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Biomedical subjects

M Tasaka

Publications and source records attributed to M Tasaka.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1990

cAMP regulation of the expression of prespore-specific genes, SP96 and Dp87, in disaggregated slug cells of Dictyostelium discoideum.

By Northern transfer and in vitro transcription assays, we examined how cAMP controlled the expression of prespore genes, Dp87 and SP96, in disaggregated slug cells of Dictyostelium discoideum. The transcription of these genes was halted soon after disaggregation and the remaining mRNAs were completely lost within 2 h. Addition of cAMP to cells which had lost the mRNAs induced the transcription of these genes, and the mRNAs were re-accumulated after a lag period of 30 min. The cAMP signal was transduced through the cell surface receptor. Protein synthesis was not needed for the induction of the transcription but was required for the accumulation of the mRNAs. We conclude that prespore gene expression is controlled by cAMP in two different ways: direct induction of transcription of the genes, and stabilization of the transcribed mRNAs by a protein(s) synthesized after addition of cAMP.

Blotting, Northern↗
PubMed1990

Isolation and characterization of spore coat protein (sp96) gene of Dictyostelium discoideum.

A cDNA library was constructed from poly(A)+ RNA isolated from slug cells of Dictyostelium discoideum, using lambda gt11 phage, and screened with an antiserum specific for the spore coat protein sp96. A positive clone was obtained and the gene product was identified as sp96. The sp96 mRNA is 2.2 kb in size, and it starts to accumulate at the tipped aggregate stage only in prespore cells. Southern analysis using nuclear DNA established that the sp96 gene is unique. Two genomic clones containing the sp96 gene were isolated and the sequence of the gene established. The coding region contains a long open reading frame interrupted by a single intron.

Amino Acid Sequence↗
PubMed1989

Structure and expression of elongation factor 2 gene during development of Dictyostelium discoideum.

A cDNA library constructed from poly(A)+ RNA isolated from Dictyostelium discoideum cells at 12 h of development was screened with the hamster elongation factor 2 (EF-2) cDNA. Several different cDNA clones which hybridized were isolated after a second screening. A cDNA clone representing the 5'-end of the mRNA was obtained by primer extension. By comparing the amino acid sequence deduced from the nucleotide sequences of these clones with that of hamster EF-2, we found enough homology between them to conclude that the isolated clones were complementary to the mRNA of D. discoideum EF-2. The N terminus which is the GTP-binding domain and the C-terminal half where it interacts with a ribosome showed a high degree of homology. The amino acid sequence of the carboxyl half includes that it contain a site of ADP-ribosylation by diphtheria toxin. From the Northern blotting analysis, the size of the mRNA was estimated to be 2.6 kilobases. The expression of the mRNA was high in vegetative cells, became maximal at the aggregation stage, and decreased thereafter through development. Upon differentiation of prespore and prestalk cells, the mRNA was highly enriched in the former over the latter. ADP-ribosylation assay of EF-2 protein by diphtheria toxin showed nearly the same developmental changes for the protein as the mRNA. However, prestalk cells were found to contain the same amount of the protein as prespore cells. The Southern blot analyses indicated that the gene encoding EF-2 is unique.

Amino Acid Sequence↗
PubMed1988

Characterization of an unusual cAMP receptor and its related polypeptides in Dictyostelium discoideum.

Several lines of evidence indicate that cAMP modulates developmental gene activity via cell-surface receptors. We describe here a novel cAMP receptor, CABP1, whose properties are consistent with the idea that this protein is involved in gene regulation. Firstly, immunological techniques using anti-CABP1 antibodies as probes showed that this cAMP receptor can be detected on the surface of developing cells. Secondly, there is a steady migration of CABP1 to the nucleus during development. Thirdly, some genetic variants exhibiting an altered pattern of development are found to possess modified CABP1. We also showed that CABP1 co-purifies with at least seven other polypeptides which share common epitopes with CABP1. Interestingly, four of the CABP1-related polypeptides can be detected on the cell surface as well as in the nucleus.

Dictyostelium↗
PubMed1988

Cell behavior during formation of prestalk/prespore pattern in submerged agglomerates of Dictyostelium discoideum.

