bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sendai virus C protein physically associates with Stat1
Authors: Takeuchi Kenji , Komatsu Takayuki , Yokoo Junko , Kato Atsushi , Shioda Tatsuo , Nagai Yoshiyuki , Gotoh Bin 
Published: March 2003

Background The P/C gene of the Sendai virus (SeV), a member of the family Paramyxoviridae, encodes C protein, which plays a crucial role in counteracting the antiviral effect of interferon (IFN). The C protein blocks IFN signalling to prevent the activation of IFN stimulated genes. However, its underlying molecular mechanism remains to be defined.
Results
Signal transducer and activator of transcription 1 (Stat1) is a critical component of IFN‐α/β and IFN‐γ signalling. We found that both unphosphorylated Stat1 and tyrosine‐phosphorylated (pY) Stat1 were present in a form of aberrant high molecular weight complexes (HMWCs) of over 2 MDa in infected cell extracts under low‐salt conditions. Of recombinant vaccinia viruses carrying each SeV gene, only those expressing the C gene induced Stat1‐HMWC. SeV infected cell extracts further displayed an in vitro ability to convert the pY‐Stat1 homodimer to pY‐Stat1‐HMWC. This cell extract activity was not seen after removal of the C protein from the extracts. C protein was therefore involved in the formation of HMWCs. The HMWCs decomposed into smaller complexes in a high‐salt buffer, and under this stringent (high‐salt) condition, as well as a physiological (isotonic) condition, both unphosphorylated Stat1 and pY‐Stat1 were co‐precipitated with anti‐C antibody.
Conclusion
The C protein physically associates with Stat1. This suggests that SeV C protein directly targets Stat1 for inhibitory control on the transcriptional activation of IFN stimulated genes.
Complex phenotype of mice homozygous for a null mutation in the Sp4 transcription factor gene
Authors: Göllner Heike , Bouwman Peter , Mangold Monika , Karis Alar , Braun Harald , Rohner Iris , Del Rey Adriana , Besedovsky Hugo‐Oskar , Meinhardt Andreas , Van Den Broek Marion , Cutforth Tyler , Grosveld Frank , Philipsen Sjaak , Suske Guntram 
Published: March 2003

Background Sp4 is a zinc finger transcription factor which is closely related to Sp1 and Sp3. All three proteins recognize the same DNA elements and can act as transcriptional activators through glutamine‐rich activation domains. Unlike Sp1 and Sp3, which are ubiquitous proteins, Sp4 is highly abundant in the central nervous system, but also detectable in many other tissues.Results We have disrupted the mouse Sp4 gene by a targeted deletion of the exons encoding the N‐terminal activation domains. Sp4 knockout mice show a complete absence of Sp4 expression. They develop until birth without obvious abnormalities. After birth, two‐thirds die within 4 weeks. Surviving mice are growth retarded. Male Sp4null mice do not breed. The cause for the breeding defect remains obscure since they show complete spermatogenesis. In addition, pheromone receptor genes in the vomeronasal organ appear unaffected. Female Sp4null mice have a smaller thymus, spleen and uterus. In addition, they exhibit a pronounced delay in sexual maturation.Conclusions The phenotype of the Sp4null mice differs significantly from those described for Sp1–/– and Sp3–/– mice. Thus, the structural similarities, the common recognition motif and the overlapping expression pattern of these three transcription factors do not reflect similar physiological functions.
HscA is involved in the dynamics of FtsZ‐ring formation in Escherichia coli K12
Authors: Uehara Tsuyoshi , Matsuzawa Hiroshi , Nishimura Akiko 
Published: March 2003

Background FtsZ, a homologue of eukaryotic tubulin, localizes throughout the cytoplasm in non‐dividing Escherichia coli. However, it assembles in cytokinetic rings at the early stages of septation. Factors controlling the dynamics of FtsZ ring formation are unknown, and the molecular mechanism governing these dynamics is yet to be determined.Results At 42 °C, JE10715 mutant bacteria formed multinucleated filaments with a highly reduced number of FtsZ‐rings at potential division sites. The JE10715 phenotype resulted from a mis‐sense mutation in the hscA gene which encodes a heat shock Hsp70 family protein, with a single alanine‐to‐valine substitution at position 192 within the ATPase domain. Both JE10715 and the hscA knockout strain of JE10715 were completely complemented by a plasmid‐born, wild‐type hscA gene, but not by a mutant‐type hscA715 gene. An hscA conditional knockout of the wild‐type strain under non‐permissive conditions exhibited longer rod cells with an abnormal localization of FtsZ. The over‐expression of dnaK partially complemented the JE10715 mutation. In vitro, the ATPase activity of the mutant protein HscA715 was reduced to 63% of wild‐type HscA activity. HscA co‐sedimented with FtsZ‐polymers in the presence of GTP.Conclusion HscA is involved in FtsZ‐ring formation, through a chaperon‐like interaction with FtsZ. Defects in hscA, however, can partially be compensated for by redundant genes, including the wild‐type dnaK.
Time course analysis of precocious separation of sister centromeres in budding yeast: continuously separated or frequently reassociated?
Authors: Goshima Gohta , Yanagida Mitsuhiro 
Published: March 2003

