bluuebunny/crossref_metadata_2025_split huggingface dataset:
Large scale isolation of osteoclast‐specific genes by an improved method involving the preparation of a subtracted cDNA library
Authors: Kobori Masato , Ikeda Yasushi , Nara Hiromi , Kato Miyuki , Kumegawa Masayoshi , Nojima Hiroshi , Kawashima Hiroyuki 
Published: March 2003

BackgroundOsteoclasts play crucial roles in bone resorption, which triggers bone remodeling. Molecular mechanisms underlying these osteoclast‐specific biological functions remain elusive because only a limited number of osteoclast‐specific genes have been identified. To circumvent this, we isolated a large number of osteoclast‐specific genes by preparing a subtracted cDNA library of high quality.ResultsWe first constructed a plasmid expression vector (pAP3neo) that allowed an efficient subtraction. Then, we improved the standard protocols for preparation of the cDNA library and the subsequent subtraction procedure. Using our protocol, we prepared a rabbit osteoclast cDNA library of high complexity. Subsequently, we prepared an osteoclast‐specific cDNA library of high complexity by subtracting it with biotin‐labelled mRNA, derived from rabbit spleen through the biotin–avidin method. The resulting library included a high proportion of full‐length cDNA inserts. Using DNA dot blot analysis, we found that the osteoclast‐specific cDNA clones were highly enriched in this subtracted cDNA library, i.e. nearly 70% of the analysed clones were primarily detected in osteoclasts but not in spleen. Multiple‐tissue Northern analysis also showed that many of these clones were expressed almost exclusively in osteoclasts. DNA sequencing of randomly selected clones showed that 424 cDNA species out of 1136 analysed were novel. DNA sequencing also showed that our subtracted cDNA library was almost equalized, suggesting that the library may contain almost all of the osteoclast‐specific genes.ConclusionFrom these data, we conclude that our subtraction protocols, and the subsequent procedure for the analysis of the isolated clones developed here, are useful for the comprehensive isolation and identification of transcriptionally up‐ or down‐regulated genes in general.
Transcription factor CP2 is essential for lens‐specific expression of the chicken αA‐crystallin gene
Authors: Murata Takuya , Nitta Masahiro , Yasuda Kunio 
Published: March 2003

Background:Lens‐specific transcriptional activation of the chicken αA‐crystallin gene is controlled by the distal and proximal enhancers, αCE1 and αCE2, respectively. Analysis using specific monoclonal antibodies against purified αCE1‐binding factor αCEF1 revealed that αCEF1 is composed of two distinct subunits.Results:We have demonstrated that one of the subunits of αCEF1 is encoded by chicken ubiquitous transcription factor CP2 (cCP2), which is homologous to mouse CP2, and human CP2/LBP‐1/LSF‐1. Electrophoretic mobility shift assays and cross‐linking experiments showed that αCEF1 and bacterially expressed cCP2 form a tetramer. Overexpression of cCP2 activates transcription through αCE1, but a mutant cCP2 lacking the DNA‐binding domain reduced the transcription to basal levels. Although cCP2 binds to the CP2 template from the mouse α‐globin promoter, it fails to promote transcription through this template. Element substitution experiments between αCE1 and the CP2 template revealed that the lens‐specific enhancer activity of αCE1 is due to the 6 bp sequence (−139/−134; lens‐specific element (LSE)) adjacent to the 3′ of the cCP2 binding site within αCE1.Conclusion:We have shown that the tetrameric transcription factor cCP2 is essential for lens‐specific transcription of the chicken αA‐crystallin gene, although it is ubiquitously expressed. We propose a model where cCP2 cooperates with a putative lens‐specific factor which binds to LSE. Fig. 6.
Molecular cloning and characterization of a novel member of the MAP kinase superfamily
Authors: Miyata Yoshihiko , Akashi Makoto , Nishida Eisuke 
Published: March 2003

