An exogenous chloroplast genome for complex sequence manipulation in algae.

We demonstrate a system for cloning and modifying the chloroplast genome from the green alga, Chlamydomonas reinhardtii. Through extensive use of sequence stabilization strategies, the ex vivo genome is assembled in yeast from a collection of overlapping fragments. The assembled genome is then moved into bacteria for large-scale preparations and transformed into C. reinhardtii cells. This system also allows for the generation of simultaneous, systematic and complex genetic modifications at multiple loci in vivo. We use this system to substitute genes encoding core subunits of the photosynthetic apparatus with orthologs from a related alga, Scenedesmus obliquus. Once transformed into algae, the substituted genome recombines with the endogenous genome, resulting in a hybrid plastome comprising modifications in disparate loci. The in vivo function of the genomes described herein demonstrates that simultaneous engineering of multiple sites within the chloroplast genome is now possible. This work represents the first steps toward a novel approach for creating genetic diversity in any or all regions of a chloroplast genome.

Investigation of the biophysical and cell biological properties of ferroportin, a multipass integral membrane protein iron exporter.

Ferroportin is a multipass membrane protein that serves as an iron exporter in many vertebrate cell types. Ferroportin-mediated iron export is controlled by the hormone hepcidin, which binds ferroportin, causing its internalization and degradation. Mutations in ferroportin cause a form of the iron overload hereditary disease hemochromatosis. Relatively little is known about ferroportin's properties or the mechanism by which mutations cause disease. In this study, we expressed and purified human ferroportin to characterize its biochemical/biophysical properties in solution and conducted cell biological studies in mammalian cells. We found that purified detergent-solubilized ferroportin is a well-folded monomer that binds hepcidin. In cell membranes, the N- and C-termini were both cytosolic, implying an even number of transmembrane regions, and ferroportin was mainly localized to the plasma membrane. Hepcidin addition resulted in a redistribution of ferroportin to intracellular compartments that labeled with early endosomal and lysosomal, but not Golgi, markers and that trafficked along microtubules. An analysis of 16 disease-related ferroportin mutants revealed that all were expressed and trafficked to the plasma membrane but that some were resistant to hepcidin-induced internalization. The characterizations reported here form a basis upon which models for ferroportin's role in regulating iron homeostasis in health and disease can be interpreted.

Hybrid yeast-bacteria cloning system used to capture and modify adenoviral and nonviral genomes.

Adenoviral vectors are widely used to express transgenes in vitro and in vivo. A major obstacle to the generation of adenoviral vectors is the manipulation of the large (35 kb) adenoviral genome. We developed a hybrid yeast-bacteria cloning system for the creation of novel adenoviral vectors. The adenovirus 5 (Ad5) genome was cloned into a shuttle vector that contains both yeast and bacterial elements for replication and therefore functions as both a yeast artificial plasmid (YAP) and as a plasmid artificial chromosome (PAC). Any sequence can be introduced into any region of the adenoviral genome via the highly efficient homologous recombination in yeast and then these recombinants are rapidly amplified in bacteria. Adenoviral vectors are generated by introduction of the PAC into the appropriate complementing mammalian cell without the need for plaque purification. Vectors were constructed with deletions in the E1, E3, and/or E4 regions. We have generated more than 100 vectors with a number of different transgenes and regulatory elements. In addition, the YAP/PAC vector was used to capture a DNA fragment encompassing the human factor IX gene, demonstrating the utility of this system to clone and analyze genomic DNA. This novel cloning strategy allows the rapid and versatile construction of adenoviral vectors for gene expression and gene therapy applications.