Genomic structure and promoter activity of the testis haploid germ cell-specific intronless genes,Tact1 and Tact2

The Tact1 and Tact2 genes, each of which encodes an actin-like protein, are exclusively expressed and translated in haploid germ cells in testis. To characterize the haploid germ cell-specific gene structure, a mouse genomic library was screened with a Tact1 cDNA as a probe, and four independent phage clones containing the Tact1 gene were isolated. Southern hybridization and sequencing analyses revealed that Tact1 and Tact2 were single copy genes contained on a common fragment in a head-to-head orientation, and that the distance between these genes was less than 2 kb. Comparison of the nucleotide sequences of genomic DNA and cDNA demonstrated that Tact1 and Tact2 lack introns, although all known actin or actin-related genes in mammals contain introns. Human Tact orthologues also lack introns and are located within 6.4 kb in a head-to-head orientation. These findings indicate that Tact1 and Tact2 or one of these genes arose by retroposition of a spliced mRNA transcribed from an actin progenitor gene prior to the divergence of rodents and primates. The Tact1 and Tact2 genes are unusual retroposons in that they have retained an open reading frame and are expressed in testicular germ cells, because almost all retroposons become pseudogenes. It was revealed that a 2kb sequence between the two genes bidirectionally controls haploid germ-cell specific expression by analyzing transgenic mice. Comparison of the murine Tact genes with their human orthologues showed a high level of identity between the two species in the 5'-upstream and non-coding sequences as well as in the coding region, indicating that conserved elements in these regions may be involved in the regulation of haploid germ cell-specific expression. The promoter region contains no TATA-, CCAAT- or GC-boxes, although there are potential cAMP response element (CRE)-like motifs in the 5'-upstream region and the 5'-untranslated region in Tact1 and Tact2, respectively. Transient promoter analyses indicate that CREMtau may activate Tact1 and Tact2 expression in germ cells.     

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.     

Atlas of the embryonic brain in the pygmy squid,Idiosepius paradoxus

Gross structural changes and neuropil formation in the brain during development were described in Idiosepius paradoxus, a sepioid that we chose as a model cephalopod. The brain originates in 4 pairs of ectodermal placodes, which occur separately in the embryonic surface undergoing epiboly. In the final period of epiboly, neuroblasts internalize from the placodes and gather into 4 pairs of ganglionic masses. The ganglionic masses assemble into a ring-like cluster encircling the inner yolk and the foregut anlage, gradually integrated into the 4 domains of a massive brain, a subesophageal mass (SBM), a supraesophageal mass (SPM), and a pair of optic lobes. In the early brain, neuropil forms a framework composed of a longitudinal ladder lying in the SBM, and a transverse arch standing on the lateral sides of the SBM and crossing the SPM. Differentiation of brain lobes proceeds from ventral to dorsal along this framework; first the magnocellular lobes and the posterior pedal lobe appear first in the SBM, the other lobes in the SBM and the basal lobes follow in the proximal region of the SPM, and the accessory lobes develop last in the most dorsal zone of the SPM. In the hatchlings, the brain lobes show almost the same arrangement as in the adults, but the accessory lobes, particularly the vertical lobe, are much smaller than those in the adults. Comparison of the present results with those in the teuthoid and the octopod indicates that developmental sequences of the brain are highly conserved in the coleoid cephalopods.     

A novel cellulolytic,anaerobic, and thermophilic bacterium,Moorella sp. strain F21

A cellulolytic and thermophilic anaerobe was isolated from soil. This bacterium made a halo on a roll-tube culture containing Avicel. Analysis of the PCR-based 16S rRNA gene sequence showed that the bacterium was closely related to Moorella thermoacetica. Scanning electron microscopy showed the bacterium is a rod and has no protuberant structure on the surface of cells growing on cellulose, suggesting that this strain is a non-cellulosomal cellulolytic bacterium. Carboxymethyl cellulase and xylanase activities were detected in the culture broth. A major fermentation product from ball-milled cellulose was acetate. This strain has a potential to convert cellulosic biomass to acetate, directly.     

