Fanconi anemia (FA) is a genetic disorder characterized by hypersensitivity to DNA damage, bone marrow failure, congenital defects, and cancer. To further investigate the in vivo function of the FA pathway, mice with a targeted deletion in the distally acting FA gene Fancd2 were created. Similar to human FA patients and other FA mouse models, Fancd2 mutant mice exhibited cellular sensitivity to DNA interstrand cross-links and germ cell loss. In addition, chromosome mispairing was seen in male meiosis. However, Fancd2 mutant mice also displayed phenotypes not observed in other mice with disruptions of proximal FA genes. These include microphthalmia, perinatal lethality, and epithelial cancers, similar to mice with Brca2/Fancd1 hypomorphic mutations. These additional phenotypes were not caused by defects in the ATM-mediated S-phase checkpoint, which was intact in primary Fancd2 mutant fibroblasts. The phenotypic overlap between Fancd2-null and Brca2/Fancd1 hypomorphic mice is consistent with a common function for both proteins in the same pathway, regulating genomic stability.[Keywords: Fanconi anemia; cancer; Fancd2; Brca2; DNA repair; chromosome pairing] Supplemental material is available at http://www.genesdev.org.

The individual and synergistic contributions of two transcription factors, EFG1 and CPH1, have been characterized with regard to adhesion to, and invasion of, human epithelia by Candida albicans. For this purpose two in vitro reconstructed tissue models were developed. A multi-layered model of human epidermis was used to simulate superficial infections of the skin, whereas a reconstructed human intestinal model was used to mimic the first steps of systemic infections. It was shown that C. albicans deleted for both transcription factors CPH1 and EFG1, in contrast to the congenic clinical isolate Sc5314, was neither able to adhere to, nor to penetrate, either of the model systems. A strain deleted for EFG1 alone showed significant reduction in adhesion and was not able to penetrate through the stratum corneum. However, strains deleted for CPH1 showed phenotypes paralleling the phenotypes of the clinical isolate Sc5314. Using different types of multilayered human tissues reconstructed in vitro the individual contributions of Efg1p and Cph1p to two important virulence factors of C. albicans, namely adhesion and invasion, could be defined.

Gene therapy applications of naked DNA constructs for genetic disorders have been limited because of lack of permanent transgene expression. This limitation, however, can be overcome by the Sleeping Beauty (SB) transposable element, which can achieve permanent transgene expression through genomic integration from plasmid DNA. To date, only one example of an in vivo gene therapy application of this system has been reported. In this report, we have further defined the activity of the SB transposon in vivo by analyzing the expression and integration of a fumarylacetoacetate hydrolase (FAH) transposon in FAH-deficient mice. In this model, stably corrected FAH(+) hepatocytes are clonally selected and stable integration events can therefore be quantified and characterized at the molecular level. Herein, we demonstrate that SB-transposon-transfected hepatocytes can support significant repopulation of the liver, resulting in long-lasting correction of the FAH-deficiency phenotype. A single, combined injection of an FAH-expressing transposon plasmid and a transposase expression construct resulted in stable FAH expression in approximately 1% of transfected hepatocytes. The average transposon copy number was determined to be approximately 1/diploid genome and expression was not silenced during serial transplantation. Molecular analysis indicated that high-efficiency DNA-mediated transposition into the mouse genome was strictly dependent on the expression of wild-type transposase.

Zinc is an essential trace element required for structural integrity and functional activity of numerous proteins, yet mechanisms by which cells regulate zinc concentration are poorly understood. Here, we identified a gene from Proteus mirabilis that encodes a 135-amino acid residue protein, PMTR (P. mirabilis transcription regulator), a new member of the MerR family of transcription activators. Transformation of Escherichia coli with PMTR-carrying vectors specifically increases cell tolerance to zinc, suggesting the role of PMTR in zinc homeostasis. In response to zinc, PMTR-containing cells robustly accumulate a 12-kDa protein, the amount of which correlates with the cells' ability to grow at high zinc concentrations. The 12-kDa protein is not induced in the presence of Ni 2؉ , Co 2؉ , Cd 2؉ , Mn 2؉ , or Fe 2؉ , indicating that the PMTR-dependent expression of the 12-kDa protein is specifically regulated by zinc. The 12-kDa protein was identified as the C-terminal fragment of E. coli protein YJAI, and was shown to contain two zincbinding motifs. Metal-affinity chromatography and 65 Zn blotting assay confirmed the ability of the 12-kDa protein to bind zinc specifically (zinc > cobalt > > cadmium). We propose that YJAI is an important component of the zinc-balancing mechanism in E. coli, the up-regulation of which with PMTR results in an increased tolerance to zinc.Heavy metals play an important role in the metabolism of eukaryotic and prokaryotic cells. Zinc, copper, cobalt, and nickel are essential for functional activity and/or structural stability of a large variety of proteins (1-3), while other metals, such as mercury and lead, are toxic, and their accumulation in the cell has inhibitory effects on various cell functions (4, 5). High concentrations of essential heavy metals could also be deleterious; consequently, cells must precisely regulate their availability (6). Two basic mechanisms of heavy metal resistance have been identified: intracellular sequestration through formation of complexes with metal-specific proteins (such as phytochelatins in plants and yeast, or metallothioneins in animals, plants, yeast, and cyanobacteria), and reduced accumulation based on an active efflux of the cation (found in both eukaryotes and prokaryotes) (7). Some of the heavy metal binding and transporting proteins, including metallothioneins and copper-transporting P-type ATPases of mammals, bacteria, and yeast were remarkably preserved during evolution, suggesting that certain ways of regulation of essential microelements are very similar for eukaryotic and prokaryotic cells (8).Zinc plays a particularly important role in cell homeostasis. More than 300 known enzymes require zinc for their catalytic functions. The essential role of zinc in protein structure stabilization and folding has been illustrated by the discovery and characterization of the eukaryotic zinc finger transcription factors and the large family of hormone receptor proteins (8). Appreciation of the importance of zinc for cell metabolism has stimulated ge...

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