bluuebunny/crossref_metadata_2025_split huggingface dataset:
Targeted disruption of ATF4 discloses its essential role in the formation of eye lens fibres
Authors: Tanaka Takashi , Tsujimura Tohru , Takeda Kiyoshi , Sugihara Ayako , Maekawa Akiko , Terada Nobuyuki , Yoshida Nobuaki , Akira Shizuo 
Published: March 2003

Background: Activating transcription factor‐4 (ATF4)—also termed CREB2, C/ATF, and TAXREB67—is a basic‐leucine zipper (bZip) transcription factor that belongs to the ATF/CREB family. In addition to its own family members, ATF4 can also form heterodimers with other related but distinct bZIP proteins such as the C/EBP, AP‐1 and Maf families, which may give rise to a variety of combinatorial diversity in gene regulation. In order to assess the in vivo essential role of ATF4, we have generated mice lacking ATF4 by gene targeting. Results: ATF4‐deficient mice exhibited severe microphthalmia. Although ATF4‐deficient eyes revealed a normal gross lens structure up to embryonic day 14.5, later on the ATF4‐deficient lens, degenerated due to apoptosis without the formation of lens secondary fibre cells. Retinal development was normal in the mutant mice. The lens‐specific expression of ATF4 in the mutant mice led not only to the recovery of lens secondary fibres but also to the induction of hyperplasia of these fibres. Conclusion: These results demonstrated that ATF4 is essential for the later stages of lens fibre cell differentiation.
Kinase‐independent activity of Cdc2/Cyclin A prevents the S phase in the Drosophila cell cycle
Authors: Hayashi Shigeo , Yamaguchi Masamitsu 
Published: March 2003

Background:The Cdc2‐dependent inhibition of S phase is required in G2 for the correct ordering of the S and M phases in yeasts and Drosophila. This function of Cdc2 has been ascribed to its ability to phosphorylate replication factors to prevent the assembly of a preinitiation complex at the origin of replication. Whether this is the sole mechanism of S phase inhibition by Cdc2 in higher metazoans is not known because the pleiotropic functions of this essential cell cycle regulator make genetic analysis difficult.Results:We show that Cdc2 co‐expressed with Cyclin A inhibits the S phase in Drosophila salivary glands and diploid abdominal histoblasts. A kinase defective mutant of Cdc2 failed to promote mitosis, but was still able to inhibit the S phase with the same efficiency as the wild‐type protein. In addition, Cdc2 and Cyclin A cooperatively inhibit transcriptional activation by the essential S phase regulator E2F. Cdc2 binds to E2F in vitro, and post‐transcriptionally promotes its accumulation in vivo. Furthermore, the inhibitory effect of Cdc2 on S phase is overridden by E2F.Conclusion:The inhibition of S phase by Cdc2 is achieved in part by a kinase‐independent mechanism, which is likely to be mediated by the inhibition of E2F.
Trans‐dominant mutations in the 3′‐terminal region of the rpoB gene define highly conserved, essential residues in the β subunit of RNA polymerase: the GEME motif
Authors: Cromie Karen D. , Ahmad Khalid , Malik Talat , Buyukuslu Nihal , Glass Robert E. 
Published: March 2003

Background:The multimeric DNA‐dependent RNA polymerases are widespread throughout nature. The RNA polymerase of Escherichia coli, which is the most well characterized, consists of a holoenzyme with subunit stoichiometry of α2ββ′σ. The β subunit is conserved and has been implicated in all stages of transcription. The extreme C‐terminus of the β subunit, which includes two well‐conserved sequence segments, contributes to the active centre and has been proposed to act in transcriptional termination. We describe a genetic system for further characterizing the role of the extreme C‐terminus of the β subunit of E. coli RNA polymerase. This involves random, PCR (Polymerase Chain Reaction)‐mediated mutagenesis of the 3′ region of rpoB encoding the C‐terminal 116 amino acids of β, followed by the isolation and characterization of trans‐dominant‐negative mutations.Results:Substitutions of conserved residues in this region were obtained that exhibited different degrees of growth inhibition in a host expressing the chromosomal‐encoded wild‐type form of the β subunit. A number of different substitutions were isolated within the highly conserved sequence motif GEME (residues 1271→1274 of the E. coliβ subunit). In addition, substitutions were obtained in the extreme C‐terminal (surface‐exposed) region of β and at two residues previously proposed to be in the active site (H1237, K1242). The properties of the purified mutant holoenzymes, assessed by transcription assays in vitro, suggested a promoter blockading action.Conclusions:We have identified an important, highly conserved motif in the β subunit, GEME (residues 1271→1274). The nature and effect of the amino acid substitutions at the Gly residue in GEME emphasize the importance of a small, uncharged residue at this position. The in vitro properties of the most extreme trans dominant‐negative mutants altered in the GEME motif (and the mutant characteristics in vivo) were similar to those of certain previously identified active‐site mutants, suggesting that the altered RNA polymerases were capable of promoter binding and RNA chain initiation but were deficient in the subsequent transcriptional stage.
Phosphorylation–dependent interaction of the N‐methyl‐ d‐aspartate receptor ε2 subunit with phosphatidylinositol 3‐kinase
Authors: Hisatsune Chihiro , Umemori Hisashi , Mishina Masayoshi , Yamamoto Tadashi 
Published: March 2003

