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Papers by William Marcotte
Insect molecular biology (Print), 2008
We demonstrate that a chimeric gene containing the 8-glucuronidase (GUS) reporter gene linked to ... more We demonstrate that a chimeric gene containing the 8-glucuronidase (GUS) reporter gene linked to a 646-base pair 5 ’ fragment (-554 to +92) from the abscisic acid (ABA)-regulated Em gene from wheat is correctly expressed in transgenic tobacco. We observe high activity only in embryos of mature seeds, and immature seeds cultured on ABA show enhanced expression. Using a rice transient assay, we identify a 260-base pair fragment (-168 to +92) that accounts for the ABA-specific 15-fold to 20-fold increase in GUS expression. A 50-base pair sequence (-152 to-103) fused 5 ‘ in either orientation to a truncated cauliflower mosaic virus promoter (35s) increases GUS activity threefold in the presence of ABA. lnsertion of the Em 5‘-untranslated region (+6 to +86) between the 35s promoter and the ATG of GUS results in a 10-fold increase in GUS activity in the absence of ABA. These results suggest the following two functional fragments of the Em 5 ‘ region: an ABA response element from-152 to-10...
Our goals for this project were to: 1. Identify the fundamental molecular biology required for th... more Our goals for this project were to: 1. Identify the fundamental molecular biology required for the clonal production of fiber forming protein polymers through genetic expression in yeast and plants. 2. Study arachnid biology for silk production and lay the foundation for the application of that biology to protein fiber wet-spinning. 3. Establish a “spider farm ” for controlled production of silk to use in analysis, and spiders for biology experiments. Our research program is de facto divided into two domains. In the molecular biology work, we have utilized published sequences of spidroin 1 and 2 genes to construct oligonucleotides which have been linked together to build repetitive gene units for protein expression studies. The structure of the protein we are using as a model is that of spider silk, in particular, the silk used for dragline. The other major effort is the design of a spinning system that is modeled after the clearly successful activities of spiders. In order better t...
On decrit la preparation et l'utilisation de fragments promoteurs de l'acide nucleique ho... more On decrit la preparation et l'utilisation de fragments promoteurs de l'acide nucleique homologues du gene Em du ble et qui servent a soumettre l'expression de genes selectionnes dans des plantes a une regulation exterieure. Le fragment promoteur Em est sensible a l'acide abscisique (ABA) et a d'autres composes qui presentent une activite similaire a celle de l'ABA. En transformant des protoplastes et des cellules vegetales avec des constructions d'ADN recombinant qui comprennent ces fragments promoteurs, on obtient l'expression de genes selectionnes fonctionnellement lies, en reponse a l'ABA et a des composes presentant une activite similaire a celle de l'ABA. On decrit egalement l'application de ces fragments promoteurs et de ces constructions dans des systemes d'essai transitoires afin de prevoir les probabilites d'une transformation stable des plantes.
Frontiers in Plant Science
[This corrects the article DOI: 10.3389/fpls.2020.00122.].
Frontiers in Plant Science
Scaffold proteins form critical biomatrices that support cell adhesion and proliferation for rege... more Scaffold proteins form critical biomatrices that support cell adhesion and proliferation for regenerative medicine and drug screening. The increasing demand for such applications urges solutions for cost effective and sustainable supplies of hypoallergenic and biocompatible scaffold proteins. Here, we summarize recent efforts in obtaining plant-derived biosynthetic spider silk analogue and the extracellular matrix protein, collagen. Both proteins are composed of a large number of tandem block repeats, which makes production in bacterial hosts challenging. Furthermore, post-translational modification of collagen is essential for its function which requires co-transformation of multiple copies of human prolyl 4-hydroxylase. We discuss our perspectives on how the GAANTRY system could potentially assist the production of native-sized spider dragline silk proteins and prolyl hydroxylated collagen. The potential of recombinant scaffold proteins in drug delivery and drug discovery is also ...
