Systematic Reverse Genetics of Transfer-DNA-Tagged Lines of Arabidopsis1 : Isolation of Mutations in the Cytochrome P450 Gene Superfamily

We have developed an efficient reverse-genetics protocol that uses expedient pooling and hybridization strategies to identify individual transfer-DNA insertion lines from a collection of 6000 independently transformed lines in as few as 36 polymerase chain reactions. We have used this protocol to systematically isolate Arabidopsis lines containing insertional mutations in individual cytochrome P450 genes. In higher plants P450 genes encode enzymes that perform an exceptionally wide range of functions, including the biosynthesis of primary metabolites necessary for normal growth and development, the biosynthesis of secondary products, and the catabolism of xenobiotics. Despite their importance, progress in assigning enzymatic function to individual P450 gene products has been slow. Here we report the isolation of the first 12 such lines, including one (CYP83B1-1) that displays a runt phenotype (small plants with hooked leaves), and three insertions in abundantly expressed genes. The DNAs used in this study are publicly available and can be used to systematically isolate mutants in Arabidopsis.

Expediting the transfer of evidence into practice: building clinical partnerships*

A librarian/clinician partnership was fostered in one hospital through the formation of the Evidence-based Practice Committee, with an ulterior goal of facilitating the transfer of evidence into practice. The paper will describe barriers to evidence-based practice and outline the committee's strategies for overcoming these barriers, including the development and promotion of a Web-based guide to evidence-based practice specifically designed for clinicians (health professionals). Educational strategies for use of the Web-based guide will also be addressed. Advantages of this partnership are that the skills of librarians in meeting the needs of clinicians are maximized. The evidence-based practice skills of clinicians are honed and librarians make a valuable contribution to the knowledgebase of the clinical staff. The knowledge acquired through the partnership by both clinicians and librarians will increase the sophistication of the dialogue between the two groups and in turn will expedite the transfer of evidence into practice.

Efficient gene transfer into human hepatocytes by baculovirus vectors.

Viral vectors are the most efficient tools for gene delivery, and the search for tissue-specific infecting viruses is important for the development of in vivo gene therapy strategies. The baculovirus Autographa californica nuclear polyhedrosis virus is widely used as a vector for expression of foreign genes in insect cells, and its host specificity is supposed to be restricted to arthropods. Here we demonstrate that recombinant A. californica nuclear polyhedrosis virus is efficiently taken up by human hepatocytes via an endosomal pathway. High-level reporter gene expression from heterologous promoters was observed in human and rabbit hepatocytes in vitro. Mouse hepatocytes and some other epithelial cell types are targeted at a considerably lower rate. The efficiency of gene transfer by baculovirus considerably exceeds that obtained by calcium phosphate or lipid transfection. These properties of baculovirus suggest a use for it as a vector for liver-directed gene transfer but highlight a potential risk in handling certain recombinant baculoviruses.