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We used the Tc1/mariner family transposable element Sleeping Beauty (SB) for transgenesis and long-term expression studies in the zebrafish (Danio rerio), a popular organism for clinical disease, vertebrate patterning, and cell biology applications. SB transposase enhanced the transgenesis and expression rate sixfold (from 5 to 31%) and more than doubled the total number of tagged chromosomes over standard, plasmid injection-based transgenesis methods. Molecular analysis of these loci demonstrated a precise integration of these elements into recipient chromosomes with genetic footprints diagnostic of transposition. GFP expression from transposase-mediated integrants was Mendelian through the eighth generation. A blue-shifted GFP variant (BFP) and a red fluorescent protein (DsRed) were also useful transgenesis markers, indicating that multiple reporters are practical for use with SB in zebrafish. We showed that SB is suitable for tissue-specific transgene applications using an abbreviated gamma-crystallin GFP cassette. Finally, we describe a general utility transposon vector for chromosomal engineering and molecular genetics experiments in zebrafish. Together, these data indicate that SB is an efficient tool for transgenesis and expression in zebrafish, and that the transposon will be useful for gene expression in cell biology applications as well as an insertional mutagen for gene discovery during development.

SR proteins, named for their multiple arginine͞serine (RS) dipeptide repeats, are critical components of the spliceosome, influencing both constitutive and alternative splicing of pre-mRNA. SR protein function is regulated through phosphorylation of their RS domains by multiple kinases, including a family of evolutionarily conserved SR protein-specific kinases (SRPKs). The SRPK family of kinases is unique in that they are capable of phosphorylating repetitive RS domains with remarkable specificity and efficiency. Here, we carried out kinetic experiments specially developed to investigate how SRPK1 phosphorylates the model human SR protein, ASF͞SF2. By using the start-trap strategy, we monitored the progress curve for ASF͞SF2 phosphorylation in the absence and presence of an inhibitor peptide directed at the active site of SRPK1. ASF͞SF2 modification is not altered when the inhibitor peptide (trap) is added with ATP (start). However, when the trap is added first and allowed to incubate for a specific delay time, the decrease in phosphate content of the enzyme-substrate complex follows a simple exponential decline corresponding to the release rate of SRPK1. These data demonstrate that SRPK1 phosphorylates a specific region within the RS domain of ASF͞SF2 by using a fully processive catalytic mechanism, in which the splicing factor remains ''locked'' onto SRPK1 during RS domain modification.B oth the assembly of the spliceosome and the specificity of splice-site selection in mammalian cells require SR proteins, which contain one or two RNA-recognition motifs (RRMs) in the N terminus and a signature domain enriched with arginine͞ serine dipeptide repeats (RS) in the C terminus (1, 2). The SR proteins are phosphorylated and activated by the SRPK and Clk͞Sty families of protein kinases (3)(4)(5). This posttranslational modification is required for translocation of the SR protein from the cytoplasm to the nucleus (6, 7) and recruitment of the SR proteins from nuclear speckles (also known as splicing-factor compartments) to nascent transcripts for cotranscriptional splicing (8-10). Phosphorylated SR proteins are believed to facilitate 5Ј splice-site recognition through interaction with the RS domain in U1-70 K (a component of the U1 small nuclear ribonucleoprotein) and 3Ј splice-site selection by means of the RS domains present in the U2AF heterodimer (11-13). Finally, reversal of the kinase action by protein phosphatases appears to be essential for spliceosome activation and the return of splicing factors to nuclear speckles (5). ASF͞SF2 is a prototypical SR protein with two RRM domains for RNA binding and a Cterminal RS domain for mediating protein-protein interactions during spliceosome assembly (see Fig. 1C). A total of 20 serine residues lie in the RS domain and are potential sites for phosphorylation. Mutational studies have verified that phosphorylation occurs exclusively in the RS domain (3,7,14).Although the importance of phosphorylation of SR proteins is well documented for their biological functions in splici...

SummaryTuberculosis continues to be a global health threat, making bicyclic nitroimidazoles an important new class of therapeutics. A deazaflavin-dependent nitroreductase (Ddn) from Mycobacterium tuberculosis catalyzes the reduction of nitroimidazoles such as PA-824, resulting in intracellular release of lethal reactive nitrogen species. The N-terminal 30 residues of Ddn are functionally important but are flexible or access multiple conformations, preventing structural characterization of the full-length, enzymatically active enzyme. Several structures were determined of a truncated, inactive Ddn protein core with and without bound F420 deazaflavin coenzyme as well as of a catalytically competent homolog from Nocardia farcinica. Mutagenesis studies based on these structures identified residues important for binding of F420 and PA-824. The proposed orientation of the tail of PA-824 toward the N terminus of Ddn is consistent with current structure-activity relationship data.