Biomacromolecules
Spider dragline silk is a proteinaceous material that combines superior toughness and biocompatib... more Spider dragline silk is a proteinaceous material that combines superior toughness and biocompatibility, which makes it a promising biomaterial. The distinct protein structure and the fiber formation process contribute to the superior toughness of dragline silk. Previously, we have produced recombinant spider silk-like proteins in transgenic tobacco that are readily purified from plant extracts. The plant-derived spidroin-like proteins consisted of native major ampullate spidroin 1 or spidroin 2 N- and C-termini flanking 8, 16, or 32 copies of their respective consensus block repeats (mini-spidroins). Here, we present the generation of fibers from mini-spidroins (rMaSp1R8 and rMaSp2R8) by polyelectrolyte complex formation using an anionic polyelectrolyte, gellan gum. Mini-spidroins, when treated with acetic acid and cross-linked by glutaraldehyde, formed a thin film at the interface when overlaid with a gellan gum solution. Immediate pulling of the film resulted in autofluorescent fibrous materials from either mini-spidroin alone or a combination of rMaSp1R8 and rMaSp2R8 (70:30). Addition of chitosan to the mini-spidroin solutions permitted continuous fiber production until the spinning dope supply was exhausted. When air-dried as-spun fibers were rehydrated and stretched in water, the fiber diameter decreased and the overall toughness improved. This study showed that spider silk-like fibers can be produced in large quantities through charge attraction that assembles chitosan, mini-spidroins, and gellan gum into fibrous complexes. We speculate that the spider silk self-assembly process in the duct may involve attraction of variously charged chitinous polymers, spidroins, and glycoproteins.
Materials Science and Engineering: C
A proposed source of stem cells for nerve regeneration are dental pulp stem cells (DPSCs), based ... more A proposed source of stem cells for nerve regeneration are dental pulp stem cells (DPSCs), based on their close embryonic origin to neurons and the ease with which DPSCs can be obtained from a donor. This study evaluated the response of human DPSCs to spider dragline silk fibers, a potential substrate material for tissue regeneration. The DPSCs' morphology and spread pattern were characterized after these cells were plated onto Nephila clavipes dragline fibers in media. In addition, the responses of two other well established cell lines, osteoblasts (7F2s), and fibroblasts (3T3s), were also studied under identical conditions. The inclusion of 3T3s and 7F2s in this study allowed for both direct comparisons to prior published work and a qualitative comparison to the morphology of the DPSCs. After twelve days, the DPSCs exhibited greater relative alignment and adherence to the spider dragline fibers than the 3T3s and 7F2s. The impact of a common sterilization method (ultraviolet light) on the spider dragline fiber surface and subsequent cell response to this modified surface was also characterized. Exposure of the silk to ultraviolet light did not have a measureable effect on cell alignment, but it did eliminate bacterial growth and changed fiber surface roughness. Spiders' exposure to stressful environments did not have an effect on silk to impair cell alignment or adhesion. Synthetic recombinant protein silk did not act as a substrate for cell adhesion or alignment but hydrogels with similar composition supported cell attachment, growth and proliferation. In all cases, natural drawn spider silk acted as an effective substrate for cellular adhesion and alignment of DPSCs and could be used in neural differentiation applications.
Journal of Biological Chemistry, 2016
Aatcc Review the Magazine of the Textile Dyeing Printing and Finishing Industry, 2011
Transgenic Research, 2016
The high tensile strength and biocompatibility of spider dragline silk makes it a desirable mater... more The high tensile strength and biocompatibility of spider dragline silk makes it a desirable material in many engineering and tissue regeneration applications. Here, we present the feasibility to produce recombinant proteins in transgenic tobacco Nicotiana tabacum with sequences representing spider silk protein building blocks . Recombinant mini-spidroins contain native N- and C-terminal domains of major ampullate spidroin 1 (rMaSp1) or rMaSp2 flanking an abbreviated number (8, 16 or 32) of consensus repeat domains. Two different expression plasmid vectors were tested and a downstream chitin binding domain and self-cleavable intein were included to facilitate protein purification. We confirmed gene insertion and RNA transcription by PCR and reverse-transcriptase PCR, respectively. Mini-spidroin production was detected by N-terminus specific antibodies. Purification of mini-spidroins was performed through chitin affinity chromatography and subsequent intein activation with reducing reagent. Mini-spidroins, when dialyzed and freeze-dried, formed viscous gelatin-like fluids.
AATCC review, Mar 1, 2011
Spider dragline silk is a proteinaceous fiber with impressive physical characteristics making it ... more Spider dragline silk is a proteinaceous fiber with impressive physical characteristics making it attractive for use in advanced materials. The fiber is composed of two proteins (spidroins MaSp1 and MaSp2), each of which contains a large central repeat array flanked by non-repetitive N- and C-terminal domains. The repeat arrays appear to be largely responsible for the tensile properties of the fiber, suggesting that the N- and C-terminal domains may be involved in self-assembly. We recently isolated the MaSp1 and MaSp2 N-terminal domains from Nephila clavipes and have incorporated these into mini-silk genes for expression in transgenic systems. Current efforts involve the development of expression vectors that will allow purification using a removable affinity tag for scalable protein purification.
