bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential roles of GATA‐1 and GATA‐2 in growth and differentiation of mast cells
Authors: Harigae Hideo , Takahashi Satoru , Suwabe Naruyoshi , Ohtsu Hiroshi , Gu Lin , Yang Zhouying , Tsai Fong‐Ying , Kitamura Yukihiko , Douglas Engel James , Yamamoto Masayuki
Published: March 2003

Background

While mast cells have been previously shown to express both GATA‐1 and GATA‐2 mRNAs, individual functions for these related factors during their course of differentiation within the mast cell lineage have not yet been defined. To address this question, the expression of GATA‐1 and GATA‐2 mRNAs and proteins were examined in three mouse mast cell progenitor lines as well as in mast cells isolated from both wild‐type and GATA‐1‐deficient mice.

Results

Both mast cell progenitor lines, as well as primary mouse bone marrow‐derived mast cells (BMMCs) and peritoneal mast cells (PMCs) were examined by RNA blotting and immunological analyses. GATA‐2 protein was abundantly expressed in all three mast cell lines and in BMMCs, but only weakly in some of PMCs. In contrast, GATA‐1 protein was expressed in PMCs and BMMCs after culture in the presence of IL‐3 and SCF. We also found the presence of Alcian blue staining‐positive but berberine staining‐negative mast cells in the skin of mice heterozygous to GATA‐1 knock‐down allele.

Conclusion

These results suggest that the expression of GATA factor‐dependent genes is regulated by GATA‐2 during mast cell development and that GATA‐1 is required for the specification of differentiated mast cell phenotypes.
Rad52 forms ring structures and co‐operates with RPA in single‐strand DNA annealing
Authors: Shinohara Akira , Shinohara Miki , Ohta Tsutomu , Matsuda Shimako , Ogawa Tomoko
Published: March 2003

Background

The RAD52 epistasis group in Saccharomyces cerevisiae is involved in various types of homologous recombination including recombinational double‐strand break (DSB) repair and meiotic recombination. A RecA homologue, Rad51, plays a pivotal role in homology search and strand exchange. Genetic analysis has shown that among members of its epistasis group, RAD52 alone is required for recombination between direct repeats yielding deletions. Very little has been discovered about the biochemical roles and structure of the Rad52 protein.

Results

Purified Rad52 protein binds to both single‐stranded DNA (ssDNA) and double‐stranded DNA (dsDNA). Electron microscope observations revealed that Rad52 molecules form multimeric rings. An increase in the intensity of fluorescence when Rad52 is bound to εDNA showed an alteration of the structure of ssDNA. RPA was binding to Rad52 and enhanced the annealing of complementary ssDNA molecules. This enhancement was not observed in Escherichia coli SSB protein or T4 phage gp32 protein.

Conclusion

Rad52 forms a ring‐like structure and binds to ssDNA. Its structure and DNA binding properties are different from those of Rad51. The interaction of Rad52 with RPA plays an important role in the enhancement of annealing of complementary ssDNAs. We therefore propose that Rad52 mediates the RAD51‐independent recombination through an ssDNA annealing, assisted by RPA.
Identification of sna41 gene, which is the suppressor of nda4 mutation and is involved in DNA replication in Schizosaccharomyces pombe
Authors: Miyake Sanae , Yamashita Shigeru
Published: March 2003

Background

The replication licensing factor limits DNA replication to once in a cell cycle and is thought to contain MCM proteins as its component parts. Six MCM subtypes have been identified in various species. These MCM proteins are thought to bind each other to make a heteromeric complex. The Nda4 protein of Schizosaccharomyces pombe is one of the MCM proteins and is involved in DNA replication.

Results

The suppressor mutant of nda4 was isolated and the mutant gene was named sna41. The sna41‐912 mutant demonstrated the ts phenotype, with an elongated cell shape at the restrictive temperature. Cells with 1C DNA content accumulated 2 h after shifting up to the restrictive temperature. This result suggests that sna41 is also involved in DNA replication. The sna41 genomic clone was isolated by a complementation of the ts phenotype of the mutant strain and was sequenced. The sna41 gene encodes a protein of 638 amino acids, which has low homology with CDC45 in S. cerevisiae. The gene disruption analysis showed that sna41 gene is essential for viability.

