bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Pho85 kinase, a member of the yeast cyclin‐ dependent kinase (Cdk) family, has a regulation mechanism different from Cdks functioning throughout the cell cycle
Authors: Nishizawa Masafumi , Suzuki Kohji , Fujino Marie , Oguchi Tomoko , Toh‐e Akio 
Published: March 2003

BackgroundThe PHO85 gene is a negative regulator of the PHO system in the yeast Saccharomyces cerevisiae and encodes a protein kinase (Pho85p) which is highly homologous to the Cdc28 kinase (Cdc28p). Although the two kinases share a 51% identity and their functional domains are well conserved, PHO85 fails to replace CDC28. Pho85p forms complexes with G1‐cyclin homologues, including Pcl1p, Pcl2p and Pcl9p, and is thought to be involved in the cell‐cycle regulation at G1 and the end of M. By analysing the genetic and biochemical properties of Pho85p, we studied whether the regulation of Pho85p activity is similar to other cyclin‐dependent kinases (Cdks) directly involved in cell cycle regulation.ResultsA functional analysis of various Pho85 mutants revealed that E53 in the PSTAIRE sequence was important for Pho85p function. On the other hand, residues in the T‐loop including S166, S167 and E168, appeared dispensable for Pho85p function, suggesting that the phosphorylation of S166, corresponding to T161 of Cdc2p and T169 of Cdc28p, was not required for the kinase activity of Pho85p. Instead, we found that phosphorylation of Y18, corresponding to Y15 of Cdc2p and Y19 of Cdc28p, may be important for the binding of Pho80p but not of Pcl1p, suggesting that tyrosine phosphorylation may function as a signal which discriminates various Pho85‐cyclins.ConclusionIn Cdks functioning throughout the cell cycle, tyrosine phosphorylation is inhibitory to the activation of kinase, whereas the phosphorylation of threonine in the T‐loop is essential for activation. Our finding indicates that the regulation mechanism of Pho85p activation appears to be distinct from these Cdks.
Heterogeneous nuclear RNA‐ribonucleoprotein F binds to DNA via an oligo(dG)‐motif and is associated with RNA polymerase II
Authors: Yoshida Tatsushi , Kokura Kenji , Makino Yasutaka , Ossipow Vincent , Tamura Taka‐aki 
Published: March 2003

BackgroundThe heterogeneous nuclear ribonucleoprotein F (hnRNP‐F) is one of the constituents of the splicing‐related hnRNP complex. Recent studies suggest that pre‐mRNA modification and splicing factors are associated with transcriptional initiation factors and RNA polymerase II (RNA pol II) at a promoter, implying that pre‐mRNA‐engaged factors might be associated with a promoter.ResultsWe isolated a cDNA of rat hnRNP‐F and expressed the recombinant protein. HnRNP‐F selectively bound to oligo(dG) in constructions with other oligohomonucleotides. The ssDNA of an SV40 promoter sequence having the GC‐boxes was bound to hnRNP‐F, while hnRNP‐F bound to the (G/C)‐stretch as dsDNA. Consequently, hnRNP‐F was designated as an oligo(dG)‐binding protein. None of the RNA‐binding domains (RBDs) in hnRNP‐F were critical for the oligo(dG)‐binding. In contrast, the GY‐rich region that exists between RBD‐II and ‐III exhibited an oligo(dG)‐binding ability. We suggest that the GY‐rich region is a novel DNA‐binding motif. HnRNP‐F was shown to contain two types of binding motifs for RNA and DNA. Additionally, we found that hnRNP‐F was co‐precipitated with RNA pol II. Interestingly, the RNA pol II holoenzyme also contained hnRNP‐F. The tissue distribution profile of hnRNP‐F was similar to that of both RNA pol II and TBP.ConclusionThe above results suggest that hnRNP‐F is associated with a transcriptional initiation apparatus that includes RNA pol II. The DNA‐binding ability of hnRNP‐F might facilitate the entry of pre‐mRNA modification/splicing factors at a promoter.
ORC1 interacts with c‐Myc to inhibit E‐box‐dependent transcription by abrogating c‐Myc–SNF5/INI1 interaction
Authors: Takayama Masa‐aki , Taira Takahiro , Tamai Katsuyuki , Iguchi‐Ariga Sanae M. M. , Ariga Hiroyoshi 
Published: March 2003

