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K Takeyasu

Publications and source records attributed to K Takeyasu.

At least 19 recordsLinked to original sources
PubMed1999

Atomic force microscopy sees nucleosome positioning and histone H1-induced compaction in reconstituted chromatin.

We addressed the question of how nuclear histones and DNA interact and form a nucleosome structure by applying atomic force microscopy to an in vitro reconstituted chromatin system. The molecular images obtained by atomic force microscopy demonstrated that oligonucleosomes reconstituted with purified core histones and DNA yielded a 'beads on a string' structure with each nucleosome trapping 158 +/- 27 bp DNA. When dinucleosomes were assembled on a DNA fragment containing two tandem repeats of the positioning sequence of the Xenopus 5S RNA gene, two nucleosomes were located around each positioning sequence. The spacing of the nucleosomes fluctuated in the absence of salt and the nucleosomes were stabilized around the range of the positioning signals in the presence of 50 mM NaCl. An addition of histone H1 to the system resulted in a tight compaction of the dinucleosomal structure.

Animals↗
PubMed1999

Long range interaction of cis-DNA elements mediated by architectural transcription factor Bach1.

BACKGROUND: A central question in vertebrate transcriptional regulation is how cis-regulatory modules, including enhancers, silencers and promoters, communicate with each other over long distances to mandate proper gene expression. In order to address this question we analysed protein/DNA interactions in the human beta-globin locus control region (LCR). One of the many proteins that are potentially implicated in LCR function is Bach1. Bach1 possesses a basic leucine zipper (bZip) domain, as well as a BTB/POZ domain that has been shown to be involved in the regulation of chromatin structure. Bach1 forms heterodimers with small Maf proteins through its leucine zipper and binds to Maf recognition elements (MARE). RESULTS: Using atomic force microscopy we visualized large looped DNA structures between MAREs located in different regulatory elements within the human beta-globin LCR that were mediated by Bach1/MafK heterodimers. The formation of these DNA loops required the Bach1 BTB/POZ protein interaction domain. Furthermore, in transfection studies we found that Bach1 repressed the enhancer activity of the LCR in a BTB/POZ domain-dependent manner. CONCLUSION: Our results suggest that Bach1 and other BTB/POZ transcription factors may represent a class of nuclear architectural proteins that mediate long range interactions between cis-regulatory elements in order to regulate gene expression.

Basic-Leucine Zipper Transcription Factors↗
PubMed1998

The Na+,K+-ATPase carrying the carboxy-terminal Ca2+/calmodulin binding domain of the Ca2+ pump has 2Na+,2K+ stoichiometry and lost charge movement in Na+/Na+ exchange.

An altered ion-transport stoichiometry from 3Na+,2K+ to 2Na+,2K+ is observed in a chimeric Na+,K+ATPase, which carries the Ca2+/calmodulin binding domain (CBD) of the plasma membrane Ca2+-ATPase at its carboxy-terminus [Zhao et al., FEBS Lett. 408 (1997) 271-2751. The ouabain-resistant mutant of this chimera (ORalpha1-CBD) was constructed to further investigate the effect of the CBD on ion-transport properties. The ORalpha1-CBD still shows the 2Na+,2K+ stoichiometry. The loss of electrogenicity is accompanied by the disappearance of transient charge movements in the Na+/Na+ exchange mode. We conclude that the binding of the third Na+ ion, but not of the two others, in 3Na+,2K+ transport mode apparently senses the electric field, and that the voltage-dependent Na+ binding is likely to be lost in the chimera with CBD.

Animals↗
PubMed1998

Molecular structure of human topoisomerase II alpha revealed by atomic force microscopy.

The entire human topoisomerase II alpha (hTopoII alpha) dimer was expressed in the yeast Saccaromyces cerevisiae, purified to homogeneity, and subjected to atomic force microscopy (AFM) under a tapping mode. Molecular images obtained exhibited a 'heart or donut-like' structure with a large axial hole. The main benefit of the application of AFM to study the hTopoII alpha is that clear images of the internal 'pore' have been achieved without crystallization, staining, or fixation of the sample. These images are consistent with the model in which topoisomerase II has a large internal gate for DNA strand passage.

Antigens, Neoplasm↗
PubMed1998

Structural studies of the vacuolar H(+)-pyrophosphatase: sequence analysis and identification of the residues modified by fluorescent cyclohexylcarbodiimide and maleimide.