Conclusions

The S. pombe sna41 mutation suppresses the nda4‐108 mutation. Sna41 is involved in DNA replication and may play some roles in the regulation of DNA replication by the MCM proteins.
Characterization of the C. elegans gap‐2 gene encoding a novel Ras‐GTPase activating protein and its possible role in larval development
Authors: Hayashizaki Seiji , Iino Yuichi , Yamamoto Masayuki
Published: March 2003

Background

The Ras signalling pathway plays several important roles in the development of the nematode Caenorhabditis elegans. So far, two types of Ras‐GTPase activating proteins (Ras‐GAPs) have been identified in this organism. To aid the study of the regulation and function of the Ras pathway, we set out to isolate a new GAP gene from C. elegans by transcomplementation of the fission yeast gap1 mutant.

Results

We isolated a C. elegans cDNA that encoded a protein which was similar to, but not exactly homologous with mammalian p120 Ras‐GAP. This gene, named gap‐2, generated at least nine distinct mRNA species through transcription from different promoters and subsequent alternative splicing involving 25 exons. These isoforms were differentially expressed among tissues. A deletion of gap‐2 caused no obvious phenotype by itself, but a loss of gap‐2 function could suppress larval lethality in both let‐23 and let‐60 reduction‐of‐function mutants, in which the Ras activity was lowered.

Conclusions

C. elegans gap‐2 encodes a novel Ras‐GAP, which is similar to vertebrate p120 but which may constitute a new GAP subfamily. gap‐2 mRNA isoforms arise by an unusually extensive variation in initiation sites and associated alternative splicing, and each isoform may play a distinct role in specific tissues. GAP‐2 appears to function as a negative regulator of LET‐60 Ras during larval development.
Myosin II activation promotes neurite retraction during the action of Rho and Rho‐kinase
Authors: Amano Mutsuki , Chihara Kazuyasu , Nakamura Nao , Fukata Yuko , Yano Takeo , Shibata Masao , Ikebe Mitsuo , Kaibuchi Kozo
Published: March 2003

Background

The Rho small GTPase regulates myosin II activity through the phosphorylation of the myosin light chain (MLC) by activating Rho‐kinase, which is a target of Rho. Several lines of evidence point to an important role of Rho in the action of lysophosphatidic acid (LPA) and thrombin in provoking neurite retraction in N1E‐115 neuroblastoma cells.

Results

Here we examined whether Rho‐kinase and myosin II are involved in neurite retraction in N1E‐115 cells. We showed that the expression of constitutively active forms of Rho‐kinase induced neurite retraction in N1E‐115 cells and MLC phosphorylation in NIH 3T3 cells, whereas the expression of dominant negative forms of Rho‐kinase inhibited the LPA‐induced neurite retraction in N1E‐115 cells and the serum‐induced MLC phosphorylation in NIH 3T3 cells. The expression of mutant MLCT18D,S19D (substitution of Thr and Ser by Asp), which is known to lead to the activation of myosin ATPase and a conformational change of myosin II when reconstituted with myosin heavy chains in vitro, also promoted neurite retraction.

Conclusion

These results indicate that Rho‐kinase is involved in the LPA‐induced neurite retraction downstream of Rho, and that myosin II activation promotes neurite retraction downstream of Rho and Rho‐kinase.
recessive spotting: a linked locus that interacts with W/Kit but is not allelic
Authors: Bennett Dorothy C. , Trayner Ian D. , Piao Xianhua , Easty David J , Klüppel Michael , Alexander Warren S. , Wagner Erwin F. , Bernstein Alan
Published: March 2003

Background

The murine coat‐colour mutation recessive spotting (rs) maps very closely to the W/Kit locus, encoding the proto‐oncoprotein Kit, the protein tyrosine kinase receptor for stem cell factor. Kit is important in the development of melanocytes, germ cells, interstitial cells of Cajal (ICC) and haemopoietic lineages, including mast cells. rs has never been genetically separated from Kit, and interacts with Kit mutations, suggesting that it is a recessive allele of Kit. Here we have tested this possibility. We have shown previously that diploid rs/rs melanocytes proliferated more slowly than did +/+ melanocytes, as did an immortal line of rs/rs melanocytes, melan‐rs.