Plant molecular biology, 1999
The biased amino acid composition and aperiodic (random coil) configuration of Group 1 late embry... more The biased amino acid composition and aperiodic (random coil) configuration of Group 1 late embryogenesis-abundant (LEA) proteins imply that these proteins are capable of binding large amounts of water. While Group 1 LEAs have been predicted to contribute to osmotic stress protection in both embryonic and vegetative tissues, biochemical support has been lacking. We have used Saccharomyces cerevisiae as a model system to test the putative osmoprotective function of a wheat Group 1 LEA protein, Em. We demonstrate that expression of Em protein in yeast cells is not deleterious to growth in media of normal osmolarity and attenuates the growth inhibition normally observed in media of high osmolarity. Enhanced growth is observed in the presence of a variety of osmotically active compounds indicating that Em protein is capable of mitigating the detrimental effect of low water potential in a relatively non-specific manner. These results are the first biochemical demonstration of an osmoprot...
Biochemical Society transactions, 1992
The Plant Journal, 1995
CDeT6-19 is an ABA-regulated gene which has been isolated from Craterostigma plantagineum. The CD... more CDeT6-19 is an ABA-regulated gene which has been isolated from Craterostigma plantagineum. The CDeT6-19 gene promoter has been fused to the beta-glucuronidase reporter gene (GUS) and used to stably transform Arabidopsis thaliana and Nicotiana tabacum. This construct has been shown to be expressed in stomatal guard cells and often in the adjacent epidermal cells of both species in response to both exogenous ABA and drought stress. These results indicate that the stomatal guard cell is competent to relay an ABA signal to the nucleus. In contrast GUS expression directed by the promoter from a predominantly seed-specific, ABA-regulated gene, Em, or the promoter from the ABA-regulated CDeT27-45 gene is not detectable in the epidermal or guard cells of tobacco or Arabidopsis in response to ABA. The fact that not all ABA-regulated gene promoters are active in stomatal guard cells suggests that effective transduction of the signal is dependent upon particular regions within the gene promoter or that guard cells lack all or part of the specific transduction apparatus required to couple the ABA signal to these promoters. This suggests that there are multiple ABA stimulus response coupling pathways. The identification of a regulatory sequence from an ABA-induced gene which is expressed in stomatal guard cells creates the possibility of examining the role of Ca2+ and other second messengers in ABA-induced gene expression.
Insect molecular biology (Print), 2008
We demonstrate that a chimeric gene containing the 8-glucuronidase (GUS) reporter gene linked to ... more We demonstrate that a chimeric gene containing the 8-glucuronidase (GUS) reporter gene linked to a 646-base pair 5 ’ fragment (-554 to +92) from the abscisic acid (ABA)-regulated Em gene from wheat is correctly expressed in transgenic tobacco. We observe high activity only in embryos of mature seeds, and immature seeds cultured on ABA show enhanced expression. Using a rice transient assay, we identify a 260-base pair fragment (-168 to +92) that accounts for the ABA-specific 15-fold to 20-fold increase in GUS expression. A 50-base pair sequence (-152 to-103) fused 5 ‘ in either orientation to a truncated cauliflower mosaic virus promoter (35s) increases GUS activity threefold in the presence of ABA. lnsertion of the Em 5‘-untranslated region (+6 to +86) between the 35s promoter and the ATG of GUS results in a 10-fold increase in GUS activity in the absence of ABA. These results suggest the following two functional fragments of the Em 5 ‘ region: an ABA response element from-152 to-10...
Our goals for this project were to: 1. Identify the fundamental molecular biology required for th... more Our goals for this project were to: 1. Identify the fundamental molecular biology required for the clonal production of fiber forming protein polymers through genetic expression in yeast and plants. 2. Study arachnid biology for silk production and lay the foundation for the application of that biology to protein fiber wet-spinning. 3. Establish a “spider farm ” for controlled production of silk to use in analysis, and spiders for biology experiments. Our research program is de facto divided into two domains. In the molecular biology work, we have utilized published sequences of spidroin 1 and 2 genes to construct oligonucleotides which have been linked together to build repetitive gene units for protein expression studies. The structure of the protein we are using as a model is that of spider silk, in particular, the silk used for dragline. The other major effort is the design of a spinning system that is modeled after the clearly successful activities of spiders. In order better t...
