Biomedical subjects

Y Shimura

Publications and source records attributed to Y Shimura.

At least 19 recordsLinked to original sources

PubMed1999

Chemical shift perturbation studies of the interactions of the second RNA-binding domain of the Drosophila sex-lethal protein with the transformer pre-mRNA polyuridine tract and 3' splice-site sequences.

The interactions of the second RNA-binding domain of the Drosophila melanogaster Sex-lethal protein (Sxl RBD2) with the oligoribonucleotides, GUUUUUUUU (GU8) and CUAGUG, representing the sequences surrounding an alternative 3'-splicing site of the transformer pre-mRNA (GU8CUAGUG), were studied using heteronuclear two-dimensional NMR techniques. The 1H and 15N chemical shifts of the backbone amide resonances upon titration of Sxl RBD2 with each of these RNAs were recorded. It was found that Sxl RBD2 can bind not only to the polyuridine tract, GU8, but also to the downstream 3' splice-site sequence, CUAGUG, with similar affinities. In contrast, a nonspecific sequence, C8, did not bind to Sxl RBD2. This result is consistent with previous in vitro RNA-selection and UV-cross-linking results which indicated that the Sex-lethal protein binds to the uridine stretch and the AG dinucleotide in the consensus sequence, AUnNnAGU. In both cases, the chemical-shift perturbations were significant for almost the same amino acid residues, including the two central beta-strands formed by the RNP2-motif and RNP1-motif with the two highly conserved aromatic residues (Y214 and F256) in the middle. As the first RNA-binding domain of Sex-lethal (Sxl RBD1) has a characteristic aliphatic residue at one of the two corresponding positions (I128 and F170), Y214 of Sxl RBD2 was replaced by Ile using site-directed mutagenesis. On the one hand, the 1H and 15N chemical-shift perturbations indicated that GU8 binds to the same interface of mutant Sxl RBD2 as of wild-type Sxl RBD2, although its binding affinity was decreased significantly. On the other hand, the specific binding of Sxl RBD2 to CUAGUG was abolished almost completely by the Y-->I mutation. Taken together, the present results indicate that the interface residues that bind with GU8 and CUAGUG are much the same, but the role of the Y214 residue is clearly different between these two target sequences.

Amides↗

PubMed1999

FILAMENTOUS FLOWER controls the formation and development of arabidopsis inflorescences and floral meristems.

Phenotypic analysis of single and multiple mutants as well as in situ localization analysis of the expression patterns of floral genes have revealed that the FIL AMENTOUS FLOWER (FIL) gene plays important roles in establishing the inflorescence in Arabidopsis. As previously reported, the fil mutant generates clusters of both filamentous structures and flowers with floral organs of altered number and shape. The structural resemblance of the filamentous structures to peduncles and the expression pattern of the APETALA1 (AP1) gene have shown that these filamentous structures are underdeveloped flowers that fail to form receptacles and floral organs, indicating that one of the roles of the FIL gene is to support the development of the floral meristem. That FIL also is involved in fate determination in the floral meristem is demonstrated by the homeotic conversion of flowers to inflorescences in fil ap1 double mutants and in fil ap1 cauliflower triple mutants. In double mutants with flowering-time loci (i.e., f t or f wa), leafy (lf y), and unusual floral organs (ufo), filamentous structures are formed, but very few or no flowers with floral organs develop. The enhanced phenotype in the fil ap1 and the fil lfy double mutants suggests that the FIL protein may work together with AP1 and LFY proteins. The FIL gene also may be involved in the cell fate determination of floral organ primordia, possibly by controlling the spatial expression patterns of the class A and C floral organ identity genes.

Arabidopsis↗

PubMed1997

The Arabidopsis HY5 gene encodes a bZIP protein that regulates stimulus-induced development of root and hypocotyl.