When cells dissociated from Dictyostelium discoideum slugs were cultured in roller tubes, they formed agglomerates in which prestalk cells were initially dispersed but soon sorted out to the center and then moved to the edge to reconstitute the prestalk/prespore pattern. To examine the mechanism of sorting out, individual prestalk cells were traced by a videotape recorder. The radial component of the rate of movement toward the center of the presumptive prestalk region was calculated. Prestalk cells did not move randomly, but rather directionally toward the center. Their movement was pulsatile, with a period of ca. 15 min, and accompanied by occasional formation of cell streams, thus resembling the movement observable during cell aggregation. These results favor the idea that prestalk cells sort out to the prestalk region due to differential chemotaxis rather than differential adhesiveness. After formation of the prestalk/prespore pattern, the prestalk region rotated along the circumference of the agglomerates. This appears comparable to migration of slugs on the substratum, the rate of rotation being similar to that of slug migration. To examine the processes of pattern formation during development, washed vegetative cells were cultured in roller tubes. Prespore cells identified by antispore immunoglobulin initially appeared randomly within the agglomerates, but then nonprespore cells accumulated in the center and finally moved to the edge to establish the prestalk/prespore pattern, the processes being similar to those of pattern reconstruction with differentiated prestalk and prespore cells.

Cell Aggregation↗
PubMed1988

Molecular cloning of cell-type-specific cDNAs exhibiting new types of developmental regulation in Dictyostelium discoideum.

By screening cDNA libraries from NC-4 cells, we have obtained five prespore-specific and two prestalk-specific cDNA clones. The two classes of prespore genes began to be expressed at the tipped aggregate stage: one class was expressed throughout development, while the other was shut off during culmination. Another class of prespore genes was expressed in vegetative cells but only in prespore cells at the later stages. Prestalk-specific genes were first expressed at the tipped aggregate stage concurrently with the two prespore gene classes, indicating the importance of this stage for transcriptional regulation in both prestalk and prespore differentiation. Except for the first prespore gene class, these represent new cell-type-specific genes as to their temporal pattern of expression.

Cloning, Molecular↗
PubMed1987

Expression of an altered cAMP binding protein by rapid-developing strains of Dictyostelium discoideum.

Immunoblotting with a monoclonal antibody raised against a novel cAMP binding protein termed CABP1 revealed that the molecular weights of the two CABP1 subunits are altered in certain strains of Dictyostelium discoideum. Cell-free translation followed by immunoprecipitation showed that the altered CABP1 polypeptides are derived from primary translation products. In addition, the affinity of the altered CABP1 for cAMP is much higher than the wild-type form. Morphologically, these strains are indistinguishable from other wild-type strains except that their developmental phase is considerably shorter. The rapid developers also exhibit a precocious appearance of CABP1. These results indicate a good correlation between an altered CABP1 and rapid development.

Antibodies, Monoclonal↗
PubMed1986

Identification of multiple cyclic AMP-binding proteins in developing Dictyostelium discoideum cells.

We have purified two cAMP-binding proteins from developing Dictyostelium discoideum cells, which we designate as CABP-1 and CABP-2. Purified CABP-1 consists of two polypeptides of Mr 41,000 and 36,000, which we refer to as CABP-1A and CABP-1B, respectively. Although CABP-1 exhibited specificity for cAMP, it was not labeled at a detectable level when mixed with 8-azidoadenosine 3':5'-monophosphate (8-N3[3H]cAMP). Unlike CABP-1, CABP-2 was labeled efficiently with 8-N3[3H]cAMP. Purified CABP-2 has a molecular weight of 41,000 and an isoelectric point of 5.8-6.0. The physical and biochemical properties of CABP-2 suggest that it is the regulatory subunit of cAMP-dependent protein kinase described by others (de Gunzburg, J., Part, D., Guiso, N., and Veron, M. (1984) Biochemistry 23, 3805-3812; Majerfeld, J. H., Leichtling, B. H., Maligeni, J. A., Spitz, E., and Rickenberg, H. V. (1984) J. Biol. Chem. 259, 654-661). Although CABP-1A and CABP-2 have the same molecular weight, they appear to be encoded by different genes. Two-dimensional gel electrophoresis revealed that the two polypeptides had different isoelectric points. Moreover, monoclonal antibodies raised against CABP-1 did not cross-react with CABP-2. Also, in vitro translation followed by immunoprecipitation showed that these two polypeptides were derived from primary translation products. Our finding of a novel cAMP-binding protein, CABP-1, suggests that cAMP-dependent protein kinase may not be the only intracellular regulator mediating the effects of cAMP in developing D. discoideum cells.

Adenosine Monophosphate↗
PubMed1986

Coupled transport of water and ions through membranes as a possible cause of cytotoxic edema.

To clarify the mechanism of cytotoxic edema, NaCl and colloid solutions were separated isoosmotically by an artificial, electronegatively charged membrane. Under control conditions, no fluid movement occurred through the membrane since it was almost impermeable, even to small ions due to high charge density of the membrane. Following elimination of the negative charge, a fluid shift through the membrane from the NaCl to the colloid compartment began, indicating that although no intercompartmental difference in chemical potential of water exists, water movement occurred coupled with ions which commenced movement along the ionic gradient across the membrane. This process is discussed from the viewpoint of non-equilibrium thermodynamics.

