Craig S. Newman

Fluorescent Proteins Expressed in Mouse Transgenic Lines Mark Subsets of Glia, Neurons, Macrophages, and Dendritic Cells for Vital Examination

To enable vital observation of glia at the neuromuscular junction, transgenic mice were generated that express proteins of the green fluorescent protein family under control of transcriptional regulatory sequences of the human S100B gene. Terminal Schwann cells were imaged repetitively in living animals of one of the transgenic lines to show that, except for extension and retraction of short processes, the glial coverings of the adult neuromuscular synapse are stable. In other lines, subsets of Schwann cells were labeled. The distribution of label suggests that Schwann cells at individual synapses are clonally related, a finding with implications for how these cells might be sorted during postnatal development. Other labeling patterns, some present in unique lines, included astrocytes, microglia, and subsets of cerebellar Bergmann glia, spinal motor neurons, macrophages, and dendritic cells. We show that lines with labeled macrophages can be used to follow the accumulation of these cells at sites of injury.

Project management system for structural and functional proteomics: Sesame

A computing infrastructure (Sesame) has been designed to manage and link individual steps in complex projects. Sesame is being developed to support a large-scale structural proteomics pilot project. When complete, the system is expected to manage all steps from target selection to data-bank deposition and report writing. We report here on the design criteria of the Sesame system and on results demonstrating successful achievement of the basic goals of its architecture. The Sesame software package, which follows the client/server paradigm, consists of a framework, which supports secure interactions among the three tiers of the system (the client, server, and database tiers), and application modules that carry out specific tasks. The framework utilizes industry standards. The client tier is written in Java2 and can be accessed anywhere through the Internet. All the development on the server tier is also carried out in Java2 so as to accommodate a wide variety of computer platforms. The database tier employs a commercial database management system. Each Sesame application module consists of a simple user interface in the client tier, corresponding objects in the server tier, and relevant data stored in the centralized database. For security, access to stored data is controlled by access privileges. The system facilitates both local and remote collaborations. Because users interact with the system using Java Web Start or through a web browser, access is limited only by the availability of an Internet connection. We describe several Sesame modules that have been developed to the point where they are being utilized routinely to support steps involved in structural and functional proteomics. This software is available to parties interested in using it and assisting to guide its further development.

Results from high-throughput DNA cloning of Arabidopsis thaliana target genes using site-specific recombination.

AbstractThe Center for Eukaryotic Structural Genomics (CESG) was founded as a collaborative effort to develop technologies for the rapid and economic determination of protein three-dimensional structures. The initial focus was on the genome of the model plant Arabidopsis thaliana. Protocols for high-throughput cloning of Arabidopsisopen reading frames into Escherichia coli expression vectors are presented along with an analysis of results from ~2000 cloning experiments. Open reading frames were chosen on the likelihood that they would represent important unknown regions of protein conformation and fold space or that they would elucidate novel fold–function relationships. The chosen open reading frames were amplified from a cDNA pool created by reverse transcription of RNA isolated from an Arabidopsis callus culture. A novel GatewayTM protocol was developed to insert the amplified open reading frames into an entry vector for storage and sequence determination. Sequence verified entry clones were then used to create expression vectors again via the GatewayTM system.

Comparison of Cell-Based and Cell-Free Protocols for Producing Target Proteins from the Arabidopsis thaliana Genome for Structural Studies

We describe a comparative study of protein production from 96 Arabidopsis thaliana open reading frames (ORFs) by cell‐based and cell‐free protocols. Each target was carried through four pipeline protocols used by the Center for Eukaryotic Structural Genomics (CESG), one for the production of unlabeled protein to be used in crystallization trials and three for the production of 15N‐labeled proteins to be analyzed by 1H‐15N NMR correlation spectroscopy. Two of the protocols involved Escherichia coli cell‐based and two involved wheat germ cell‐free technology. The progress of each target through each of the protocols was followed with all failures and successes noted. Failures were of the following types: ORF not cloned, protein not expressed, low protein yield, no cleavage of fusion protein, insoluble protein, protein not purified, NMR sample too dilute. Those targets that reached the goal of analysis by 1H‐15N NMR correlation spectroscopy were scored as HSQC+ (protein folded and suitable for NMR structural analysis), HSQC± (protein partially disordered or not in a single stable conformational state), HSQC− (protein unfolded, misfolded, or aggregated and thus unsuitable for NMR structural analysis). Targets were also scored as X− for failing to crystallize and X+ for successful crystallization. The results constitute a rich database for understanding differences between targets and protocols. In general, the wheat germ cell‐free platform offers the advantage of greater genome coverage for NMR‐based structural proteomics whereas the E. coli platform when successful yields more protein, as currently needed for crystallization trials for X‐ray structure determination. Proteins 2005.

Control of cell migration during Caenorhabditis elegans development

In Caenorhabditis elegans, cell migration is guided by localized cues, including molecules such as EGL-17/FGF and UNC-6/netrin. These external cues are linked to an intracellular response to migrate, at least in part, by CED-5, a homolog of DOCK180/MBC, and MIG-2, a Rac-like GTPase. In addition, metalloproteases are required for a cell migration that controls organ shape.

