Rapid generation of gene disruption constructs by in vitro transposition and identification of a Dictyostelium protein kinase that regulates its rate of growth and development

We describe a rapid method for creating Dictyo stelium gene disruption constructs, whereby the target gene is interrupted by a drug resistance cassette using in vitro transposition. A fragment of genomic DNA containing the gene to be disrupted is amplified by PCR, cloned into a plasmid vector using topoisomerase and then employed as the substrate in an in vitro Tn5 transposition reaction. The transposing species is a fragment of DNA containing a Dictyostelium blasticidin S resistance (bs^r) cassette linked to a bacterial tetracycline resistance (tet^r) cassette. After transposition the plasmid DNA is transformed into Escherichia coli and clones in which the bs^r-tet^r cassette is inserted into the Dictyostelium target DNA are identified. To demonstrate its utility we have employed the method to disrupt the gene encoding QkgA, a novel protein kinase identified from the Dictyostelium genome sequencing project. QkgA is structurally homologous to two previously identified Dictyostelium kinases, GbpC and pats1. Like them it contains a leucine-rich repeat domain, a small GTP-binding (ras) domain and a MEKK domain. Disruption of the qkgA gene causes a marked increase in growth rate and, during development, aggregation occurs relatively slowly to form abnormally large multicellular structures.     

Selection based on the folding properties of proteins with ribosome display

Ribosome display is a powerful tool for selecting and evolving protein functions through ligand-binding. Here, this in vitro system was used to perform selection based on the folding properties of proteins, independent of specific ligand-binding. The selection is based on two properties of misfolded proteins: (1) increased sensitivity to proteolysis and (2) greater exposure of hydrophobic area. By targeting these properties, we show that compactly folded and soluble proteins can be enriched over insoluble and random coil proteins. This approach may be especially useful for selection and evolution of folded proteins from random sequence libraries.     

Redox-coupled conformational alternations in cytochrome c(3) from D. vulgaris Miyazaki F on the basis of its reduced solution structure

Heteronuclear NMR spectroscopy was performed to determine the solution structure of (15)N-labeled ferrocytochrome c(3) from Desulfovibrio vulgaris Miyazaki F (DvMF). Although the folding of the reduced cytochrome c(3) in solution was similar to that of the oxidized one in the crystal structure, the region involving hemes 1 and 2 was different. The redox-coupled conformational change is consistent with the reported solution structure of D. vulgaris Hildenborough ferrocytochrome c(3), but is different from those of other cytochromes c(3). The former is homologous with DvMF cytochrome c(3) in amino acid sequence. Small displacements of hemes 1 and 2 relative to hemes 3 and 4 were observed. This observation is consistent with the unusual behavior of the 2(1)CH(3) signal of heme 3 reported previously. As shown by the (15)N relaxation parameters of the backbone, a region between hemes 1 and 2 has more flexibility than the other regions. The results of this work strongly suggest that the cooperative reduction of hemes 1 and 2 is based on the conformational changes of the C-13 propionate of heme 1 and the aromatic ring of Tyr43, and the interaction between His34 and His 35 through covalent and coordination bonds.     

Inhibition by ajoene of skin-tumor promotion in mice

Ajoene, a major compound containing sulfur in oil-macerated garlic products, inhibited in a two-stage carcinogenesis test on mouse skin. Treatment with ajoene suppressed skin tumor formation, depending on the amount. In particular, the group treated with 250 microg of ajoene had only 4.9% the number of tumors per mouse compared with the control group at 18 weeks.     

Drosophila myosin phosphatase and its role in dorsal closure

Myosin phosphatase negatively regulates nonmuscle myosin II through dephosphorylation of the myosin regulatory light chain (MRLC). Its regulatory myosin-binding subunit, MBS, is responsible for regulating the catalytic subunit in response to upstream signals and for determining the substrate specificity. DMBS, the Drosophila homolog of MBS, was identified to study the roles of myosin phosphatase in morphogenesis. The embryos defective for both maternal and zygotic DMBS demonstrated a failure in dorsal closure. In the mutant embryos, the defects were mainly confined to the leading edge cells which failed to fully elongate. Ectopic accumulation of phosphorylated MRLC was detected in lateral region of the leading edge cells, suggesting that the role of DMBS is to repress the activation of nonmuscle myosin II at the subcellular location for coordinated cell shape change. Aberrant accumulation of F-actin within the leading edge cells may correspond to the morphological aberrations of such cells. Similar defects were seen in embryos overexpressing Rho-kinase, suggesting that myosin phosphatase and Rho-kinase function antagonistically. The genetic interaction of DMBS with mutations in the components of the Rho signaling cascade also indicates that DMBS functions antagonistically to the Rho signal transduction pathway. The results indicate an important role for myosin phosphatase in morphogenesis.     