Plant developmental processes are controlled by both endogenous programs and environmental stimuli. As a photomorphogenetic mutant, hy5 of Arabidopsis has been isolated and characterized. Our detailed characterization has revealed that the mutant is deficient in a variety of stimulus responses, including gravitropic response and waving growth of roots, as well as light-dependent hypocotyl elongation. In the roots and hypocotyl, the hy5 mutation also affects greening and specific cell proliferation such as lateral root formation and secondary thickening. Those phenotypes indicate that the HY5 gene is responsible for the regulation of fundamental developmental processes of the plant cell: cell elongation, cell proliferation, and chloroplast development. Molecular cloning of the HY5 gene using a T-DNA-tagged mutant has revealed that the gene encodes a protein with a bZIP motif, one of the motifs found in transcriptional regulators. Nuclear localization of the HY5 protein strongly suggests that the HY5 gene modulates the signal transduction pathways under the HY5-related development by controlling expression of genes downstream of these pathways.

Amino Acid Sequence↗

PubMed1997

A characteristic arrangement of aromatic amino acid residues in the solution structure of the amino-terminal RNA-binding domain of Drosophila sex-lethal.

The Sex-lethal (Sxl) protein from Drosophila melanogaster has two RNA-binding domains (RBDs). As the amino-terminal RBD (RBD1) of the Sxl protein exhibits low sequence homology to the typical RBDs, particularly at the putative functional residues, it was difficult to unambiguously locate the RNP1 and RNP2 motifs. Therefore, in the present study, we defined the amino and carboxy-terminal borders of the first RNA-binding domain (RBD1) of the Sxl protein by limited tryptic digestion. By replacement of Phe166 by Tyr, we constructed a highly soluble mutant, which exhibits the same RNA-binding properties as those of the wild-type. Using this mutant protein, we performed NMR measurements, and elucidated the secondary and tertiary structures of the Sxl RBD1 in solution. The betaalphabetabetaalphabeta folding pattern is conserved in the solution structure of the Sxl RBD1, as in other reported RBD structures. This allowed us to identify both the RNP1 and RNP2 motifs of the Sxl RBD1 unambiguously. Intriguingly, the RNP2 motif of the Sxl RBD1 has an Ile residue at the second position, which is generally occupied by an aromatic amino acid residue in RBDs and has been suggested to be involved in their RNA binding. Furthermore, the loop region between beta2 and beta3 of the Sxl RBD1 has an exceptional cluster of aromatic amino acid residues, in place of the normal basic amino acid cluster. In contrast, the second RBD of Sxl does not exhibit these characteristic features.

Amino Acid Sequence↗

PubMed1997

Epidermal cell differentiation in Arabidopsis determined by a Myb homolog, CPC.

The roots of plants normally carry small hairs arranged in a regular pattern. Transfer DNA-tagged lines of Arabidopsis thaliana included a mutant with few, randomly distributed root hairs. The mutated gene CAPRICE (CPC) encoded a protein with a Myb-like DNA binding domain typical of transcription factors involved in animal and plant development. Analysis in combination with other root hair mutations showed that CPC may work together with the TTG gene and upstream of the GL2 gene. Transgenic plants overexpressing CPC had more root hairs and fewer trichomes than normal. Thus, the CPC gene determines the fate of epidermal cell differentiation in Arabidopsis.

Amino Acid Sequence↗

PubMed1997

NMR analysis of the hydrogen bonding interactions of the RNA-binding domains of the Drosophila sex-lethal protein with target RNA fragments with site-specific [3-15N]uridine substitutions.

It has been reported that a 183 residue fragment, consisting of the two RNA-binding domains (RBD1- RBD2) of the Drosophila melanogster Sex-lethal (Sxl) protein, strongly binds an oligonucleotide of the target RNA sequence (5'-GUUUUUUUUC-3') that regulates alternative splicing, and forms four or five hydrogen bonds with the imino groups of the RNA. In the present study, we used site-directed mutagenesis to improve the solubility of the didomain fragment of Sxl, and confirmed that this mutant fragment forms hydrogen bonds with the target RNA in the same manner as that of the wild-type fragment. The mutant fragment was shown to bind the cognate RNA sequences GUUUUUUUUC and AUUUUUUUUC more tightly than UUUUUUUUC. By using a [3-15N]uridine phosphoramidite, we synthesized a series of15N-labeled target RNAs, in which one of the uridine residues was specifically replaced by [3-15N]uridine. By observing the imino1H-15N coupling of the labeled uridine residue, we assigned all four of the hydrogen-bonded imino protons to U1, U2, U5 and U6, respectively, of the target RNA. The imino protons of U2 and U6 exhibited nuclear Overhauser effects with aliphatic protons of the protein. All these results indicate that the A/G, U1, U2, U5 and U6 residues in the target sequence of (G/A)UUUUUUUU are specifically recognized by the two RNA-binding domains of the Sxl protein.

