as an evolutionary mechanism to generate biodiversity and adaptation in yeasts

Scientific Reports, 2015

The evolutionary history of the characters underlying the adaptation of microorganisms to food an... more The evolutionary history of the characters underlying the adaptation of microorganisms to food and biotechnological uses is poorly understood. We undertook comparative genomics to investigate evolutionary relationships of the dairy yeast Geotrichum candidum within Saccharomycotina. Surprisingly, a remarkable proportion of genes showed discordant phylogenies, clustering with the filamentous fungus subphylum (Pezizomycotina), rather than the yeast subphylum (Saccharomycotina), of the Ascomycota. These genes appear not to be the result of Horizontal Gene Transfer (HGT), but to have been specifically retained by G. candidum after the filamentous fungiyeasts split concomitant with the yeasts' genome contraction. We refer to these genes as SRAGs (Specifically Retained Ancestral Genes), having been lost by all or nearly all other yeasts, and thus contributing to the phenotypic specificity of lineages. SRAG functions include lipases consistent with a role in cheese making and novel endoglucanases associated with degradation of plant material. Similar gene retention was observed in three other distantly related yeasts representative of this ecologically diverse subphylum. The phenomenon thus appears to be widespread in the Saccharomycotina and argues that, alongside neo-functionalization following gene duplication and HGT, specific gene retention must be recognized as an important mechanism for generation of biodiversity and adaptation in yeasts.

Research paper thumbnail of Overview of a surface-ripened cheese community functioning by meta-omics analyses

PloS one, 2015

Cheese ripening is a complex biochemical process driven by microbial communities composed of both... more Cheese ripening is a complex biochemical process driven by microbial communities composed of both eukaryotes and prokaryotes. Surface-ripened cheeses are widely consumed all over the world and are appreciated for their characteristic flavor. Microbial community composition has been studied for a long time on surface-ripened cheeses, but only limited knowledge has been acquired about its in situ metabolic activities. We applied metagenomic, metatranscriptomic and biochemical analyses to an experimental surface-ripened cheese composed of nine microbial species during four weeks of ripening. By combining all of the data, we were able to obtain an overview of the cheese maturation process and to better understand the metabolic activities of the different community members and their possible interactions. Furthermore, differential expression analysis was used to select a set of biomarker genes, providing a valuable tool that can be used to monitor the cheese-making process.

Research paper thumbnail of Growth and adaptation of microorganisms on the cheese surface

FEMS microbiology letters, 2015

Microbial communities living on cheese surfaces are composed of various bacteria, yeasts and mold... more Microbial communities living on cheese surfaces are composed of various bacteria, yeasts and molds that interact together, thus generating the typical sensory properties of a cheese. Physiological and genomic investigations have revealed important functions involved in the ability of microorganisms to establish themselves at the cheese surface. These functions include the ability to use the cheese's main energy sources, to acquire iron, to tolerate low pH at the beginning of ripening and to adapt to high salt concentrations and moisture levels. Horizontal gene transfer events involved in the adaptation to the cheese habitat have been described, both for bacteria and fungi. In the future, in situ microbial gene expression profiling and identification of genes that contribute to strain fitness by massive sequencing of transposon libraries will help us to better understand how cheese surface communities function.

Research paper thumbnail of Investigation of Geotrichum candidum gene expression during the ripening of Reblochon-type cheese by reverse transcription-quantitative PCR