Results

The Kit mRNA level in rs/rs melanocytes was indistinguishable from that of other melanocyte lines. The Kit cDNA sequence from rs/rs melanocytes and the kinase activity of Kit in rs/rs mast cells appeared to be normal. No deficiency of mast cells or ICC was observed in rs/rs mice. Moreover, following the overexpression of a normal Kit cDNA, proliferation of rs/rs melanocytes was retarded further, but that of +/+ melanocytes was increased, indicating an intracellular interaction between rs and Kit. Of other closely linked tyrosine kinase genes, melanocytes and melanoblasts did not express mRNA for Pdgfra, Flk‐1 or Txk, but both expressed Tec, encoding a nonreceptor kinase that interacts with Kit.

Conclusions

rs is not a mutation in Kit, although we have confirmed that rs interacts with Kit. It seems unlikely that rs affects Pdgfra, Flk‐1 or Txk, but Tec remains a candidate for rs.
Cell cycle dependent topological changes of chromosomal replication origins in Saccharomyces cerevisiae
Authors: Fujita Masako , Hori Yuji , Shirahige Katsuhiko , Tsurimoto Toshiki , Yoshikawa Hiroshi , Obuse Chikashi
Published: March 2003

Background

The ORC (Origin Recognition Complex) of Saccharomyces cerevisiae is a protein complex for the initiation of replication which interacts with a cis‐element, ACS (ARS Consensus Sequence), essential for DNA replication. The protein–DNA complex detected by the DNase I genomic footprinting method has been shown to vary depending on cell cycle progression. Further studies on topological changes of replication origin in vivo caused by ORC association are crucial for an understanding of chromosomal DNA replication in S. cerevisiae.

Results

Topological changes in the replication origins of the S. cerevisiae chromosome were studied by an in vivo UV photofootprinting method which is capable of detecting the change in the flexibility of DNA caused by protein binding. The footprinting method detected the inhibition and enhancement of UV‐induced pyrimidine dimer formation in A and B1 elements of a chromosomal origin, ARS1, depending on the activity of native ORC subunits. Furthermore, footprint patterns were reproduced in vitro with purified ORC. The inhibition regarding the A element was stronger during the S to late M phase than that during the progression through the G1 phase. Functional CDC6 and MCM5 were required for maintaining the weaker inhibition state in G1‐arrested cells.

Conclusion

The application of in vivo UV photofootprinting in studies of topological changes of S. cerevisiae replication origins revealed the presence of two modes of topological ORC‐ACS interaction. The weaker footprint in the G1 phase represents a specific topology of ACS, resulting from an alteration of the ORC–ACS interaction aided by CDC6 and MCM5, and this topological change may make the replication origin competent for initiating DNA replication.
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

Background

The 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.

Results

We 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.

Conclusions

The 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

Background

ELAV‐like neuronal RNA‐binding proteins are highly conserved in many neurone‐containing organisms and have been implicated in neuronal development and differentiation.

Results

Mammalian 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.

Conclusions

Our 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

Background

Bax 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.

Results

We 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.

Conclusions

The 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

Background

Mouse 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.

Results

The 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.

