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Synthetic biologists rely on databases of biological parts to design genetic devices and systems. The sequences and descriptions of genetic parts are often derived from features of previously described plasmids using ad hoc, error-prone... more
Synthetic biologists rely on databases of biological parts to design genetic devices and systems. The sequences and descriptions of genetic parts are often derived from features of previously described plasmids using ad hoc, error-prone and time-consuming curation processes because existing databases of plasmids and features are loosely organized. These databases often lack consistency in the way they identify and describe sequences. Furthermore, legacy bioinformatics file formats like GenBank do not provide enough information about the purpose of features. We have analyzed the annotations of a library of ∼2000 widely used plasmids to build a non-redundant database of plasmid features. We looked at the variability of plasmid features, their usage statistics and their distributions by feature type. We segmented the plasmid features by expression hosts. We derived a library of biological parts from the database of plasmid features. The library was formatted using the Synthetic Biology Open Language, an emerging standard developed to better organize libraries of genetic parts to facilitate synthetic biology workflows. As proof, the library was converted into GenoCAD grammar files to allow users to import and customize the library based on the needs of their research projects.
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A typical vertebrate cell contains several hundred sites of transitional ER (tER). Presumably, tER sites generate elements of the ER-Golgi intermediate compartment (ERGIC), and ERGIC elements then generate Golgi cisternae. Therefore,... more
A typical vertebrate cell contains several hundred sites of transitional ER (tER). Presumably, tER sites generate elements of the ER-Golgi intermediate compartment (ERGIC), and ERGIC elements then generate Golgi cisternae. Therefore, characterizing the mechanisms that influence tER distribution may shed light on the dynamic behavior of the Golgi. We explored the properties of tER sites using Sec13 as a marker protein. Fluorescence microscopy confirmed that tER sites are long-lived ER subdomains. tER sites proliferate during interphase but lose Sec13 during mitosis. Unlike ERGIC elements, tER sites move very little. Nevertheless, when microtubules are depolymerized with nocodazole, tER sites redistribute rapidly to form clusters next to Golgi structures. Hence, tER sites have the unusual property of being immobile, yet dynamic. These findings can be explained by a model in which new tER sites are created by retrograde membrane traffic from the Golgi. We propose that the tER-Golgi sys...
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Protein import into the mitochondria of Saccharomyces cerevisiae depends on two receptor subcomplexes composed of integral outer-membrane proteins. One subcomplex is the MAS37-MAS70 heterodimer, which preferentially recognizes the mature... more
Protein import into the mitochondria of Saccharomyces cerevisiae depends on two receptor subcomplexes composed of integral outer-membrane proteins. One subcomplex is the MAS37-MAS70 heterodimer, which preferentially recognizes the mature regions of precursor proteins associated with ATP-dependent cytosolic chaperones. The other subcomplex contains the acidic proteins MAS20 and MAS22, which recognize the positively charged targeting sequences of a wide variety of mitochondrial precursors. We propose that the two subcomplexes can act together as a single, multifunctional receptor that binds simultaneously to different regions of a precursor molecule.
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We have developed a method to visualize fluorescent protein-labeled !-cells in the intact pancreas through combined,reflection and confocal imaging. This method,provides a three- dimensional view of the !-cells in situ. Imaging of the... more
We have developed a method to visualize fluorescent protein-labeled !-cells in the intact pancreas through combined,reflection and confocal imaging. This method,provides a three- dimensional view of the !-cells in situ. Imaging of the pancreas from mouse insulin I promoter (MIP)-green (GFP) and red fluorescent protein (RFP) transgenic mice shows that islets, !-cell clusters and single !-cells are not evenly
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Research Interests: Functional Analysis, Mitochondria, Multidisciplinary, Structural Analysis, Saccharomyces cerevisiae, and 12 moreSequence alignment, Escherichia coli, Animals, Potassium, Proteins, Heat Shock Proteins, Protein Conformation, Biological activity, Amino Acid Sequence, Chaperone, Thallophyta, and Molecular Sequence Data
The transport of proteins between subcellular compartments is a vectorial, energy-requiring process mediated by the budding and fusion of a series of vesicular carriers. As yet, nothing is known of the chemical reactions that underlie... more
The transport of proteins between subcellular compartments is a vectorial, energy-requiring process mediated by the budding and fusion of a series of vesicular carriers. As yet, nothing is known of the chemical reactions that underlie these events, or how or in exactly what forms energy is used to sustain such movements. Here we report that fatty acyl-CoA acts as cofactor to a Golgi-associated protein factor (termed NSF) that is required for transport between cisternae of the Golgi stack in a cell-free system. This previously unsuspected connection may offer a link between the complex process of protein transport and a single, well-defined type of chemical reaction. We suggest that an ATP-dependent cycle of fatty acylation and deacylation may play an important role in driving rounds of vectorial protein transport.
