bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification and characterization of E‐APC, a novel Drosophila homologue of the tumour suppressor APC
Authors: Hamada Fumihiko , Murata Yoji , Nishida Ayumu , Fujita Fumitaka , Tomoyasu Yoshinori , Nakamura Makoto , Toyoshima Kumao , Tabata Tetsuya , Ueno Naoto , Akiyama Tetsu 
Published: March 2003

BackgroundMutations in the adenomatous polyposis coli (APC) tumour suppressor gene are implicated in the genesis of colorectal cancers. The product of the APC gene forms a complex with β‐catenin, glycogen synthase kinase 3β (GSK‐3β) and Axin/conductin, and induces the degradation of β‐catenin.ResultsWe have identified a novel Drosophila homologue of APC, E‐APC, which is similar to but differs in several respects from D‐APC. The E‐APC cDNA encodes a protein of predicted 1067 amino acids, with seven armadillo repeats, two copies of the 15‐amino acid repeat, five copies of the 20‐amino acid repeat, and one Axin/conductin binding site. E‐APC directly interacts with D‐Axin and Armadillo (Arm, the Drosophila homologue of β‐catenin) in vitro, destabilizes intracellular β‐catenin, and suppresses β‐catenin/TCF‐regulated transcription in APC−/− colon cancer cells. The E‐APC mRNA is ubiquitously expressed throughout all developmental stages in Drosophila.ConclusionOur findings suggest that E‐APC may be universally involved in the regulation of the Wingless signalling pathway by down‐regulating the level of Arm in Drosophila.
Intragenic suppression of trans‐dominant lethal substitutions in the conserved GEME motif of the β subunit of RNA polymerase: evidence for functional cooperativity within the C‐terminus
Authors: Malik Talat , Ahmad Khalid , Buyukuslu Nihal , Cromie Karen , Glass Robert E. 
Published: March 2003

BackgroundThe ubiquitous multimeric RNA polymerases contain two large, conserved subunits, of which the β subunit has been implicated in all three stages of transcription. We have previously described a genetic system involving random, PCR‐mediated mutagenesis of the region of rpoB encoding the C‐terminal 116 amino acids of the β subunit of Escherichia coli RNA polymerase and the characterization of dominant‐negative mutations. This study identified the invariant motif GEME (residues 1271→1274;
Cromie et al. 1999
). Starting with three of these GEME‐motif lethal mutations (G1271E, G1271V, M1273V), we have selected for
intragenic suppressors, located within the same 3′‐region, that prevent expression of the trans‐dominant phenotype.ResultsWe isolated a total of 24 missense mutants and a further 14 frameshift alleles (the latter generating a nested set of C‐terminal deletions of the β subunit) and studied the effect of the missense suppressors in vivo and in vitro. The majority of the second‐site substitutions pinpoint highly conserved residues and were allele‐specific. In contrast, one particular missense substitution (S1332P) acted on all three primary site mutations whilst not appreciably affecting assembly proficiency, suggesting motif‐specific suppression. Two missense substitutions were found to perturb assembly of the β subunit (M1232T and L1233P) and define a small conserved region (1228→1233) adjacent to one of the active‐site residues identified by affinity‐labelling, H1237. The majority of primary mutations were located in three main clusters within the 116 amino acid region.ConclusionsThe importance and functional co‐operativity of the three main clusters pinpointed is supported by the present isolation of suppressors of three different GEME primary mutations in the same three regions (whereas the suppressors of G1271V and M1273V are located in all three clusters, those for G1271E are all C‐terminal of this residue). Moreover, the location of the suppressors suggests that the GEME and HLVDDK regions are present as α‐helices in holoenzyme, and that functional co‐operativity is through one particular face of each helix.
Hsc70/Hsp40 chaperone system mediates the Hsp90‐dependent refolding of firefly luciferase
Authors: Minami Yasufumi , Minami Michiko 
Published: March 2003