Conclusion

These observations suggest that humans and mice have acquired distinct strategies during evolution for determining the asymmetric expression of LEFTY and lefty genes.
Thermodynamic and kinetic analyses for understanding sequence‐specific DNA recognition
Authors: Oda Masayuki , Nakamura Haruki
Published: March 2003

Thermodynamic and kinetic analyses of biomolecular interactions reveal details of the energetic and dynamic features of molecular recognition processes, and complement structural analyses of the free and complexed conformations. The recent improvements in both isothermal titration calorimetry and surface plasmon resonance sensoring provide powerful tools for analysing biomolecular interactions in thermodynamic and kinetic approaches. The thermodynamic and kinetic parameters obtained for binding between protein and DNA indicate the mechanism of specific DNA recognition, in the high‐resolution structures of the protein–DNA complexes. The effects of temperature and ionic strength reflect the conformational changes of the protein and DNA molecules upon complex formation, including important contributions of water and solutes. When combined with mutational studies, the interactions can be reduced to several energetic contributions from individual contacts. These studies should be useful to determine general features of protein functions in genetic regulation.
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

Background

The 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.

Results

The 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.

Conclusion

The 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

Background

Cryptochromes (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.

Results

We 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.

Conclusions

These 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.

Results

We 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.

Conclusion

p90rsk1 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.
Functional analysis of the C‐terminal cytoplasmic region of the M‐factor receptor in fission yeast
Authors: Hirota Kouji , Tanaka Kayoko , Watanabe Yoshinori , Yamamoto Masayuki
Published: March 2003

Background

Yeast mating‐pheromone receptors facilitate the study of G protein‐coupled signal transduction. To date, molecular dissection of the budding yeast α‐factor receptor has been done extensively, but little analysis has been performed with pheromone receptors of fission yeast, another genetically tractable yeast species.

Results

We analysed the fission yeast M‐factor receptor Map3p. Truncation of the C‐terminal 54 amino acids made Map3p dominant‐negative over the wild‐type. This form, called Map3‐dn9p, was competent in the induction of pheromone‐dependent gene expression, although it could not direct proper conjugation. Map3‐dn9p failed both to provoke the orientated projection of conjugation tubes and to induce adaptation to the pheromone signal associated with endocytosis of the receptor. Deletion and substitution analyses suggested that the integrity of the C‐terminal region, rather than a specific subgroup of amino acid residues therein, was vital for the respective Map3p activities. Ubiquitination of the C‐terminus was not absolutely essential for Map3p function.

Conclusions

The C‐terminal region of Map3p is dispensable for the pheromone signalling per se, but is pivotal for adaptation and pheromone‐induced conjugation tube formation, as is true with the budding yeast α‐factor receptor. However, the mechanisms which induce adaptation appear to differ between fission and budding yeast concerning the necessity of ubiquitination.
AAA+ superfamily ATPases: common structure–diverse function
Authors: Ogura Teru , Wilkinson Anthony J.
Published: March 2003

The AAA+ superfamily of ATPases, which contain a homologous ATPase module, are found in all kingdoms of living organisms where they participate in diverse cellular processes including membrane fusion, proteolysis and DNA replication. Recent structural studies have revealed that they usually form ring‐shaped oligomers, which are crucial for their ATPase activities and mechanisms of action. These ring‐shaped oligomeric complexes are versatile in their mode of action, which collectively seem to involve some form of disruption of molecular or macromolecular structure; unfolding of proteins, disassembly of protein complexes, unwinding of DNA, or alteration of the state of DNA–protein complexes. Thus, the AAA+ proteins represent a novel type of molecular chaperone. Comparative analyses have also revealed significant similarities and differences in structure and molecular mechanism between AAA+ ATPases and other ring‐shaped ATPases.
Initial process of polyglutamine aggregate formation in vivo
Authors: Kimura Yoko , Koitabashi Sumiko , Kakizuka Akira , Fujita Takashi
Published: March 2003

Background Polyglutamine expansion in protein is responsible for several inherited neuro‐degenerative diseases. The expansion has toxic effects on neural cells as well as results in forming aggregates. Using yeast, we examined the initial process of polyglutamine aggregate formation in vivo.

Results Following expression, polyglutamine tracts were of a soluble form during a lag period, and then formed insoluble complexes. The lag was prolonged and the formation of insoluble complex became slower by decreasing the number of polyglutamine tracts and by a treatment with guanidine hydrochloride. Gel filtration analysis revealed that the soluble polyglutamine existed in a small form. Formation of polyglutamine aggregates appeared to follow similar kinetics reported in the in vitro studies, where polyglutamine tracts self‐aggregate in a length‐, concentration‐ and time‐dependent manner. However, in vivo, Hsp104 was required for the conversion from a soluble to an insoluble state. Without Hsp104, polyglutamine tracts tended to remain in a small soluble form, prolonging the lag. Moreover, the dependency on Hsp104 for aggregate formation was strong with the short polyglutamine tract, and decreased with the long polyglutamine tract.