On decrit la preparation et l'utilisation de fragments promoteurs de l'acide nucleique ho... more On decrit la preparation et l'utilisation de fragments promoteurs de l'acide nucleique homologues du gene Em du ble et qui servent a soumettre l'expression de genes selectionnes dans des plantes a une regulation exterieure. Le fragment promoteur Em est sensible a l'acide abscisique (ABA) et a d'autres composes qui presentent une activite similaire a celle de l'ABA. En transformant des protoplastes et des cellules vegetales avec des constructions d'ADN recombinant qui comprennent ces fragments promoteurs, on obtient l'expression de genes selectionnes fonctionnellement lies, en reponse a l'ABA et a des composes presentant une activite similaire a celle de l'ABA. On decrit egalement l'application de ces fragments promoteurs et de ces constructions dans des systemes d'essai transitoires afin de prevoir les probabilites d'une transformation stable des plantes.
Frontiers in Plant Science
[This corrects the article DOI: 10.3389/fpls.2020.00122.].
Frontiers in Plant Science
Scaffold proteins form critical biomatrices that support cell adhesion and proliferation for rege... more Scaffold proteins form critical biomatrices that support cell adhesion and proliferation for regenerative medicine and drug screening. The increasing demand for such applications urges solutions for cost effective and sustainable supplies of hypoallergenic and biocompatible scaffold proteins. Here, we summarize recent efforts in obtaining plant-derived biosynthetic spider silk analogue and the extracellular matrix protein, collagen. Both proteins are composed of a large number of tandem block repeats, which makes production in bacterial hosts challenging. Furthermore, post-translational modification of collagen is essential for its function which requires co-transformation of multiple copies of human prolyl 4-hydroxylase. We discuss our perspectives on how the GAANTRY system could potentially assist the production of native-sized spider dragline silk proteins and prolyl hydroxylated collagen. The potential of recombinant scaffold proteins in drug delivery and drug discovery is also ...
Biomacromolecules
Spider dragline silk is a proteinaceous material that combines superior toughness and biocompatib... more Spider dragline silk is a proteinaceous material that combines superior toughness and biocompatibility, which makes it a promising biomaterial. The distinct protein structure and the fiber formation process contribute to the superior toughness of dragline silk. Previously, we have produced recombinant spider silk-like proteins in transgenic tobacco that are readily purified from plant extracts. The plant-derived spidroin-like proteins consisted of native major ampullate spidroin 1 or spidroin 2 N- and C-termini flanking 8, 16, or 32 copies of their respective consensus block repeats (mini-spidroins). Here, we present the generation of fibers from mini-spidroins (rMaSp1R8 and rMaSp2R8) by polyelectrolyte complex formation using an anionic polyelectrolyte, gellan gum. Mini-spidroins, when treated with acetic acid and cross-linked by glutaraldehyde, formed a thin film at the interface when overlaid with a gellan gum solution. Immediate pulling of the film resulted in autofluorescent fibrous materials from either mini-spidroin alone or a combination of rMaSp1R8 and rMaSp2R8 (70:30). Addition of chitosan to the mini-spidroin solutions permitted continuous fiber production until the spinning dope supply was exhausted. When air-dried as-spun fibers were rehydrated and stretched in water, the fiber diameter decreased and the overall toughness improved. This study showed that spider silk-like fibers can be produced in large quantities through charge attraction that assembles chitosan, mini-spidroins, and gellan gum into fibrous complexes. We speculate that the spider silk self-assembly process in the duct may involve attraction of variously charged chitinous polymers, spidroins, and glycoproteins.
Materials Science and Engineering: C
A proposed source of stem cells for nerve regeneration are dental pulp stem cells (DPSCs), based ... more A proposed source of stem cells for nerve regeneration are dental pulp stem cells (DPSCs), based on their close embryonic origin to neurons and the ease with which DPSCs can be obtained from a donor. This study evaluated the response of human DPSCs to spider dragline silk fibers, a potential substrate material for tissue regeneration. The DPSCs' morphology and spread pattern were characterized after these cells were plated onto Nephila clavipes dragline fibers in media. In addition, the responses of two other well established cell lines, osteoblasts (7F2s), and fibroblasts (3T3s), were also studied under identical conditions. The inclusion of 3T3s and 7F2s in this study allowed for both direct comparisons to prior published work and a qualitative comparison to the morphology of the DPSCs. After twelve days, the DPSCs exhibited greater relative alignment and adherence to the spider dragline fibers than the 3T3s and 7F2s. The impact of a common sterilization method (ultraviolet light) on the spider dragline fiber surface and subsequent cell response to this modified surface was also characterized. Exposure of the silk to ultraviolet light did not have a measureable effect on cell alignment, but it did eliminate bacterial growth and changed fiber surface roughness. Spiders' exposure to stressful environments did not have an effect on silk to impair cell alignment or adhesion. Synthetic recombinant protein silk did not act as a substrate for cell adhesion or alignment but hydrogels with similar composition supported cell attachment, growth and proliferation. In all cases, natural drawn spider silk acted as an effective substrate for cellular adhesion and alignment of DPSCs and could be used in neural differentiation applications.