BackgroundThe 90‐kDa heat shock protein, Hsp90, was previously shown to capture firefly luciferase during thermal inactivation, thereby preventing its irreversible off‐pathway aggregation and maintaining it in a folding‐competent state. However, subsequent refolding of the luciferase required addition of rabbit reticulocyte lysate.ResultsHere we demonstrate that Hsc70 (cytosolic Hsp70) and Hsp40/Hdj1 (cytosolic DnaJ homologue) are the effective components in a reticulocyte lysate, while other unidentified factor in the lysate is also required for the refolding of Hsp90‐captured luciferase. Though another cytosolic DnaJ homologue, Hdj2/HSDJ, was more efficient than Hsp40 in suppressing the aggregation of rhodanese, Hdj2 was less effective for the refolding of luciferase than Hsp40. In the absence of the third factor, Hsp40 could bind to the luciferase captured by Hsp90, which suggested that Hsp40 on its own was able to bind the substrate protein, but Hsc70 could not.ConclusionsHsc70, Hsp40 and at least another additional component in the reticulocyte lysate are necessary for full accomplishment of the refolding of Hsp90‐captured luciferase. The third factor may be required for the loading of Hsc70 on to the substrate protein bound to Hsp90.
A novel RNA motif that binds efficiently and specifically to the Tat protein of HIV and inhibits the trans‐activation by Tat of transcription in vitro and in vivo
Authors: Yamamoto Rika , Katahira Masato , Nishikawa Satoshi , Baba Tadashi , Taira Kazunari , Kumar Penmetcha K. R. 
Published: March 2003

BackgroundTo find a novel RNA that would bind efficiently and specifically to Tat protein but not to other cellular factors, we used an in vitro selection method and isolated a novel aptamer RNATat, a 37‐mer RNA oligomer, that binds efficiently to the Tat protein of HIV‐1. In the present study, we analysed various properties of aptamer RNATat, including binding kinetics, identification of functional groups for Tat binding, and inhibition of Tat function.ResultsThe binding affinity of the isolated aptamer RNATat to Tat‐1 was 133 times higher than that of authentic TAR‐1 RNA. RNATat is composed of inverted repeats of two TAR‐like motifs, and even though RNATat had two Tat‐binding core elements, the interaction with Tat took place at a molar ratio of 1 : 1. Several functional groups of aptamer RNATat responsible for Tat binding were identified. The selected aptamer RNATat competed effectively for binding to Tat even in the presence of a large excess of TAR‐1 or TAR‐2 RNA in vitro, and specifically prevented Tat‐dependent trans‐activation both in vitro and in vivo.ConclusionsOur results indicate that a novel aptamer, RNATat, retained strong affinity for Tat even in the presence of a large excess of HIV TAR. RNATat binds efficiently to Tat proteins or peptides derived from either HIV‐1 or HIV‐2. Unlike TAR RNA, RNATat affinity does not depend upon cellular proteins such as cyclin T1, thus RNATat has the potential for use as a molecular recognition element in biosensors.
Ste20‐like kinase (SLK), a regulatory kinase for polo‐like kinase (Plk) during the G2/M transition in somatic cells
Authors: Ellinger‐Ziegelbauer Heidrun , Karasuyama Hajime , Yamada Eitaro , Tsujikawa Kazutake , Todokoro Kazuo , Nishida Eisuke 
Published: March 2003

