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M Tasaka

Publications and source records attributed to M Tasaka.

At least 19 recordsLinked to original sources
PubMed1997

Mutations in the SGR4, SGR5 and SGR6 loci of Arabidopsis thaliana alter the shoot gravitropism.

Shoots of higher plants grow upward in response to gravity. To elucidate the molecular mechanism of this response, we have isolated shoot gravitropism (sgr) mutants in Arabidopsis thaliana. In this report, we describe three novel mutants, sgr4-1, sgr5-1 and sgr6-1 whose inflorescence stems showed abnormal gravitropic responses as previously reported for sgr1, sgr2 and sgr3. These new sgr mutations were recessive and occurred at three independent genetic loci. The sgr4-1 mutant showed severe defect in gravitropism of both inflorescence stem and hypocotyl but were normal in root gravitropism as were sgr1 and sgr2. The sgr5-1 and sgr6-1 mutants showed reduced gravitropism only in inflorescence stems but normal in both hypocotyls and roots as sgr3. These results support the hypothesis that some mechanisms of gravitropism are genetically different in these three organs in A. thaliana. In addition, these mutants showed normal phototropic responses, suggesting that SGR4, SGR5 and SGR6 genes are specifically involved in gravity perception and/or gravity signal transduction for the shoot gravitropic response.

Arabidopsis↗
PubMed1997

Genes involved in organ separation in Arabidopsis: an analysis of the cup-shaped cotyledon mutant.

Mutations in CUC1 and CUC2 (for CUP-SHAPED COTYLEDON), which are newly identified genes of Arabidopsis, caused defects in the separation of cotyledons (embryonic organs), sepals, and stamens (floral organs) as well as in the formation of shoot apical meristems. These defects were most apparent in the double mutant. Phenotypes of the mutants suggest a common mechanism for separating adjacent organs within the same whorl in both embryos and flowers. We cloned the CUC2 gene and found that the encoded protein was homologous to the petunia NO APICAL MERISTEM (NAM) protein, which is thought to act in the development of embryos and flowers.

Amino Acid Sequence↗
PubMed1996

DNA sequences responsible for specificity of DNA packaging and phage growth interference of bacteriophages T3 and T7.

T3 and T7 phages package recombinant plasmids carrying DNA necessary for DNA packaging (the pac sequences) of T3 and T7, respectively. Packaging is specific between T3 and T7. The pac sequence has a bipartite structure, consisting of target sequences for processing of concatemeric DNA (pac C) and its left side flanking sequence containing a promoter for phage RNA polymerase (pac B). To determine the sequences responsible for the specificity of plasmid DNA packaging, plasmids chimeric for the pac B and pac C sequences of T3 and T7 were constructed. Analysis of packaging of the chimeric plasmid DNAs showed that pac B is responsible for the packaging specificity of T3 and T7 DNAs. Plasmids carrying the genetic right end of T3 and T7 DNA interfered with the growth of T3 and T7 phages, respectively. Interference was specific between T3 and T7. pac B and sequences between pac B and pac C, but not pac C, were responsible for the interference. The specificity of interference was determined by pac B and sequences responsible for interference were partially defined.

Bacteriophage T3↗
PubMed1996

Gravitropic response of inflorescence stems in Arabidopsis thaliana.

We have characterized the gravitropic response of inflorescence stems in Arabidopsis thaliana. When the inflorescence stems were placed horizontally, they curved upward about 90 degrees within 90 min in darkness at 23 degrees C, exhibiting strong negative gravitropism. Decapitated stem segments (without all flowers, flower buds, and apical apices) also showed gravitropic responses when they included the elongation zone. This result indicates that the minimum elements needed for the gravitropic response exist in the decapitated inflorescence stem segments. At least the 3-min gravistimulation time was sufficient to induce the initial curvature at 23 degrees C after a lag time of about 30 min. In the gravitropic response of inflorescence stems, (a) the gravity perception site exists through the elongating zone, (b) auxin is involved in this response, (c) the gravitropic curvature was inhibited at 4 degrees C but at least the gravity perception step could occur, and (d) two curvatures could be induced in sequence at 23 degrees C by two opposite directional horizontal gravistimulations at 4 degrees C.