BackgroundThe NMDA receptors (NMDARs) are ion channels through which Ca2+ influx triggers various intracellular responses. Tyrosine phosphorylation of NMDARs regulates NMDA channel activities, which may be important in neuronal plasticity. The biological significance of the tyrosine phosphorylation events, however, differs among NMDAR subunits: tyrosine phosphorylation of NMDARε1 increases NMDA channel activities, but that of NMDARε2 does not. Since signal transductions from various cell surface receptors are mediated by protein–protein interaction through phosphotyrosine and the Src homology 2 (SH2) domain, we examined the possibility that phosphotyrosines in NMDARε2 contribute to the intracellular signalling events.ResultsWe first show that Fyn is deeply involved in the phosphorylation of NMDARε2 and second that a phosphotyrosine in NMDARε2 interacts with the p85 regulatory subunit of phosphatidylinositol 3‐kinase (PI3‐kinase). Both the level of tyrosine phosphorylation on NMDARε2 and the amounts of the p85 subunit (p85) bound to NMDARε2 are decreased in Fyn‐deficient mice. Moreover, we show that ischaemia stimulates the binding of p85 to phosphorylated NMDARε2, suggesting a physiological role of the phosphotyrosine/SH2–based interaction between NMDARε2 and p85 in the brain.ConclusionsThe tyrosine phosphorylation event on NMDARs is important in not only the regulation of its channel activity but also intracellular signalling mediated through the interaction of the NMDAR with SH2 domain‐containing molecules.
Cytoplasmic localization is required for the mammalian ELAV‐like protein HuD to induce neuronal differentiation
Authors: Kasashima Katsumi , Terashima Kazuhiro , Yamamoto Koichi , Sakashita Eiji , Sakamoto Hiroshi 
Published: March 2003

BackgroundELAV‐like neuronal RNA‐binding proteins are highly conserved in many neurone‐containing organisms and have been implicated in neuronal development and differentiation.ResultsMammalian neurone‐specific ELAV‐like Hu proteins (HuB, HuC and HuD) and Drosophila ELAV, but not HuR, were found to induce neurite outgrowth when over‐expressed in rat PC12 cells. Functional analysis of HuD deletion mutants demonstrated the importance of two conserved RNA‐binding domains (RBDs 1 and 3) and the indispensability of the linker region between RBDs 2 and 3 for the neurite‐inducing activity. Further analyses suggested the importance of nucleocytoplasmic shuttling of HuD mediated by a novel nuclear export signal (NES) sequence in the linker region for the neurite‐inducing activity. Moreover, two HuD deletion mutants containing the linker region dominantly inhibited the wild‐type neurite‐inducing activity, although they had no neurite‐inducing activity per se, suggesting that saturable intracellular trafficking mediated by the linker region is required for the neurite induction by HuD. Interestingly, the same dominant negative mutants significantly inhibited retinoic acid‐induced neuronal differentiation of mouse embryonal carcinoma P19 cells.ConclusionsOur results suggest the presence of a novel NES in neurone‐specific Hu proteins and the importance of their cytoplasmic localization, through nucleocytoplasmic shuttling, for the initiation of neuronal differentiation.
Bacterial cell death induced by human pro‐apoptotic Bax is blocked by an RNase E mutant that functions in an anti‐oxidant pathway
Authors: Nanbu‐Wakao Rika , Asoh Sadamitsu , Nishimaki Kiyomi , Tanaka Rie , Ohta Shigeo 
Published: March 2003

