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K Akama

Publications and source records attributed to K Akama.

At least 19 recordsLinked to original sources
PubMed2001

Rice (Oryza sativa) contains a novel isoform of glutamate decarboxylase that lacks an authentic calmodulin-binding domain at the C-terminus.

We have isolated full-length cDNAs for two distinct isoforms of glutamate decarboxylase (GAD), designated OsGAD1 and OsGAD2 from a rice shoot cDNA library. Open reading frames found in OsGAD1 and OsGAD2 cDNAs encode putative proteins of 501 (56.7 kDa) and 500 amino acids (55.6 kDa), respectively. They show 69% identity to each other and 67-78% identity to dicotyledonous counterpart sequences determined so far. Comparative analysis of relevant genomic clones obtained from the rice genomic library with these cDNAs as probes demonstrated that the number and sizes of introns deduced for these two genes differ considerably. Interestingly, in the regions in the putative gene products corresponding to the C-terminal 30-amino-acid peptide known as the calmodulin-binding domain of plant GADs, OsGAD1 possesses a typical motif, while OsGAD2 contains several substitutions of amino acids that contribute strongly to the binding of calmodulin (CaM). An in vitro CaM-binding assay of these proteins over-expressed in Escherichia coli revealed that OsGAD1 can in fact bind specifically to bovine CaM but OsGAD2 cannot. RNA analysis showed that transcripts of OsGAD1 and OsGAD2 were present in all tissues examined, but their expression was differentially regulated, at least in roots and maturing seeds.

Amino Acid Sequence↗
PubMed2001

Tracking the estrogen receptor in neurons: implications for estrogen-induced synapse formation.

Estrogens (E) and progestins regulate synaptogenesis in the CA1 region of the dorsal hippocampus during the estrous cycle of the female rat, and the functional consequences include changes in neurotransmission and memory. Synapse formation has been demonstrated by using the Golgi technique, dye filling of cells, electron microscopy, and radioimmunocytochemistry. N-methyl-d-aspartate (NMDA) receptor activation is required, and inhibitory interneurons play a pivotal role as they express nuclear estrogen receptor alpha (ERalpha) and show E-induced decreases of GABAergic activity. Although global decreases in inhibitory tone may be important, a more local role for E in CA1 neurons seems likely. The rat hippocampus expresses both ERalpha and ERbeta mRNA. At the light microscopic level, autoradiography shows cell nuclear [3H]estrogen and [125I]estrogen uptake according to a distribution that primarily reflects the localization of ERalpha-immunoreactive interneurons in the hippocampus. However, recent ultrastructural studies have revealed extranuclear ERalpha immunoreactivity (IR) within select dendritic spines on hippocampal principal cells, axon terminals, and glial processes, localizations that would not be detectable by using standard light microscopic methods. Based on recent studies showing that both types of ER are expressed in a form that activates second messenger systems, these findings support a testable model in which local, non-genomic regulation by estrogen participates along with genomic actions of estrogens in the regulation of synapse formation.

Animals↗
PubMed2001

EXAFS analysis of the zinc-binding domain of boar spermatidal transition protein 2.

Boar Spermatidal Transition Protein 2 (TP2; 137 amino acid residues) is supposed to play an important role in initiation of chromatin condensation and cessation of transcriptional activity during mammalian spermniogenesis. Boar TP2 has three potential zinc finger motifs and binds three atoms of zinc per molecule. However the structure of the zinc-binding domain of boar TP2 has not been completely determined. To elucidate the local structure around the zinc atoms of boar TP2, we performed an X-ray absorption fine structure (XAFS) measurement on the zinc-binding domain of TP2(TP2Z)(residues 1-103) in the fluorescence mode. By EXAFS analyses we have demonstrated that each of the three zinc atoms is coordinated by approximately two sulfur and two nitrogen atoms on average. The average Zn-S and Zn-N distances were found to be 2.36 and 2.01 A, respectively. The sulfur and nitrogen atoms are attributed to cysteine and histidine residues, respectively, from comparison of the EXAFS spectra with model compounds ZnS and ZnTPP zinc(II) tetraphenylporphyrin).