BackgroundMembers of the MAP kinase superfamily play important roles in a wide variety of signal transduction pathways, and several members have been identified. However, the diversity and complexity of cellular responses in mammalian systems may imply existence of hitherto unidentified members of the MAP kinase superfamily.ResultsWe report the molecular cloning and characterization of a novel member of the MAP kinase superfamily. We isolated full‐length mouse and human cDNAs that encode complete open reading frames of a novel protein kinase, termed MOK. MOK consists of 419 (human) and 420 (mouse) amino acids, with a calculated molecular weight of 48kDa. MOK contains all of the protein serine/threonine kinase consensus motifs and shows a modest similarity to members of the MAP kinase superfamily and MAK and MAK‐related kinase (MRK). In addition, MOK possesses a Thr‐Glu‐Tyr (TEY) motif in the activation loop domain, as do classical MAP kinases. MOK is widely expressed in normal tissues and organs and localizes to the cytoplasm. MOK is able to phosphorylate several known MAP kinase substrates and to undergo autophosphorylation. A mutation in the TEY motif to AEF abolished the kinase activity of MOK, and the treatment of cells with a phosphatase inhibitor, okadaic acid, enhanced the kinase activity of MOK, suggesting the existence of an upstream kinase. Phorbol ester TPA was found to stimulate the kinase activity of MOK, whereas serum stimulation, osmotic shock, or anisomycin treatment did not significantly activate MOK.ConclusionThese results indicate that MOK is distantly related to members of known subfamilies of the MAP kinase superfamily and can therefore be classified as a novel member.
Hyperosmotic stress‐induced reorganization of actin bundles in Dictyostelium cells over‐expressing cofilin
Authors: Aizawa Hiroyuki , Katadae Maiko , Maruya Mikako , Sameshima Masazumi , Murakami‐Murofushi Kimiko , Yahara Ichiro 
Published: March 2003

BackgroundCofilin is a low‐molecular weight actin‐modulating protein, which binds to, severs, and depolymerizes actin filaments in vitro. Aip1, an actin‐interacting protein, was recently identified as a product of a gene on a multicopy plasmid which suppresses the temperature‐sensitive phenotype of a cofilin mutant in Saccharomyces cerevisiae. Actin cytoskeleton plays an essential role in resistance to hyperosmotic stress in Dictyostelium discoideum. The roles of cofilin and Aip1 in this resistance are not known.ResultsIn response to hyperosmotic stress, D. discoideum cells round up. This stress‐induced morphological change involves the redistribution of cofilin, together with actin filaments, into cortical contractile portions of the cells, followed by their contraction. Over‐expression of cofilin increases and thickens cortical actin bundles in cells. The bundles become tight and are reorganized into a ring‐shaped structure in response to hyperosmotic stress. The ring structure of actin bundles had two characteristic bands across them; bright and dark bands, heavily stained and not stained with phalloidin. In the bundles, straight filaments with a diameter of 5.3‐nm were aligned parallel by cross‐bridge structures. In cells lacking the myosin‐II heavy chain, the bundles, which were induced by an over‐expression of cofilin, shortened and became straight following hyperosmotic stress, forming a polygonal structure. D. discoideum Aip1/Wrp2 enhanced the severing of actin filaments by cofilin in vitro and colocalized with cofilin in cells, including those that were over‐expressing cofilin before and after exposure to hyperosmotic stress.ConclusionsCofilin plays a pivotal role in concert with Aip1/Wrp2 in the reorganization of actin architectures into bundles that contract in a myosin‐II‐independent manner, in response to hyperosmotic stress.
Multiple mammalian proteasomal ATPases, but not proteasome itself, are associated with TATA‐binding protein and a novel transcriptional activator, TIP120
Authors: Makino Yasutaka , Yoshida Tatsushi , Yogosawa Shingo , Tanaka Keiji , Muramatsu Masami , Tamura Taka‐aki 
Published: March 2003

BackgroundSUG1 belongs to proteasomal ATPase. Previous studies have demonstrated that SUG1 is associated with TBP. It is assumed to be involved in transcriptional regulation in addition to proteolysis. In this study, we investigated the association of mammalian SUG1 with TBP in more detail.ResultsPull‐down experiments with TBP revealed multiple TBP‐interacting proteins (TIPs) that were recovered dependent upon the presence of C‐terminal conserved domain of TBP. By 2‐D electrophoresis, we identified SUG1 in TIPs. By using far‐Western analysis, we identified two proteins that could directly bind to TBP: SUG1 and another proteasomal ATPase (S4). Protein microsequencing and Western blotting identified all the remaining proteasomal ATPases (MSS1, TBP1, TBP7, and SUG2) in the TIP preparations. We present evidence that TBP and at least SUG1, MSS1, and S4 form a complex in the cell. However, no evidence of association of TBP with the 26S proteasome or its 19S regulatory unit was obtained. The molecular mass of the TBP/ATPases‐complex, which also included a novel transcription regulatory factor, TIP120, was estimated to be ≈800 kDa.ConclusionThese results suggest that there is a novel multisubunit complex containing TBP and proteasomal ATPases. Based on our findings, we hypothesize that proteasomal ATPases are involved in transcriptional regulation in addition to proteolysis.
Rho‐dependence of Schizosaccharomyces pombe Pck2
Authors: Sayers Lee G. , Katayama Satoshi , Nakano Kentaro , Mellor Harry , Mabuchi Issei , Toda Takashi , Parker Peter J. 
Published: March 2003