Computational studies on prion proteins: effect of Ala(117)-->Val mutation

Molecular dynamics calculations demonstrated the conformational change in the prion protein due to Ala(117)-->Val mutation, which is related to Gerstmann-Str?ussler-Sheinker disease, one of the familial prion diseases. Three kinds of model structures of human and mouse prion proteins were examined: (model 1) nuclear magnetic resonance structures of human prion protein HuPrP (125-228) and mouse prion protein MoPrP (124-224), each having a globular domain consisting of three alpha-helices and an antiparallel beta-sheet; (model 2) extra peptides including Ala(117) (109-124 in HuPrP and 109-123 in MoPrP) plus the nuclear magnetic resonance structures of model 1; and (model 3) extra peptides including Val(117) (109-124 in HuPrP and 109-123 in MoPrP) plus the nuclear magnetic resonance structures of model 1. The results of molecular dynamics calculations indicated that the globular domains of models 1 and 2 were stable and that the extra peptide in model 2 tended to form a new alpha-helix. On the other hand, the globular domain of model 3 was unstable, and the beta-sheet region increased especially in HuPrP.     

Role of the conserved DRY motif on G protein activation of rat angiotensin II receptor type 1A

To delineate the functional importance of the highly conserved triplet amino acid sequence, Asp-Arg-Tyr (DRY) among G protein-coupled receptors in the second intracellular loop, these residues of rat angiotensin II (Ang II) receptor type 1A (AT(1A)) were changed by alanine or glycine by site-directed mutagenesis. These mutant receptors were stably expressed in CHO-K1 cells, and the binding of Ang II, GTP effect, InsP(3) production, and the acidification of the medium in response to Ang II were determined. The effects of GTPgammaS on Ang II binding in the mutant receptors D125A and D125G were markedly reduced. InsP(3) production of the mutant D125A, D125G, R126A, and R126G was markedly reduced. Extracellular acidification of D125A was not distinguishable from untransfected CHO-K1 cells. Mutant Y127A was able to produce InsP(3) and acidify medium comparable with wild type AT(1A). These results indicate as follows; Asp(125) is essential for intracellular signal transduction involving G protein coupling, Arg(126) is essential for coupling of G(q) protein but not other G proteins, and Tyr(127) is not important for G protein coupling.     

Simple detection of point mutations in DNA oligonucleotides using SYBR Green I

A novel and simple method for detection of mutations in DNA oligonucleotides using a double-stranded DNA specific dye (SYBR Green I) is reported. The SYBR Green I is bound specifically with a duplex DNA oligonucleotide (intercalation). This intercalation induces fluorescent emission at 525 nm with excitation at 494 nm. The fluorescence intensity of mismatched oligonucleotides (40-mer) decreases (by more than 13%) in comparison with the perfectly matched oligonucleotides. Moreover, fluorescence measurement of the SYBR Green I can distinguish various types of single-base mismatches, except for the T-G terminal mismatch. The addition of 20% (v/v) formamide, however, to an oligonucleotide solution improved the sensitivity of detection and also enabled the detection of the T-G terminal-mismatch. This detection method requires only a normal fluorescence spectrophotometer, an inexpensive dye and just 50 pmol of sample DNA.     

Development of rhythmic melatonin secretion from the pineal gland of embryonic mummichog (Fundulus heteroclitus)

The pineal gland of vertebrates produces and secretes the hormone melatonin in response to changes in the light-dark cycle, with high production at night and low production during the day. Melatonin is thought to play an important role in synchronizing daily and/or seasonal physiological, behavioral, and developmental rhythms in vertebrates. In this study, the functional development of the pineal melatonin-generating system was examined in the mummichog, Fundulus heteroclitus, an euryhaline teleost. In this species, the pineal gland contains an endogenous oscillator, ultimately responsible for timing the melatonin rhythm. Oocytes from gravid females were collected and fertilized in vitro from sperm collected from mature males. Skull caps containing attached pineal glands were obtained from F. heteroclitus embryos at different embryonic stages and placed in static or perfusion culture under various photoperiodic regimes. Rhythmic melatonin secretion from pineal glands of embryonic F. heteroclitus embryos exposed to a 12L:12D cycle in static culture was observed at five days post-fertilization. The ontogeny of circadian-controlled melatonin production from F. heteroclitus pineal glands exposed to constant darkness for five days was also seen at day five post-fertilization. These data show that early development of the pineal melatonin-generating system in this teleost occurs prior to hatching. Pre-hatching development of the melatonin-generating system may confer some selective advantage in this species in its interactions with the environment.     

Genetic manipulation of gibberellin metabolism in transgenic rice

The 'green revolution' was fueled by the introduction of the semi-dwarf trait into cereal crop cultivars. The semi-dwarf cultivars--which respond abnormally to the plant growth hormone gibberellin (GA)--are more resistant to wind and rain damage and thus yield more grain when fertilized. To generate dwarf rice plants using a biotechnological approach, we modified the level of GA by overproduction of a GA catabolic enzyme, GA 2-oxidase. When the gene encoding GA 2-oxidase, OsGA2ox1, was constitutively expressed by the actin promoter, transgenic rice showed severe dwarfism but failed to set grain because GA is involved in both shoot elongation and reproductive development. In contrast, OsGA2ox1 ectopic expression at the site of bioactive GA synthesis in shoots under the control of the promoter of a GA biosynthesis gene, OsGA3ox2 (D18), resulted in a semi-dwarf phenotype that is normal in flowering and grain development. The stability and inheritance of these traits shows the feasibility of genetic improvement of cereal crops by modulation of GA catabolism and bioactive GA content.