Arabidopsis↗
PubMed1996

SGR1, SGR2, SGR3: novel genetic loci involved in shoot gravitropism in Arabidopsis thaliana.

In higher plants shoots show a negative gravitropic response but little is known about its mechanism. To elucidate this phenomenon, we have isolated a number of mutants with abnormal shoot gravitropic responses in Arabidopsis thaliana. Here we describe mainly three mutants: sgr1-1, sgr2-1, and sgr3-1 (shoot gravitropism). Genetic analysis confirmed that these mutations were recessive and occurred at three independent loci, named SGR1, SGR2, and SGR3, respectively. In wild type, both inflorescence stems and hypocotyls show negative gravitropic responses. The sgr1-1 mutants showed no response to gravity either by inflorescence stems or by hypocotyls. The sgr2-1 mutants also showed no gravitropic response in inflorescence stems but showed a reduced gravitropic response in hypocotyls. In contrast, the sgr3-1 mutant was found to have reduced gravitropic responses in inflorescence stems but normal gravitropic responses in hypocotyls. These results suggest that some genetic components of the regulatory mechanisms for gravitropic responses are common between inflorescence stems and hypocotyls, but others are not. In addition, these sgr mutants were normal with respect to root gravitropism, and their inflorescence stems and hypocotyls could carry out phototropism. We conclude that SGR1, SGR2, and SGR3 are novel genetic loci specifically involved in the regulatory mechanisms of shoot gravitropism in A. thaliana.

Arabidopsis↗
PubMed1995

Analysis of the fine structure of the prohead binding domain of the packaging protein of bacteriophage T3 using a hexapeptide, an analog of a prohead binding site.

A large subunit of bacteriophage T3 packaging enzyme, a product of gene 19 (gp19, 586 amino acid residues), binds a prohead prior to DNA translocation in DNA packaging. Its C-terminal region (571 to 576, Region I) is of crucial importance for prohead binding. To elucidate the functional role(s) of Region I in DNA packaging, a hexapeptide (6pT3) corresponding to the Region I sequence and its variants were synthesized and their effects on DNA packaging in a defined in vitro system were examined. 6pT3 did not inhibit gp19wt (wild type)-prohead binding but interfered with their functional interaction, resulting in inhibition of DNA packaging. The inhibitory effect of 6pT3 on gp19wt was reversible. The effect of 6pT3 was examined with gp19 delta C10, which was active in DNA packaging in spite of lacking the extreme C-terminal 10 amino acids (Region II). The inhibitory effect on gp19 delta C10 was more severe than that on gp19wt and was irreversible. From these results, we concluded that the prohead binding domain is composed of two subdomains: Region I is a "core" domain, and its binding to the prohead is crucial for DNA packaging, and Region II is an "anchor" domain stabilizing the binding by Region I.

Amino Acid Sequence↗
PubMed1995

Structural and functional domains of the large subunit of the bacteriophage T3 DNA packaging enzyme: importance of the C-terminal region in prohead binding.

During head assembly of phage T3, DNA is packaged into a preformed protein shell, called the prohead, with the aid of non-capsid packaging proteins, the products of genes 18 and 19 (gp18 and gp19). We have developed a defined system, composed of purified gp18,gp19 and proheads for in vitro packaging of T3 DNA. Our previous results using the defined in vitro system indicate the sequential events in DNA packaging: the packaging proteins, gp18 and gp19, bind DNA and proheads, respectively. These complexes associate to form a direct precursor complexes for DNA translocation into the head. The formation of the precursor complexes requires ATP as an allosteric effector. Subsequent DNA translocation is driven by ATP hydrolysis. gp19 is an ATP binding protein that plays multiple roles in DNA packaging through interaction with ATP. gp19 changes its conformation by binding to ATP, as judged from the analysis of limited proteolysis. Sites cleaved by limited proteolysis were determined and mapped on the gp19 polypeptide (586 amino acid residues) to image the conformational change of gp19 induced by ATP. C-Terminal fragments generated by trypsin digestion bound the prohead and inhibited DNA packaging by intact gp19 in a competitive manner. On the other hand, N-terminal fragments did not bind the prohead nor did they inhibit DNA packaging. These results define a prohead binding domain at the C terminus of gp19. To identify the prohead binding domain more precisely, deletion mutants lacking the last 10 and 15 amino acids (gp19-delta C10 and gp19-delta C15, respectively) of the extreme C terminus of gp19 were constructed. Limited tryptic digestion patterns of these mutant proteins in the presence or absence of ATP were basically the same as those of gp19-wt, indicating that the conformation and its ATP response were not changed by these deletions. gp19-delta C15 lacked prohead binding activity and, therefore, DNA packaging activity. gp19-delta C10 had significant DNA packaging activity although it was reduced to one-tenth of that of gp19-wt. These results indicate that a C-terminal region of residues L571 to D576 of gp19 is crucial for prohead binding and that the last ten residues D577 to W586 of the C terminus seems to be important in stable binding of gp19 to the prohead.