Animals↗

PubMed1997

An estimate of divergence time of Parazoa and Eumetazoa and that of Cephalochordata and Vertebrata by aldolase and triose phosphate isomerase clocks.

Previously we suggested that four proteins including aldolase and triose phosphate isomerase (TPI) evolved with approximately constant rates over long periods covering the whole animal phyla. The constant rates of aldolase and TPI evolution were reexamined based on three different models for estimating evolutionary distances. It was shown that the evolutionary rates remain essentially unchanged in comparisons not only between different classes of vertebrates but also between vertebrates and arthropods and even between animals and plants, irrespective of the models used. Thus these enzymes might be useful molecular clocks for inferring divergence times of animal phyla. To know the divergence time of Parazoa and Eumetazoa and that of Cephalochordata and Vertebrata, the aldolase cDNAs from Ephydatia fluviatilis, a freshwater sponge, and the TPI cDNAs from Ephydatia fluviatilis and Branchiostoma belcheri, an amphioxus, have been cloned and sequenced. Comparisons of the deduced amino acid sequences of aldolase and TPI from the freshwater sponge with known sequences revealed that the Parazoa-Eumetazoa split occurred about 940 million years ago (Ma) as determined by the average of two proteins and three models. Similarly, the aldolase and TPI clocks suggest that vertebrates and amphioxus last shared a common ancestor around 700 Ma and they possibly diverged shortly after the divergence of deuterostomes and protostomes.

Amino Acid Sequence↗

PubMed1997

A serine/threonine protein kinase gene isolated by an in vivo binding procedure using the Arabidopsis floral homeotic gene product, AGAMOUS.

During the course of characterizing fragments bound to an Arabidopsis floral protein AGAMOUS in vivo, a gene encoding a putative serine/threonine protein kinase was found on one of the fragments. The deduced 426 amino acid residues of the gene, named APK2a, are 65% identical to a previously reported Arabidopsis serine/threonine protein kinase, APK1a. The gene is composed of 6 exons and maps at 10 cM from the upper end of chromosome 1. Northern hybridization experiments indicated that the gene is strongly expressed in leaves, moderately in roots, and very weakly in flowers. Further in situ analysis of the expression in floral buds showed that the APK2a gene is expressed at pedicels, is not expressed at the floral organ primordia of wild type floral buds, but is moderately expressed in the floral organ primordia of the agamous mutant. In vitro binding assay suggest that the AGAMOUS protein binds to a sequence similar to, but different from, the known MADS-binding consensus sequences, the CArG box, located 3' downstream of the APK2a gene. These results suggest that APK2a expression is negatively regulated by the AG protein. A close homologue of the APK2a gene, named APK2b, was also isolated from the Arabidopsis cDNA library. The expression pattern of the APK2b gene differs from that of APK2a. It is strongly expressed in leaves, moderately in flowers, and weakly in roots.

AGAMOUS Protein, Arabidopsis↗

PubMed1997

Restoration of seminiferous tubular function after discontinuation of long-term gonadotropin-releasing hormone agonist administration in premature male rats.