Amino Acid Sequence↗
PubMed2001

A tobacco nuclear extract supporting transcription, processing, splicing and modification of plant intron-containing tRNA precursors.

Nuclear tRNA genes are transcribed by RNA polymerase III (Pol III) and pre-tRNAs are processed into mature tRNAs via complex processes in the nucleus. We have developed an in vitro Pol III-dependent transcription system derived from tobacco cultured cells, which supports efficiently not only transcription of a variety of plant tRNA genes but also 5'-and 3'-end processing, nucleotide modification and splicing of intron-containing pre-tRNAs. The structures of in vitro transcripts have been confirmed by primer extension analysis and by RNase T1 fingerprinting. The optimal Mg2+ concentration differed for each step so that each reaction can be controlled by adjusting the Mg2+ concentration. At 1 mm Mg2+, only transcription occurs so that pre-tRNAs accumulate. The splicing reaction can be initiated by raising Mg2+ ions (> 5 mm) and enhanced by adding 1 mm hexamminecobalt chloride. Using the optimized system for the Nicotiana intron-containing tRNATyr gene, the precise initiation and termination sites of transcription and the splice sites were determined. The presence of 1 mm NAD+ in the reaction mixture leads to the removal of the 2' phosphate at the splice junction of tRNATyr, demonstrating the activity of a 2'-phosphotransferase in the tobacco nuclear extract. Many modified nucleosides such as m2G, m22G, m1A, phi27 and phi35 are introduced in either of the studied transcripts. As shown in other systems, the conversion of U35 to phi requires an intron-containing substrate.

Base Sequence↗
PubMed2000

Identification of two catalytic subunits of tRNA splicing endonuclease from Arabidopsis thaliana.

tRNA splicing endonuclease is essential for the correct removal of introns from precursor tRNA molecules of Archaea and Eucarya. The only well-characterized eucaryotic enzyme until now is the endonuclease from yeast (Saccharomyces cerevisiae). This protein has a heterotetrameric structure. Two of the four subunits, i.e. Sen34 and Sen44, contain the active sites for cleavage at the 3'- and 5'-splice sites, respectively. We have identified three novel genes from Arabidopsis thaliana, encoding putative subunits of tRNA splicing endonuclease. They are designated as AtSen1, AtSen2, and AtpsSen1. Both genes AtSen1 and AtSen2 seem to be functionally active, as deduced from corresponding cDNA sequences. Comparison of the amino acid sequences of the these two Arabidopsis proteins revealed 72% identity. However, AtpsSen1 is more similar to AtSen1, but is very likely a pseudogene, as concluded from extended stretches of deletions and the presence of in-frame stop codons. All putative proteins contain a conserved domain at their C-terminus common to counterparts from other organisms. Interestingly, they are more similar to the yeast catalytic subunit Sen44 than to Sen34. Southern analysis with various probes revealed that each gene is present as single copies in the nuclear genome. The evolutionary implications of these findings are discussed.

Amino Acid Sequence↗
PubMed2000

Splicing of arabidopsis tRNA(Met) precursors in tobacco cell and wheat germ extracts.