BackgroundBax is a member of the Bcl‐2 family and induces apoptosis of mammalian cells. We have shown that a trace amount of human Bax induces the cell death of Escherichia coli, accompanied by damage to DNA, and that the region of Bax which is lethal to E. coli is also responsible for apoptosis‐inducing activity in the mammalian cells.ResultsWe isolated a Bax‐resistant mutant from E. coli cells that survive in the presence of paraquat, a generator of superoxide, by screening a library constructed from the random insertion of a transposon. Psb1 (paraquat‐resistant, suppressor of Bax‐1) mutant had a Tn 10 transposon inserted in the rne gene of E. coli, splitting the RNase E gene (rne) into N‐ and C‐terminal halves. The introduction of the truncated 5′ end of rne specifically enhanced resistance to paraquat, prevented cell death induced by Bax and decreased the intracellular H2O2 concentration. The region responsible for the paraquat‐ and Bax‐resistance was not the catalytic site for the endoribonuclease activity of RNase E.ConclusionsThe N‐terminal region of the RNase E protein inhibits bacterial death induced by human Bax as well as paraquat through a unique mechanism that is distinct from RNA digestion. This study implies that the protection of bacterial death induced by Bax is associated with an anti‐oxidant pathway and that a mutant RNase E has a novel function as an anti‐oxidant.
Distinct transcriptional regulation and phylogenetic divergence of human LEFTY genes
Authors: Yashiro Kenta , Saijoh Yukio , Sakuma Rui , Tada Masatomo , Tomita Naohiro , Amano Kenji , Matsuda Youichi , Monden Morito , Okada Shintaro , Hamada Hiroshi 
Published: March 2003

BackgroundMouse lefty1 and lefty2 genes are expressed on the left side of developing embryos and are required for left‐right determination. Here we have studied expression and transcriptional regulatory mechanisms of human LEFTY genes.ResultsThe human LEFTY locus comprises two functional genes (LEFTY1 and LEFTY2) and a putative pseudogene. LEFTY1 is expressed in colon crypts. However, whereas LEFTY1 mRNA is present in basal cells of the crypts, LEFTY1 protein is localized in the apical region, suggesting that this secreted protein undergoes long‐range transport. Human LEFTY2 possesses a left side‐specific enhancer (ASE) like mouse lefty2; however, the LEFTY2 ASE shows markedly higher activity in the floor plate than does the lefty2 ASE. In contrast to mouse lefty1, which is expressed predominantly in the floor plate under the control of a right side‐specific silencer, human LEFTY1 is expressed mainly in left lateral plate mesoderm under the control of an ASE‐like left side‐specific enhancer. The presence of FAST‐binding sites in the LEFTY1 enhancer (and their absence in lefty1) contributes to the difference.ConclusionThese observations suggest that humans and mice have acquired distinct strategies during evolution for determining the asymmetric expression of LEFTY and lefty genes.
Two types of localization of the DNA‐binding proteins within the Escherichia coli nucleoid
Authors: Azam Talukder Ali , Hiraga Sota , Ishihama Akira 
Published: March 2003

BackgroundThe genome DNA of Escherichia coli is folded into the nucleosome‐like structure, often called a nucleoid, by the binding of several DNA‐binding proteins. We previously determined the specificity and affinity of DNA‐binding for 12 species of the E. coli DNA‐binding protein, and their intracellular concentrations at various growth phases. The intracellular localization of these proteins in E. coli could be predicted from these data, but no attempt has been made thus far to directly observe the intracellular distribution of the DNA‐binding proteins.ResultsThe intracellular localization in Escherichia coli of 10 species of the nucleoid‐associated protein, three components of the transcripton apparatus, and three components of the translation machinery was investigated by indirect immuno‐fluorescence microscopy. The DNA‐binding proteins could be classified into two groups. The group‐I proteins, including the major nucleoid‐structural proteins, H‐NS, HU, IHF, StpA and Dps, are distributed uniformly within the entire nucleoid. In contrast, the group‐II proteins, which are presumed to possess regulatory activities of DNA functions accumulate at specific loci within the nucleoid, forming 2 (SeqA), 3‐4 (CbpA and CbpB) and 6‐10 (Fis and IciA) immuno‐stained dots. Each immuno‐stained dot may represent either the association of a hundred to one thousand molecules of each DNA‐binding protein at a specific locus of the genome DNA or the assembly of protein‐associated DNA segments from different domains of the folded genome. Both the RNA polymerase core enzyme and the σ70 subunit are mainly associated with the nucleoid, but the anti‐σ70 factor (Rsd) appears to be accumulated at the boundary between the nucleoid and the cytosol in the stationary‐phase cells. Here we show that the majority of Hfq is present in cytoplasm together with ribosomal proteins L7/L12 and RMF.ConclusionThe DNA‐binding proteins of E. coli could be classified into two groups. One group proteins was distributed uniformly within the nucleoid, but the other group of proteins showed an irregular distribution, forming immuno‐stained spots or clumps.
Molecular analysis of zebrafish photolyase/cryptochrome family: two types of cryptochromes present in zebrafish
Authors: Kobayashi Yuri , Ishikawa Tomoko , Hirayama Jun , Daiyasu Hiromi , Kanai Satoru , Toh Hiroyuki , Fukuda Itsuki , Tsujimura Tohru , Terada Nobuyuki , Kamei Yasuhiro , Yuba Shunsuke , Iwai Shigenori , Todo Takeshi 
Published: March 2003

