Functional cloning of a cDNA encoding Mei2-like protein from Arabidopsis thaliana using a fission yeast pheromone receptor deficient mutant.

To isolate Arabidopsis cDNAs that encode signal transducers and components involved in the regulation of meiosis, a trans-complementation analysis was performed using a Schizosaccharomyces pombe meiosis-defective mutant in which the genes for pheromone receptors were disabled. One cDNA obtained in this screening encodes a polypeptide, named AML1, that shows significant similarity to S. pombe Mei2 protein and has three putative RNA-recognition motifs like as Mei2.

Rapid construction of a transcription map for a cosmid contig of Arabidopsis thaliana genome using a novel cDNA selection method.

Significant progress has been made on the random sequencing of cDNAs (ESTs) and the genetic and physical mapping of the Arabidopsis thaliana genome. New techniques are now required to identify and map the expressed genes efficiently on A. thaliana chromosomes.

Activation of p38 MAP kinase pathway by erythropoietin and interleukin-3.

Activation of p38 MAP kinase (p38) as well as JNK/SAPK has been described as being induced by a variety of environmental stresses such as osmotic shock, ultraviolet radiation, and heat shock, or the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-1 (IL-1). We found that the hematopoietic cytokines erythropoietin (Epo) and IL-3, which regulate growth and differentiation of erythroids and hematopoietic progenitors, respectively, also activate a p38 cascade. Immunoblot analyses and in vitro kinase assay clearly showed that Epo and IL-3 rapidly and transiently phosphorylated and activated p38 in Epo- or IL-3-dependent mouse hematopoietic progenitor cells.

Disruption of hippocampal development in vivo by CR-50 mAb against reelin.

We previously generated a monoclonal alloantibody, CR-50, by immunizing reeler mutant mice with homogenates of normal embryonic brains. This antibody recently was shown to recognize a Reelin protein, which is coded by the recently identified candidate gene for the reeler mutation. However, it is still unclear whether Reelin, especially the CR-50 epitope region, is indeed responsible for the reeler phenotype in vivo.

Characterization of the pleckstrin homology domain of Btk as an inositol polyphosphate and phosphoinositide binding domain.

We previously reported that the pleckstrin homology (PH) domain of Bruton's tyrosine kinase (Btk) binds Ins(1,3,4,5)P4 and that missense mutations in this domain which cause either human X-linked agammaglobulinemia (XLA) or murine X-linked immunodeficiency (Xid) also dramatically reduce the Ins(1,3,4,5)P4 binding activity. In this paper, we describe the inositol phosphate binding specificity of the Btk PH domain and different inositol polyphosphate binding properties among the PH domains of Tec family kinases. Our results suggest that certain inositol phosphates and/or phosphoinositides are physiological ligands of some Tec family kinases and that Tec family members are differently regulated by inositol molecules.

Activation of heat shock transcription factor 3 by c-Myb in the absence of cellular stress.

In vertebrates, the presence of multiple heat shock transcription factors (HSFs) indicates that these factors may be regulated by distinct stress signals. HSF3 was specifically activated in unstressed proliferating cells by direct binding to the c-myb proto-oncogene product (c-Myb). These factors formed a complex through their DNA binding domains that stimulated the nuclear entry and formation of the transcriptionally active trimer of HSF3.

The function of inositol high polyphosphate binding proteins.

The inositol phosphate metabolism network has been found to be much more complex than previously thought, as more and more inositol phosphates and their metabolizing enzymes have been discovered. Some of the inositol phosphates have been shown to have biological activities, but little is known about their signal transduction mechanisms except for that of inositol 1,4,5-trisphosphate. The recent discovery, however, of a number of binding proteins for inositol high polyphosphate [inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate, or inositol hexakisphosphate] enables us to speculate on the physiological function of these compounds.

Assignment of the human gene for KBF2/RBP-Jk to chromosome 9p12-13 and 9q13 by fluorescence in situ hybridization.

The transcription factor KBF2 has been characterized as a factor that binds to the NFkB site of mouse major histocompatibility complex (MHC) class I genes and its amino acid sequence has been shwn to be identical to those of members of the recombination signal-sequence binding protein (RBP-Jk) family. Previous studies by Amakawa et al. (Genomics 17, 306-315, 1993) demonstrated that the functional gene is localized at human chromosome 3q25.

Regulation of Purkinje cell alignment by reelin as revealed with CR-50 antibody.