GON-1 and fibulin have antagonistic roles in control of organ shape.

Most developing organs are surrounded by an extracellular matrix (ECM), which must be remodeled to accommodate growth and morphogenesis. In C. elegans, the GON-1 ADAMTS metalloprotease regulates both elongation and shape of the developing gonad . Here, we report that either human ADAMTS-4 or ADAMTS-9 can substitute for GON-1 in transgenic worms, suggesting functional conservation between human and nematode homologs. We further identify fibulin (FBL-1), a widely conserved ECM component , as critical for gonadal morphogenesis. FBL-1 is expressed in nongonadal tissues but is present at the surface of the elongating gonad. A fibulin deletion mutant has a wider than normal gonad as well as body size defects. We find that GON-1 and fibulin have antagonistic roles in controlling gonadal shape. Depletion of fbl-1, but not other ECM components, rescues gon-1 elongation defects, and removal of gon-1 rescues fbl-1 width defects. Therefore, the GON-1 protease normally promotes tissue elongation and expansion, whereas the fibulin ECM protein blocks these key morphogenetic processes. We suggest that control of organ shape by GON-1 and fibulin in C. elegans may provide a model for similar cellular processes, including vasculogenesis, in humans.

Crystal structure of At2g03760, a putative steroid sulfotransferase from Arabidopsis thaliana

David W. Smith, Kenneth A. Johnson, Craig A. Bingman, David J. Aceti, Paul G. Blommel, Russell L. Wrobel, Ronnie O. Frederick, Qin Zhao, Hassan Sreenath, Brian G. Fox, Brian F. Volkman, Won Bae Jeon, Craig S. Newman, Eldon L. Ulrich, Adrian D. Hegeman, Todd Kimball, Sandy Thao, Michael R. Sussman, John L. Markley, and George N. Phillips, Jr.* Center for Eukaryotic Structural Genomics, Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin Center for Eukaryotic Structural Genomics, Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin

X‐ray structure of Arabidopsis At1g77680, 12‐oxophytodienoate reductase isoform 1

Brian G. Fox,* Thomas E. Malone, Kenneth A. Johnson, Stacey E. Madson, David Aceti, Craig A. Bingman, Paul G. Blommel, Blake Buchan, Brendan Burns, John Cao, Claudia Cornilescu, Jurgen Doreleijers, Jason Ellefson, Ronnie Frederick, Holokere Geetha, David Hruby, Won Bae Jeon, Todd Kimball, John Kunert, John L. Markley, Craig Newman, Andrew Olson, Francis C. Peterson, George N. Phillips Jr., John Primm, Bryan Ramirez, Nathan S. Rosenberg, Mike Runnels, Kory Seder, Jeff Shaw, David W. Smith, Hassan Sreenath, Jikui Song, Michael R. Sussman, Sandy Thao, Donna Troestler, Ejan Tyler, Robert Tyler, Eldon Ulrich, Dimitriy Vinarov, Frank Vojtik, Brian F. Volkman, Gary Wesenberg, Russell L. Wrobel, Jie Zhang, Qin Zhao, and Zolt Zolnai University of Wisconsin Center for Eukaryotic Structural Genomics, University of Wisconsin–Madison, Madison, Wisconsin Molecular and Environmental Toxicology Program, University of Wisconsin–Madison, Madison, Wisconsin Biophysics Doctoral Program, University of Wisconsin–Madison, Madison, Wisconsin

Crystal structure of the protein from gene At3g17210 of Arabidopsis thaliana

Craig A. Bingman, Kenneth A. Johnson, Francis C. Peterson, Ronnie O. Frederick, Qin Zhao, Sandy Thao, Brian G. Fox, Brian F. Volkman, Won Bae Jeon, David W. Smith, Craig S. Newman, Eldon L. Ulrich, Adrian Hegeman, Michael R. Sussman, John L. Markley, and George N. Phillips, Jr.* Department of Biochemistry, University of Wisconsin–Madison, Madison, Wisconsin Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin Department of Biochemistry, University of Wisconsin–Madison, Madison, Wisconsin

The structure at 2.4 A resolution of the protein from gene locus At3g21360, a putative Fe(II)/2-oxoglutarate-dependent enzyme from Arabidopsis thaliana.

The crystal structure of the gene product of At3g21360 from Arabidopsis thaliana was determined by the single-wavelength anomalous dispersion method and refined to an R factor of 19.3% (Rfree = 24.1%) at 2.4 A resolution. The crystal structure includes two monomers in the asymmetric unit that differ in the conformation of a flexible domain that spans residues 178-230. The crystal structure confirmed that At3g21360 encodes a protein belonging to the clavaminate synthase-like superfamily of iron(II) and 2-oxoglutarate-dependent enzymes. The metal-binding site was defined and is similar to the iron(II) binding sites found in other members of the superfamily.