Intron-containing tRNA genes are exceptional within nuclear plant genomes. It appears that merely two tRNA gene families coding for tRNA(GpsiA(Tyr)) and elongator tRNA(CmAU(Met)) contain intervening sequences. We have previously investigated the features required by wheat germ splicing endonuclease for efficient and accurate intron excision from Arabidopsis pre-tRNA(Tyr). Here we have studied the expression of an Arabidopsis elongator tRNA(Met) gene in two plant extracts of different origin. This gene was first transcribed either in HeLa or in tobacco cell nuclear extract and splicing of intron-containing tRNA(Met) precursors was then examined in wheat germ S23 extract and in the tobacco system. The results show that conversion of pre-tRNA(Met) to mature tRNA proceeds very efficiently in both plant extracts. In order to elucidate the potential role of specific nucleotides at the 3' and 5' splice sites and of a structured intron for pre-tRNA(Met) splicing in either extract, we have performed a systematic survey by mutational analyses. The results show that cytidine residues at intron-exon boundaries impair pre-tRNA(Met) splicing and that a highly structured intron is indispensable for pre-tRNA(Met) splicing. tRNA precursors with an extended anticodon stem of three to four base pairs are readily accepted as substrates by wheat and tobacco splicing endonuclease, whereas pre-tRNA molecules that can form an extended anticodon stem of only two putative base pairs are not spliced at all. An amber suppressor, generated from the intron-containing elongator tRNA(Met) gene, is efficiently processed and spliced in both plant extracts.

Arabidopsis↗
PubMed2000

Protamine dissociation before decondensation of sperm nuclei during in vitro fertilization of pig oocytes.

The correlation between morphological changes and the dynamics of protamine in boar sperm chromatin during in vitro fertilization of pig oocytes matured in vitro was assessed. For this purpose, protamine was purified from boar sperm nuclei and an antiserum against protamine was developed. After affinity purification, the antiserum reacted exclusively with boar protamine during western blotting, showing no crossreactivity with core histones. Immunohistochemical evaluation revealed that only fully developed spermatid nuclei in boar testes stained strongly with the antiserum. When pig oocytes matured in vitro were fertilized in vitro, sperm penetration was observed in 37% of oocytes at 2 h after insemination and the penetration rate increased to 99% by 5 h after insemination, accompanied by an increase in polyspermic penetration. Paraffin wax sections of the inseminated oocytes were examined by immunohistochemical analysis with the antiserum. The proportion of condensed sperm nuclei that reacted with the antiserum was 87% of the sperm nuclei that penetrated by 2 h after insemination, and this decreased to 20 and 13% at 3 and 5 h after insemination, respectively. However, none of the decondensing sperm nuclei or male pronuclei reacted with the antiserum during the entire insemination period. These results indicate that a specific antiserum against boar protamine can be raised and, using this serum, it has been demonstrated that protamine is dissociated from boar sperm nuclei before decondensation during in vitro fertilization.

Analysis of Variance↗
PubMed1999

Transition protein 4 from boar late spermatid nuclei is a topological factor that stimulates DNA-relaxing activity of topoisomerase I.

Transition protein 4 (TP4) from boar late spermatid nuclei, having higher affinity for double-stranded DNA and a local melting activity of DNA, stimulated SV40 DNA-relaxing activity of eukaryotic topoisomerase I at TP4/DNA molar ratios of 6.6-11. A TP4-spermidine mixture stimulated the activity of topoisomerase I much more than spermidine alone, but no more than TP4 alone, and poly-L-arginine did not. These results suggest that TP4 contributes to the chromatin reorganization in the late spermatid nuclei from nucleosomal-type structure with negatively supercoiled DNA to nucleoprotamine structure with no supercoiled DNA.

Animals↗
PubMed1999

Expression of a zinc-binding domain of boar spermatidal transition protein 2 in Escherichia coli.

Transition protein 2 (TP2; 137 amino acid residues) from boar late spermatid nuclei has three potential zinc finger motifs in the N-terminal 34 region. Gel shift assays revealed that boar TP2 recognized a CpG island sequence in a zinc-dependent manner. However, there was some nonspecific recognition of the oligonucleotide. Then, we constructed the expression system of zinc-binding domain of TP2 (TP2Z) (residues 1-103) in Escherichia coli. Double-stranded DNA fragments encoding TP2Z were synthesized as 18 fragments with 103 residues, annealed, and cloned into the expression plasmid pET11d. TP2Z was expressed upon induction with 1 mM isopropylthiogalactoside and extracted with acid including 0.71 M 2-mercaptoethanol. TP2Z was purified by ion-exchange chromatography on Fractogel EMD SO(-)(3) and HPLC on Nucleosil 300 7C18 and on Diol-120. Atomic absorption and CD spectroscopy showed that TP2Z bound three atoms of zinc per molecule of the protein and underwent a zinc-dependent conformational change in a manner similar to that for intact TP2. Gel shift assays indicated that TP2Z recognized a CpG island sequence more specifically than intact TP2 and that the specificity is dependent on zinc.