We examined whether seminiferous tubular function develops normally after discontinuation of long-term gonadal suppression in premature male rats. Wistar male rats 4 weeks old were subjected to the injection of gonadotropin-releasing hormone (Gn-RH) agonist or normal saline solution as control for 12 weeks. The rats were sacrificed 0 and 6 weeks after discontinuation of the treatment. Histological examinations of the seminiferous tubules immediately after cessation of the Gn-RH agonist treatment demonstrated a stage-related change in specific germ cells. Seminiferous tubules of Gn-RH agonist-treated rats were narrow and irregular in shape, and contained significantly fewer spermatids and pachytene spermatocytes at stages VII to XIV than those in controls. A complete development of spermatogenesis was histologically observed 6 weeks after cessation of the treatment. Leydig cells became atrophic without any reduction in cell number immediately after the treatment, but Leydig cells grew rapidly and were similar in appearance to those in control rats 6 weeks after cessation of the treatment. Serum testosterone concentrations were noticeably suppressed immediately after cessation of the treatment (P < 0.01 vs. control) and reached a similar level to those of controls 6 weeks after the cessation. Testes weights were significantly lower in Gn-RH agonist-treated rats than in control rats and had not fully developed 6 weeks after cessation of the treatment (P < 0.01 vs. control). These results suggest that the testicular function develops normally after cessation of the long-term gonadal suppression in premature rats, although the increase in testicular weight may be slightly influenced.

Animals↗

PubMed1997

[Lipoprotein(a) concentrations and apolipoprotein(a) phenotype frequencies in cord blood of newborn infants and sera of children in Japan].

Lipoprotein(a) [Lp(a)] is considered to be an additional, independent, and largely genetically risk factor for the development of premature coronary heart disease. We measured Lp(a) levels and apo(a) phenotype in cord blood samples from 140(72 males, 68 females) Japanese newborn infants, and those in serum samples from seventy-nine normolipidemic out-patient children from the pediatrics. In the present study, we determined the relations between cord blood Lp(a) levels and apo(a) phenotypes. Maternal age, gestational age, weight and height were analyzed as covariates. The distribution of Lp(a) levels in cord blood was highly skewed towards the lower levels, as previously described. The mean Lp(a) concentration was 4.6 mg/dl(SD of 2.22 mg/dl) and median of 4.0 mg/dl, approximately three-fold lower than that observed in Japanese adult subjects. We did not find differences in the mean and median cord blood Lp(a) levels between male and female. Phenotyping of apo(a) was performed for 140 sera. The mean Lp(a) level in the B type was almost three times higher than that for the S4 type. A strong inverse relationship was found between the apparent molecular weight of apo(a) isoforms and serum Lp(a) concentration. The concentrations of Lp(a) reached to adult levels within first five years of life.

Adult↗

PubMed1996

A human RNA helicase-like protein, HRH1, facilitates nuclear export of spliced mRNA by releasing the RNA from the spliceosome.

Because the nuclear export of mRNA occurs only after the splicing reaction is completed, intron-containing pre-mRNA does not normally appear in the cytoplasm. As a mechanism to secure this, intron-containing RNA is retained in the nucleus via formation of the spliceosome. Therefore, the process of releasing spliced mRNA from the spliceosome after completion of splicing is an essential step for triggering the nuclear export of the spliced mRNA. In budding yeast, RNA helicase-like protein Prp22 is implicated in this process. Here we demonstrate the function of HRH1, a human protein homologous to Prp22, in mammalian cells using dominant-negative HRH1++ mutants (dn-HRH1). dn-HRH1 protein stalls on the spliceosome and prevents release of the spliced RNA from the spliceosome in vitro. Expression of dn-HRH1 in mammalian cells leads to inhibition of splicing and to extensive nuclear export of unspliced pre-mRNA, probably because of the incapability of recycling spliceosome components that normally retain the pre-mRNA in the nucleus. The arginine/serine-rich domain (RS domain) of HRH1, which is missing in Prp22, confers a nuclear localization signal, and appears to facilitate the interaction of HRH1 with the spliceosome. This is the first report on a bona fide mammalian homolog of yeast Prp splicing factor, and also on a mammalian RNA helicase-like splicing factor.

Amino Acid Sequence↗

PubMed1996

Factors involved in the activation of pre-mRNA splicing from downstream splicing enhancers.

The excision of introns with weak polypyrimidine tracts at their 3' splice sites can be enhanced by sequence elements in the downstream exon or by a downstream 5' splice site. The enhancers inside the exon do not conform to a strict consensus, but they are generally rich in purines. Here, we show that members of the family of SR proteins recognize these elements. Not only does SF2/ASF activate many different polypurine enhancers, but also at least one other SR protein, most likely SC35, is active as well. The degree of splicing activation varies with the polypurine enhancers and the SR proteins. Further, we show that the similar activation by downstream 5' splice sites requires U1 snRNP, which is not the case with purine-rich enhancers. These results are consistent with a model showing that U1 snRNP binds to the 5' splice site and SR proteins to exonic sequences upstream of the 5' splice site. Both interact with U2AF at the 3' splice site. This represents a molecular explanation for the exon recognition which is important for splice site selection in mammals.