We determined the amino acid residues of the H(+)-translocating inorganic pyrophosphatase (H(+)-PPase) of pumpkin which are covalently labeled by two fluorescent labeling reagents; N-cyclohexyl-N'-[4-(dimethyl amino)-alpha-naphthyl] carbodiimide (NCD) and N-pyrenylmaleimide (NPM). NCD and NPM are fluorescent analogues of N,N-dicycrohexylcarbodiimide and N-ethylmaleimide, respectively, and inactivate H(+)-PPase activity. Excess Mg2+ protected the H(+)-PPase from the inactivation by these reagents. Furthermore, we identified the cDNA clone encoding the pumpkin H(+)-PPase in order to determine the position of labeled residues. The nucleotide sequence of the cDNA clone contains a 2,304 bp open reading frame encoding a polypeptide with 768 amino acids. Chemical sequence analysis of fluorescent peptide fragments revealed that Glu749 located in the C-terminal putative transmembrane alpha-helix was a NCD-labeled residue, and Cys632 was a NPM-labeled residue located in a putative cytosolic domain. The amino acid sequence of the region that includes Glu749 is highly conserved in H(+)-PPases from other plants and it also shows some sequence similarity with the region of the carbodiimide-reactive Glu (or Asp) of F0F1-ATPase c-subunit. The reactive glutamic acids in these proteins are located at the last C-terminal transmembrane alpha-helix. We also found that the H(+)-PPase shows significant amino acid sequence similarity to Kdp-ATPase A chain of E. coli. This similarity between the two different proteins suggest that they have evolved from a common ancestor and may utilize a common basic mechanism for ion transport.

Amino Acid Sequence↗
PubMed1997

The Ca2+/calmodulin binding domain of the Ca2+-ATPase linked to the Na+,K+-ATPase alters transport stoichiometry.

Using Xenopus oocytes as an expression system, we have investigated ion-transport and ouabain-binding properties of a chimeric ATPase (alpha1-CBD; Ishii and Takeyasu (1995) EMBO J. 14, 58-67) formed by the alpha1-subunit of chicken Na+,K(+)-ATPase (alpha1) and the calmodulin binding domain (CBD) of the rat plasma membrane Ca2(+)-ATPase. alpha1-CBD can be expressed and transported to the oocyte plasma membrane without the beta-subunit, and shows ouabain binding. In contrast to ouabain binding, this chimera requires the beta-subunit for its cation (Na+ and K+) transport activity. alpha1-CBD exhibits an altered stoichiometry of Na(+)-K+ exchange. A detailed analysis of 22Na+ efflux, 86Rb+ uptake, pump current and ouabain binding suggests that the chimeric molecule can operate in an electrically silent 2Na(+)-2K+ exchange mode and, with much lower probability, in its normal 3Na(+)-2K+ exchange mode.

Animals↗
PubMed1997

Carboxy-terminal regions of the sarcoplasmic/endoplasmic reticulum Ca(2+)- and the Na+/K(+)-ATPases control their K+ sensitivity.

The Na+,K(+)-ATPase and the sarcoplasmic/endoplasmic reticulum Ca(2+)-(SERCA-) ATPase belong to a family of P-type ATPases that undergo a cycle of conformational changes between the phosphorylated and dephosphorylated stages in an ion-specific manner. The ouabain-inhibitable Na+,K(+)-ATPase activity requires Na+ and K+. On the other hand, the Ca(2+)-dependent and thapsigargin-inhibitable activity of the SERCA-ATPase does not depend upon Na+ and K+ for its basal activity. However, the SERCA-ATPase and Ca(2+)-transport activities can be further activated either by K+ in a two-step fashion with high (ED50 approximately 20 mM) and low affinity (ED50 approximately 70 mM) or by Na+ in a one-step fashion with an ED50 value of approximately 50 mM. A chimera, in which the carboxy-terminal region (Leu861-COOH) of the Na+,K(+)-ATPase alpha 1 subunit replaced the corresponding region (Ser830-COOH) of the SERCA1-ATPase, lacked the low-affinity K+ activation of the SERCA-ATPase but displayed a higher-affinity (ED50 < 10 mM) activation by K+, similar to that of the Na+,K(+)-ATPase, whereas activation by Na+ was not affected. The replacement of the large cytosolic loop (Gly354-Lys712) and the amino-terminal regions (Met1-Asp162) of the SERCA1-ATPase with the corresponding portions of the Na+,K(+)-ATPase alpha 1 subunit did not affect the sensitivity of the SERCA-ATPase activity to K+. Thus, the carboxy-terminal regions of both the SERCA1 and the Na+,K(+)-ATPase alpha 1 subunit are critical for K+ sensitivity. Analysis of additional (Ca2+/Na+,K+)-ATPase chimeras demonstrated that the carboxy-terminal 102 amino acids (Phe920-Tyr1021) of the Na+/K(+)-ATPase alpha 1 subunit are sufficient to shift the K+ affinity for activation of the SERCA-ATPase without the beta subunit. No change in the two-step activation of SERCA-ATPase by K+ was seen when residues Thr871-Thr898 of the SERCA1-ATPase were replaced with residues Asn894-Ala919 of the Na+,K(+)-ATPase alpha 1 subunit, a region known to bind the Na+,K(+)-ATPase beta subunit [Lemas, M. V., et al. (1994) J. Biol. Chem. 269, 8255-8259]. Thus, the Na+,K(+)-ATPase subunit-assembly domain and the K(+)-sensitive region are distinct within the carboxy-terminal 161 amino acids of the Na+,K(+)-ATPase.

