Hypoxia-inducible factor-1␣ (HIF-1␣) plays an essential role in cellular and systemic O 2 homeostasis by regulating the expression of genes important in glycolysis, erythropoiesis, angiogenesis, and catecholamine metabolism. It is also believed to be a key component of the cellular response to hypoxia and ischemia under pathophysiological conditions, such as stroke. To clarify the function of HIF-1␣ in the brain, we exposed adult mice with late-stage brain deletion of HIF-1␣ to hypoxic injuries. Contrary to expectations, the brains from the HIF-1␣-deficient mice were protected from hypoxia-induced cell death. These surprising findings suggest that decreasing the level of HIF-1␣ can be neuroprotective. Gene chip expression analysis revealed that, contrary to expectations, the majority of hypoxia-dependent gene-expression changes were unaltered, whereas a specific downregulation of apoptotic genes was observed in the HIF-1␣-deficient mice. Although the role of HIF-1␣ has been extensively characterized in vitro, in cancer models, and in chronic preconditioning paradigms, this is the first study to evaluate the role of HIF-1␣ in vivo in the brain in response to acute hypoxia/ischemia. Our data suggest, that in acute hypoxia, the neuroprotection found in the HIF-1␣-deficient mice is mechanistically consistent with a predominant role of HIF-1␣ as proapoptotic and loss of function leads to neuroprotection. Furthermore, our data suggest that functional redundancy develops after excluding HIF-1␣, leading to the preservation of gene expression regulating the majority of other previously characterized HIF-dependent genes.

Deinococcus radiodurans Rl and other members of this genus share extraordinary resistance to the lethal and mutagenic effects of ionizing radiation. We have recently identified a RecA homolog in strain RI and have shown that mutation of the corresponding gene causes marked radiosensitivity. We show here that following high-level exposure to gamma irradiation (1.75 megarads, the dose required to yield 37% of CFU for plateau-phase wild-type R1), the wild-type strain repairs >150 double-strand breaks per chromosome, whereas a recA-defective mutant (rec30) repairs very few or none. A heterologous Escherichia coli-D. radiodurans shuttle plasmid (pMD68) was constructed and found to be retained in surviving D. radiodurans Rl and rec30 following any radiation exposure up to the highest dose tested, 3 megarads. Plasmid repair was monitored in vivo following irradiation with 1.75 megarads in both R1/pMD68 and rec30/pMD68. Immediately after irradiation, plasmids from both strains contained numerous breaks and failed to transform E. coli. While irradiation with 1.75 megarads was lethal to rec30 cultures, a small amount of supercoiled plasmid was regenerated, but it lacked the ability to transform E. coli. In contrast, wild-type cultures showed a cell division arrest of about 10 h, followed by exponential growth. Supercoiled plasmid was regenerated at normal levels, and it readily transformed E. coli. These studies show that D. radiodurans retains a heterologous plasmid following irradiation and repairs it with the same high efficiency as its chromosomal DNA, while the repair defect in rec30 prevents repair of the plasmid. Taken together, the results of this study suggest that plasmid DNA damaged in vivo in D. radiodurans is repaired by recA-dependent mechanisms similar to those employed in the repair of chromosomal DNA.

Lentivirus vectors can transduce dividing and nondividing cells. Using three-plasmid transient transfections, high-titer (>109 IU/ml) recombinant lentivirus vectors pseudotyped with vesicular stomatitis virus G (VSV-G) protein can be generated (T. Kafri et al., Nat. Genet. 17:314–317, 1997; H. Miyoshi et al., Proc. Natl. Acad. Sci. USA 94:10319–10323, 1997; L. Naldini et al., Science 272:263–267, 1996). The recombinant lentiviruses can efficiently infect brain, liver, muscle, and retinal tissue in vivo. Furthermore, the transduced tissues demonstrated long-term expression of reporter genes in immunocompetent rodents. We now report the generation of a tetracycline-inducible VSV-G pseudotyped lentivirus packaging cell line which can generate virus particles at titers greater than 106 IU/ml for at least 3 to 4 days. The vector produced by the inducible cell line can be concentrated to titers of 109 IU/ml and can efficiently transduce nondividing cells in vitro and in vivo. The availability of a lentivirus packaging cell line will significantly facilitate the production of high-titer lentivirus vectors for gene therapy and study of human immunodeficiency virus biology.

Deinococcus radiodurans and other species of the same genus share extreme resistance to ionizing radiation and many other agents that damage DNA. Two different DNA damage-sensitive strains generated by chemical mutagenesis were found to be defective in a gene that has extended DNA and protein sequence homology with poL4 of Escherichia coli. Both mutant strains lacked DNA polymerase, as measured in activity gels. Transformation of this gene from wild-type D. radiodurans restored to the mutants both polymerase activity and DNA damage resistance. A technique for targeted insertional mutagenesis in D. radiodurans is presented. This technique was employed to construct a pol mutant isogenic with the wild type (the first example of targeted mutagenesis in this eubacterial family). This insertional mutant lacked DNA polymerase activity and was even more sensitive to DNA damage than the mutants derived by chemical mutagenesis. In the case of ionizing radiation, the survival of the wild type after receiving 1 Mrad was 100%o while survival of the insertional mutant extrapolated to 10-24. These results demonstrate that the gene described here encodes a DNA polymerase and that defects in this pol gene cause a dramatic loss of resistance of D. radiodurans to DNA damage.

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