RAE28, BMI1, and M33 are members of heterogeneous multimeric mammalian Polycomb group complexes.

The Polycomb group loci in Drosophila encode chromatin proteins required for repression of homeotic loci in embryonic development. We show that mouse Polycomb group homologues, RAE28, BMI1 and M33, have overlapping but not identical expression patterns during embryogenesis and in adult tissues. These three proteins coimmunoprecipitate from embryonic nuclear extracts.

Possible role of tau protein kinases in pathogenesis of Alzheimer's disease.

Tau protein kinases (TPK) I and II were isolated as candidate enzymes responsible for the hyperphosphorylation observed in PHF-tau. Four phosphorylation sites of tau were identified for each kinase, accounting for most, but not all, of the major phosphorylation sites of PHF-tau. Immunostaining with anti-TPKI antibody indicated that this kinase is up-regulated in AD brain.

Early patterning of the rat cerebral wall for regional organization of a neuronal population expressing latexin.

The exact timing of regional patterning in the developing cerebral cortex and other telencephalic structures remains to be elucidated. In the present study, we addressed this issue by comparing the distribution and density of neuronal population expressing latexin in the adult rat telencephalon, with the regional pattern in the fetal cerebral wall as to the potential to generate latexin-expressing neurons. Immunohistochemical analyses on adult animals have shown that latexin-expressing neurons are restricted to a lateral cortical field, within which they are most abundant at the middle level, decreasing in number rostrally and caudally.

Dhh1p, a putative RNA helicase, associates with the general transcription factors Pop2p and Ccr4p from Saccharomyces cerevisiae.

The POP2 (Caf1) protein in Saccharomyces cerevisiae affects a variety of transcriptional processes and is a component of the Ccr4p complex. We have isolated five multicopy suppressor genes of a pop2 deletion mutation: CCR4, DHH1 (a putative RNA helicase), PKC1, STM1, and MPT5 (multicopy suppressor of pop two). Overexpression of either the CCR4 or DHH1 genes effectively suppressed phenotypes associated with pop2 mutant cells; overexpression of PKC1, STM1, or MPT5 genes produced only partial suppression.

Alpha helix content of G protein alpha subunit is decreased upon activation by receptor mimetics.

To elucidate the mechanism whereby liganded receptor molecules enhance nucleotide exchange of GTP-binding regulatory proteins (G proteins), changes in the secondary structure of the recombinant Gi1 alpha subunit (Gi1alpha) upon binding with receptor mimetics, compound 48/80 and mastoparan, were analyzed by circular dichroism spectroscopy. Compound 48/80 enhanced the initial rate of GTPgammaS binding to soluble Gi1alpha 2.6-fold with an EC50 of 30 microg/ml.

Protein anatomy: C-tail region of human tau protein as a crucial structural element in Alzheimer's paired helical filament formation in vitro.

Tau is a microtubule-associated protein in mammalian brain. In Alzheimer's disease, this protein is present in the somatodendritic compartment of certain nerve cells, where it forms a portion of paired helical filament, the major constituent of the neurofibrillary tangle. For clarification of the mechanism of this formation, recombinant human tau and its fragments (N-terminal half, C-terminal half, and 4-repeats) expressed in Escherichia coli were prepared, eight peptide fragments (C-tails 1-8) of the C-tail region were synthesized, and the conformation and capacity for aggregation essential for filamentous structure formation in vitro were examined.

Physical map of the Bacillus subtilis 166 genome: evidence for the inversion of an approximately 1900 kb continuous DNA segment, the translocation of an approximately 100 kb segment and the duplication of a 5 kb segment.

An I-CeuI-NotI-SfiI endonuclease map of the Bacillus subtilis 166 genome was constructed. It was almost identical to that of B. subtilis 168 except for the inversion of an approximately 1900 kb DNA segment, the translocation of an approximately 100 kb segment and the duplication of a 5 kb segment.

Expression patterns of Brx1 (Rieg gene), Sonic hedgehog, Nkx2.2, Dlx1 and Arx during zona limitans intrathalamica and embryonic ventral lateral geniculate nuclear formation.