Alpharetrovirus↗

PubMed1996

Listener and context dependency in the perception of emotional aspects of infant voice.

Developmental aspects of an infant's ability to express emotions through vocalizations were studied based on perceptual rating experiments against 12 vocalization- and emotion-related reference words. Three groups of listeners, students, mothers with infants, and nursery governesses, rated 28 voice samples recorded from a male infant at 6, 9, 12 and 17 months of age, under a positive or negative context. Among three factors extracted by a factor analysis, one representing the emotional contrast of frightened/angry versus happy was found to be independent of listener group, infant age and context. The other two, one representing contrast between pleased/happy versus demanding/sad and the other seeking affection and rejecting/angry were dependent on infant age and context. These results may indicate that infants even at 6 months of age can express the emotional contrast of 'pleasure' versus 'discomfort' through vocalization, which listeners perceive independently in context.

Anger↗

PubMed1996

A Y-box protein is a suppressor factor that decreases thyrotropin receptor gene expression.

The decanucleotides in a tandem repeat, -162 to -140 bp, are suppressor elements that decrease TSH receptor (TSHR) gene expression by different mechanisms. A factor(s) interacting with the 3'-decanucleotide compete for proteins that bind the cAMP response element, -139 to -132 bp, a constitutive enhancer necessary for efficient TSHR expression. The 5'-decanucleotide is in a CT-rich, S1 nuclease-sensitive region of the promoter; its suppressor activity has been related to its ability to bind a nonthyroid-specific protein to its coding strand. In this report we clone a complementary DNA encoding a single strand DNA-binding protein that forms a specific protein-DNA complex with the coding strand of the 5'- but not the 3'-decanucleotide and not with the 5'-decanucleotide noncoding or double strand. We show, by cotransfection with TSHR promoter-chloramphenicol acetyltransferase chimeras, that the protein is a suppressor that regulates the function of the 5'- but not the 3'-decanucleotide. The protein is a Y-box protein that was previously cloned as an enhancer factor from the rat liver; it is, however, 95% identical to human YB-1, which suppresses major histocompatibility class II gene expression, and to human nuclease-sensitive element protein-1, a Y-box protein identified by its ability to bind single strand, CT-rich, nuclease-sensitive elements of genes that, like the TSHR, have GC-rich promoters. Unexpectedly, the Y-box protein binds two other sites in the minimal TSHR promoter in a single strand-specific fashion and acts a suppressor at each of these sites. One is associated with the insulin response element of the minimal TSHR promoter and is not in an overtly CT-rich region. The other is located 3' to the cAMP response element in a region termed the S-box, -120 to -113 bp, because of its homology to the S-box of the major histocompatibility class II promoter; this site is in a CT-rich area and, as in the class II promoter, is linked to cAMP-induced gene suppression. A conserved CCTC sequence in each site is important for the binding and suppressor function of the Y-box protein.

Amino Acid Sequence↗

PubMed1995

Comparison of primary structures and substrate specificities of two pullulan-hydrolyzing alpha-amylases, TVA I and TVA II, from Thermoactinomyces vulgaris R-47.

Thermoactinomyces vulgaris R-47 produces two alpha-amylases, TVA I, an extracellular enzyme, and TVA II, an intracellular enzyme. Both enzymes hydrolyze pullulan to produce panose, and also hydrolyze cyclodextrins. We cloned and sequenced the TVA I gene. The TVA I gene consisted of 1833 base pairs, and the deduced primary structure was composed of 611 amino-acid residues, including an N-terminal signal sequence consisting of 29 amino-acid residues. The similarity between the amino-acid sequence of mature TVA I with those of other pullulan/cyclodextrin-hydrolyzing enzymes, such as TVA II and Bacillus stearothermophilus neopullulanase, was only 30%, although that of TVA II with neopullulanase was 48%. TVA II prefers specific small oligosaccharides and alpha- and beta-cyclodextrins. Whereas kcat/Km values of TVA I for pullulan were larger than that of TVA II, and TVA II could not hydrolyze starch completely. TVA II was inhibited by maltose, the hydrolysate of starch, which seems to be the reason for inefficient hydrolysis of starch. These kinetic properties indicate that TVA I and TVA II have differential physiological roles in sugar metabolism extracellularly and intracellularly, respectively.