Animals↗
PubMed1997

1,25-Dihydroxyvitamin D3 selectively induces increased expression of the Na,K-ATPase beta 1 subunit in avian myelomonocytic cells without a concomitant change in Na,K-ATPase activity.

Treatment of avian myelomonocytic cells with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) results in an approximately two fold increase in levels of Na,K-ATPase beta 1 subunit mRNA and protein (both total and plasma membrane-associated). The changes in beta 1 subunit expression occur in the absence of a detectable increase in expression of any of the three alpha subunit isoforms or in Na,K-ATPase activity. The selective induction of the expression of the beta subunit in avian myelomonocytic cells by 1,25(OH)2D3 reveals a previously unobserved feature of the regulation of Na,K-ATPase expression, while the targeting of beta subunit polypeptides to the plasma membrane in the absence of a corresponding increase in active Na,K-ATPase suggests that, in these cells, transport of the beta subunit to the plasma membrane may be independent of its binding to the alpha subunit.

Animals↗
PubMed1997

The Drosophila Na,K-ATPase alpha-subunit gene: gene structure, promoter function and analysis of a cold-sensitive recessive-lethal mutation.

The Drosophila Na,K-ATPase (or sodium pump) alpha-subunit gene was found to contain 10 exons and span approx. 25 kb. Two nearly adjacent transcriptional initiation sites were identified, and the 2085-nucleotide sequence upstream of the first transcriptional start was analysed for promoter activity in transfected Drosophila SL2 cells. This region was found to contain many cis-acting elements that influence promoter activity, including elements that confer 2- to 3-fold higher activity in SL2 cells cultured at 30 degrees C versus 22 degrees C. Temperature-sensitive transcriptional regulation of the Na,K-ATPase alpha-subunit in Drosophila is a plausible mechanistic candidate for the factor driving temperature-dependent up-regulation of the Na,K-ATPase alpha-subunit described here for fly strains homozygous for single P-element insertions in the alpha-subunit gene. Four new P-element insertion strains were identified in this study, each insertion site lying within the first intron of the Na,K-ATPase alpha-subunit gene. The insertion in strain 0462 resulted in cold-sensitive recessive lethality; flies homozygous for the 0462 mutation could be rescued by growth at 29-30 degrees C, a condition that partially corrected a deficiency in the level of Na,K-ATPase alpha-subunit. The high-temperature rescue of homozygous 0462 flies appeared to result primarily from improved Na,K-ATPase expression rather than an increase in the rate of ion transport per Na,K-ATPase molecule. These observations point to a role for sodium-pump activity in determining the range of temperature tolerance in Drosophila and demonstrate that relatively subtle changes in sodium-pump expression can have major consequences in whole organisms.

Adaptation, Physiological↗
PubMed1997

Primary structure and evolution of the ATP-binding domains of the P-type ATPases in Tetrahymena thermophila.