Cerebellar Purkinje cells are generated in the ventricular zone, migrate outward, and finally form a monolayer in the cortex. In reeler mice, however, most Purkinje cells cluster abnormally in subcortical areas. Reelin, the candidate reeler gene product recognized by the CR-50 monoclonal antibody, is concentrated in a cortical zone along which Purkinje cells are aligned linearly, implying that it may regulate their alignment.

Serum thrombopoietin level is not regulated by transcription but by the total counts of both megakaryocytes and platelets during thrombocytopenia and thrombocytosis.

Thrombopoietin (Tpo) regulates platelet production, but the mechanisms regulating the serum Tpo level and platelet count in circulation have been a subject of debate. Tpo was reported to be expressed mainly in liver and kidney, but we found that Tpo is expressed in all tissues examined: abundantly in liver, kidney, muscle, colon, brain and intestine, and moderately in bone marrow, spleen, lung, stomach, heart, thymus, ovary, and endothelial and leukemic cell lines. The levels of Tpo transcripts in major Tpo producing organs, liver and kidney, and in the platelet production sites bone marrow and spleen, were constant during acute thrombocytopenia induced by anti-platelet monoclonal antibody administration in mice, and during thrombocytosis induced by Tpo injection.

Drosophila CBP is a co-activator of cubitus interruptus in hedgehog signalling.

The transcription factor CBP, originally identified as a coactivator for CREB, enhances transcription mediated by many other transcription factors. Mutations in the human CBP gene are associated with Rubinstein-Taybi syndrome, a haploinsufficiency disorder characterized by abnormal pattern formation, but the mechanism by which decreased CBP levels affect pattern formation is unclear. The hedgehog (hh) signalling pathway is an important determinant of pattern formation.

Regulation by bivalent cations of phospholipid binding to the C2A domain of synaptotagmin III.

Synaptotagmins are Ca2+-and phospholipid-binding proteins of synaptic vesicles that might function as Ca2+ receptors for neurotransmitter release via their first C2 (C2A) domain. Here we describe the effect of Mg2+ on phospholipid binding to the C2A domains of multiple synaptotagmins (II-VI), and demonstrate that only synaptotagmin III can bind negatively charged phospholipids [phosphatidylserine (PS) and phosphatidylinositol] in a Mg2+-dependent manner. The Mg2+-dependent interaction with PS was found to have an EC50 of approx.

Activation of JNK signaling pathway by erythropoietin, thrombopoietin, and interleukin-3.

A variety of environmental stresses, such as osmotic shock, UV radiation, and heat shock, or the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-1 reportedly induce activation of c-Jun amino-terminal kinases (JNK), which are usually activated by SEK1/MKK4. We report here that the hematopoietic cytokines interleukin-3 (IL-3), erythropoietin (Epo), and thrombopoietin (Tpo), which regulate growth and differentiation of hematopoietic progenitor cells, erythroids, and megakaryocytes/platelets, respectively, also activate a JNK signaling cascade. In-gel kinase assay as well as in vitro kinase assay clearly showed that IL-3, Epo, and Tpo rapidly and transiently activated both JNK1 and JNK2 in IL-3-, Epo-, or Tpo-dependent mouse hematopoietic progenitor cells.

Dimerization of midkine by tissue transglutaminase and its functional implication.

Midkine (MK), a retinoic acid-inducible growth/differentiation factor, serves as a substrate for tissue transglutaminase (Kojima, S. , Muramatsu, H., Amanuma, H.

Up-regulation of cell cycle-associated genes by p53 in apoptosis of an erythroleukemic cell line.

A murine erythroleukemic cell line (1-2-3) which expresses only the temperature-sensitive mutant p53 gene (Ala-to-Val substitution at codon 135) was established. When these cells were cultured at 32 degrees C, the growth rate was reduced significantly and DNA fragmentation, a typical character of apoptosis, was observed. In this process, p53 migrated from cytoplasm to nucleus and protein complexes binding to the p53-responsive element were detected in nuclear extracts of the cells cultured at 32 degrees C by gel-shift assay and transactivation from the p53-responsive element was detected.

Point mutation of p53 tumor suppressor gene in bovine leukemia virus-induced lymphosarcoma.

To elucidate the mechanism of leukemogenesis induced by bovine leukemia virus (BLV), the abnormality of p53 tumor suppressor gene was examined using the sequencing method of polymerase chain reaction (PCR)-amplified DNA from peripheral blood lymphocytes (PBL) and tumors from BLV-infected cattle that showed evidence of different stages during the progression of enzootic bovine leukosis (EBL). Mutations of the p53 gene were found in tumor cells from cattle with EBL, but not in PBL from BLV-free normal cattle or BLV-infected cattle without any evidence of tumor, suggesting mutation of p53 gene occurred specifically at the lymphoma stage of the disease. Twelve of eighteen cattle with EBL had seven missense and five silent mutations.