Background Sister kinetochores are bioriented toward the spindle poles in eukaryotic metaphase before chromosome segregation. In the budding yeast Saccharomyces cerevisiae, sister centromeres/kinetochores are separated in the early spindle, while the sister arms remain associated. Biorientation is thought to be established in this organism with precocious separation of sister centromeres in early stages of the cell cycle. It is not, however, settled whether this pre‐anaphase separation is continuous or only transient and whether the transient separation has any physiological significance.Results Time‐lapse observation of the behaviour of budding yeast centromeres in living cells was performed using GFP alone or in combination with CFP marking. Sixty‐three per cent of the cell population showed permanent separation of centromeres for a long period of time from the small‐budded stage to the onset of anaphase in the single‐colour GFP‐CEN construct. The remaining cell population (6 of 16) showed brief apparent reassociation of centromere signals before anaphase, but the frequency of the association was very low. In a time‐lapse observation of the double‐colour marked cells by GFP‐CEN and CFP‐SPB (the spindle pole body), the continuous separation of sister centromeres in the short medial spindle was firmly established.Conclusions In the budding yeast, once sister centromeres separate, they rarely reassociate in pre‐anaphase. Sister centromere cohesion at this stage appears to be irrelevant for normal chromosome segregation. Whether abundant cohesin in the centromere regions has any role in anaphase remains to be determined.
Expression of human and mouse genes encoding polκ: testis‐specific developmental regulation and AhR‐dependent inducible transcription
Authors: Ogi Tomoo , Mimura Junsei , Hikida Masaki , Fujimoto Hirokazu , Fujii‐Kuriyama Yoshiaki , Ohmori Haruo 
Published: March 2003

Backgrounds Human polκ is a newly identified low‐fidelity DNA polymerase. While the enzyme bypasses an abasic site and acetylaminofluorene‐adduct in an error‐prone manner, it bypasses benzo[a]pyrene‐N2‐dG lesions in a mostly error‐free manner by incorporating predominantly dC opposite the bulky lesions. Benzo[a]pyrene (B[a]P) is activated through intracellular process mediated by the arylhydrocarbon receptor (AhR, also called the dioxin receptor), which is a ligand‐activated transcription factor with high affinities for aromatic compounds such as B[a]P and dioxin.Results We examined promoter structures of the human POLK and mouse Polk genes to study how their expressions are regulated. The mouse Polk gene is developmentally regulated in testis and utilizes two transcription start sites during spermatogenesis, while it utilizes only one site in tissues other than testis. Both of the mouse Polk and the human POLK genes have two AhR‐binding sites in the promoter regions and the expression of the mouse Polk gene is indeed enhanced upon AhR‐activation.Conclusions The AhR activation increases expression of the mouse Polk gene and probably the human POLK gene, the product of which bypasses benzo[a]pyrene‐N2‐dG lesions in a mostly accurate manner. Thus, polκ seems to function to reduce mutagenesis at benzo[a]pyrene‐adducts, although it may also have a role related to spermatogenesis.
Characterization of GTPase‐activating proteins for the function of the Rho‐family small GTPases in the fission yeastSchizosaccharomyces pombe
Authors: Nakano Kentaro , Mutoh Tadashi , Mabuchi Issei 
Published: March 2003