BackgroundThe c‐myc oncogene product (c‐Myc) is a transcription factor that forms a complex with Max and recognizes the E‐box sequence. c‐Myc plays key functions in cell proliferation, differentiation and apoptosis. As for its activity towards cell proliferation, it is generally thought that c‐Myc transactivates the E‐box‐containing genes that encode proteins essential to cell‐cycle progression. Despite the characterization of candidate genes regulated by c‐Myc in culture cells, these have still not been firmly recognized as real target genes for c‐Myc.ResultsWe found that c‐Myc directly bound to the N‐terminal region of origin recognition complex‐1 (ORC1), a region that is responsible for gene silencing, in a state of complex containing other ORC subunits and Max in vivo and in vitro. Furthermore, ORC1 inhibited E‐box‐dependent transcription activity of c‐Myc by competitive binding to the C‐terminal region of c‐Myc with SNF5, a component of chromatin remodelling complex SNF/Swi1.ConclusionsThese results suggest that ORC1 suppresses the transcription activity of c‐Myc by its recruitment into an inactive form of chromatin during some stage of the cell cycle.
The Schizosaccharomyces pombe spo6+ gene encoding a nuclear protein with sequence similarity to budding yeast Dbf4 is required for meiotic second division and sporulation
Authors: Nakamura Tomohiro , Kishida Masao , Shimoda Chikashi 
Published: March 2003

BackgroundSporulation of the fission yeast Schizosaccharomyces pombe is a cell differentiation process which accompanies meiosis. The spo6+ gene was identified as a sporulation‐specific gene, whose transcription was regulated by the forkhead family transcription factor Mei4.Results spo6
+ encodes a protein with sequence similarity to Saccharomyces cerevisiae Dbf4p, which is required for the initiation of DNA replication. However, doubling time and cell morphology of spo6 deletion mutants and spo6‐cDNA over‐expressing cells were indistinguishable from wild‐type cells. Spliced mature mRNAs of spo6+ appeared when diploid cells committed to meiosis. Spo6p fused to green fluorescent protein (GFP) preferentially localized in a nucleus. Although spo6Δ diploids normally underwent premeiotic DNA replication and meiosis‐I, approximately 80% of cells were blocked at the binucleate stage during meiosis and virtually no asci were formed. Anti‐tubulin staining revealed that only 25% of the binucleate cells assembled spindle microtubules for meiosis‐II. In a small number of tetranucleate cells, sister nuclei insufficiently separated and spindles were frequently fragmented. The meiosis‐II arrest phenotype was exaggerated at low temperature and in the presence of caffeine.ConclusionsThese results indicate that Spo6p is a novel Dbf4‐related nuclear protein, which is expressed during meiosis and is indispensable for normal progression of meiosis‐II and sporulation.
Interactions between Mcm10p and other replication factors are required for proper initiation and elongation of chromosomal DNA replication in Saccharomyces cerevisiae
Authors: Kawasaki Yasuo , Hiraga Shin‐ichiro , Sugino Akio 
Published: March 2003

MCM10 is essential for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae. Previous work showed that Mcm10p interacts with the Mcm2–7 protein complex that may be functioning as the replication‐licensing factor. In addition, Mcm10p is required during origin activation and disassembly of the prereplicative complex, which allows smooth passage of replication forks.We show that an mcm10 mutation causes a slow progression of DNA synthesis and a loss of chromosome integrity during the S phase and prevents entry into mitosis, despite apparent completion of chromosomal DNA replication at nonpermissive temperatures. Furthermore, Mcm10p interacts genetically with the origin recognition complex (ORC) and various replication elongation factors, including a subunit of DNA polymerases ε and δ. Mcm10p is an abundant protein (approximately 4 × 104 copies per haploid cell) that is almost exclusively localized in the chromatin and/or nuclear matrix fractions during all phases of the cell cycle. When it is visualized by the chromosome‐spreading method followed by immunostaining, Mcm10p forms punctate foci on chromatin throughout the cell cycle and these foci mostly overlap with those of Orc1p, a component of ORC.These results suggest that Mcm10p, like the Mcm2–7 proteins, is a critical component of the prereplication chromatin and acts together with ORC during the initiation of chromosomal DNA replication; in addition, Mcm10p plays an important role during the elongation of DNA replication.
Identification of Tetrahymena hsp60 as a 14‐nm filament protein/citrate synthase‐binding protein and its possible involvement in the oral apparatus formation
Authors: Takeda Tetsuya , Yoshihama Isao , Numata Osamu 
Published: March 2003