Isolation and characterization of an elongation factor-1α gene in Porphyra yezoensis (Rhodophyta)

A gene of Porphyra yezoensis, coding for the translation elongation factor 1 (EF-1), was isolated from a P. yezoensis genomic library. The coding of 1347 nucleotides encodes a polypeptide of 449 amino acids which exhibits sequence similarity as the known EF-1. An intron is located in the 5 untranslated region. Comparison of the deduced amino acid sequence showed higher similarity to the Porphyra purpurea EF-1tef-c (97%) than to the P. purpurea EF-1tef-s (61%). The mRNA was detected both in the leafy gametophyte and filamentous sporophyte by RT-PCR. The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank databases under accession number AB098024.     

A genomewide survey of developmentally relevant genes in Ciona intestinalis. X. Genes for cell junctions and extracellular matrix

Cell junctions and the extracellular matrix (ECM) are crucial components in intercellular communication. These systems are thought to have become highly diversified during the course of vertebrate evolution. In the present study, we have examined whether the ancestral chordate already had such vertebrate systems for intercellular communication, for which we have searched the genome of the ascidian Ciona intestinalis. From this molecular perspective, the Ciona genome contains genes that encode protein components of tight junctions, hemidesmosomes and connexin-based gap junctions, as well as of adherens junctions and focal adhesions, but it does not have those for desmosomes. The latter omission is curious, and the ascidian type-I cadherins may represent an ancestral form of the vertebrate type-I cadherins and desmosomal cadherins, while Ci-Plakin may represent an ancestral protein of the vertebrate desmoplakins and plectins. If this is the case, then ascidians may have retained ancestral desmosome-like structures, as suggested by previous electron-microscopic observations. In addition, ECM genes that have been regarded as vertebrate-specific were also found in the Ciona genome. These results suggest that the last common ancestor shared by ascidians and vertebrates, the ancestor of the entire chordate clade, had essentially the same systems of cell junctions as those in extant vertebrates. However, the number of such genes for each family in the Ciona genome is far smaller than that in vertebrate genomes. In vertebrates these ancestral cell junctions appear to have evolved into more diverse, and possibly more complex, forms, compared with those in their urochordate siblings.     

Thyroid-hormone-dependent and fibroblast-specific expression of BMP-4 correlates with adult epithelial development during amphibian intestinal remodeling

We have identified one of the genes that are up-regulated by thyroid hormone (TH) in Xenopus laevis small intestine as the Xenopus homolog of bone morphogenetic protein-4 (BMP-4). To clarify possible roles of BMP-4 in intestinal remodeling during metamorphosis, we have examined its expression in X. laevis intestine by using in situ hybridization and organ culture techniques. At the beginning of metamorphic climax, BMP-4 mRNA first becomes detectable in the connective tissue, concurrently with the appearance of adult epithelial primordia. Subsequently, when the adult epithelial primordia are actively proliferating, BMP-4 mRNA becomes more abundant only in the connective tissue with a gradient toward the epithelium. Thereafter, as the adult primordia differentiate, the level of BMP-4 mRNA gradually decreases. Thus, BMP-4 expression correlates well with cell proliferation and/or initial differentiation of the adult epithelium, but not with apoptosis of the larval epithelium. Furthermore, the present culture study indicates that (1) TH-induced expression of BMP-4 mRNA is higher in the anterior part of the intestine than in the posterior part, which agrees with the better development of the adult epithelium in the more anterior part, and that (2) the expression of BMP-4 mRNA is up-regulated by TH in the presence of epithelium, but not in its absence. Therefore, BMP-4, which is indirectly induced by TH through some epithelial factor(s), probably plays important roles in adult epithelial development during amphibian intestinal remodeling.     