The Brx1 homeobox gene has been isolated and shown to be expressed in the zona limitans intrathalamica (ZLI) of the mouse embryo. Brx1 is a member of the Brx gene family and comprises the genes for Brx1a and Brx1b, which differ in the sequence in the region located on the 5'-terminal side of the homeobox. The complete amino acid sequences of the open reading frame of Brx1a and Brx1b were determined and each was found to be similar to that of Rgs, the mouse homologue of the Rieger syndrome associated human RIEG gene (RGS), to the extent that the sequence of Rgs has been clarified.

Comparison of exocytotic mechanisms between acetylcholine- and catecholamine-containing vesicles in rat pheochromocytoma cells.

The molecular mechanisms of exocytosis from two types of secretory organelles, synaptic-like microvesicles and secretory vesicles, were compared by measuring acetylcholine (ACh) and catecholamine (CA) release from a newly isolated PC12 subclone, PC12-C3 which contains a high level of Ach. Digitonin-permeabilized PC12-C3 cells released both transmitters with similar Ca(2+)-dependency. Ca(2+)-evoked Ach and CA release from permeabilized cells were increased in the presence of MgATP, suggesting the existence of a MgATP-dependent priming step prior to the Ca(2+)-triggered fusion step in both ACh release and CA release.

Cloning of cDNA and expression of the gene encoding rat presenilin-2.

We have cloned the rat homologue of the presenilin-2 (PS-2) cDNA. PS-2 is responsible for chromosome 1-linked familial Alzheimer's disease. Sequence analysis predicted that the rat PS-2 encodes a 448 amino acid (aa) protein, and there was a very high degree of amino acid identity between rat and human PS-2 (95%).

Binding of chimeric analogs of omega-conotoxin MVIIA and MVIIC to the N- and P/Q-type calcium channels.

Despite their high sequence homology, the peptide neurotoxins omega-conotoxin MVIIA and MVIIC selectively block N- and P/Q-type calcium channels, respectively. To study the recognition mechanism of calcium channel subtypes, two chimeric analogs of omega-conotoxin MVIIA and MVIIC were synthesized by exchanging their N- and C-terminal halves. Binding assay for both N- and P/Q-type calcium channels showed that amino acid residues restricted to the N-terminal half are important for the recognition of N-type channels, whereas essential residues for P/Q-type channel recognition are widely spread over the whole omega-conotoxin molecule.

In vitro virus: bonding of mRNA bearing puromycin at the 3'-terminal end to the C-terminal end of its encoded protein on the ribosome in vitro.

Adequate means for genotype assignment to phenotype is essential in evolutionary molecular engineering. In this study, construction of 'in vitro virus' was carried out in which a genotype molecule (mRNA) covalently binds to the phenotype molecule (protein) through puromycin on the ribosome in a cell-free translation system. Bonding efficiency was approximately 10%, thus indicating a population of the in vitro virus to have approximately 10(12) protein variants, this number being 10(4) that in the phage display.

A DNA helicase activity is associated with an MCM4, -6, and -7 protein complex.

All six minichromosome maintenance (MCM) proteins have DNA-dependent ATPase motifs in the central domain which is conserved from yeast to mammals. Our group purified MCM protein complexes consisting of MCM2, -4 (Cdc21), -6 (Mis5), and -7 (CDC47) proteins from HeLa cells by using histone-Sepharose column chromatography (Ishimi, Y., Ichinose, S.

Imaging of cAMP-dependent protein kinase activity in living neural cells using a novel fluorescent substrate.

In order to visualize the activity of the cAMP-dependent protein kinase (PKA) in living cells, we have constructed a new fluorescence PKA substrate by conjugating a fluorescence probe to a partial amino acid sequence of PKA regulatory domain II which contains a specific autophosphorylation site. The fluorescent peptide was cell-permeable and became phosphorylated when the intracellular cAMP concentration was increased, resulting in a decrease in its fluorescence intensity. In NG108-15 cells, PKA activity was localized to the cytosol around the nucleus.