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Synthetic biologists rely on databases of biological parts to design genetic devices and systems. The sequences and descriptions of genetic parts are often derived from features of previously described plasmids using ad hoc, error-prone... more
Synthetic biologists rely on databases of biological parts to design genetic devices and systems. The sequences and descriptions of genetic parts are often derived from features of previously described plasmids using ad hoc, error-prone and time-consuming curation processes because existing databases of plasmids and features are loosely organized. These databases often lack consistency in the way they identify and describe sequences. Furthermore, legacy bioinformatics file formats like GenBank do not provide enough information about the purpose of features. We have analyzed the annotations of a library of ∼2000 widely used plasmids to build a non-redundant database of plasmid features. We looked at the variability of plasmid features, their usage statistics and their distributions by feature type. We segmented the plasmid features by expression hosts. We derived a library of biological parts from the database of plasmid features. The library was formatted using the Synthetic Biology Open Language, an emerging standard developed to better organize libraries of genetic parts to facilitate synthetic biology workflows. As proof, the library was converted into GenoCAD grammar files to allow users to import and customize the library based on the needs of their research projects.
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The red fluorescent protein DsRed has spectral properties that are ideal for dual-color experiments with green fluorescent protein (GFP). But wild-type DsRed has several drawbacks, including slow chromophore maturation and poor... more
The red fluorescent protein DsRed has spectral properties that are ideal for dual-color experiments with green fluorescent protein (GFP). But wild-type DsRed has several drawbacks, including slow chromophore maturation and poor solubility. To overcome the slow maturation, we used random and directed mutagenesis to create DsRed variants that mature 10-15 times faster than the wild-type protein. An asparagine-to-glutamine substitution at position 42 greatly accelerates the maturation of DsRed, but also increases the level of green emission. Additional amino acid substitutions suppress this green emission while further accelerating the maturation. To enhance the solubility of DsRed, we reduced the net charge near the N terminus of the protein. The optimized DsRed variants yield bright fluorescence even in rapidly growing organisms such as yeast.
Research Interests: Kinetics, Fluorescence Microscopy, Multidisciplinary, Cnidaria, Saccharomyces cerevisiae, and 11 moreMutagenesis, Animals, Temperature, Green Fluorescent Protein, Glutamine, Time Factors, Amino Acid Profile, Spectral Properties, Amino Acid Substitution Rates, Asparagine, and Site-directed Mutagenesis
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Research Interests: Genetics, Fluorescence Microscopy, Cell Cycle, Membrane Proteins, Cell Biology, and 12 moreTranscription Factors, Biological Sciences, Saccharomyces cerevisiae, Mutagenesis, Microbial genetic and drug resistance, Actin Cytoskeleton, Phenotype, Biological markers, The cell, Thiazoles, Golgi Apparatus, and Cell Cycle Proteins
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Cyclophilins are a family of ubiquitous proteins that are the intracellular target of the immunosuppressant drug cyclosporin A. Although cyclophilins catalyze peptidylprolyl cis-trans isomerization in vitro, it has remained open whether... more
Cyclophilins are a family of ubiquitous proteins that are the intracellular target of the immunosuppressant drug cyclosporin A. Although cyclophilins catalyze peptidylprolyl cis-trans isomerization in vitro, it has remained open whether they also perform this function in vivo. Here we show that Cpr3p, a cyclophilin in the matrix of yeast mitochondria, accelerates the refolding of a fusion protein that was synthesized in a reticulocyte lysate and imported into the matrix of isolated yeast mitochondria. The fusion protein consisted of the matrix-targeting sequence of subunit 9 of F1F0-ATPase fused to mouse dihydrofolate reductase. Refolding of the dihydrofolate reductase moiety in the matrix was monitored by acquisition of resistance to proteinase K. The rate of refolding was reduced by a factor of 2-6 by 2.5 microM cyclosporin A. This reduced rate of folding was also observed with mitochondria lacking Cpr3p. In these mitochondria, protein folding was insensitive to cyclosporin A. The rate of protein import was not affected by cyclosporin A or by deletion of Cpr3p.