Background Tetrahymena 14‐nm filament protein (14FP) is bifunctional, with roles as a citrate synthase in mitochondria and as a cytoskeletal protein in nuclear events during fertilization and in oral morphogenesis. In this study, to further our understanding of the bifunctional property of 14FP, we attempted to screen 14FP‐binding proteins using affinity column chromatography.ResultsThrough the screening of 14FP‐binding proteins using 14FP‐affinity chromatography, we detected 65 kDa and 70 kDa proteins that bound to 14FP in an ATP dependent manner. From the N‐terminal amino acid sequence, these proteins were identified as the Tetrahymena mitochondrial chaperones, hsp60 and mthsp70, respectively. Tetrahymena hsp60 was recognized with a monoclonal antibody raised against human hsp60. Immunofluorescence and immunoelectron microscopy using the monoclonal antibody showed that Tetrahymena hsp60 was localized to mitochondria. Moreover, Tetrahymena hsp60 was also present at extramitochondrial sites including basal bodies of cilia and oral apparatus, and particularly at the developing oral apparatus during cell division.ConclusionThese results suggest that Tetrahymena hsp60 is localized in basal bodies and is involved in cortical patterning such as the formation of the oral apparatus as well as having a role in the folding of mitochondrial proteins in mitochondria.
Tumour suppressor activity of human imprinted gene PEG3 in a glioma cell line
Authors: Kohda Takashi , Asai Akio , Kuroiwa Yoshimi , Kobayashi Shin , Aisaka Kohzoh , Nagashima Goro , Yoshida Michihiro C. , Kondo Yasumitsu , Kagiyama Naoto , Kirino Takaaki , Kaneko‐Ishino Tomoko , Ishino Fumitoshi 
Published: March 2003

BackgroundMouse imprinted gene Peg3 encodes a large C2H2 type zinc finger protein with unique characteristics. Peg3 knockout mice were found to show an impairment in maternal behaviour of the adult female. Mouse Peg3 is located on the proximal region of chromosome 7 which is syntenic to the long arm of human chromosome 19. It has been reported that a loss of heterozygosity (LOH) of chromosome 19q occurs frequently in several glioma types.ResultsWe isolated human PEG3 cDNA. Both human and mouse PEG3 were strongly expressed in the adult brain and the Peg3 protein was localized in the nuclei of both neurones and glial cells. A significant decrease in PEG3 expression was more commonly observed in glioma cell lines as compared with that in primary cultures of astrocytes. Transfection of PEG3 cDNA in a glioma cell line resulted in a loss of tumorigenicity in nude mice.ConclusionsThe human PEG3 gene is a paternally expressed imprinted gene. Introduction of PEG3 cDNA into the glioma cells suggests that human PEG3 protein functions as a tumour suppressor. Human PEG3 is located on 19q13.4 and is one of the candidates for tumour suppressor genes that are predicted to be sited in gliomas.
Cofilin‐2, a novel type of cofilin, is expressed specifically at aggregation stage of Dictyostelium discoideum development
Authors: Aizawa Hiroyuki , Kishi Yoshiro , Iida Kazuko , Sameshima Masazumi , Yahara Ichiro 
Published: March 2003

Background A conventional cofilin, cofilin‐1 in Dictyostelium discoideum plays significant roles in cell proliferation, phagocytosis, chemotactic movement and macropinocytosis.Results We identified a new member of the cofilin family, named cofilin‐2 in D. discoideum. Cofilin‐2 shows significant homology to a conventional Dictyostelium cofilin, cofilin‐1, through its entire sequence, and contains residues conserved among the cofilin family that are responsible for actin‐binding. On the other hand, several residues that are conserved among the cofilin family are missing from cofilin‐2. Purified cofilin‐2 depolymerized actin filaments in a dose‐ and pH‐dependent manner and reduced the apparent viscosity of an actin solution, although they did not co‐sediment with actin filaments at all. Cofilin‐2 was not expressed in vegetative cells, but was transiently induced during the aggregation stage of development, whereas cofilin‐1 was predominantly expressed in vegetative cells. Immunocytochemistry revealed that cofilin‐2 localizes at substrate adhesion sites, where cofilin‐1 is almost completely excluded. Disruption of the cofilin‐2 gene caused an increase in actin accumulation at the substrate adhesion sites. We also found that cofilin‐2 did not rescue Δcof1 yeast cells, whereas cofilin‐1 did.Conclusions Cofilin‐2 may play a distinct role from that of cofilin‐1 in destabilization of the actin cytoskeleton during Dictyostelium development.
Level of the RNA polymerase II in the fission yeast stays constant but phosphorylation of its carboxyl terminal domain varies depending on the phase and rate of cell growth
Authors: Sakurai Hitomi , Ishihama Akira 
Published: March 2003

