Characterization of novel GPCR gene coding locus in amphioxus genome: gene structure, expression, and phylogenetic analysis with implications for its involvement in chemoreception

Chemosensation is the primary sensory modality in almost all metazoans. The vertebrate olfactory receptor genes exist as tandem clusters in the genome, so that identifying their evolutionary origin would be useful for understanding the expansion of the sensory world in relation to a large-scale genomic duplication event in a lineage leading to the vertebrates. In this study, I characterized a novel GPCR (G-protein-coupled receptor) gene-coding locus from the amphioxus genome. The genomic DNA contains an intronless ORF whose deduced amino acid sequence encodes a seven-transmembrane protein with some amino acid residues characteristic of vertebrate olfactory receptors (ORs). Surveying counterparts in the Ciona intestinalis (Asidiacea, Urochordata) genome by querying BLAST programs against the Ciona genomic DNA sequence database resulted in the identification of a remotely related gene. In situ hybridization analysis labeled primary sensory neurons in the rostral epithelium of amphioxus adults. Based on these findings, together with comparison of the developmental gene expression between amphioxus and vertebrates, I postulate that chemoreceptive primary sensory neurons in the rostrum are an ancient cell population traceable at least as far back in phylogeny as the common ancestor of amphioxus and vertebrates.     

Possible involvement of leaf gibberellins in the clock-controlled expression of XSP30, a gene encoding a xylem sap lectin, in cucumber roots

Root-produced organic compounds in xylem sap, such as hormones and amino acids, are known to be important in plant development. Recently, biochemical approaches have revealed the identities of several xylem sap proteins, but the biological functions and the regulation of the production of these proteins are not fully understood. XYLEM SAP PROTEIN 30 kD (XSP30), which is specifically expressed in the roots of cucumber (Cucumis sativus), encodes a lectin and is hypothesized as affecting the development of above-ground organs. In this report, we demonstrate that XSP30 gene expression and the level of XSP30 protein fluctuate in a diurnal rhythm in cucumber roots. The rhythmic gene expression continues for at least two or three cycles, even under continuous light or dark conditions, demonstrating that the expression of this gene is controlled by a circadian clock. Removal of mature leaves or treatment of shoots with uniconazole-P, an inhibitor of gibberellic acid (GA) biosynthesis, dampens the amplitude of the rhythmic expression; the application of GA negates these effects. These results suggest that light signals perceived by above-ground organs, as well as GA that is produced, possibly, in mature leaves, are important for the rhythmic expression of XSP30 in roots. This is the first demonstration of the regulation of the expression of a clock-controlled gene by GA.     

Identification of the functional expression of adenosine A<Subscript>3</Subscript> receptor in pancreas using transgenic mice expressing jellyfish apoaequorin

To investigate the functional expression of adenosine A₃ receptor (A3AR) in mammalian living tissues, we generated an apoaequorin-transgenic mouse that expresses jellyfish apoaequorin throughout its body. The expression of apoaequorin under the control of a strong CAG promoter was detected in various tissues, including the abdominal skin, adipose, ear, brain, esophagus, heart, inferior vena cava vessel, kidney, lens, liver, lung, pancreas, skeletal muscle, spleen, tail, testis, and thymus. The transgene was mapped to the C1–2 region of chromosome 16 by Fluorescence in situ hybridization analysis. Among these transgenic mouse tissues, we succeeded in detecting elevated responses of intracellular Ca²⁺ as a light emission of aequorin induced by the A3AR agonist in the pancreas, brain, and testis, the last two of which are known to be main tissues abundantly expressing A3AR. The A3AR agonist led to the phosphorylation of both extracellular signal-regulated kinase 1/2 and protein kinase B in mouse pancreas, and all the intracellular responses via A3AR were antagonized by the A3AR-specific antagonist. In addition, the mRNA expression of A3AR and the A3AR-induced intracellular responses were also found in the rat pancreatic acinar cell line AR42J. These results suggest that pancreas is one of the main tissues functionally expressing A3AR in mammalians in vivo, and that the present approach using transgenic mice that express apoaequorin throughout their bodies will facilitate the functional analysis of proteins of interest. Kazuya Yamano and Katsuhiro Mori contributed equally to this work     

Sexing of rat embryos with antisera specific for male rats.

Male-specific antigenicity (H-Y antigen) of rat embryos has been examined, and the feasibility of sexing rat embryos by use of H-Y antibodies has been studied. Rat H-Y antisera were produced by immunization of female Wistar rats with a homogenate of testes from male Wistar neonates. Male specificity of the antiserum (H-Y antibody) was determined by retention of cytotoxicity to male epidermal cells after absorption with female cells. After cultivation of rat embryos for 5 to 6 hr in the presence of antibody, half of the embryos were arrested at the morula stage. However, these embryos developed into blastocysts after removal of the antiserum, and then they grew into male young in recipient foster mothers. Eighty percent of the embryos that developed to blastocysts in the presence of the antiserum grew into female young.     

