Vsx-1 is a paired-like:CVC homeobox gene whose expression is linked to bipolar cell differentiation during zebrafish retinogenesis. We used a yeast two-hybrid screen to identify proteins interacting with Vsx-1 and isolated Ubc9, an enzyme that conjugates the small ubiquitin-like modifier SUMO-1. Despite its interaction with Ubc9, we show that Vsx-1 is not a substrate for SUMO-1 in COS-7 cells or in vitro. When a yeast two-hybrid assay is used, deletion analysis of the interacting domain on Vsx-1 shows that Ubc9 binds to a nuclear localization signal (NLS) at the NH 2 terminus of the homeodomain. In SW13 cells, Vsx-1 localizes to the nucleus and is excluded from nucleoli. Deletion of the NLS disrupts this nuclear localization, resulting in a diffuse cytoplasmic distribution of Vsx-1. In SW13 AK1 cells that express low levels of endogenous Ubc9, Vsx-1 accumulates in a perinuclear ring and colocalizes with an endoplasmic reticulum marker. However, NLStagged STAT1 protein exhibits normal nuclear localization in both SW13 and SW13 AK1 cells, suggesting that nuclear import is not globally disrupted. Cotransfection of Vsx-1 with Ubc9 restores Vsx-1 nuclear localization in SW3 AK1 cells and demonstrates that Ubc9 is required for the nuclear localization of Vsx-1. Ubc9 continues to restore nuclear localization even after a C93S active site mutation has eliminated its SUMO-1-conjugating ability. These results suggest that Ubc9 mediates the nuclear localization of Vsx-1, and possibly other proteins, through a nonenzymatic mechanism that is independent of SUMO-1 conjugation.A s regulators of retinal progenitor cell proliferation and cell specification, homeobox proteins are one set of transcription factors that are essential to vertebrate eye development (1-5). Of these, the paired-like homeobox proteins (3, 6, 7) are particularly important during retinogenesis. Vsx-1 and Vsx-2 are two paired-like homeobox proteins that were originally discovered in the regenerating visual pathway of goldfish (8 -10) and further studied in the developing zebrafish (11,12). During retinal development, the complementary expression of Vsx-1 and Vsx-2 mRNA suggests that Vsx-1 is involved in bipolar cell differentiation whereas Vsx-2 is involved in cell proliferation (12).Vsx-1 and Vsx-2 are denoted as paired-like:CVC homeobox proteins because their primary structures contains a conserved 54-aa CVC domain that is downstream and adjacent to the homeodomain. Orthologs of Vsx-1 and Vsx-2 have been isolated and characterized from other organisms, including Caenorhabditis elegans (13) and chicken (14), as well as mammals (15-17). More specifically, Chx10 is the mouse and human ortholog of Vsx-2 (18), whereas the bovine Vsx-1, named RINX͞Vsx-1, has recently been identified (16). The functional importance of these genes is clear because a human CHX10 gene mutation results in microphthalmia (19). Furthermore, a homozygous null allele for Chx10 gives rise to an ocular retardation phenotype in mice that can be partially rescued (20).Such a functional role...

Damage from UVB radiation (280-320 nm) in the form of cyclobutane pyrimidine dimers (CPD) in DNA and the capacity for their repair were measured in newly spawned eggs and yolk-sac larvae of northern anchovy, Engraulis mordax, exposed to natural diel cycles of sunlight. The CPD were measured by a newly developed chemiluminescent immunoblot assay capable of measuring CPD in samples as small as 50 ng DNA. Eggs and yolk-sac larvae exposed to full irradiance levels died. At lower dose rates, equivalent to deeper more natural locations in the water column, there was a diel cycle of dimer concentration that tracked solar intensity. This die1 cycle was due to the interaction of damage and repair processes. Repair of CPD in anchovy eggs and larvae could be attributed to true photodependent repair that could be stopped by moving samples into the dark.The CPD present at sunset remained until the following morning. The diel cycles of damage and repair were maintained over at least 4 days without a long-term upward or downward trend in dimer concentration. This indicates that at the UVB doses used for these experiments, there was no long-term accumulation of CPD nor an induction of increased repair capacity. Unhatched embryos spawned in the dark also exhibited a strong photorepair response, suggesting that photolyase expression was innate and not dependent on previous light exposure. The diel cycle observed here indicates that, at least for northern anchovy, the CPD concentration at the time of sampling is a good indicator of dose rate but a poor indicator of cumulative dose (i.e. late afternoon samples have the highest cumulative dose but relatively low CPD concentrations). The CPD immunoassay described here has the required sensitivity for measuring DNA damage in wild populations of ichthyoplankton exposed to natural sunlight. These results will guide the collection and interpretation of field data on natural levels of CPD in