Th2 polarization enhanced by oral administration of higher doses of antigen

Although oral administration of a soluble proteinantigen can induce various immune responses, theeffect of the dosage of oral antigen on thepredominance of Th2-type cytokine and antibodyresponses has not been well clarified yet. In thepresent study, we fed T cell receptor (TCR) transgenic(tg) mice various amounts of chicken ovalbumin (0.1,5, and 250 mg) and examined the resulting immuneresponses to this antigen. In these TCR tg mice, theresponses of antigen-specific T cells were greatlyamplified concomitantly with significantantigen-specific cytokine secretion. We found that ahigh dose (250 mg) of antigen significantlyupregulated the serum antigen-specific IgG1 and IgAantibody responses in mice later intraperitoneallyinjected with antigen plus adjuvant. The miceadministered the same oral dose but not immunizedshowed upregulation of Th2-type IL-4 and IL-5secretion and downregulation of Th1-type IL-2 andIFN-. This enhancement of Th2-type cytokineand antibody responses was more marked when largerdoses of antigen orally administered. These resultsdemonstrated that antigen feeding induces thedevelopment of T cells secreting Th2-type cytokines ina dose-dependent manner and that these T cells have ahelper function for the production of antibodies ofthe Th2-type isotypes. This experimental system shouldbe useful to screen foods and other substances thatcan modulate Th2-type responses relating to allergy.     

Purification of a 72-kDa protein-tyrosine kinase from rat liver and its identification as Syk: involvement of Syk in signaling events of hepatocytes

Syk protein-tyrosine kinase (PTK) has been implicated in a variety of hematopoietic cell responses including immunoreceptor signaling. However, so far, there has been no evidence of the expression of Syk or Syk-related PTK in non-hematopoietic tissues. In this study, we have purified from blood cell-depleted rat liver a 72-kDa cytoplasmic PTK which shows cross-reactivity with anti-Syk antibody. Partial amino acid sequence analysis revealed that this 72-kDa PTK is identical to Syk. Immunohistochemical and RT-PCR analyses demonstrated that Syk is expressed in human hepatocytes and two rat liver-derived cell lines, JTC-27 and RLC-16. Furthermore, Syk is significantly tyrosine-phosphorylated in response to angiotensin II in JTC-27 cells, and angiotensin II-induced MAP kinase activation is blocked by the treatment of cells with a Syk-selective inhibitor, piceatannol. These results suggest that Syk plays an important role in signaling events of hepatocytes, such as signaling steps leading to MAP kinase activation by G-protein-coupled receptors. This is the first report of the expression of Syk in non-hematopoietic tissue.     

Photoreduction of methemoglobin by irradiation in the near-ultraviolet region

Ferric metHb can be photoreduced to the ferrous state by direct photoexcitation in the near-ultraviolet region. In this research, we studied the mechanism and facilitating conditions for the photoreduction and the resulting restoration of O(2) binding. MetHb in phosphate-buffered saline or pure water in a CO atmosphere was photoreduced to form HbCO by illuminating the N band (365 nm), one of the porphyrin pi --> pi transitions, whereas the photoreduction did not occur in Ar, N(2), or O(2). The transient absorption spectrum exhibited the generation of deoxyHb within 30 ns in both the CO and Ar atmospheres; however, only in CO did the subsequent CO binding inhibit the back reaction. The photoreduction rate was dependent on the pH and ligand anions, showing that aquametHb in the high-spin state was predominant for the photoreduction. Axial ligand-to-metal charge-transfer (LMCT) bands overlap with the Soret and Q bands in metHb; however, the excitation of these bands showed little photoreduction, indicating that the contribution of these LMCT bands is minimal. Excitation of the N band significantly contributes to the photoreduction, and this is facilitated by the external addition of mannitol, hyaluronic acid, Trp, Tyr, etc. Especially, Trp allowed the photoreduction even in an Ar atmosphere, and the reduced Hb can be converted to HbO(2) by O(2) bubbling. One mechanism of the metHb photoreduction that is proposed on the basis of these results consists of a charge transfer from the porphyrin ring to the central ferric iron to form the porphyrin pi cation radical and ferrous iron by the N band excitation, and the contribution of the amino acid residues in the globin chain as an electron donor or an electron pathway.