BackgroundThe small GTPase Rho1 has been shown to regulate the organization of the actin cytoskeleton and formation of the cell wall in the fission yeastSchizosaccharomyces pombe. Activity of Rho1 must be precisely regulatedin vivo, since both increases and decreases in its activity affect cell growth and shape. Thus, it is important to clarify the mechanism by which the activity of Rho1 is regulatedin vivo.ResultsSeven genes encoding putative GAPs, GTPase‐activating proteins, for the function of the Rho‐family proteins were isolated fromS. pombe. After disruption of these genes,rga1+was found to play important roles in cell growth and morphogenesis. Inrga1null cells, delocalized F‐actin patches and extraordinary thickening of the cell wall and the septum were observed. On the other hand, over‐expression of Rga1 produced shrunken or dumpy cells. The phenotype of therga1null cells or the Rga1‐over‐expressing cells was similar to that of cells containing abnormally high or low Rho1 activity, respectively. Moreover, direct association of Rga1 with Rho1 was shown. Rga1 was localized to the cell ends and septum where Rho1 is known to function.ConclusionsInS. pombe, Rga1 is involved in the F‐actin patch localization, cell morphogenesis, regulation of septation, and cell wall synthesis, probably functioning as a GAP for the function of Rho1.
Over‐expression of PAR‐3 suppresses contact‐mediated inhibition of cell migration in MDCK cells
Authors: Mishima Aki , Suzuki Atsushi , Enaka Makiko , Hirose Tomonori , Mizuno Keiko , Ohnishi Tetsuo , Mohri Hiroshi , Ishigatsubo Yoshiaki , Ohno Shigeo 
Published: March 2003

AbstractBackground: PAR‐3 is one of the PAR proteins, previously named ASIP, which are indispensable for the establishment of cell polarity in the embryo as well as differentiated epithelial cells. In mammalian epithelial cells, it forms a ternary complex with aPKC and PAR‐6, and is localized to the tight junction that has been suggested as being important for creating cell polarity.Results: To gain insights into the mode of PAR‐3 function in mammalian epithelial cells, we examined the effect of PAR‐3 over‐expression in MDCK cells. Although exogenous PAR‐3‐expression does not affect the epithelial polarity of confluent cells, it drastically transforms the morphology of cells at low density into a fibroblastic form with developed membrane protrusions. Time‐lapse observations have revealed that PAR‐3 over‐expressing cells show intense motility, even after they have assembled into loose colonies, suggesting that the contact‐mediated inhibition of cell migration (CIM) is suppressed. The expressions of E‐cadherin and vimentin do not change with PAR‐3 over‐expression, suggesting that exogenous PAR‐3 only disturbs the endogenous equilibrium of cellular states between a fundamental fibroblastic structure and an epithelial one. The co‐expression of a dominant negative mutant of Rac1 and the addition of nocodazole strongly antagonize the effect of PAR‐3 over‐expression, suggesting the involvement of Rac1 activation and microtubule polymerizations.Conclusions:

The data presented here suggest an intriguing link between the contact‐mediated inhibition of cell migration and the regulation of cell polarity. The putative PAR‐3 activities demonstrated here may function endogenously in the epithelial cell polarization process by being sequestered from the cytosol to the cell‐cell junctional regions with aPKC and PAR‐6 upon cell‐cell adhesion.
Characterization of HscC (Hsc62), homologue of Hsp70 in Escherichia coli: over‐expression of HscC modulates the activity of house keeping sigma factor σ70
Authors: Arifuzzaman Mohammad , Oshima Taku , Nakade Shinsuke , Mori Hirotada 
Published: March 2003

AbstractBackground: HscC, the third member of the Hsp70 family in Escherichia coli, shares 33% identity with the other two homologues, DnaK and HscA, and displays ATPase activity. Genetic and biochemical evidence indicates that the DnaK‐DnaJ chaperone system interacts with σ32 and is involved in the negative regulation of the heat shock response. Although HscC is a highly conserved protein in the Hsp70 family, its function is still unknown.Results: We observed that the over‐expression of HscC caused severe growth inhibition. To explore this effect, we performed primer extension analysis and a β‐galactosidase assay and found that HscC reduced the σ70‐dependent promoter activity. An in vitro transcription assay revealed that HscC inhibited σ70‐dependent transcription. In addition, the co‐purification analysis showed that σ70 co‐eluted with HscC.Conclusion: These results indicate that HscC forms a complex with σ70 and may function as its negative modulator.
Mapping of early firing origins on a replication profile of budding yeast
Authors: Yabuki Nami , Terashima Hiromichi , Kitada Kunio 
Published: March 2003