International Journal of Food Microbiology, 2015

Cheese ripening involves the activity of various bacteria, yeasts or molds, which contribute to t... more Cheese ripening involves the activity of various bacteria, yeasts or molds, which contribute to the development of the typical color, flavor and texture of the final product. In situ measurements of gene expression are increasingly being used to improve our understanding of the microbial flora activity in cheeses. The objective of the present study was to investigate the physiology and metabolic activity of Geotrichum candidum during the ripening of Reblochon-type cheeses by quantifying mRNA transcripts at various ripening times. The expression of 80 genes involved in various functions could be quantified with a correct level of biological repeatability using a set of three stable reference genes. As ripening progresses, a decrease in expression was observed for genes involved in cell wall organization, translation, vesicular mediated transport, and in cytoskeleton constituents and ribosomal protein genes. There was also a decrease in the expression of mitochondrial F1F0 ATP synthase and plasma membrane H(+) ATPase genes. Some genes involved in the catabolism of lactate, acetate and ethanol were expressed to a greater extent at the beginning of ripening. During the second part of ripening, there was an increased expression of genes involved in the transport and catabolism of amino acids, which could be attributed to a change in the energy source. There was also an increase in the expression of genes involved in autophagy and of genes possibly involved in lifespan determination. Quantification of mRNA transcripts may also be used to produce bioindicators relevant for cheesemaking, for example when considering genes encoding enzymes involved in the catabolism of amino acids.

Research paper thumbnail of Secretion of active anti-Ras single-chain Fv antibody by the yeasts Yarrowia lipolytica and Kluyveromyces lactis

Microbiology (Reading, England), 2002

Yarrowia lipolytica and Kluyveromyces lactis secretion vectors were constructed and assessed for ... more Yarrowia lipolytica and Kluyveromyces lactis secretion vectors were constructed and assessed for the expression of heterologous proteins. An anti-Ras single-chain antibody fragment (scFv) coding sequence was fused in-frame to different pre- or prepro-regions, or downstream from a reporter secretory gene (Arxula adeninivorans glucoamylase), separated by a Kex2 protease (Kex2p)-like processing sequence. Both organisms are able to secrete soluble scFv, with yields depending on the nature of the expression cassette, up to levels ranging from 10 to 20 mg l(-1). N-terminal sequence analysis of the purified scFv showed that fusions are correctly processed to the mature scFv by a signal peptidase or a Kex2p-type endoprotease present in Y. lipolytica and K. lactis. The scFv protein also retains the capacity to bind to a glutathioneS-transferase (GST)-Harvey-Ras(Val12) fusion, indicating that the antibody is functional. These results indicate that the yeasts Y. lipolytica and K. lactis have p...

Research paper thumbnail of pIN-III- ompA secretion vectors: modification of the ompA signal peptide sequence for easier insert cloning

Nucleic Acids Research, 1988

Research paper thumbnail of Genome evolution in yeasts

Research paper thumbnail of Production and purification of biologically active recombinant tilapia (Oreochromis niloticus) prolactins

Journal of Endocrinology, 1991

Recombinant expression vectors carrying tilapia prolactin-I or -II (tiPRL-I or tiPRL-II) cDNA wer... more Recombinant expression vectors carrying tilapia prolactin-I or -II (tiPRL-I or tiPRL-II) cDNA were constructed and the tiPRL-I and II proteins were produced in E. coli as inclusion bodies. These inclusion bodies were dissolved in 6 mol urea/l. Refolding of the proteins was followed by SDS-PAGE under non-reducing conditions so as to visualize the oxidized state of the molecules. Proteins tiPRL-I and tiPRL-II were purified by gel filtration and ion-exchange chromatography. The N-terminal sequence and bioactivities of both purified proteins were then analysed. Recombinant tiPRL-I and tiPRL-II induced a significant rise in plasma calcium levels as well as in mucocyte density in the abdominal skin epithelium. When tested on kidney membrane, both proteins exhibited potency in competing with 125I-labelled tiPRL-I for binding sites, but tiPRL-I seemed to be more potent than tiPRL-II in competing for these sites. The results obtained for the biological activities tested suggest that both recombinant prolactins were correctly refolded and had retained the full biological activity previously observed with the natural hormone preparations extracted from the animals.

Research paper thumbnail of Structure of the Tilapia ( Oreochromis mossambicus ) Prolactin I Gene

DNA and Cell Biology, 1992

The tilapia (Oreochromis mossambicus) prolactin-I (PRL-I) gene has been cloned and sequenced. Its... more The tilapia (Oreochromis mossambicus) prolactin-I (PRL-I) gene has been cloned and sequenced. Its transcript (3,677 bases long) begins with a guanine and is organized in five exons and four introns like the other known prolactin genes. Analysis of the 1,555-bp 5'-flanking region suggests that pituitary-specific expression of the gene could be regulated through a trans-factor related to the mammalian pituitary-specific factor Pit-1. Two potential binding sites for such a factor were found in the first intron, suggesting a possible regulatory role for this region. Moreover, two potential Z-DNA regions are located at positions -837 to -812 and -246 to -179 from the transcription start site. These two regions could play an important role in the regulation of PRL gene expression.