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The protein import system of the yeast mitochondrial inner membrane includes at least three membrane proteins that presumably form a transmembrane channel as well as several chaperone proteins that mediate the import and refolding of... more
The protein import system of the yeast mitochondrial inner membrane includes at least three membrane proteins that presumably form a transmembrane channel as well as several chaperone proteins that mediate the import and refolding of precursor proteins. We show that one of the membrane proteins, Isp45, spans the mitochondrial inner membrane yet is extracted from this membrane at high pH. Solubilization of mitochondria with a nonionic detergent releases Isp45 as a complex with the chaperones mitochondrial hsp70 (mhsp70) and GrpEp. Both chaperones reversibly dissociate from Isp45 upon addition of ATP or adenosine 5'-[gamma-thio]triphosphate, suggesting that dissociation requires the binding of ATP. Control experiments indicate that the interaction between mhsp70 and Isp45 occurs in the intact mitochondria. We propose that Isp45 lines the inside of a proteinaceous channel across the inner membrane and that it is the membrane anchor for an ATP-driven "import motor" composed of mhsp70 and GrpEp. This arrangement is reminiscent of the protein transport systems of the yeast endoplasmic reticulum and the bacterial plasma membrane.
Research Interests: Membrane Proteins, Mitochondria, Multidisciplinary, Molecular chaperones, Saccharomyces cerevisiae, and 10 moreEndoplasmic Reticulum, Solubility, Heat Shock Proteins, Base Sequence, Membrane Protein, Protein Binding, Plasma Membrane, Protein Transport, Adenosine Triphosphate, and Molecular Sequence Data
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Transitional ER (tER) sites are ER subdomains that are functionally, biochemically and morphologically distinct from the surrounding rough ER. Here we have used confocal video microscopy to study the dynamics of tER sites and Golgi... more
Transitional ER (tER) sites are ER subdomains that are functionally, biochemically and morphologically distinct from the surrounding rough ER. Here we have used confocal video microscopy to study the dynamics of tER sites and Golgi structures in the budding yeast Pichia pastoris. The biogenesis of tER sites is tightly linked to the biogenesis of Golgi, and both compartments can apparently form de novo. tER sites often fuse with one another, but they maintain a consistent average size through shrinkage after fusion and growth after de novo formation. Golgi dynamics are similar, although late Golgi elements often move away from tER sites towards regions of polarized growth. Our results can be explained by assuming that tER sites give rise to Golgi cisternae that continually mature.
Research Interests: Biochemistry, Genetics, Biophysics, Molecular Biology, Stem Cells, and 27 moreMigration, MicroRNA, Cancer, Cell Cycle, Membrane Proteins, Cell Biology, Confocal Microscopy, DNA damage, Apoptosis, Adhesion, Nature, Cytoskeleton, Signal Transduction, Transcription, Budding Yeast, Biological Sciences, Cell Differentiation, Video microscopy, Endoplasmic Reticulum, Signalling, Pichia pastoris, Chromatin, Nuclear envelope, Time Factors, Polarity, Zebrafish, and Golgi Apparatus
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Research Interests: Pharmacology, Biochemistry, Bioinformatics, Evolutionary Biology, Genetics, and 52 moreMarine Biology, Neuroscience, Environmental Science, Geophysics, Physics, Materials Science, Quantum Physics, Developmental Biology, Immunology, Climate Change, Molecular Biology, Structural Biology, Genomics, RNA, Computational Biology, Transcriptomics, Kinetics, Biotechnology, Systems Biology, Cancer, Fluorescence Microscopy, Biology, Metabolomics, Cell Cycle, Proteomics, Ecology, Drug Discovery, Evolution, Nanotechnology, Confocal Microscopy, Astrophysics, Neurobiology, Medicine, Multidisciplinary, Palaeobiology, Functional Genomics, Nature, Signal Transduction, Maturity Model, Astronomy, DNA, Saccharomyces cerevisiae, Video microscopy, Secretory Pathway, Cell Signalling, Medical Research, Membrane transport proteins, Three Dimensional, Carboxypeptidases, Golgi Apparatus, Earth Science, and Protein Transport
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Proteins are exported from the ER at transitional ER (tER) sites, which produce COPII vesicles. However, little is known about how COPII components are concentrated at tER sites. The budding yeast Pichia pastoris contains discrete tER... more
Proteins are exported from the ER at transitional ER (tER) sites, which produce COPII vesicles. However, little is known about how COPII components are concentrated at tER sites. The budding yeast Pichia pastoris contains discrete tER sites and is, therefore, an ideal system for studying tER organization. We show that the integrity of tER sites in P. pastoris requires the peripheral membrane protein Sec16. P. pastoris Sec16 is an order of magnitude less abundant than a COPII-coat protein at tER sites and seems to show a saturable association with these sites. A temperature-sensitive mutation in Sec16 causes tER fragmentation at elevated temperature. This effect is specific because when COPII assembly is inhibited with a dominant-negative form of the Sar1 GTPase, tER sites remain intact. The tER fragmentation in the sec16 mutant is accompanied by disruption of Golgi stacks. Our data suggest that Sec16 helps to organize patches of COPII-coat proteins into clusters that represent tER sites. The Golgi disruption that occurs in the sec16 mutant provides evidence that Golgi structure in budding yeasts depends on tER organization.