BackgroundIn metazoans, the HR1 domain, a motif found in a number of proteins including the protein kinase C‐related PRKs, is responsible for an interaction with Rho‐GTPases. The structural similarity between the Schizosaccaromyces pombe Pck proteins and the mammalian Rho‐dependent protein kinase C‐related family, has led us to investigate the relationship between the function of Rho and that of Pck1/2.ResultsRho1 is shown to interact with the conserved N‐terminal HR1 domain of Pck1/2 in vitro and in vivo. Lethal overproduction of Rho1 is neutralized by co‐expression of the Pck2 HR1 domain, which by itself compromises growth when overproduced. The Pck2–Rho1 interaction has a profound effect on the steady state expression of Pck2 and this is shown to parallel the immunoprecipitated activity and phosphorylation of Pck2 at its activation loop site. It is further shown that Pck2 becomes localized at the septum, where Rho1 is also located.ConclusionsThe results demonstrate that the Pck proteins are Rho1 effectors in fission yeast and that the HR1 domain is a universal motif for the Rho‐GTPase interaction. Furthermore, the evidence supports the contention that the yeast Pck1 and Pck2 proteins are primitive protein kinases, which in vertebrates have evolved into the two distinct PKC and PRK families.
Regulation of interaction of the acetyltransferase region of p300 and the DNA‐binding domain of Sp1 on and through DNA binding
Authors: Suzuki Toru , Kimura Akatsuki , Nagai Ryozo , Horikoshi Masami 
Published: March 2003

BackgroundThe coactivator p300 acts as a transcriptional adaptor for many DNA‐binding activators. The finding that p300 possesses intrinsic acetyltransferase activity which, by chemically modifying histone tails affects the nucleosomal environment and transcription, has greatly advanced our understanding of its function. Subsequent recent studies have shown that non‐histone proteins are also acetylated. However, one central question which has remained unanswered is how the coactivator/acetyltransferase interacts with DNA‐binding activators to modulate their actions.ResultsHere we have demonstrated physical and functional interaction between the acetyltransferase region of p300 and the DNA‐binding domain (DBD) of the transcription factor Sp1. This interaction stimulates DNA binding by the DBD of Sp1, which is mediated primarily by physical interaction rather than acetylation, despite acetylation of the DBD of Sp1 by the acetyltransferase region of p300. Furthermore, DNA binding by the DBD of Sp1 inhibits both its association and acetylation by the acetyltransferase region of p300.ConclusionsThese findings suggest a new role for p300 in regulating promoter access by DNA‐binding activators through multiple regulatory interactions.
Temporal and selective association of multiple sigma factors with RNA polymerase during sporulation in Bacillus subtilis
Authors: Fujita Masaya 
Published: March 2003

BackgroundDuring sporulation in Bacillus subtilis, an asymmetric division produces two cells, a forespore and mother cell, with which follow different developmental paths. The highly ordered programme of temporal and spatial gene activation during sporulation is governed by the principal RNA polymerase holoenzyme (EσA) and alternative holoenzyme forms containing the developmental sigma factors σH, σF, σE, σG and σK, which appear successively during development. The control mechanism(s) of temporal and selective association of multiple sigma factors with core RNA polymerase is unclear. As a first step to addressing these issues, this report quantifies the amount of each subunit of RNA polymerase that is present in the sporangium during sporulation, and analyses in vitro the relative affinities of each sigma subunit for core RNA polymerase.ResultsUsing quantitative immunoblot analysis, the amounts of EσA, EσH, EσE and EσK in relation to the total amount of RNA polymerase at appropriate time‐points were found to be 15%, 1%, 6% and 2%, respectively. Therefore, the core RNA polymerase is predicted to be in excess. The level of core RNA polymerase and σA remained constant during the transition from vegetative growth to sporulation, whereas the sporulation‐specific sigma factors appeared successively, in the order σH, σE and σK. Competition experiments between sigma factors in an in vitro transcription system revealed the dominance of σA over σH and σE for open promoter complex formation. These results are inconsistent with the idea that late appearing sigma factors can displace earlier appearing sigmas from the core enzyme.ConclusionsAs the core RNA polymerase is in excess, the results suggest that successive sigma factors can bind to core RNA polymerase without having to displace earlier appearing sigma factors. Thus, the programme of gene expression during sporulation might not require mechanisms for the substitution of one sigma factor by another on the core RNA polymerase.
MSSP promotes ras/myc cooperative cell transforming activity by binding to c‐Myc
Authors: Niki Takeshi , Izumi Sachiyo , Saëgusa Yumiko , Taira Takahiro , Takai Toshiki , Iguchi‐Ariga Sanae M. M. , Ariga Hiroyoshi 
Published: March 2003