Research paper thumbnail of Tilapia Growth Hormone: Molecular Cloning of cDNA and Expression in Escherichia coli

DNA, 1989

A cDNA library was prepared from poly(A)+RNA extracted from tilapia Oreochromis niloticus anterio... more A cDNA library was prepared from poly(A)+RNA extracted from tilapia Oreochromis niloticus anterior pituitaries. The recombinant clones carrying the cDNA sequence of tilapia growth hormone (tiGH) were selected using a fragment of the trout growth hormone (tGH) cDNA as hybridization probe. The nucleotide sequence of the full-length tiGH cDNA was determined. This cDNA encodes a protein of 204 amino acids, including the putative signal peptide of 17 amino acids. Mature tiGH cDNA was inserted in an Escherichia coli expression vector which led to the production of tiGH protein with a yield estimated to be 20% of the total bacterial proteins.

Research paper thumbnail of Rainbow Trout Prolactin cDNA Cloning in Escherichia coli

DNA, 1989

We describe the isolation and characterization of a cDNA for trout prolactin (tPrl). An extensive... more We describe the isolation and characterization of a cDNA for trout prolactin (tPrl). An extensive analysis of tPrl recombinant clones by restriction analysis and sequencing revealed the presence of only one form of tPrl mRNA. The deduced protein sequence consists of 210 amino acids, including a signal peptide of 23 amino acids. The amino acid sequence of the mature protein is compared among teleosts and mammals, showing two domains of strong similarity that may be involved in biological activity.

Research paper thumbnail of Yarrowia lipolytica vesicle-mediated protein transport pathways

BMC Evolutionary Biology, 2007

Background: Protein secretion is a universal cellular process involving vesicles which bud and fu... more Background: Protein secretion is a universal cellular process involving vesicles which bud and fuse between organelles to bring proteins to their final destination. Vesicle budding is mediated by protein coats; vesicle targeting and fusion depend on Rab GTPase, tethering factors and SNARE complexes. The Génolevures II sequencing project made available entire genome sequences of four hemiascomycetous yeasts, Yarrowia lipolytica, Debaryomyces hansenii, Kluyveromyces lactis and Candida glabrata. Y. lipolytica is a dimorphic yeast and has good capacities to secrete proteins. The translocation of nascent protein through the endoplasmic reticulum membrane was well studied in Y. lipolytica and is largely co-translational as in the mammalian protein secretion pathway.

Research paper thumbnail of Folding Proteome of Yarrowia lipolytica Targeting with Uracil Permease Mutants

Journal of Proteome Research, 2010

The acquisition of the correct folding of membrane proteins is a crucial process that involves se... more The acquisition of the correct folding of membrane proteins is a crucial process that involves several steps from the recognition of nascent protein, its targeting to the endoplasmic reticulum membrane, its insertion, and its sorting to its final destination. Yarrowia lipolytica is a hemiascomycetous dimorphic yeast and an alternative eukaryotic yeast model with an efficient secretion pathway. To better understand the quality control of membrane proteins, we constructed a model system based on the uracil permease. Mutated forms of the permease were stabilized and retained in the cell and made the strains resistant to the 5-fluorouracil drug. To identify proteins involved in the quality control, we separated proteins extracted in nondenaturing conditions on blue native gels to keep proteins associated in complexes. Some gel fragments where the model protein was immunodetected were subjected to mass spectrometry analysis. The proteins identified gave a picture of the folding proteome, from the translocation across the endoplasmic reticulum membrane, the folding of the proteins, to the vesicle transport to Golgi or the degradation via the proteasome. For example, EMC complex, Gsf2p or Yet3p, chaperone membrane proteins of the endoplasmic reticulum were identified in the Y. lipolytica native proteome.