Adenosine Triphosphate↗
PubMed1995

Lipid-regulating action of gemfibrozil in the stroke-prone spontaneously hypertensive rat.

1. Gemfibrozil (Lopid) is extensively used as lipid-regulating agent in the Western World, and its beneficial effect is demonstrated in human studies such as the Helsinki Heart Study. However, the mechanism of its hypolipidaemic action is not fully understood. In the present paper, to elucidate the hypolipidaemic mechanism, we examined the effects of gemfibrozil on lipid metabolism in the normocholesterolaemic and hypercholesterolaemic stroke-prone spontaneously hypersensitive rat (SHRSP). 2. Gemfibrozil effectively increased high density lipoprotein (HDL) subfraction rich in apoE (apoE-HDL) and significantly decreased very low density lipoprotein (VLDL) in normocholesterolaemic SHRSP. In the liver of normocholesterolaemic SHRSP, gemfibrozil significantly reduced the activity of microsomal acyl-CoA:cholesterol acyltransferase. 3. Gemfibrozil markedly reduced atherogenic beta-very low density lipoprotein (beta-VLDL) and low density lipoprotein (LDL) in hypercholesterolaemic SHRSP fed a high-fat and high-cholesterol diet (HFC diet). On the other hand, it significantly increased the contents of apoA-I, A-IV and E in the HDL fraction compared with the control group, suggesting that gemfibrozil effectively increases anti-atherogenic HDL subfractions rich in apoA-I, A-IV or E. In the liver of hypercholesterolaemic SHRSP, gemfibrozil markedly prevented lipid accumulation.

Animals↗
PubMed1994

Analysis of functional domains of the packaging proteins of bacteriophage T3 by site-directed mutagenesis.

Intracellular phage T3 DNA is synthesized as a concatemer in which unit-length molecules are jointed together in head-to-tail fashion through terminally redundant sequences. The concatemeric DNA is processed and packaged into the prohead with the aid of non-capsid proteins, gp18 and gp19. We have developed a defined system, composed of purified gp18, gp19 and proheads, and a crude system, composed of lysates of T3 infected cells, for in vitro packaging of T3 DNA. The defined system displays an ATPase activity which is composed of DNA packaging-dependent and -independent ATPases (pac- and nonpac-ATPases, respectively). In the crude system, DNA is packaged by a way of concatemer as an intermediate. gp19 has ATP binding activity and three ATP binding and two Mg2+ binding consensus motifs in its amino acid sequence. We have expanded the previous studies on the roles of these domains in the DNA packaging reaction by more extensive analysis by site-directed mutagenesis. gp19 mutants, including the previously isolated four mutants, were divided into four groups according to the DNA packaging activity in the defined and crude systems: group 1 mutants were defective in both systems (gp19-G61D, which is a gp19 mutant with Gly to Asp at amino acid 61 and so on, and gp19-H344D); the group 2 mutant had decreased activity in both systems (gp19-G429R); group 3 mutants were active in the defined system but defective in the crude system (gp19-G63D, gp19-H347R, gp19-G367D, gp19-G369D, gp19-G424E); group 4 mutants had almost the same activity as gp19-wt (gp19-K64T, gp19-K370I, gp19-G429L, gp19-K430T and gp19-H553L). Group 1 mutants had an altered conformation, resulting in defective interaction with ATP and in abortive binding to the prohead, and lost specifically the pac-ATPase activity. The group 2 mutant had an increased pac-ATPase activity in spite of the decreased DNA packaging activity, indicating that this mutant is inefficient in coupling of ATP hydrolysis to DNA translocation. The inability of the group 3 mutants except gp19-H347R to package DNA in the crude system would be due to a defect in processing of concatemer DNA. gp19-H347R would be a mutant defective in the initiation event(s) of DNA packaging.