AbstractBackground: Understanding of the firing time determination of replication origins in the entire genome will require a genome‐wide survey of replication origins and their mapping on chromosomes. A microarray technology was applied to obtain a genome‐wide profile of DNA replication and to classify early firing origins.Results: A total of 260 potential replication origins (PROs) were identified in the entire budding yeast genome: 247 as defined peaks on the replication profile and 13 as regions located in the chromosomal termini. Based on the firing time, the 247 PROs were classified into 143 early PROs and 104 late PROs, that were not randomly distributed on chromosomes but formed separated clusters. Most of the early PROs were found to fire in the presence of hydroxyurea, indicating that they were free from the control of the intra‐S‐checkpoint mediated by Mec1 and Rad53.Conclusions: The monitoring method of DNA replication and the analysis method of microarray data used in this study proved powerful for obtaining a genome‐wide view of the initiation and progression of DNA replication.
Clamp and clamp loader structures of the human checkpoint protein complexes, Rad9‐1‐1 and Rad17‐RFC
Authors: Shiomi Yasushi , Shinozaki Ayako , Nakada Daisuke , Sugimoto Katsunori , Usukura Jiro , Obuse Chikashi , Tsurimoto Toshiki 
Published: March 2003

AbstractBackground: We have reported that protein imaging by transmission electron microscope observation based on low‐angle platinum shadowing can reproduce characteristic ring structures of the replication clamp, proliferating cell nuclear antigen (PCNA), and the clamp loader protein, replication factor C (RFC). The checkpoint protein complexes, Rad9‐Hus1‐Rad1 (Rad9‐1‐1) and Rad17‐RFCs2‐5 (Rad17‐RFC), have been predicted to function as novel clamp and clamp loader proteins, respectively, due to their amino acid sequence similarities with PCNA and RFC.Results: We reconstituted human Rad9‐1‐1 and Rad17‐RFC complexes in insect cells using a baculovirus expression system and showed purified Rad9‐1‐1 to be composed of equimolar amounts of Rad9, Hus1 and Rad1 proteins, exhibiting a native molecular mass of 100 kDa, in line with a trimeric complex. When Rad17 was co‐expressed with the four small subunits of RFC in insect cells, these proteins formed a complex of 240 kDa that displayed DNA binding, ATPase activity and binding to its predicted target protein, Rad9‐1‐1. Analyses of the molecular architecture of Rad9‐1‐1 and Rad17‐RFC using transmission electron microscopy, in comparison with PCNA and RFC, revealed the Rad9‐1‐1 complex to have a characteristic ring structure indistinguishable from that of PCNA in shape and size. In addition, the Rad17–RFC complex was found to be oval in structure and 26 × 22 nm in size with a cleft, reminiscent of the structure of RFC.Conclusion: Our direct comparison of images from the two sets of clamp and clamp loader proteins indicated that Rad9‐1‐1 and Rad17‐RFC are, respectively, structural orthologs of PCNA and RFC, with presumed functions as novel clamp and clamp‐loader proteins in eukaryotes.
Cut1/separase C‐terminus affects spindle pole body positioning in interphase of fission yeast: pointed nuclear formation
Authors: Nakamura Takahiro , Nagao Koji , Nakaseko Yukinobu , Yanagida Mitsuhiro 
Published: March 2003

AbstractBackground: The separase‐securin complex is required for anaphase. Separase activated by securin destruction cleaves the cohesin subunit Scc1/Rad21 enriched in kinetochores. Fission yeast Cut1/separase resides in interphase cytoplasm and mobilizes to the spindle and the spindle pole bodies (SPBs) in mitosis, while Cut2/securin remains in the nucleus from interphase to metaphase, and temporarily locates at the short spindle.Results: We here report a novel SPB‐led dynamic nuclear movement in fission yeast, when the Cut1 C‐terminal fragment is over‐expressed. The tip of the pointed nucleus contained both SPB and centromeric DNA, and rapidly moved along the bundled cytoplasmic microtubules. The same pointed nucleus was produced when the human separase C‐fragment was over‐expressed. The pointed nuclear formation did not require the protease site of separase, but required the conserved C‐terminus and a microtubule‐ and kinetochore‐binding protein Mtc1/Alp14, a homologue of frog XMAP215 and budding yeast Stu2. The movement‐inducing C‐fragment should be cytoplasmic, as the pointed nucleus was abolished when the fragment contained the NLS (nuclear localization signal).Conclusions: Overproduced separase C‐fragment abolishes correct SPB‐positioning in interphase. Resulting pointed nuclear formation (alternatively called ‘pigtail movement’) requires cytoplasmic microtubules and Mtc1/Alp14.
lal‐1: a differentially expressed novel gene during proliferation in liver regeneration and in hepatoma cells
Authors: Della Fazia Maria Agnese , Piobbico Danilo , Bartoli Daniela , Castelli Marilena , Brancorsini Stefano , Viola Magni Mariapia , Servillo Giuseppe 
Published: March 2003