Biological Transport↗
PubMed1983

Prestalk and prespore differentiation in Dictyostelium as detected by cell type-specific monoclonal antibodies.

Monoclonal antibodies specifically reactive against prestalk and prespore cells of the cellular slime mold Dictyostelium discoideum were obtained. By the use of these antibodies, we examined processes of differentiation of the two cell types during development. Cells stained with prespore-specific antibodies first appeared after 12-14 hr of starvation within cell aggregates with tips, coincidentally with the appearance of other prespore markers. The number of prespore cells then increased to a level of 70-80% of total cells at the slug stage. By contrast, cells stained with prestalk-specific antibodies began to appear after 3 hr of starvation and thereafter increased in number to a maximum of ca. 80% after 12 hr of starvation. Stained cells appeared at random in the aggregation field and were not morphologically distinguishable from unstained cells. Furthermore, cells showed a considerable heterogeneity in the amount of antigen they contain. Concomitantly with the increase in prespore cells, the number of cells stained by the prestalk antibodies decreased to a level of ca. 20% by the slug stage. From these experiments, we suggest that the prestalk antigen is synthesized in the majority of cells during the early period of aggregation. Within tight cell aggregates, some of these cells lose the antigen to become prespore cells and the normal proportion between the two cell types will eventually result within slugs.

Journal Article↗
PubMed1981

Role of cell sorting in pattern formation in Dictyostelium discoideum.

To examine the relationship between cell sorting and cell differentiation in the development of Dictyostelium discoideum, labeled cells grown in the absence of glucose [G(-)cells] and unlabeled cells grown in its presence [G(+)cells] were mixed and either allowed to undergo normal morphogenesis or cultured under submerged conditions. Changes in the distributions within a cell aggregate of labeled cells and cells stained with the conjugated antispore serum (prespore cells) were followed on the same sections by the methods of autoradiography and immunohistochemistry. In normal morphogenesis, differentiation of prespore cells apparently initiated and proceeded coincident with sorting out between G(+) and G(-) cells, during formation of standing slug. By contrast, within an aggregate formed under submerged conditions, prespore cells began to differentiate long before G(+) and G(-) cells were sorted out, indicating that the cell sorting is not a prerequisite for the cell differentiation. The sorting out, however, brought about an accumulation of prespore cells in a hemisphere, thus producing a prestalk-prespore pattern within the aggregate.

Cell Aggregation↗
PubMed1979

Sorting out behaviour of disaggregated cells in the absence of morphogenesis in Dictyostelium discoideum.

Cells disaggregated from slugs of Dictyostelium discoideum were cultured in Bonner's salt solution in roller tubes. Cells rapidly stuck together to form an amorphous loose agglutinate which was later transformed into a spheroidal tight agglutinate surrounded by slime sheath material. Prespore cells in the loose agglutinate underwent partial dedifferentiation by starting to decompose their specific antigen until formation of the tight agglutinate, in which the antigen was resynthesized. During the process, there was some decrease in the proportion of prespore cells. Changes in the distribution of prespore and prestalk cells in the agglutinates were examined by using immunocytochemical staining. They were randomly distributed in the early agglutinates, but became well separated in 4 h agglutinates in such a way that prestalk cells were completely enveloped by prespore cells. Prestalk cells later came outside to be partially enveloped and finally occupied a hemisphere side by side with prespore cells. During the process, cells in one or two outer layers differentiated into prestalk or stalk cells. Similar changes in the distribution pattern were observed, when labelled prestalk cells were cultured with unlabelled prespore cells and their distribution in co-agglutinates was followed by autoradiography. It was concluded from these results that the majorities of prestalk and prespore cells isolated from slugs are sorted out in agglutinates without changing their original cell types, and that the sorting-out occurs in the complete absence of polar structures and movement of the cell mass. The distribution patterns in agglutinates of prestalk and prespore cells were discussed with reference to intercellular adhesion among/between them.

Agglutination↗
PubMed1978

Thermoosmosis through charged membranes. Theoretical analysis of concentration dependence.

A theoretical equation for thermoosmosis through charged membranes in electrolyte solutions is derived from nonequilibrium thermodynamics. The theory shows that the volume flux through the membrane is proportional to the temperature difference across the membrane. The proportionality constant, i.e., the thermoosmotic coefficient is a function of electrolyte concentration. The electrolyte concentration dependence of the thermoosmotic coefficient calculated is compared with our previous experimental results. Agreement between theory and experiments is satisfactory.

Electrolytes↗