BackgroundActivation of the cyclin‐dependent kinase cdc2‐cyclin B1 at the G2/M transition of the cell cycle requires dephosphorylation of threonine‐14 and tyrosine‐15 in cdc2, which in higher eukaryotes is brought about by the Cdc25C phosphatase. In Xenopus, there is evidence that a kinase cascade comprised of xPlkk1 and Plx1, the Xenopus polo‐like kinase 1, plays a key role in the activation of Cdc25C during oocyte maturation. In the mammalian somatic cell cycle, a polo‐like kinase homologue (Plk1) also functions during mitosis, but a kinase upstream of Plk is still unknown.ResultsWe show here that human Ste20‐like kinase (SLK), which is a ubiquitously expressed mammalian protein related to xPlkk1, can phosphorylate and activate murine Plk1. During progression through the G2 phase of the mammalian cell cycle, the activity of endogenous SLK is increased. The amount of SLK protein is decreased in quiescent and differentiating cells. Treatment with okadaic acid induces a phosphorylation‐dependent enhancement of SLK activity.ConclusionsWe propose that SLK has a role in the regulation of Plk1 activity in actively dividing cells during the somatic cell cycle. SLK itself is suggested to be regulated by phosphorylation.
Ebp2p, yeast homologue of a human protein that interacts with Epstein–Barr virus Nuclear Antigen 1, is required for pre‐rRNA processing and ribosomal subunit assembly
Authors: Tsujii Rota , Miyoshi Keita , Tsuno Akiko , Matsui Yasushi , Toh‐e Akio , Miyakawa Tokichi , Mizuta Keiko 
Published: March 2003

BackgroundA defect in the secretory pathway causes the transcriptional repression of both rRNA and ribosomal protein genes in Saccharomyces cerevisiae, suggesting a coupling of ribosome synthesis and plasma membrane synthesis. Rrs1p, an essential nuclear protein, is required for the secretory response.Results EBP2, encoding the yeast homologue of a human protein that interacts with Epstein–Barr virus Nuclear Antigen 1, was cloned in a two‐hybrid screen using RRS1 as a bait. The rrs1‐1 mutation, which produces Rrs1p without the C‐terminal half and causes a defect in the secretory response, almost abolished the interaction with Ebp2p. Ebp2p is essential for growth and is mainly localized in the nucleolus. The effects of Ebp2p depletion on ribosome biogenesis is quite similar to that of Rrs1p depletion; in the Ebp2p‐depleted cells, the rate of pre‐rRNA processing is slower, and significantly less mature 25S rRNA is produced compared to those in wild‐type cells. The polysome pattern indicates that Ebp2p‐depletion causes a decrease of 80S monosomes and polysomes, an accumulation of 40S subunits, and the appearance of half‐mer polysomes.ConclusionsEbp2p is required for the maturation of 25S rRNA and 60S subunit assembly. Ebp2p may be one of the target proteins of Rrs1p for executing the signal to regulate ribosome biogenesis.
Phosphorylation of ERM proteins at filopodia induced by Cdc42
Authors: Nakamura Nao , Oshiro Noriko , Fukata Yuko , Amano Mutsuki , Fukata Masaki , Kuroda Shinya , Matsuura Yoshiharu , Leung Thomas , Lim Louis , Kaibuchi Kozo 
Published: March 2003

BackgroundERM (ezrin, radixin, and moesin) proteins function as membrane‐cytoskeletal linkers, and are known to be localized at filopodia and microvilli‐like structures. We have shown that Rho‐associated kinase (Rho‐kinase)/ROKα/ROCK II phosphorylates moesin at Thr‐558 at the lower stream of Rho, and the phosphorylation is crucial to the formation of microvilli‐like structures (Oshiro, N., Fukata, Y. & Kaibuchi, K. (1998) Phosphorylation of moesin by Rho‐associated kinase (Rho‐kinase) plays a crucial role in the formation of microvilli‐like structures. J. Biol. Chem. 273, 34663– 34666). However, the role of ERM proteins in the formation of filopodia is less well characterized.ResultsHere we examined the phosphorylation state of ERM during filopodia formation induced by Cdc42 using the antibody recognizing ERM proteins phosphorylated at COOH (C)‐terminal threonine. When NIH 3T3 cells were transfected with constitutively active Cdc42 (Cdc42V12), filopodia formation was induced and phosphorylation of ERM at C‐terminal threonine was observed at the tip of filopodia, while the phosphorylation levels of ERM were lower and phosphorylated ERM was distributed throughout the cytoplasm in the control cells. We also showed that
Myotonic dystrophy kinase‐
related Cdc42‐binding
kinase (MRCK) which has been identified as an effector of Cdc42, phosphorylated moesin at C‐terminal threonine in a cell‐free system. Coexpression of the dominant negative form of MRCK inhibited both the formation of filopodia and accumulation of C‐terminal threonine‐phosphorylated ERM proteins at filopodia induced by Cdc42V12.ConclusionThe formation of filopodia induced by Cdc42 is accompanied by phosphorylation of ERM proteins, and MRCK is a candidate for the kinase that phosphorylates ERM proteins at filopodia.
MBD2‐MBD3 complex binds to hemi‐methylated DNA and forms a complex containing DNMT1 at the replication foci in late S phase
Authors: Tatematsu Ken‐ichiro , Yamazaki Tetsu , Ishikawa Fuyuki 
Published: March 2003