Amino Acid Sequence↗
PubMed1999

Molecular characterization of a gene for alanine aminotransferase from rice (Oryza sativa).

A cDNA clone encoding alanine aminotransferase (AlaAT) has isolated from randomly sequenced clones derived from a cDNA library of maturing rice seeds by comparison to previously identified genes. The deduced amino acid sequence was 88% and 91% homologous to those of the enzymes from barley and broomcorn millet (Panicum miliaceum), respectively. Using this cDNA as a probe, we isolated and sequenced the corresponding genomic clone. Comparison of the sequences of the cDNA and the genomic gene revealed that the coding region of the gene was interrupted by 14 introns 66 to 1547 bp long. Northern and western blotting analyses showed that the gene was expressed at high levels in developing seeds. When the 5'-flanking region between -930 and +85 from the site of initiation of transcription was fused to a reporter gene for beta-glucuronidase (GUS) and then introduced into the rice genome, histochemical staining revealed strong GUS activity in the inner endosperm tissue of developing seeds and weak activity in root tips. Similar tissue-specific expression was also detected by in situ hybridization. These results suggest that AlaAT is involved in nitrogen metabolism during the maturation of rice seed.

Alanine Transaminase↗
PubMed1999

Molecular cloning and characterization of a nuclear gene encoding a putative subunit of tRNA splicing endonuclease from Arabidopsis thaliana.

tRNA splicing endonuclease is required to produce mature tRNAs from intron-containing tRNA precursors. To characterize the structural features of plant endonuclease, we have isolated a cDNA and a corresponding genomic DNA clone from libraries of Arabidopsis thaliana which encode a putative subunit of the endonuclease. The gene product has an apparent mass of 27 kDa and contains a homologous domain of approximately 130 amino acids at the C-terminal region commonly found in other eucaryal and archaeal counterparts. Southern hybridization analysis of Arabidopsis genomic DNA utilizing the cDNA clone as probe indicates the presence of at least two related genes.

Arabidopsis↗
PubMed1998

Plant cytosolic tRNAHis possesses an exceptional C54 in the canonical TPsiC loop.

A nuclear gene coding for tRNAHis from Arabidopsis has been reported to contain C54in the TPsiC loop, although the corresponding nucleotide is an invariant U or a derivative in nearly all other tRNAs. The only previously reported plant cytosolic tRNAHis sequence, from lupin, has U54. To re-examine plant cytosolic tRNAsHis and their genes we have used DNA and RNA sequence analyses, restriction enzyme digestion of PCR-amplified tRNA genes, RNA hybridization and in vivo aminoacylation assays. Our results suggest that Arabidopsis nuclear tRNAHis genes ubiquitously contain C54, as do those from tobacco, lupin and pea. The C54 nucleotide is maintained in the mature tRNAHis, which is aminoacylated in vivo , but to a relatively low level compared with other tRNAs examined. Finally, it was shown that an Arabidopsis tRNAHis gene with T54in place of C54 is over 5-fold more transcriptionally active than the wild-type gene using an in vitro system derived from plant nuclei. A possible role for this apparently sub-optimal tRNAHis sequence is suggested.

Acylation↗
PubMed1998

Transition protein 1 from boar late spermatid nuclei having DNA-melting activity is a dimeric protein.