BackgroundCryptochromes (CRY), members of the DNA photolyase/cryptochrome protein family, regulate the circadian clock in animals and plants. Two types of animal CRYs are known, mammalian CRY and Drosophila CRY. Both CRYs participate in the regulation of circadian rhythm, but they have different light dependencies for their reactions and have different effects on the negative feedback loop which generates a circadian oscillation of gene expression. Mammalian CRYs act as a potent inhibitor of transcriptional activator whose reactions do not depend on light, but Drosophila CRY functions as a light‐dependent suppressor of transcriptional inhibitor.ResultsWe cloned seven zebrafish genes that carry members of the DNA photolyase/cryptochrome protein family; one (6‐4)photolyase and six cry genes. A sequence analysis and determination of their in vitro functions showed that these zebrafish cry genes constitute two groups. One has a high sequence similarity to mammalian cry genes and inhibits CLOCK:BMAL1 mediated transcription. The other, which has a higher sequence similarity to the Drosophila cry gene rather than the mammalian cry genes, does not carry transcription inhibitor activity. The expressions of these cry genes oscillate in a circadian manner, but their patterns differ.ConclusionsThese findings suggest that functionally diverse cry genes are present in zebrafish and each gene has different role in the molecular clock.
Competition between the replication initiator DnaA and the sequestration factor SeqA for binding to the hemimethylated chromosomal origin of E. coli in vitro
Authors: Taghbalout Aziz , Landoulsi Ahmed , Kern Renée , Yamazoe Mitsuyoshi , Hiraga Sota , Holland Barry , Kohiyama Masamichi , Malki Abderrahim 
Published: March 2003

Following replication initiation, the replication origin (oriC) in Escherichia coli enters a hemimethylated state at Dam methylation sites which are recognized by the SeqA protein. SeqA binds preferentially to hemimethylated GATC sequences of DNA in vitro. SeqA is essential for the synchronous initiation of chromosome replication from oriC copies in vivo.We show that: (i) purified SeqA binds AT‐rich and 13‐mers regions and two DnaA boxes, R1 and M, of hemimethylated oriC. (ii) SeqA inhibits the in vitro replication of a hemimethylated oriC plasmid more efficiently than the fully methylated, (iii) SeqA inhibits competitive binding of DnaA protein to the regions of the hemimethylated oriC plasmid, explaining the mechanism of its inhibitory effect. The inhibition of DnaA binding by SeqA also occurs efficiently on a small hemimethylated oriC fragment containing both R1 and M DnaA boxes, but not the 13‐mer region.SeqA binds strongly the long region from the AT‐rich region to the M DnaA box of the hemimethylated oriC DNA and releases DnaA molecules from the long region.
Selenium metabolism in zebrafish: multiplicity of selenoprotein genes and expression of a protein containing 17 selenocysteine residues
Authors: Kryukov Gregory V. , Gladyshev Vadim N. 
Published: March 2003