Conclusion For polyglutamine aggregate formation, a balance of parameters including the length of the polyglutamine tract, Hsp104, and level of polyglutamine expression determined its efficiency.
Cell culture analysis of the regulatory frameshift event required for the expression of mammalian antizymes
Authors: Howard Michael T. , Shirts Brian H. , Zhou Jiadong , Carlson C. Lance , Matsufuji Senya , Gesteland Raymond F. , Weeks Reitha S. , Atkins John F.
Published: March 2003

Background Antizyme is a critical regulator of cellular polyamine levels due to its effect on polyamine transport and its ability to target ornithine decarboxylase for degradation. Antizyme expression is autoregulatory, through dependence on an unusual +1 translational frameshift mechanism that responds to polyamine levels.

Results HEK293 cells were depleted of polyamines by treatment with an ornithine decarboxylase inhibitor, difluoromethylornithine (DFMO), and grown in the presence or absence of exogenous polyamines prior to the analysis of ribosomal frameshifting levels. Results obtained using an optimized dual luciferase assay system reveal a 10‐fold dynamic range of frameshifting, which correlates positively with polyamine addition. Polyamine addition to cells, which have not been pre‐treated with DFMO, also resulted in an increase in antizyme frameshifting but to a lesser degree (1.3 to 1.5‐fold). In addition, the constructs with the 3′ deletion were more responsive to stimulation by polyamine addition than those with the 5′ deletion.

Conclusions The observed regulation of antizyme frameshifting demonstrates the efficiency of a polyamine homeostatic mechanism, and illustrates the utility of a quantifiable cell‐based assay for the analysis of polyamines or their analogues on translational frameshifting.
Circadian rhythm of aromatic l‐amino acid decarboxylase in the rat suprachiasmatic nucleus: gene expression and decarboxylating activity in clock oscillating cells
Authors: Ishida Yoshiki , Yokoyama Chihiro , Inatomi Tsutomu , Yagita Kazuhiro , Dong Xin , Yan Lily , Yamaguchi Shun , Nagatsu Ikuko , Komori Takahide , Kitahama Kunio , Okamura Hitoshi
Published: March 2003

Background: Aromatic l‐amino acid decarboxylase (AADC) is the enzyme responsible for the decarboxylation step in both the catecholamine and indoleamine synthetic pathways. In the brain, however, a group of AADC containing neurones is found outside the classical monoaminergic cell groups. Since such non‐monoaminergic AADC is expressed abundantly in the suprachiasmatic nucleus (SCN), the mammalian circadian centre, we characterized the role of AADC in circadian oscillation.

Results :
AADC gene expression was observed in neurones of the dorsomedial subdivision of the SCN and its dorsal continuant in the anterior hypothalamic area. These AADC neurones could uptake exogenously applied L‐DOPA and formed dopamine. AADC was co‐expressed with vasopressin and the clock gene Per1 in the neurones of the SCN. Circadian gene expression of AADC was observed with a peak at subjective day and a trough at subjective night. The circadian rhythm of AADC enzyme activity in the SCN reflects the expression of the gene.

Conclusions: Non‐monoaminergic AADC in the SCN is expressed in clock oscillating cells, and the decarboxylating activity of master clock cells are under the control of the circadian rhythm.
SsrA‐mediated protein tagging in the presence of miscoding drugs and its physiological role in Escherichia coli
Authors: Abo Tatsuhiko , Ueda Koji , Sunohara Takafumi , Ogawa Kazuko , Aiba Hiroji
Published: March 2003

Background: We have shown recently that read‐through of a normal stop codon by a suppressor tRNA in specific genes possessing a Rho‐independent terminator leads to SsrA‐mediated tagging of extended proteins in Escherichia coli cells. Miscoding antibiotics such as kanamycin and streptomycin reduce translational fidelity by binding to the 30S ribosomal subunit. The aim of the present study was to address how miscoding antibiotics affect the read‐through of stop codons and SsrA‐mediated protein tagging.