Polyacrylamide gel electrophoretic behavior of boar transition protein 1, TP1, under dissociating and non-dissociating buffer conditions, and titration of fluorescently labeled TP1 with increasing amounts of TP1 showed that TP1 formed a dimer without intermolecular disulfide bond. TP1 dimer with intermolecular disulfide bond had similar DNA-melting activity to TP1, but was not detected in extracts from boar late spermatid nuclei. These results suggest that TP1 dimer without intermolecular disulfide bond induces local destabilization of DNA in the late spermatid nuclei.

Animals↗
PubMed1997

Characterization of nuclear tRNA(Tyr) introns: their evolution from red algae to higher plants.

We have previously isolated numerous intron-containing nuclear tRNA(Tyr) genes derived from either monocotyledonous (Triticum) or dicotyledonous (Arabidopsis, Nicotiana) plants by screening the corresponding genomic phage libraries with a synthetic tRNA(Tyr)-specific oligonucleotide. Here we have characterized additional tRNA(Tyr) genes from phylogenetically divergent plant species representing red algae (Champia), brown algae (Cystophyllum), green algae (Ulva), stonewort (Chara), liverwort (Marchantia), moss (Polytrichum), fern (Rumohra) and gymnosperms (Ginkgo) using amplification of the coding sequences from the corresponding genomic DNAs by polymerase chain reaction (PCR). All novel tRNA(Tyr) genes contain intervening sequences of variable sequence and length ranging in size from 11 to 21 bp. However, two features are conserved in all plant pre-tRNA(Tyr) introns: they possess a uridine and less frequently an adenosine at the 5' boundary and can adopt similar intron secondary structures in which an extended anticodon helix of 4-5 bp is formed by base-pairing between nucleotides of the intron and the anticodon loop. In order to elucidate the potential role of the highly conserved uridine at the first intron position, we have replaced it by all other nucleosides in an Arabidopsis pre-tRNA(Tyr) and have studied in wheat germ extract its effect on splicing and on conversion of U to psi in the GpsiA anticodon. Furthermore, we discuss the putative acquisition of tRNA(Tyr) introns at an early step of evolution after the separation of Archaea and Eucarya.

Anticodon↗
PubMed1997

Isolation of intact transition protein 2 with three zinc finger motifs from boar late spermatid nuclei.

Boar intact transition protein 2, TP2, was isolated from the late spermatid nuclei by chromatography on Fractogel EMD SO3- 650 (M), and HPLCs on Nucleosil 300 7C18, Diol-120 and Chemcosorb 3C18H. CD spectroscopy study showed that TP2 underwent a small but significant zinc dependent secondary structural change. TP2, having three potential zinc finger motifs, was shown to be contain 3 atoms of zinc bound per molecule of the protein by atomic absorption spectroscopy. These results together with the amino acid sequence of TP2 suggest that TP2 is a zinc metalloprotein with three zinc finger structures.

Amino Acid Sequence↗
PubMed1996

Striking differences in mitochondrial tRNA import between different plant species.

A systematic comparison of the tRNAs imported into the mitochondria of larch, maize and potato reveals considerable differences among the three species. Larch mitochondria import at least eleven different tRNAs (more than half of those tested) corresponding to ten different amino acids. For five of these tRNAs [tRNA(Phe(GAA)), tRNA(Lys(CUU)), tRNA(Pro(UGG)), tRNA(Ser(GCU)) and tRNA(Ser(UGA))] this is the first report of import into mitochondria in any plant species. There are also differences in import between relatively closely related plants; wheat mitochondria, unlike maize mitochondria import tRNA(His), and sunflower mitochondria, unlike mitochondria from other angiosperms tested, import tRNA(Ser(GCU)) and tRNA(Ser(UGA)). These results suggest that the ability to import each tRNA has been acquired independently at different times during the evolution of higher plants, and that there are few apparent restrictions on which tRNAs can or cannot be imported. The implications for the mechanisms of mitochondrial tRNA import in plants are discussed.

Base Sequence↗