Adenosine Triphosphatases↗
PubMed1994

Intracellular free calcium level and its response to cAMP stimulation in developing Dictyostelium cells transformed with jellyfish apoaequorin cDNA.

A new method is described for measuring intracellular free calcium concentrations, [(Ca2+)i], in the cells of Dictyostelium discoideum transformed with apoaequorin cDNA of the jellyfish, Aequorea victoria. Aequorin, a calcium-specific indicator, was regenerated in vivo from apoaequorin produced in the cells by incubation with coelenterazine. The results showed that [(Ca2+)i] in developing cells markedly increases at the aggregation stage and again at the culmination stage after a temporary drop at the migration stage. Except for the vegetative stage, the cells at all stages of development exhibit a sharp transient increase in [(Ca2+)i] upon stimulation with a cAMP (50 nM) pulse, high responses being observed at the migration and culmination stages. Separated prestalk cells of migrating slugs contain more than twice as much [(Ca2+)i] and show three times as large a response to cAMP stimulation as prespore cells.

Aequorin↗
PubMed1994

Cooperation of positively and negatively acting promoter elements determines prespore-specific transcription of Dp87 gene in Dictyostelium.

Dp87 gene in Dictyostelium is a novel prespore-specific gene, whose expression is first observed when the aggregation stream is formed, the earliest among prespore-specific genes so far isolated. By 5'-sequential deletion analyses, we had previously indicated that the region between -447 and -356 is important for transcription. Here we show by detailed analyses that the regulatory mechanism of the gene is more complex in that multiple positive and negative regulatory regions including the previously identified region act cooperatively. In addition, we show that the region including the putative TATA box and the transcriptional start site is required for proper negative regulation of the gene.

Animals↗
PubMed1994

Dictyostelium prespore-specific gene Dp87 encodes a sorus matrix protein.

In this paper we report on the characteristics of the product of a prespore-specific gene (Dp87) of Dictyostelium discoideum. Polyclonal antibody was made against a bacterially synthesized Dp87-encoded protein fragment. Using this antibody, the product was characterized by immunochemical and immunocytological methods. It was shown that the Dp87-encoded protein is a prespore-specific protein with a molecular mass of 83 kDa, which first appears at the standing slug stage and persists in mature fruiting bodies. Western blot studies revealed the presence of an additional 81 kDa protein prior to the appearance of the 83 kDa protein from the tipped aggregate to the standing slug stage, thus indicating the former to be a precursor protein. Immunocytochemical and immunoelectron microscopic studies showed that the protein is bound to ER at the early stages of development when only the 81 kDa protein is present. At the later stages when the 83 kDa protein predominates, however, it becomes localized in prespore-specific vacuoles (PSVs) and is associated with the inner fibrous material of PSVs, but not with the peripheral membranous material. This is in contrast to spore coat proteins, which are localized in PSVs from the beginning of their appearance and associated with both structures of PSVs. In mature fruiting bodies, most Dp87 protein is localized to the interspore space (matrix) of the sori, with some left on the surface of the stalk tube. Disruptants of the Dp87 gene were also produced. Although they contained neither 81 kDa nor 83 kDa protein, they showed no phenotypic defects as compared to the parental strain.

Animals↗
PubMed1994

Regulation of cell differentiation and pattern formation in Dictyostelium development.