The P-type ATPases (e.g., Na+-K+-ATPase and Ca2+-ATPase) occur widely in living cells of fungi, Protozoa, plants, and animals. These ion pumps show a high degree of divergence in their primary structures but share a limited number of common amino acid residues for their ATP-catalytic function. Particularly, the amino acid sequences for the phosphorylation site (DKTGTLT) and the binding site for ATP (and its analogs; GDGVND) are conserved throughout evolution. Using two degenerate oligonucleotides corresponding to these regions, we applied a polymerase chain reaction (PCR) technique to the search for P-type ATPase isoforms, which will provide a clue to the evolutionary mechanisms of ion pumps in Tetrahymena thermophila. A total of 12 distinct P-type ATPase genes were identified. Sequence comparisons revealed that seven of them can be compiled into a multigene family, which is similar to animal Na+-K+- and H+-K+-ATPase genes. One of them is close to the sarco(endo)plasmic reticulum Ca2+-ATPase gene, and the other four share a significant homology with the gene encoding Plasmodium ATPase-1 whose function is unknown. A Northern blot analysis and reverse transcriptase-PCR demonstrated that all identified genes are expressed, but the expression levels vary widely under different culture conditions. A Southern blot analysis after pulse-field gel electrophoresis showed that all of these genes exist in T. thermophila macronuclei. The Na+-K+- and H+-K+-ATPase gene family has a high multiplicity (at least 10 different genes detected on genomic Southern blot analysis) and is distributed on four different macronuclear chromosomes. On the basis of a calculation with the amino acid sequences of the cloned cytoplasmic loop region (between the phosphorylation and the gamma-[4-(N-2-chloroethyl-N-methylamino)]-benzylamido ATP sites), the genes with >80% identity form a cognate linkage group within the same macronuclei chromosome, whereas the genes with <70% identity are separated in different chromosomes. The phylogenetic analysis showed that this multigene family is the result of a series of gene duplications.

Adenosine Triphosphatases↗
PubMed1996

Negative transcriptional regulation of the chicken Na+/K(+)-ATPase alpha 1-subunit gene.

Although the Na+/K(+)-ATPase alpha 1-subunit gene is ubiquitously expressed in vertebrates, its level of expression varies among tissue and cell types. In spite of similar mRNA distribution in tissues of mammals and birds, the 5'-flanking regions of alpha 1-subunit genes exhibit remarkable diversity; i.e., the core promoter activity of the TATA-less chicken alpha 1 gene strongly depends upon multiple Sp1-based regulation (six Sp1 sites), whereas the promoter activity of the TATA-like rat alpha 1-subunit gene relies on the two Sp1 and additional positive regulatory factors. Further analysis of the regulatory regions of the Na+/K(+)-ATPase alpha 1-subunit genes revealed that the vertebrate alpha 1-subunit genes may share common inhibitory mechanisms for subtle transcriptional regulation; the core promoter activities can be either enhanced or repressed depending on the availability of inhibitory factors. Two potential candidates for such inhibitory elements in both avian and mammalian Na+/K(+)-ATPase alpha 1-subunit genes are (1) a newly identified element, GCCCTC, and (2) a GCF-binding sequence, NN[G/c]CG[G/c][G/c][G/c]CN, or its reverse complement. Gel retardation assays using the inhibitory region of the chicken gene and crude nuclear extracts from tissue-cultured chicken and mouse cells showed the existence of a set of proteins that bind to this region. The amounts of individual regulatory proteins in different cell types seem to vary, resulting in differential formation of DNA/protein complexes in different cell types. Thus, the regulation of Na+/K(+)-ATPase alpha 1-subunit gene expression under different cellular environment as well as in different cell types can be achieved by a shared mechanism; modulation of the ratio of the abundance of individual inhibitory factors.

Animals↗
PubMed1996

Quantitative analysis of the transcription factor AP2 binding to DNA by atomic force microscopy.

Atomic force microscopy (AFM) allows to study the molecular structure of biological macromolecules with nm to A resolutions without crystallization. We show here the applicability of AFM in the quantitative analysis of the molecular mechanisms of DNA/protein interaction: (i) Protein-binding sites can be mapped over a few kilobases of target DNA. (ii) Multimerization state of DNA-binding proteins can be determined simply by measuring the sizes of proteins bound to the DNA. These features are significant advantages over the capabilities provided by conventional techniques in biochemistry and molecular and structural biology.

Animals↗
PubMed1996

Atomic force microscopy proposes a novel model for stem-loop structure that binds a heat shock protein in the Staphylococcus aureus HSP70 operon.

The Staphylococcus aureus HSP70 operon produces a polycistronic RNA in response to heat shock, and ORF37 is the first protein to be translated. The promoter of this operon contains a palindromic nucleotide sequence that may form a stem-loop structure. Structural analysis of the promoter regions by atomic force microscopy (AFM) revealed a quadruplet that consists of a pair of stem-loops. A novel "SL2S' (Stem-Loop-Loop-Stem) model was proposed for this structure. AFM also revealed the binding of ORF37 to the quadruplet, establishing a molecular mechanism for this heat shock gene expression; ORF37 acts as a regulator by binding to the SL2S structure in the promoter.

Base Sequence↗