AbstractBackground: The RNA polymerase II of the fission yeast Schizosaccharomyces pombe consists of 12 Rpb subunits, of which four (Rpb1, Rpb2, Rpb3 and Rpb11) form the assembly and catalytic core and five (Rpb5, Rpb6, Rpb8, Rpb10 and Rpb12) are shared among RNA polymerases I, II and III. The intracellular levels of three RNA polymerase forms should be interrelated, but the control of RNA polymerase formation remains mostly unknown.Results: To reveal the physiological role and the synthesis control of each Rpb subunit, the intracellular levels of the Rpb proteins were examined in S. pombe growing at various phases under various conditions. Results indicate that the intracellular concentrations of the Rpb proteins stay constant at levels characteristic of the rate and phase of cell growth, and the relative level between the 12 subunits also remains constant, together implying that the intracellular concentration of RNA polymerase II stays constant, as in the case of prokaryotes. As an attempt to gain insights into the activity control of RNA polymerase II, we also analysed the phosphorylation level of the carboxyl‐terminal domain (CTD) of the largest subunit Rpb1. Phosphorylated forms of Tyr1 and Thr4 within 29 repeats of the YSPTSPS heptapeptide were detected in both slow‐migrating IIo and fast‐migrating IIa forms of Rpb1 on SDS‐PAGE (polyacrylamide gel electrophoresis). However, phosphorylated Ser2 and Ser5 were identified only in the IIo form, indicating that Ser phosphorylation contributes to the conformational change in CTD. The phosphorylation levels of Ser, Thr and Tyr all vary depending on the cell culture conditions.Conclusion: The intracellular level of RNA polymerase II stays constant, but the amount engaged in transcription cycle varies depending on the culture conditions, as estimated from the sites and levels of phosphorylation of Rpb1 CTD.
Mispaired P3 region in the hierarchical folding pathway of the Tetrahymena ribozyme
Authors: Ohki Yasushi , Ikawa Yoshiya , Shiraishi Hideaki , Inoue Tan 
Published: March 2003

AbstractBackground: The Tetrahymena group I ribozyme folds into a complex three‐dimensional structure for performing catalytic reactions. The catalysis depends on its catalytic core consisting of two helical domains, P4–P6 and P3–P7, connected by single stranded regions. In the folding process, most of this ribozyme folds in a hierarchical manner in which a kinetically stable intermediate determines the overall folding rate.Results: Although the nature of this intermediate has not yet been elucidated, a mispaired P3 stem (alt‐P3) appears a likely candidate. To examine the effects of the alt‐P3 structure on the kinetic and thermodynamic properties of the active structure of the ribozyme or its P3–P7 domain formation, we prepared and analysed variant ribozymes in which relative stabilities of the original P3 and alt‐P3 structure were altered systematically.Conclusion: The results indicate that the alt‐P3 structure is not the major rate‐limiting factor in the folding process.
Structural requirements of the mRNA for intracistronic translation initiation of the enterobacterial infB gene
Authors: Laursen Brian Søgaard , De A. Steffensen Søren A. , Hedegaard Jakob , Moreno Juan Manuel Palacios , Mortensen Kim Kusk , Sperling‐Petersen Hans Uffe 
Published: March 2003

