A radiation hybrid map of the zebrafish genome.

Recent large-scale mutagenesis screens have made the zebrafish the first vertebrate organism to allow a forward genetic approach to the discovery of developmental control genes. Mutations can be cloned positionally, or placed on a simple sequence length polymorphism (SSLP) map to match them with mapped candidate genes and expressed sequence tags (ESTs). To facilitate the mapping of candidate genes and to increase the density of markers available for positional cloning, we have created a radiation hybrid (RH) map of the zebrafish genome. This technique is based on somatic cell hybrid lines produced by fusion of lethally irradiated cells of the species of interest with a rodent cell line. Random fragments of the donor chromosomes are integrated into recipient chromosomes or retained as separate minichromosomes. The radiation-induced breakpoints can be used for mapping in a manner analogous to genetic mapping, but at higher resolution and without a need for polymorphism. Genome-wide maps exist for the human, based on three RH panels of different resolutions, as well as for the dog, rat and mouse. For our map of the zebrafish genome, we used an existing RH panel and 1,451 sequence tagged site (STS) markers, including SSLPs, cloned candidate genes and ESTs. Of these, 1,275 (87.9%) have significant linkage to at least one other marker. The fraction of ESTs with significant linkage, which can be used as an estimate of map coverage, is 81.9%. We found the average marker retention frequency to be 18.4%. One cR3000 is equivalent to 61 kb, resulting in a potential resolution of approximately 350 kb.

Zebrafish vasa RNA but not its protein is a component of the germ plasm and segregates asymmetrically before germline specification.

Work in different organisms revealed that the vasa gene product is essential for germline specification. Here, we describe the asymmetric segregation of zebrafish vasa RNA, which distinguishes germ cell precursors from somatic cells in cleavage stage embryos. At the late blastula (sphere) stage, vasa mRNA segregation changes from asymmetric to symmetric, a process that precedes primordial germ cell proliferation and perinuclear localization of Vasa protein. Analysis of hybrid fish between Danio rerio and Danio feegradei demonstrates that zygotic vasa transcription is initiated shortly after the loss of unequal vasa mRNA segregation. Blocking DNA replication indicates that the change in vasa RNA segregation is dependent on a maternal program. Asymmetric segregation is impaired in embryos mutant for the maternal effect gene nebel. Furthermore, ultrastructural analysis of vasa RNA particles reveals that vasa RNA, but not Vasa protein, localizes to a subcellular structure that resembles nuage, a germ plasm organelle. The structure is initially associated with the actin cortex, and subsequent aggregation is inhibited by actin depolymerization. Later, the structure is found in close proximity of microtubules. We previously showed that its translocation to the distal furrows is microtubule dependent. We propose that vasa RNA but not Vasa protein is a component of the zebrafish germ plasm. Triggered by maternal signals, the pattern of germ plasm segregation changes, which results in the expression of primordial germ cell-specific genes such as vasa and, consequently, in germline fate commitment.

A mutation in the zebrafish maternal-effect gene nebel affects furrow formation and vasa RNA localization.

BACKGROUND: In many animals, embryonic patterning depends on a careful interplay between cell division and the segregation of localized cellular components. Both of these processes in turn rely on cytoskeletal elements and motor proteins. A type of localized cellular component found in most animals is the germ plasm, a specialized region of cytoplasm that specifies the germ-cell fate. The gene vasa has been shown in Drosophila to encode an essential component of the germ plasm and is thought to have a similar function in other organisms. In the zebrafish embryo, the vasa RNA is localized to the furrows of the early cellular divisions. RESULTS: We identified the gene nebel in a pilot screen for zebrafish maternal-effect mutations. Embryos from females homozygous for a mutation in nebel exhibit defects in cell adhesion. Our analysis provides genetic evidence for a function of the microtubule array that normally develops at the furrow in the deposition of adhesive membrane at the cleavage plane. In addition, nebel mutant embryos show defects in the early localization of vasa RNA. The vasa RNA localization phenotype could be mimicked with microtubule-inhibiting drugs, and confocal microscopy suggests an interaction between microtubules and vasa-RNA-containing aggregates. CONCLUSIONS: Our data support two functions for the microtubule reorganization at the furrow, one for the exocytosis of adhesive membrane, and another for the translocation of vasa RNA along the forming furrow.

The reductive half-reaction of xanthine oxidase. The involvement of prototropic equilibria in the course of the catalytic sequence.

The pH dependence and solvent isotope sensitivity of three discrete steps in the reductive half-reaction of xanthine oxidase have been investigated. The pH dependence of both kcat/Km from steady-state experiments and kred/Kdfrom rapid reaction experiments with xanthine as substrate indicate that enzyme reacts preferentially with the neutral form of substrate and that an ionizable group in the active site having a pKa of approximately 6.6 must be unprotonated for reaction to take place. The solvent kinetic isotope effect on kred/Kd is 2.4, once a uniform shift on going to D2O of approximately 1 unit for both pKa values is taken into account. The pH dependence of the formation and decay of Ered-P formed in the course the reaction of xanthine oxidase with lumazine has also been examined. Formation of this complex exhibits bell-shaped pH dependence, with pKa values of 6.5 and 7.8, consistent with the results obtained with xanthine. Decay of the Ered-P complex is base-catalyzed with a pKa > 11 and exhibits a small solvent kinetic isotope effect of 1.7 at pH/D 8.5. By contrast, the catalytic intermediate giving rise to the "very rapid" EPR signal that is transiently observed in the course of the reaction of enzyme with the substrate 2-hydroxy-6-methylpurine is found to undergo acid-catalyzed breakdown with an associated pKa < 6. Formation and decay of this species exhibit solvent kinetic isotope effects of 2.0 and 3.5 at pH 10. The results are discussed in the context of a specific reaction mechanism for the reductive half-reaction of xanthine oxidase, in which discrete ionizations associated with the molybdenum center of the active site play critical roles in determining the magnitude of the rate constants by which the Mo(IV)-P and Mo(V)-P intermediates form and decay.