BackgroundIn vertebrates and plants, DNA methylation is one of the major mechanisms regulating gene expression. Recently, a family of methyl‐CpG‐binding proteins has been identified, and some members, such as MeCP2 and MBD2, were shown to mediate gene repression by recruiting histone deacetylase complexes to methylated genes. However, the function of another member of this family, MBD3, remained elusive.ResultsIt was shown that MBD2 and MBD3 form homo‐ and hetero‐dimers (or multimers) in vitro and in vivo. Significantly, the MBD2‐MBD3 complex showed an affinity to hemi‐methylated DNAs, a property that has never been reported with any member of the family proteins. MBD2 and MBD3 were co‐localized with DNMT1 at replication foci in 293 cell nuclei at late S phase. Moreover, by a co‐immunoprecipitation experiment, DNMT1 was shown to form a complex with MBD2 and MBD3. Finally, the abundance of MBD3 was highest in the late S phase when the DNMT1 is also most abundant, whereas the MBD2 level was largely constant throughout the cell cycle.ConclusionsThe results suggest that MBD3 may play an important role in the S phase. We hypothesize that the MBD2‐MBD3 complex recognizes hemi‐methylated DNA concurrent with DNA replication and recruits histone deacetylase complexes, as well as DNMT1, to establish and/or maintain the transcriptionally repressed chromatin.
Identification and characterization of human Wee1B, a new member of the Wee1 family of Cdk‐inhibitory kinases
Authors: Nakanishi Makoto , Ando Hitomi , Watanabe Nobumoto , Kitamura Kenzo , Ito Kenji , Okayama Hiroto , Miyamoto Tomomi , Agui Takashi , Sasaki Makoto 
Published: March 2003

BackgroundIn eukaryotic cells, the kinase activity of the mitosis‐promoting complex composed of cyclin B and Cdc2 (Cdk1) is negatively regulated by the phosphorylation of Cdk1 on threonine or tyrosine residues within its ATP binding domain.ResultsWe identified human Wee1B by searching a sequence database. The predicted human Wee1B protein comprises 561 amino acids. Northern blot analysis revealed that human Wee1B mRNA is particularly abundant in testis. Interestingly, RT‐PCR using early embryos revealed that the Wee1B product was readily detectable at the mature oocyte, but abruptly disappeared at embryonic day 2.5, suggesting that the amount of Wee1B mRNA is dependent on the maternal expression. GFP‐Wee1B showed a predominantly nuclear localization in HeLa cells. Human Wee1B was able to rescue the lethal phenotype of the fission yeast wee1‐50Δmik1 mutant, and over‐expression of the human protein in these cells resulted in cell elongation as a result of arrest of the cell cycle at the G2‐M transition. Recombinant Wee1B effectively phosphorylated cyclin B‐associated Cdk1 on tyrosine‐15, resulting in an inactivation of the kinase activity of Cdk1.ConclusionWe identified human Wee1B as a novel Cdk1‐inhibitory kinase. The identification of this new member of the Wee1 family suggests that inhibition of Cdk1 is mediated at multiple levels in mammals.
Membrane‐associated guanylate kinase with inverted orientation (MAGI)‐1/brain angiogenesis inhibitor 1‐associated protein (BAP1) as a scaffolding molecule for Rap small G protein GDP/GTP exchange protein at tight junctions
Authors: Mino Akihisa , Ohtsuka Toshihisa , Inoue Eiji , Takai Yoshimi 
Published: March 2003