Amino Acid Sequence↗

PubMed1995

Identification of the factors that interact with NCBP, an 80 kDa nuclear cap binding protein.

It has been shown that the monomethylated cap structure plays important roles in pre-mRNA splicing and nuclear export of RNA. As a candidate for the factor involved in these nuclear events we have previously purified an 80 kDa nuclear cap binding protein (NCBP) from a HeLa cell nuclear extract and isolated its full-length cDNA. In this report, in order to obtain a clue to the cellular functions of NCBP, we attempted to identify a factor(s) that interacts with NCBP. Using the yeast two-hybrid system we isolated three clones from a HeLa cell cDNA library. We designated the proteins encoded by these clones NIPs (NCBP interacting proteins). NIP1 and NIP2 have an RNP consensus-type RNA binding domain, whereas NIP3 contains a unique domain of Arg-Glu or Lys-Glu dipeptide repeats. We also show that NCBP requires NIP1 for binding to the cap structure. Possible roles of NIPs in cap-dependent nuclear processes are discussed.

Amino Acid Sequence↗

PubMed1995

Developmental and tissue-specific regulation of the gene for the wheat basic/leucine zipper protein HBP-1a(17) in transgenic Arabidopsis plants.

Wheat basic/leucine zipper protein HBP-1a(17) binds in vitro specifically to ACGT motif-containing cis-acting elements, such as the type I element of plant histone promoters and the G-box of hormone- and light-inducible promoters. To address the in vivo function of HBP-1a(17), we isolated and structurally analyzed the HBP-1a(17) gene and examined its expression in transgenic Arabidopsis plants. The HBP-1a(17) gene is composed of 14 exons; the basic region and leucine zipper are encoded by separate small exons, as is the case for other bZIP protein genes. The G-box of the HBP-1a(17) promoter bound specifically to HBP-1a(17) and its related HBP-1a isoforms, suggesting that the HBP-1a(17) gene may be autoregulated, although the binding affinity of these proteins in vitro is very low. In Arabidopsis plants, activation of the HBP-1a(17) promoter was highly restricted to photosynthetically active mesophyll, and guard cells and vascular bundles of vegetative leaves. Etiolation of transgenic plants resulted in inhibition of expression of the HBP-1a(17) promoter. Indeed, the HBP-1a(17) promoter contains several sequence elements homologous to cis-acting elements conserved in light-inducible promoters. It is, therefore, assumed that the HBP-1a(17) gene is light regulated and that HBP-1a(17) is involved in light-responsive gene transcription via the G-box.

Amino Acid Sequence↗

PubMed1995

Transcriptional regulation of the Sex-lethal gene by helix-loop-helix proteins.

Somatic sex determination in Drosophila depends on the expression of Sex-lethal (Sxl), whose level is determined by the relative number of X chromosomes and sets of autosomes (X:A ratio). The first step in regulation of Sxl expression is transcriptional control from its early promoter and several genes encoding transcription factors of the helix-loop-helix (HLH) family such as daughterless (da), sisterless-b (sis-b), deadpan (dpn) and extramacrochaetae (emc) have been implicated. By the use of transfection assays and in vitro binding experiments, here we show that da/sis-b heterodimers bind several sites on the Sxl early promoter with different affinities and consequently tune the level of active transcription from this promoter. Interestingly, our data indicate that repression by the dpn product of da/sis-b dependent activation results from specific binding of dpn protein to a unique site within the promoter. This contrasts with the mode of emc repression, which inhibits the formation of the da/sis-b heterodimers. These results reveal the molecular mechanisms by which Sxl gene transcription is positively or negatively regulated to control somatic sex determination.

Animals↗