Fish are an important source of selenium in human nutrition and the zebrafish is a potentially useful model organism for the study of selenium metabolism and its role in biology and medicine. Selenium is present in vertebrate proteins in the form of selenocysteine (Sec), the 21st natural amino acid in protein which is encoded by UGA.We report here the detection of 18 zebrafish genes for Sec‐containing proteins. We found two zebrafish orthologs of human SelT, glutathione peroxidase 1 and glutathione peroxidase 4, and single orthologs of several other selenoproteins. In addition, new zebrafish selenoproteins were identified that were distant homologues of SelP, SelT and SelW, but their direct orthologs in other species are not known. This multiplicity of selenoprotein genes appeared to result from gene and genome duplications, followed by the retention of new selenoprotein genes. We found a zebrafish selenoprotein P gene (designated zSelPa) that contained two Sec insertion sequence (SECIS) elements and encoded a protein containing 17 Sec residues, the largest number of Sec residues found in any known protein. In contrast, a second SelP gene (designated zSelPb) was also identified that contained one SECIS element and encoded a protein with a single Sec. We found that zSelPa could be expressed and secreted by mammalian cells.The occurrence of zSelPa and zSelPb suggested that the function of the N‐terminal domain of mammalian SelP proteins may be separated from that of the C‐terminal Sec‐rich sequence: the N‐terminal domain containing the UxxC motif is likely involved in oxidoreduction, whereas the C‐terminal portion of the protein may function in selenium transport or storage. Our data also suggest that the utilization of Sec is more common in zebrafish than in previously characterized species, including mammals.
Transcription organization and mRNA levels of the genes for all 12 subunits of the fission yeast RNA polymerase II
Authors: Sakurai Hitomi , Ishihama Akira 
Published: March 2003

The RNA polymerase II (Pol II) of eukaryotes is composed of 12 subunits, of which five are shared among Pol I, Pol II and Pol III. At present, however, little is known about the regulation of synthesis and assembly of the 12 Pol II subunits. To obtain an insight into the regulation of synthesis of these 12 Pol II subunits, Rpb1 to Rpb12, in the fission yeast Schizosaccharomyces pombe, we analysed the transcriptional organization of the rpb genes by use of the oligo capping method, and determined mRNA levels by quantitative competitive PCR assay.The intracellular concentrations of the 12 Rpb subunits in growing S. pombe cells are different, within a range of 15‐fold difference between the least abundant Rpb3 and the most abundant Rpb12. The transcription of one group of genes including rpb3, rpb4, rpb5, rpb6, rpb7 and rpb10 is mainly initiated at a single site, while that of the other group of genes for rpb1, rpb2, rpb8, rpb9, rpb11 and rpb12 is initiated at multiple sites. The promoters of the first group of genes contain the TATA box sequence between −26 and −62, while the second group of genes carry TATA‐less promoters. Several common sequence segments, tentatively designated ‘Rpb motifs’, were identified in the promoter regions of the rpb genes. Competitive PCR analysis indicated that mRNAs for Rpb1, Rpb3, Rpb7 and Rpb9 were among the group which had a low abundance, while the levels of Rpb6 and Rpb10 mRNAs were about fivefold, and that of Rpb2 mRNA was about 40‐fold higher than the Rpb3 mRNA level.The levels of rpb mRNAs do not correlate with those of Rpb proteins. The protein‐to‐mRNA ratio or the translation efficiency is low for the rpb1, rpb2, rpb3 and rpb11 genes, encoding the homologues of subunits β′, β, α and α, respectively, of the prokaryotic RNA polymerase core enzyme.
A serine/threonine kinase p90rsk1 phosphorylates the anti‐proliferative protein Tob
Authors: Suzuki Toru , Matsuda Satoru , Tsuzuku Junko K. , Yoshida Yutaka , Yamamoto Tadashi 
Published: March 2003

Background tob is a member of a gene family with anti‐proliferative function. Over‐expression of Tob in NIH3T3 cells results in the suppression of cell proliferation. The growth suppression is hampered by the presence of activated ErbB2 kinase. The molecular mechanisms by which Tob suppresses cell growth and by which ErbB2 abrogates Tob function remain to be elucidated.ResultsWe show that Tob is phosphorylated on serines and threonines, but not tyrosines, by a kinase(s) that associates with Tob in the lysates of various cells, including ErbB2‐over‐expressed cells. We also show that a 95 kDa kinase associates with Tob in vitro. The autophosphorylation activity of this kinase co‐chromatographes with Tob‐phosphorylating activity, suggesting that the 95 kDa kinase phosphorylates Tob. Among the known kinases with molecular mass around 95 kDa, p90rsk1 associates with Tob in vitro and in vivo, and phosphorylates Tob at least in vitro. Therefore, it is likely that p90rsk1 represents the 95 kDa kinase and is involved in the regulation of Tob function through phosphorylation.Conclusionp90rsk1 associates with and phosphorylates Tob. Because p90rsk1 is activated downstream of receptor tyrosine kinases, we propose that Tob function is at least in part under the control of growth factor‐stimulated tyrosine kinases through its phosphorylation by p90rsk1.