Membrane‐associated guanylate kinase (MAGUK) with inverted orientation (MAGI)‐1/brain angiogenesis inhibitor 1‐associated protein (BAP1), is a member of the MAGUK family that has multiple PDZ domains and interacts with many transmembrane proteins, including receptors and channels, through these domains. MAGI‐1/BAP1 is ubiquitously expressed and localized at tight junctions in epithelial cells. It is an isoform of the neurone‐specific synaptic scaffolding molecule (S‐SCAM), which is known to interact with NMDA receptors and neuroligins. We have recently found that S‐SCAM also interacts with a signalling molecule, a GDP/GTP exchange protein (GEP) that is specific for Rap1 small G protein, Rap GEP, which has also recently been referred to as RA‐GEF/PDZ‐GEFI/CNras‐GEF. In this study, we have examined whether MAGI‐1/BAP1 also interacts with and serves as a scaffolding molecule for Rap GEP at tight junctions in epithelial cells.MAGI‐1/BAP1 similarly interacted with Rap GEP in cell‐free and intact cell systems. A Northern blot analysis revealed that Rap GEP was expressed in most tissues examined. However, neither postsynaptic density (PSD)‐95/synapse‐associated protein (SAP) 90 (another member of the MAGUK family) nor SAP97/human discs‐large tumour suppressor gene product (another ubiquitously expressed MAGUK localizing to adherens junctions in epithelial cells and the isoform of PSD‐95/SAP90) interacted with Rap GEP.These results indicate that MAGI‐1/BAP1 serves as a scaffolding molecule for Rap GEP at tight junctions in epithelial cells.
E2F regulates growth‐dependent transcription of genes encoding both catalytic and regulatory subunits of mouse primase
Authors: Nishikawa Naoko S. , Izumi Masako , Yokoi Masayuki , Miyazawa Hiroshi , Hanaoka Fumio 
Published: March 2003

DNA polymerase α‐primase is one of the principal enzymes involved in eukaryotic chromosomal DNA replication. Mouse DNA polymerase α‐primase consists of four subunits with molecular masses of 180, 68, 54 and 46 kDa. Protein and mRNA expression levels of the four subunits are up‐regulated in a coordinated manner in response to growth stimulation. We have previously analysed the transcription of the 180 kDa (p180) and 68 kDa (p68) subunits, which form the DNA polymerase catalytic complex, and found that growth‐dependent regulation of transcription of the mouse p180 and p68 genes is mediated by a common factor, E2F, while the basal transcription of the genes is regulated by different transcription factors.We characterized the transcriptional regulation of the 54 kDa (p54) and 46 kDa (p46) subunits, which form the DNA primase catalytic complex. We isolated genomic clones spanning the 5′‐flanking regions of the p54 and p46 genes and showed, using transient expression and gel mobility shift assays, that the basal transcription of p54 is controlled by Sp1 and GA‐binding protein, as is the basal transcription of the p180 gene. The basal transcription of p46 is controlled by unknown factor(s) which were bound to the upstream sequence. The variant E2F sites close to the transcription initiation sites of the p54 and p46 genes had no basal promoter activity, but were essential for the growth‐dependent transcription of both genes.The promoter regions of the four subunits of mouse DNA polymerase d‐primase complex share several common features. The coordinated transcription of all four subunits in response to growth stimulation appears to be controlled by E2F.
Involvement of RNase G in in vivo mRNA metabolism in Escherichia coli
Authors: Umitsuki Genryou , Wachi Masaaki , Takada Ayako , Hikichi Takafusa , Nagai Kazuo 
Published: March 2003