AbstractBackground: During liver regeneration, 95% of the resting hepatocytes enter in G1/S phase of the cell cycle. A number of hormones, growth factors and cytokines were identified that activate signal transduction pathways playing a primary role in hepatocyte proliferation. A wide and representative cDNA library containing 1.5 × 106 independent clones was constructed from regenerating liver in order to identify and characterize gene the products which play a crucial role in the first hours of the proliferative process of liver regeneration.Results: A novel gene expressed in liver regeneration was cloned by subtractive hybridization. The putative protein displays in the N′‐terminal a annexin‐like domain and an aminopeptidase domain. We named the novel gene Liver Annexin Like‐1 (lal‐1). The lal‐1 gene is modulated during liver regeneration, in hepatoma cells following physiological stimulation and after cAMP induction.Conclusion: The results indicate that lal‐1 is involved in liver regeneration and that its expression is finely regulated during proliferative process. The isolation of lal‐1 paves the way for a further characterization helping to assess lal‐1 involvement in cell function and proliferation.
A comparative cDNA microarray analysis reveals a spectrum of genes regulated by Pax6 in mouse lens
Authors: Chauhan Bharesh K. , Reed Nathan A. , Yang Ying , Čermák Lukáš , Reneker Lixing , Duncan Melinda K. , Cvekl Aleš 
Published: March 2003

AbstractBackground: Pax6 is a transcription factor that is required for induction, growth, and maintenance of the lens; however, few direct target genes of Pax6 are known.Results: In this report, we describe the results of a cDNA microarray analysis of lens transcripts from transgenic mice over‐expressing Pax6 in lens fibre cells in order to narrow the field of potential direct Pax6 target genes. This study revealed that the transcript levels were significantly altered for 508 of the 9700 genes analysed, including five genes encoding the cell adhesion molecules β1‐integrin, JAM1, L1 CAM, NCAM‐140 and neogenin. Notably, comparisons between the genes differentially expressed in Pax6 heterozygous and Pax6 over‐expressing lenses identified 13 common genes, including paralemmin, GDIβ, ATF1, Hrp12 and Brg1. Immunohistochemistry and Western blotting demonstrated that Brg1 is expressed in the embryonic and neonatal (2‐week‐old) but not in 14‐week adult lenses, and confirmed altered expression in transgenic lenses over‐expressing Pax6. Furthermore, EMSA demonstrated that the BRG1 promoter contains Pax6 binding sites, further supporting the proposition that it is directly regulated by Pax6.Conclusions: These results provide a list of genes with possible roles in lens biology and cataracts that are directly or indirectly regulated by Pax6.
Mechanism of H‐8 inhibition of Cyclin‐dependent kinase 9: study using inhibitor‐immobilized matrices
Authors: Shima Daisuke , Yugami Masato , Tatsuno Michiko , Wada Tadashi , Yamaguchi Yuki , Handa Hiroshi 
Published: March 2003

AbstractBackground: Positive transcription elongation factor b (P‐TEFb), which phosphorylates the carboxyl‐terminal domain (CTD) of RNA polymerase II (RNAPII), is comprised of the catalytic subunit cyclin‐dependent kinase 9 (CDK9) and the regulatory subunit cyclin T. The kinase activity and transcriptional activation potential of P‐TEFb is sensitive to various compounds, including H‐8, 5,6‐dichloro‐1‐β‐d‐ribofuranosylbenzimidazole (DRB), and flavopiridol.Results: We investigated the molecular mechanism of the H‐8 inhibition of CDK9 using matrices to which H‐9, an amino derivative of H‐8, was immobilized. CDK9 bound specifically to H‐9, and this interaction was competitively inhibited by ATP and DRB, but not by flavopiridol. Mutational analyses demonstrated that the central region of CDK9, which encompasses the T‐loop region, was important for its binding to H‐9.Conclusions: H‐9‐immobilized latex beads are useful for trapping CDK9 and a subset of kinases from crude cell extracts. The flavopiridol‐binding region of CDK9 is most likely different from its H‐9‐binding region. These biochemical data support previously reported observations which were based on crystallographic data.