AbstractBackground: The gene infB encodes the prokaryotic translation initiation factor IF2, a central macromolecular component in the formation of the ribosomal 70S initiation complex. In Escherichia coli, infB encodes three forms of IF2: IF2α, IF2β and IF2γ. The expression of IF2β and IF2γ is a tandem translation from intact infB mRNA and not merely a translation of post‐transcriptionally truncated mRNA. The molecular mechanism responsible for the ribosomal recognition of the two intracistronic translation initiation sites in E. coli infB is not well characterized.Results: We found three different forms of IF2 in Enterobacter cloacae, Klebsiella oxytoca, Salmonella enterica, Salmonella typhimurium, and two different forms in Proteus vulgaris. We identified the intracistronic translation initiation sites of the mRNA by isolation and N‐terminal sequencing of the shorter isoforms of IF2 in S. enterica and S. typhimurium. A further search in the readily available public sequence databases revealed that infB from Yersinia pestis also contains an intracistronic in‐frame initiation site used for the translation of IF2β. The base composition in a part of the 5′ end of the DNA coding strand of the enterobacterial infB gene shows a strong preference for adenine (A) over thymine (T) with a maximum ratio of A‐to‐T around the intracistronic initiation sites. We demonstrate that the mRNA has an open structure around the ribosomal binding region.Conclusion: Efficient intracistronic translation initiation of the infB gene is suggested to require an mRNA with this special base composition that results in an open, single‐stranded structure at the ribosomal binding region.Schematic view of the translation process of infB mRNA. The 5′‐end of the infB mRNA preceding the intracistronic translation initiation codon(s) has a number of rare codons. This stalls the translation in this region of the mRNA, providing space for the ribosomes to initiate at the intracistronic initiation site(s). The region around the intracistronic sites(s) has a high content of adenine and a low content of uracil. This leads to an open single‐stranded structure of the mRNA in the region, which is a necessity for the intracistronic translation initiation of the infB gene.image
Physical interaction between proliferating cell nuclear antigen and replication factor C fromPyrococcus furiosus
Authors: Matsumiya Shigeki , Ishino Sonoko , Ishino Yoshizumi , Morikawa Kosuke 
Published: March 2003

AbstractBackground: Proliferating cell nuclear antigen (PCNA), which is recognized as a DNA polymerase processivity factor, has direct interactions with various proteins involved in the important genetic information processes in Eukarya. We determined the crystal structure of PCNA from the hyperthermophilic archaeon,Pyrococcus furiosus(PfuPCNA) at 2.1 Å resolution, and found that the toroidal ring‐shaped structure, which consists of homotrimeric molecules, is highly conserved between the Eukarya and Archaea. This allowed us to examine its interaction with the loading factor at the atomic level.Results:The replication factor C (RFC) is known as the loading factor of PCNA on to the DNA strand.P. furiosusRFC (PfuRFC) has a PCNA binding motif (PIP‐box) at the C‐terminus of the large subunit (RFCL). An 11 residue‐peptide containing a PIP‐box sequence of RFCL inhibited the PCNA‐dependent primer extension ability ofP. furiosus PolI in a concentration‐dependent manner. To understand the molecular interaction mechanism of PCNA with PCNA binding proteins, we solved the crystal structure ofPfuPCNA complexed with the PIP‐box peptide. The interaction mode of the two molecules is remarkably similar to that of human PCNA and a peptide containing the PIP‐box of p21WAF1/CIP1. Moreover, the PIP‐box binding may have some effect on the stability of the ring structure ofPfuPCNA by some domain shift.Conclusions:Our structural analysis onPfuPCNA suggests that the interaction mode of the PIP‐box with PCNA is generally conserved among the PCNA interacting proteins and that the functional meaning of the interaction via the PIP‐box possibly depends on each protein. A movement of the C‐terminal region of the PCNA monomer by PIP‐box binding may cause the PCNA ring to be more rigid, suitable for its functions.
Artificial recruitment of certain Mediator components affects requirement of basal transcription factor IIE
Authors: Sakurai Hiroshi , Fukasawa Toshio 
Published: March 2003

AbstractBackground: Basal transcription factors are essential for RNA polymerase II (RNAPII)‐catalysed transcription of many but not all the mRNA‐encoding genes in vivo as well as in vitro. For example, copper‐inducible transcription of the copper metallothionein gene CUP1 occurs independently of basal factor TFIIE in budding yeast. To gain insight into the mechanism by which the requirement for TFIIE is bypassed, we artificially recruited certain constituents of Mediator, a large protein complex transmitting signals from various activators to the RNAPII machinery, to the CUP1 promoter by protein fusions with Ace1, the copper‐inducible activator.Results: Fusions with Med2 or Pgd1 activated CUP1 independently of TFIIE. Surprisingly, fusions with neither Srb5 nor Med9 circumvented TFIIE requirement for the CUP1 activation. Components of TFIID were similarly recruited to the CUP1 promoter without activation. By using a chromatin immunoprecipitation technique, we found that TFIIE is necessary for stable binding of TFIIH and RNAPII to the ADH1 promoter, whose activation requires TFIIE. However, binding of TFIIH and RNAPII to CUP1 upon its activation did not require TFIIE.Conclusions: Our results strongly suggest that the TFIIE requirement of a gene is determined by a target(s) in Mediator through which the signal of the cognate activator is transmitted.