Results: Miscoding antibiotics caused translational read‐through of stop codons when added to the culture medium at sublethal concentrations. Under the same conditions, the drugs enhanced SsrA‐mediated tagging of bulk cellular proteins, as observed in cells carrying an ochre suppressor tRNA. Translational read‐through products generated from the crp gene in the presence of the antibiotics was efficiently tagged by the SsrA system, presumably because the ribosome reached the 3′ end of the mRNA defined by the terminator hairpin. The SsrA‐defective cells were more sensitive to the miscoding antibiotics compared to the wild‐type cells.

Conclusion: We conclude that the SsrA system contributes to the survival of cells by dealing with translational errors in the presence of low concentrations of miscoding antibiotics.
The VAR1 locus of Arabidopsis encodes a chloroplastic FtsH and is responsible for leaf variegation in the mutant alleles
Authors: Sakamoto Wataru , Tamura Takayuki , Hanba‐Tomita Yuko ,   Sodmergen, Murata Minoru
Published: March 2003

Background: A leaf‐variegated mutation var1 of Arabidopsis results in the development of abnormal plastids and the formation of a green/white sector. Genetic analysis of the var1 mutant indicated that it acts synergistically with another mutation var2, suggesting that the two genes are relevant. The VAR2 locus has been shown to encode a chloroplastic FtsH, an ATP‐dependent protease which is possibly involved in the degradation of thylakoid proteins and plastid development.

Results: In this study we show that the VAR1 locus encodes a chloroplastic FtsH protein homologous to VAR2. VAR1 contains a conserved motif for ATPase and a metalloprotease characteristic to FtsH proteins, and is targeted into chloroplasts. A VAR1‐fusion protein synthesized in vitro exhibited ATPase activity and partial metalloprotease activity. The maximum yield of photochemistry, measured by chlorophyll fluorescence, showed that the var1 mutants were sensitive to photoinhibitory light exposure at 800 µmol/m2/s.

Conclusion: VAR1 and VAR2 comprise an FtsH small gene family together with other FtsH genes in Arabidopsis. VAR1 as well as VAR2 may play an important role in degrading photodamaged subunits in photosystem II. Loss of VAR1 and VAR2 perhaps impairs the photoprotection mechanism and thylakoid development, causing leaf variegation as a consequence.
Cyclin G1 associates with MDM2 and regulates accumulation and degradation of p53 protein
Authors: Kimura Shinya H. , Nojima Hiroshi
Published: March 2003

Background: Cyclin G1 is a transcriptional target of p53 and is induced by DNA damage in a p53 dependent manner. Analysis of cyclin G1 disrupted mice demonstrated that cyclin G1 is involved in many of the functions regulated by p53 such as apoptosis, growth control and check point regulation in response to DNA damage. The results suggest that the main role of cyclin G1 is to mediate or regulate the function of p53.

Results: Western blot analysis revealed that the accumulation of p53 protein during the initial 24 h period following DNA damage is reduced in cyclin G1–/– cells compared to wild‐type cells. This decrease in p53 accumulation could be recovered by introducing a cDNA expressing cyclin G1. Cyclin G1 interacted directly with MDM2 and promoted the formation of the ARF/MDM2 complex within the initial 24 h period following DNA damage. Furthermore, 48 h after irradiation, accumulation of p53 protein was enhanced in cyclin G1–/– cells compared to wild‐type cells. In contrast, in 48 h postirradiated wild‐type cells, the cyclin G1‐MDM2 complex was found not to be associated with ARF but with the B′α subunit of protein phosphatase A.

Conclusion: These results suggest that cyclin G1 stabilizes and promotes the degradation of p53 protein by associating, respectively, with MDM2 complexes containing ARF and PP2A.