Effects of Monensin and Veratridine on Acetylcholine Release and Cytosolic Free Ca2+ Levels in Cerebrocortical Synaptosomes of Rats

High-affinity specific receptors of endothelin (ET-1) were identified on primary cultures of mouse embryo striatal astrocytes by binding experiments performed with 125I-ET-1. Stimulation of production of inositol phosphates, a biphasic increase of the intracellular calcium concentration, and inhibition of cyclic AMP accumulation were observed in the same cells under ET-1 stimulation. Pretreatment of these cells with Bordetella pertussis toxin affected these effects to different extends, an observation suggesting that they are mediated by multiple transduction pathways, possibly involving several guanine nucleotide-binding proteins.     

A Nitrate-Inducible Plasma Membrane Protein of a Marine Alga, Heterosigma akashiwo

The rate of nitrate uptake by Heterosigma akashiwo cells thathad been cultured in medium with nitrate or ammonium ions asthe source of nitrogen was measured using¹⁵NO_3– The ratioof ¹⁵N/¹⁴N increased dramatically in nitrate-grown cells. Inammonium-grown cells, the ratio of ¹⁵N/¹⁴N did not increasefor 3 h but then it began to increase. Even when nitrate reductaseactivity was inhibited by tungstate, nitrate-grown cells couldtake up nitrate. Plasma membranes from nitrate-grown and ammonium-grown cellswere purified by the silica-microbead method, and polypeptidesassociated with the membranes were analyzed by SDS-PAGE andimmunostaining. A major polypeptide with a molecular mass of26 kDa appeared 3 h after the transfer of ammonium-grown cellsto nitrate-containing medium, and it disappeared 2 d after thetransfer of nitrate-grown cells to ammonium-containing medium.The 26 kDa polypeptide also appeared when cell growth shiftedfrom the logarithmic phase to the stationary phase and the ammoniumcontent of the medium decreased, even when the cells were culturedin ammonium-containing medium. (Received April 10, 1992; Accepted July 30, 1992)     

Geranylgeranyltransferase I of Candida albicans: null mutants or enzyme inhibitors produce unexpected phenotypes

Geranylgeranyltransferase I (GGTase I) catalyzes the transfer of a prenyl group from geranylgeranyl diphosphate to the carboxy-terminal cysteine of proteins with a motif referred to as a CaaX box (C, cysteine; a, usually aliphatic amino acid; X, usually L). The alpha and beta subunits of GGTase I from Saccharomyces cerevisiae are encoded by RAM2 and CDC43, respectively, and each is essential for viability. We are evaluating GGTase I as a potential target for antimycotic therapy of the related yeast, Candida albicans, which is the major human pathogen for disseminated fungal infections. Recently we cloned CaCDC43, the C. albicans homolog of S. cerevisiae CDC43. To study its role in C. albicans, both alleles were sequentially disrupted in strain CAI4. Null Cacdc43 mutants were viable despite the lack of detectable GGTase I activity but were morphologically abnormal. The subcellular distribution of two GGTase I substrates, Rho1p and Cdc42p, was shifted from the membranous fraction to the cytosolic fraction in the cdc43 mutants, and levels of these two proteins were elevated compared to those in the parent strain. Two compounds that are potent GGTase I inhibitors in vitro but that have poor antifungal activity, J-109,390 and L-269,289, caused similar changes in the distribution and quantity of the substrate. The lethality of an S. cerevisiae cdc43 mutant can be suppressed by simultaneous overexpression of RHO1 and CDC42 on high-copy-number plasmids (Y. Ohya et al., Mol. Biol. Cell 4:1017, 1991; C. A. Trueblood, Y. Ohya, and J. Rine, Mol. Cell. Biol. 13:4260, 1993). Prenylation presumably occurs by farnesyltransferase (FTase). We hypothesize that Cdc42p and Rho1p of C. albicans can be prenylated by FTase when GGTase I is absent or limiting and that elevation of these two substrates enables them to compete with FTase substrates for prenylation and thus allows sustained growth.     

A Sialylated Voltage-Dependent Ca2+ Channel Binds Hemagglutinin and Mediates Influenza A Virus Entry into Mammalian Cells

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Role of three chitin synthase genes in the growth of Candida albicans.

The CHS2 and CHS3 genes of Candida albicans were disrupted. The double disruptant was still viable. Assessment of chitin and of calcofluor white resistance shows that CHS1 is responsible for septum formation and CHS3 is responsible for overall chitin synthesis otherwise. There were only small differences in virulence to immunocompromised mice of homozygous chs2 delta amd chs3 delta null mutants.