BackgroundMSSPs, mycsingle
strand binding
proteins, were originally identified as proteins recognizing a putative replication origin/transcriptional enhancer in the human c‐Myc gene. The cDNAs encoding four of the family proteins, MSSP‐1, MSSP‐2, Scr2 and Scr3, were cloned. These proteins carry two copies of the putative RNA binding domains, RNP‐A and RNP‐B, and have been suggested to participate in DNA replication and cell cycle progression from the G1 to the S phase.ResultsWe report that MSSP‐1 and MSSP‐2 bound directly to the C‐terminal portion of c‐Myc, along with Max, side by side. MSSP, c‐Myc and Max formed a ternary complex in vivo, although MSSP did not directly associate with Max. The MSSP/Myc/Max ternary complex lost the binding activity to the E‐box sequence—the recognition sequence of c‐Myc/Max complex—thereby abrogating the E‐box‐dependent transcription activity of c‐Myc. MSSP specifically stimulated the cooperative transforming activity of c‐myc with ras, in a manner dependent upon the RNP sequences, while mssp itself showed no transforming activity in mouse NIH3T3 cells. The NIH3T3 transformants, together with ras, myc and mssp, grew to form very large colonies in soft agar, as compared to those with ras plus myc or ras alone.ConclusionsMSSP is a modulator of c‐Myc and the c‐Myc/MSSP complex may deregulate cell cycle controls and lead cells towards transforming pathways.
Bidirectional migration of SeqA‐bound hemimethylated DNA clusters and pairing of oriC copies in Escherichia coli
Authors: Hiraga Sota , Ichinose Chiyome , Onogi Toshinari , Niki Hironori , Yamazoe Mitsuyoshi 
Published: March 2003

BackgroundWe previously found that SeqA protein, which binds preferentially to newly replicated hemimethylated DNA, is localized as discrete fluorescent foci in Escherichia coli cells. A single SeqA focus, localized at midcell, separates into two foci and these foci migrate abruptly in opposite directions.ResultsThe present study shows that (i) appearance of SeqA foci depends on continuous DNA replication, suggesting that the SeqA foci represent clusters consisting of SeqA and newly replicated hemimethylated DNA, (ii) in a synchronous round of replication, a single SeqA focus at midcell separates into two foci and these foci abruptly migrate in opposite directions midway through replication from oriC to the terminus, and (iii) oriC is replicated at midcell but replicated oriC copies remain linked with each other at midcell for 40 min after replication at 30 °C. Subsequently, the linked oriC copies separate and migrate gradually towards both borders of the nucleoid before cell division.ConclusionsA single cluster of SeqA‐bound hemimethylated DNA segment separates into two clusters and these clusters migrate abruptly in a bipolar fashion during progress of replication and prior to separation of linked sister oriC copies.
Targeted disruption of the homeobox transcription factor Bapx1 results in lethal skeletal dysplasia with asplenia and gastroduodenal malformation
Authors: Akazawa Hiroshi , Komuro Issei , Sugitani Yoshinobu , Yazaki Yoshio , Nagai Ryozo , Noda Tetsuo 
Published: March 2003