Organogenesis of the liver, thymus and spleen is affected in jumonji mutant mice.

The recessive mutant mouse jumonji (jmj), obtained by a gene trap strategy, shows neural tube defects in approximately half of homozygotes with a Balb/cA and 129/Ola mixed background. Here, we show that no neural tube defects are observed with a Balb/cA background. We also found hypoplasia of the liver, thymus and spleen with full penetrance with a Balb/cA background.

Cellular distribution of mammalian DNA topoisomerase II is determined by its catalytically dispensable C-terminal domain.

Mammalian cells express two genetically distinct isoforms of DNA topoisomerase II, designated topoisomerase IIalphaand topoisomerase IIbeta. We have recently shown that mouse topoisomerase IIalpha can substitute for the yeast topoisomerase II enzyme and complement yeast top2 mutations. This functional complementation allowed functional analysis of the C-terminal domain (CTD) of mammalian topoisomerase II, where the amino acid sequences are divergent and species-specific, in contrast to the highly conserved N-terminal and central domains.

vesl, a gene encoding VASP/Ena family related protein, is upregulated during seizure, long-term potentiation and synaptogenesis.

We have isolated a novel cDNA, vesl, that was induced during convulsive seizure in the rat hippocampus. The vesl gene encodes a protein of 186 amino acids that has significant homology to the EVH1 domain of the VASP/Ena family of proteins implicated in the control of microfilament dynamics. The expression of vesl mRNA was induced in the granule cell layer during persistent long-term potentiation (LTP) of the dentate gyrus in an NMDA receptor-dependent manner.

BIT, an immune antigen receptor-like molecule in the brain.

We previously found a brain-specific glycoprotein in the rat brain. It postnatally increases and is rich in the mature brain. We cloned cDNA of this protein.

Expression of a novel aristaless related homeobox gene 'Arx' in the vertebrate telencephalon, diencephalon and floor plate.

We have isolated a novel homeobox gene that is expressed in the vertebrate central nervous system and which shows striking similarity to the Drosophila al gene in the homeodomain (85% identity) and in a 17 amino acid-sequence near the carboxyl-terminus. This gene was designated Arx (aristaless related homeobox gene) in consideration of its structural similarity to the al gene. Arx was highly conserved between mouse and zebrafish.

Isolation and characterization of kar2-404 mutation in Saccharomyces cerevisiae.

We have devised a direct screening method to isolate mutations in the KAR2 gene, and have isolated a BiP/KAR2 mutant, kar2-404, from Saccharomyces cerevisiae as a small halo-forming mutant of secreted mouse alpha-amylase. The mutation site was identified as a point mutation at t1337 to c1337 resulting in the Ile-404Thr mutation of mature Kar2-404p, located at the most NH2-terminal first beta-sheet structure (beta 1) of the putative peptide-binding domain. This isoleucine is highly conserved in the Hsp70 family.

Regulation of cone cell formation by Canoe and Ras in the developing Drosophila eye.

Cone cells are lens-secreting cells in ommatidia, the unit eyes that compose the compound eye of Drosophila. Each ommatidium contains four cone cells derived from precursor cells of the R7 equivalence group which express the gene sevenless (sev). When a constitutively active form of Ras1 (Ras1V12) is expressed in the R7 equivalence group cells using the sev promoter (sev-Ras1V12), additional cone cells are formed in the ommatidium.

Different effects of Alzheimer-associated mutations of presenilin 1 on its processing.

Presenilin 1 (PS 1) shows missense mutations in most early-onset familial Alzheimer's disease (FAD). Transfection of cDNA for wild type PS 1 into rat pheochromocytoma PC12 cells generated a 47 kDa full-size PS 1 protein, which was processed into a 28 kDa N-terminal fragment and a 19 kDa C-terminal fragment. We prepared selected Alzheimer-associated mutations (Gly384Ala, Leu392Val, and Cys410Tyr) of PS 1, which localized after a possible cleavage site.