Background Escherichia coli rng gene (previously called cafA) encodes a novel RNase, named RNase G, which is involved in the 5′ end‐processing of 16S rRNA. In rng mutant cells, a precursor form of 16S rRNA, 16.3S rRNA, is accumulated. Here we report a role of RNase G in the in vivo mRNA metabolism.ResultsWe found that rng::cat mutant strains overproduced a protein of about 100 kDa. N‐terminal amino acid sequencing of this protein showed that it was identical to the fermentative alcohol dehydrogenase, the product of the adhE gene located at 28 min on the E. coli genetic map. The level of adhE mRNA was significantly higher in the rng::cat mutant strain than that in its parental strain, while such differences were not seen in other genes we examined. A rifampicin‐chase experiment revealed that the half‐life of adhE mRNA was 2.5‐fold longer in the rng::cat disruptant than in the wild‐type.ConclusionThese results indicate that, in addition to rRNA processing, RNase G is involved in in vivo mRNA degradation in E. coli.
Conformational switching of Escherichia coli RNA polymerase‐promoter binary complex is facilitated by elongation factor GreA and GreB
Authors: Sen Ranjan , Nagai Hiroki , Shimamoto Nobuo 
Published: March 2003

BackgroundThe initiation arrest at a modified λPR promoter is caused by irreversible divergence of the reaction pathway into productive and arrested branches. Escherichia coli GreA and GreB induce cleavage of the nascent transcript and relieve arrest in elongation. They also reduce abortive synthesis at several promoters and relieve initiation arrest. Their mechanism of action during initiation, and its relationship to the branched initiation pathway are unknown.ResultsThe Gre factors mitigated initiation arrest only when they were added to the binary complex of the holoenzyme bound to the λPR promoter, prior to RNA synthesis. They exerted little effect when they were added to ternary initiation complexes. They accelerated the exchange of the binary complex with its free components by 6–9‐fold. When they are present, a high concentration of the initiating nucleotide increased yield of the full‐length transcript, whereas a low concentration did not.ConclusionsAll the results presented above can be explained by a model where the productive and arrested pathways diverge at the binary complex stage. The Gre factors relieve the initiation arrest by introducing reversibility between subspecies of the binary complex that are precursors of the two pathways. RNA cleavage is unlikely to cause relief of initiation arrest.
Terminal deoxynucleotidyltransferase directly interacts with a novel nuclear protein that is homologous to p65
Authors: Yamashita Nobuyuki , Shimazaki Noriko , Ibe Shiro , Kaneko Reo , Tanabe Akiko , Toyomoto Tsuguri , Fujita Kiyoko , Hasegawa Tomohiko , Toji Shingo , Tamai Katsuyuki , Yamamoto Hiroko , Koiwai Osamu 
Published: March 2003

Background Terminal deoxynucleotidyltransferase (TdT) is a DNA polymerase that enhances Ig and TcR gene diversity in the N region in B‐ and T‐cells. TdT is found as a member of a large protein complex in the lysate of the thymocytes. To elucidate the molecular mechanism of the synthesis of the N region, we first attempted to isolate the genes with products that are interacting directly with TdT.Results Using a yeast two‐hybrid system, we isolated a cDNA clone encoding a novel nuclear protein that interacts with TdT. This protein was designated as TdT interacting factor 1 (TdIF1). TdIF1 has a high degree of homology to the transcription factor p65, which belongs to the nuclear receptor superfamily. TdIF1 contains HMG‐I and HMG‐Y DNA binding domains (AT‐hooks) and can bind to single‐ and double‐stranded DNA. TdT and TdIF1 were co‐eluted at position 232 kDa by gel filtration of MOLT4 lysate. TdIF1 can enhance TdT activity fourfold in vitro assay system using oligo(dT)16 as primers.Conclusions TdIF1 binds directly to TdT, both in vitro and in vivo. TdIF1 and TdT exist as the members of a 232 kDa protein complex. TdIF1 can enhance TdT activity maximum fourfold in vitro assay system, suggesting that it positively regulates the synthesis of the N region during V(D)J recombination in the Ig and TcR genes.