BackgroundNK homeobox genes have been shown to play important roles in cell‐type specification and organogenesis. Murine Bapx1, a member of NK homeobox gene family, is expressed in all the cartilageous tissues that undergo endochondral bone formation, and in gut mesentery during embryogenesis, suggesting that Bapx1 may be a key transcription factor ragulating the development of these organs.ResultsWe generated Bapx1‐deficient mice by gene targeting. Bapx1−/− mice exhibited lethal skeletal dysplasia, with abnormal development of the vertebral column and some craniofacial bones, accompanied with asplenia and gastroduodenal malformation. We showed that the proliferative activity of the sclerotome cells, forming the vertebral column, was significantly reduced in Bapx1−/− embryos. The sclerotome cells of the mutants appeared to migrate and condense normally, but subsequent differentiation into the mature vertebral bodies and intervertebral discs were affected. The sclerotome cells in the vertebral bodies failed to differentiate into hypertrophic chondrocytes, as revealed by the undetected expression of Col10a1 and Osteopontin, and the sclerotome cells in the intervertebral discs failed to express the typical extracellular matrix proteins Col2a1, Col9a2 and aggrecan. Furthermore, we investigated the effect of loss of Bapx1 on the expression of some transcription factors, identified to be expressed in the developing sclerotome and be required for normal development of the vertebral column. Among them, we found that the expression of MFH‐1 (mesenchyme forkhead‐1), which was reported to regulate the proliferation and differentiation of sclerotome cells, was significantly reduced in ventromedial sclerotome cells in Bapx1−/− mice.ConclusionOur analysis provided evidence that Bapx1 was indispensable for normal development of ventromedial structure of vertebral column and some of craniofacial bones, splenogenesis and morphogenesis of gastroduodenal tract.
Transactivation mechanisms of mouse clock transcription factors, mClock and mArnt3
Authors: Takahata Sho , Ozaki Takahiro , Mimura Junsei , Kikuchi Yasuo , Sogawa Kazuhiro , Fujii‐Kuriyama Yoshiaki 
Published: March 2003

BackgroundThe Arnt3 (also termed as BMAL1 or MOP3)/Clock heterodimer is a positive regulator of circadian rhythm and activates the transcription of target genes such as per1 and vasopressin.ResultsWe investigated the transcriptional mechanism of mArnt3/mClock heterodimer. While mClock did not possess any distinct activation domain, mArnt3 contained a transcriptional activation domain at the most C‐terminal end, the activity of which was not expressed, even in the one hybrid system, until it was bound by mClock. It has been suggested that mClock plays a regulatory or structural role in exerting a transcription enhancing effect of the mArnt3/mClock heterodimer. Deletion proceeding from amino acids 559–492 of mClock markedly reduced the transactivation activity of mArnt3/mClock heterodimer, in consistence with the results of the Clock‐Δ19 mutant. Yeast and mammalian two‐hybrid systems revealed that CBP and p300 interacted with mArnt3 via the CREB binding domain. The In vivo interaction between mArnt3 and CBP was confirmed by the GST pull down assay.ConclusionTaken together, these results suggest that the mArnt3/mClock heterodimer exerted its transactivation activity via CBP or p300 interacting with mArnt3 in the heterodimer with mClock playing a structural or regulatory role in the transactivation process.
Extracellular matrix tenascin‐X in combination with vascular endothelial growth factor B enhances endothelial cell proliferation
Authors: Ikuta Tomoki , Ariga Hiroyoshi , Matsumoto Ken‐ichi 
Published: March 2003

An extracellular matrix tenascin‐X (TNX) is highly expressed in muscular tissues, especially heart and skeletal muscle, and is also prominent around blood vessels. The precise in vivo role of TNX remains to be elucidated. To identify proteins that interact with TNX in the extracellular environment, we searched for TNX‐binding proteins using a yeast two‐hybrid system.We used mouse TNX‐specific fibronectin type III repeats (mTNX/FNIII13‐25) as a bait for the screening. We found that vascular endothelial growth factor B (VEGF‐B) binds to mTNX/FNIII13‐25. This interaction was confirmed by pull‐down assays and co‐immunoprecipitation assays. The full‐length mTNX, as well as mTNX/FNIII13‐25, interacted with both alternative splice isoforms VEGF‐B186 and VEGF‐B167. Furthermore, the full‐length mTNX also bound to VEGF‐A. The minimal region of TNX that interacts with VEGF‐B was mapped to the FNIII repeats (FNIII13‐25) but not to the other characteristic domains of TNX. The TNX‐binding site of VEGF‐B was located in the N‐terminal 115‐amino acid region. mTNX/FNIII13‐25 did not prevent the interaction of VEGF‐B with VEGFR‐1 (VEGF receptor 1), and VEGF‐B could simultaneously bind to both mTNX/FNIII13‐25 and VEGFR‐1. A conditioned medium from transfected 293T cells coexpressing full‐length TNX and VEGF‐B could promote DNA synthesis in bovine endothelial cells in which VEGFR‐1 were expressed. VEGFR‐1 phosphorylation triggered by VEGF‐B186 were increased in cells plated with mTNX/FNIII13‐25 or full‐length mTNX, compared with cells plated with VEGF‐B186 alone.TNX interacts with VEGF‐B and enhances the ability of VEGF‐B to stimulate cell proliferation. This enhanced mitogenecity is caused by increased signals mediated by the VEGFR‐1 receptor. This finding suggests a role for TNX in the regulation of the development of blood vessels such as vasculogenesis and angiogenesis.