Free-living cells of Dictyostelium discoideum aggregate to form a slug-shaped cell mass and differentiate into prestalk and prespore cells. The differentiation of prespore cells is characterized by expression of Dp87 gene, the earliest event of prespore differentiation. It encodes a protein which first appears in ER of aggregating cells in a precursor form, is then translocated to prespore vacuoles and modified to a mature form and finally exocytosed to constitute the sorus matrix. The transcription of Dp87 is regulated by the cis-acting region consisting of positive, prespore-specific, negative, non-prespore-specific and positive, cell-type-non-specific elements. Cells expressing Dp87 appear at random in early aggregation streams and centers and then sort out to the posterior part of the slug. Intercellular signals required for prestalk and prespore differentiation were investigated by incubation at a low cell density of disaggregated cells. cAMP is inhibitory at the first and second stages of prespore differentiation, while it is required at the third stage. The stalk differentiation is divided into four stages: cAMP is required at the second stage and differentiation inducing factor (DIF) at the third stage, where a low molecular weight secretory substance is also required. At the third stage, cAMP inhibits both ecmA and ecmB expression, while 8-Br-cAMP specifically induces ecmB and maturation of prestalk to stalk cells. The relationship between the differentiation tendency of preaggregative cells and the cell-cycle phase at the initiation of development was studied by the use of cells synchronized for growth by a temperature-shift method.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

DNA packaging ATPase of bacteriophage T3.

A defined in vitro DNA packaging system of phage T3, which is composed of purified proheads and two packaging proteins, the products of genes 18 and 19 (gp18 and gp19, respectively), displayed a DNA-dependent ATPase activity. ATP was hydrolyzed to ADP and Pi. The ATPase activity was stimulated by nonpackageable DNA, such as single-stranded or circular DNA, or RNA (nonpac-ATPase). Among the inhibitors of DNA packaging, actinomycin D specifically inhibited the ATPase activity that was tightly coupled to DNA packaging (pac-ATPase), but did not inhibit the nonpac-ATPase activity. Both activities depended upon a functional packaging complex, but the nonpac-ATPase, once activated, did not require DNA. Unpackageable pUC18 DNA inhibited the pac-ATPase and the phage yield in parallel. Approximately one molecule of ATP was hydrolyzed during the translocation of 1.8 bp of T3 DNA.

Adenosine Triphosphatases↗
PubMed1993

Developmental regulation of transcription of a novel prespore-specific gene (Dp87) in Dictyostelium discoideum.

The Dp87 is a novel prespore specific gene of Dictyostelium discoideum which has a long open reading frame of 555 amino acids. The entire amino acid sequence had low but significant homology to the spore coat proteins, SP96 and SP70, of this organism. When a chimeric gene, containing a 1380 bp of the 5' upstream region of this gene fused with CAT gene, as reporter, was introduced into cells of this organism, it was expressed only in prespore cells of the slug. Transformation experiments, using chimeric genes, containing a series of 5' deletions of the upstream region, showed that -447 bp to -357 bp is an important cis-acting regulatory region for transcription. A nuclear factor(s) that specifically bind to this cis-acting region were detected from slug cell nuclei. Transformation experiments using a chimeric gene consisting of the 5' region between -666 bp and +149 bp of this gene, a beta-galactosidase reporter and an actin 8 terminator, showed that the reporter gene was expressed as early as in aggregation streams, indicating that Dp87 become transcribed a few hours earlier than the other prespore-specific genes so far reported. This was confirmed by northern hybridization detected using an image plate analyzer. The fact that cells expressing Dp87 appeared at random in aggregation streams gives solid support to the idea that position-independent differentiation of prespore and prestalk cells, followed by their sorting, brings about pattern formation in this organism.

Amino Acid Sequence↗
PubMed1992

Protein binding and DNase-I-hypersensitive sites in the cis-acting regulatory region of the spore-coat SP96 gene of Dictyostelium.

The spore-coat protein gene (SP96) of Dictyostelium discoideum is transcribed only in prespore cells. To identify the cis-acting region of this gene, mutant mini-genes which contained different lengths of 5' upstream region, the partially deleted SP96 coding region and ca. 600 bp of 3' flanking sequence were transformed into D. discoideum cells. Expression of the mini-genes was analysed by Northern hybridization. Our results indicate that the 5' upstream region from -686 to -494 contains an important cis-acting element for the temporal and cell type-specific transcription. A nuclear factor which specifically bound the cis-acting region was identified by gel retardation assay. DNase-I-hypersensitivity of the 5' upstream region was examined and it was shown that the appearance of two new hypersensitive sites correlates with transcriptional activation of the gene. One of the two sites maps to the TATA region and the other was located in the cis-acting region identified by deletion analysis. Our results suggest that gene activation occurs by conformational changes in the chromatin structure of the cis-acting region followed by subsequent binding of regulatory factors and the TATA-binding protein.

Animals↗