Journal of Biological Chemistry, 2016
Aatcc Review the Magazine of the Textile Dyeing Printing and Finishing Industry, 2011
Transgenic Research, 2016
The high tensile strength and biocompatibility of spider dragline silk makes it a desirable mater... more The high tensile strength and biocompatibility of spider dragline silk makes it a desirable material in many engineering and tissue regeneration applications. Here, we present the feasibility to produce recombinant proteins in transgenic tobacco Nicotiana tabacum with sequences representing spider silk protein building blocks . Recombinant mini-spidroins contain native N- and C-terminal domains of major ampullate spidroin 1 (rMaSp1) or rMaSp2 flanking an abbreviated number (8, 16 or 32) of consensus repeat domains. Two different expression plasmid vectors were tested and a downstream chitin binding domain and self-cleavable intein were included to facilitate protein purification. We confirmed gene insertion and RNA transcription by PCR and reverse-transcriptase PCR, respectively. Mini-spidroin production was detected by N-terminus specific antibodies. Purification of mini-spidroins was performed through chitin affinity chromatography and subsequent intein activation with reducing reagent. Mini-spidroins, when dialyzed and freeze-dried, formed viscous gelatin-like fluids.
AATCC review, Mar 1, 2011
Spider dragline silk is a proteinaceous fiber with impressive physical characteristics making it ... more Spider dragline silk is a proteinaceous fiber with impressive physical characteristics making it attractive for use in advanced materials. The fiber is composed of two proteins (spidroins MaSp1 and MaSp2), each of which contains a large central repeat array flanked by non-repetitive N- and C-terminal domains. The repeat arrays appear to be largely responsible for the tensile properties of the fiber, suggesting that the N- and C-terminal domains may be involved in self-assembly. We recently isolated the MaSp1 and MaSp2 N-terminal domains from Nephila clavipes and have incorporated these into mini-silk genes for expression in transgenic systems. Current efforts involve the development of expression vectors that will allow purification using a removable affinity tag for scalable protein purification.
Plant molecular biology, 1999
The biased amino acid composition and aperiodic (random coil) configuration of Group 1 late embry... more The biased amino acid composition and aperiodic (random coil) configuration of Group 1 late embryogenesis-abundant (LEA) proteins imply that these proteins are capable of binding large amounts of water. While Group 1 LEAs have been predicted to contribute to osmotic stress protection in both embryonic and vegetative tissues, biochemical support has been lacking. We have used Saccharomyces cerevisiae as a model system to test the putative osmoprotective function of a wheat Group 1 LEA protein, Em. We demonstrate that expression of Em protein in yeast cells is not deleterious to growth in media of normal osmolarity and attenuates the growth inhibition normally observed in media of high osmolarity. Enhanced growth is observed in the presence of a variety of osmotically active compounds indicating that Em protein is capable of mitigating the detrimental effect of low water potential in a relatively non-specific manner. These results are the first biochemical demonstration of an osmoprot...
Biochemical Society transactions, 1992
The Plant Journal, 1995
CDeT6-19 is an ABA-regulated gene which has been isolated from Craterostigma plantagineum. The CD... more CDeT6-19 is an ABA-regulated gene which has been isolated from Craterostigma plantagineum. The CDeT6-19 gene promoter has been fused to the beta-glucuronidase reporter gene (GUS) and used to stably transform Arabidopsis thaliana and Nicotiana tabacum. This construct has been shown to be expressed in stomatal guard cells and often in the adjacent epidermal cells of both species in response to both exogenous ABA and drought stress. These results indicate that the stomatal guard cell is competent to relay an ABA signal to the nucleus. In contrast GUS expression directed by the promoter from a predominantly seed-specific, ABA-regulated gene, Em, or the promoter from the ABA-regulated CDeT27-45 gene is not detectable in the epidermal or guard cells of tobacco or Arabidopsis in response to ABA. The fact that not all ABA-regulated gene promoters are active in stomatal guard cells suggests that effective transduction of the signal is dependent upon particular regions within the gene promoter or that guard cells lack all or part of the specific transduction apparatus required to couple the ABA signal to these promoters. This suggests that there are multiple ABA stimulus response coupling pathways. The identification of a regulatory sequence from an ABA-induced gene which is expressed in stomatal guard cells creates the possibility of examining the role of Ca2+ and other second messengers in ABA-induced gene expression.