Peptide-based bovine leukemia virus (BLV) vaccine that induces BLV-Env specific Th-1 type immunity.

In controlling retrovirus infection and replication, cell-mediated immunity (CMI) is considered to be effective. To develop a synthetic peptide vaccine capable of inducing CMI, mannan-coated liposome encapsulating 20-mer synthetic peptide, spanning the 98-117 amino acids of bovine leukemia virus (BLV) envelope glycoprotein (Env) gp51 was constructed and inoculated to BALB/c mice. The liposome induced specific delayed-type hypersensitivity, lymphocyte proliferative responses, and a weak cytotoxic lymphocyte response.

Mutational analysis of the structure-function relationship of the leukemogenic membrane glycoprotein (GP55) of Friend spleen focus-forming virus (F-SFFV).

Friend spleen focus-forming virus (F-SFFV) causes acute erythroleukemia in adult mice. F-SFFV encodes an envelope protein-like membrane glycoprotein called gp55 in its defective env gene. Gp55 is responsible for the early stage of leukemogenesis by F-SFFV by specifically binding to and activating the murine erythropoietin receptor (EPO-R).

Simple and rapid preparation of plasmid template by a filtration method using microtiter filter plates.

We developed a new simple high-throughput plasmid DNA extraction procedure, based on a modified alkaline lysis method, using only one 96-well microtiter glassfilter plate. In this method, cell harvesting, lysis by alkaline and plasmid purification are performed on only one microtiter glassfilter plate. After washing out RNAs or other contaminants, plasmid DNA is eluted by low-ion strength solution, although precipitated chromosomal DNA is not eluted.

The expression of the mouse Zic1, Zic2, and Zic3 gene suggests an essential role for Zic genes in body pattern formation.

We examined the expression of Zic1, Zic2, and Zic3 genes in the mouse embryo by means of in situ hybridization. Zic genes were found as a group of genes coding for zinc finger proteins that are expressed in a restricted manner in the adult mouse cerebellum. We showed that the genes are the vertebrate homologues of Drosophila odd-paired, which may play an essential role in parasegmental subdivision and in visceral mesoderm development.

Linkage map of Syrian hamster with restriction landmark genomic scanning.

We have constructed the linkage map with precise genetic analysis of the Syrian hamster, Mesocricetus auratus, according to the restriction landmark genomic scanning (RLGS) spot mapping method. Although only 3.2-6.

A unique downregulation of h2-calponin gene expression in Down syndrome: a possible attenuation mechanism for fetal survival by methylation at the CpG island in the trisomic chromosome 21.

To understand the effect of trisomic chromosome 21 on the cause of Down syndrome (DS), DNA methylation in the CpG island, which regulates the expression of adjacent genes, was investigated with the DNAs of chromosome 21 isolated from DS patients and their parents. A methylation-sensitive enzyme, BssHII, was used to digest DNAs of chromosome 21, and the resulting DNA fragments were subjected to RLGS (restriction landmark genomic scanning). Surprisingly, the CpG island of the h2-calponin gene was shown to be specifically methylated by comparative studies with RLGS and Southern blot analysis.

Effect of chemical modification of oligohomopyrimidine on triplex formation: thermodynamic and kinetic studies.

To investigate the effect of chemical modification of the third strand on the stability of triplex DNA, we have examined the thermodynamic properties of the triplex formation between a 23-mer double-stranded homopurine-homopyrimidine and each of five kinds of 15-mer chemically modified single-stranded homopyrimidines by isothermal titration calorimetry, and the kinetic properties by interaction analysis system. The modifications of the third strand included two base modifications, two sugar moiety modifications, and one phosphate backbone modification. The thermodynamic and kinetic parameters for the triplex formation were similar in magnitude among the two base-modified and two sugar-modified single strands.

Design of hydrophobic core of E. coli malate dehydrogenase based on the side-chain packing.

We have developed computational programs for the de novo design of hydrophobic cores of proteins. The first program optimizes side-chain conformations using an updated rotamer library for potential hydrophobic residues, based on the backbone structure of the protein of interest. The second program selects candidates to be engineered among the sequences by estimating changes in Gibbs free energy between the folded and unfolded structure of the proteins with new sequence.