bluuebunny/crossref_metadata_2025_split huggingface dataset:
NAP‐I is a functional homologue of TAF‐I that is required for replication and transcription of the adenovirus genome in a chromatin‐like structure
Authors: Kawase Hiroyuki , Okuwaki Mitsuru , Miyaji Mary , Ohba Reiko , Handa Hiroshi , Ishimi Yukio , Fujii‐Nakata Tomoko , Kikuchi Akihiko , Nagata Kyosuke 
Published: November 2003

Background:
For the activation of replication and transcription from DNA in a chromatin structure, a variety of factors are thought to be needed that alter the chromatin structure. Template activating factor‐I (TAF‐I) has been identified as such a host factor required for replication of the adenovirus (Ad) genome complexed with viral basic core proteins (Ad core). TAF‐I also stimulates transcription from the Ad core DNA.
Results:
Using mutant TAF‐I proteins, we have demonstrated that the acidic stretch present in the carboxyl terminal region is essential for the stimulation of transcription from the Ad core. A genomic footprinting experiment with restriction endonuclease has revealed that TAF‐I causes a structural change in the Ad core. TAF‐I has been shown to have significant amino acid similarity to nucleosome assembly protein‐I (NAP‐I), which is involved in the formation of the chromatin structure. We have shown that TAF‐I can be substituted by NAP‐I in the activation of the cell‐free Ad core transcription system. Two of the tripartite acidic regions and the region homologous to TAF‐I in NAP‐I are required for the maximal TAF‐I activity of NAP‐I. Furthermore, TAF‐I has been shown to have NAP‐I activity, and the acidic region of TAF‐I is required for this activity.
Conclusions:
Since TAF‐I causes the structural change of the Ad core and thereby activates transcription, TAF‐I is thought to be one of the proteins which is involved in chromatin remodeling. NAP‐I is structurally related to TAF‐I and functionally substitutes for TAF‐I. Furthermore, TAF‐I has NAP‐I activity. These observations suggest that this type of molecule has dual functions, possibly by participating in facilitating the assembly of the chromatin structure as well as perturbing the chromatin structure to allow transcription to proceed.
Mesodermal‐ vs. neuronal‐specific expression of MafK is elicited by different promoters
Authors: Motohashi Hozumi , Igarashi Kazuhiko , Onodera Kou , Takahashi Satoru , Ohtani Haruo , Nakafuku Masato , Nishizawa Makoto , Engel James Douglas , Yamamoto Masayuki 
Published: November 2003

Background: Small members of the Maf family of transcriptional regulatory proteins share similar basic‐leucine zipper domains but have no intrinsic ability to activate transcription. One member of the family (MafK) has been shown to mediate both negative and positive regulation: in addition to forming a homodimer which represses transcription, MafK can also form a heterodimer with p45 (the large subunit of erythroid transcription factor NF‐E2) to activate transcription.Results: We examined the expression of mafK during murine development. mafK mRNA was first detected in 7.5 days post coitus (dpc) embryonic mesoderm and persisted in mesodermal derivatives (mesenchymal and haematopoietic cells) thereafter. However, around 13 dpc mafK was also strongly induced in neuronal cells and it is broadly expressed in neurones in postnatal mouse. The neuronal expression of mafK is directed by a distinct promoter located 6 kbp 3′ to the mesoderm‐specific promoter. mafK in neurones associates with a different partner molecule from p45. In transgenic mice, a regulatory domain in the immediate vicinity of the mesodermal promoter was found to direct mesenchymal, but not haematopoietic, expression of mafK.Conclusion: The cell type‐ and developmental stage‐specific expression of MafK suggests that, in addition to its demonstrated role in erythroid transcriptional regulation, MafK also plays an important regulatory role in other mesodermally and neuroectodermally derived tissues during mouse embryonic development.
Differential thermoregulation of two highly homologous cold‐shock genes, cspA and cspB, of Escherichia coli
Authors: Etchegaray Jean‐Pierre , Jones Pamela G. , Inouye Masayori 
Published: November 2003

Background The major cold‐shock protein in Escherichia coli is CspA, a 7.4 kDa protein. A CspA family has been found which consists of four additional proteins, CspB, CspC, CspD and CspE. The expression of cspB, unlike the other homologues, is cold‐shock inducible like cspAResults: We examined the cold‐shock induction of CspA and CspB at various temperatures. The cspA induction is observed by temperature shift from 37 to 30 °C and high levels of CspA production are observed between 24 and 10 °C. In contrast, CspB production occurs only by temperature shift to below 20 °C, with maximum induction at 15 °C. Both cspA and cspB expressions were found to be induced at the level of transcription as determined by primer extension.Conclusions: These results show that cspA and cspB expressions are differentially regulated at low temperature indicating that E. coli contains at least two different biothermostats or thermoregulators that are likely to play important roles in cellular adaptation to low temperature. The cspB promoter shows sequence similarity to the cspA promoter. Furthermore, both cspA and cspB mRNAs have unusually long 5′ untranslated regions (159 and 161 bases, respectively), both of which are able to form similar extensive secondary structures. These features are considered to contribute to the nature of the thermostats for cspA and cspB
Specification of posterior midbrain region in zebrafish neuroepithelium
Authors: Miyagawa Terumi , Amanuma Hiroshi , Kuroiwa Atsushi , Takeda Hiroyuki 
Published: November 2003

Background: The developing vertebrate nervous system displays a pronounced anterior–posterior (A‐P) pattern, but the mechanism that generates this pattern is poorly understood. We examined through cell‐transplantation experiments, when and how the cells in the zebrafish posterior midbrain acquire regional specificity along the A‐P axis as shown by pax[b] gene expression. Results: Labelled donor cells from the presumptive midbrain region at various stages were transplanted into more anterior part of unlabelled host embryos of the same developmental stage, and the expression of pax[b] in the donor cells were examined by in situ hybridization. The results indicated that, in the cells from the presumptive midbrain region, expression of pax[b] was determined as early as the 55%‐epiboly (6.5 h, early gastrulation) when the underlying hypoblastic layer reached the presumptive midbrain region. We also found that when transplanted heterotopically, anterior, but not posterior, hypoblast cells induced expression of pax[b] in the overlying ectoderm. Conclusions: Expression of a midbrain specific gene is determined during early gastrulation and the hypoblastic layer plays an important role in this determination process.
Schizosaccharomyces pombe gad7+ encodes a phosphoprotein with a bZIP domain, which is required for proper G1 arrest and gene expression under nitrogen starvation
Authors: Kanoh Junko , Watanabe Yoshinori , Ohsugi Miho , Iino Yuichi , Yamamoto Masayuki 
Published: November 2003

Background: Fission yeast cells arrest at G1 phase when starved of nitrogen. The molecular mechanism that ensures this arrest is poorly understood. We took a genetic approach to this problem. Results: The fission yeast gad7‐1 mutant failed to arrest at G1 when starved of nitrogen, and was poor in mating and sporulation. The gad7 gene was cloned by complementation. The deduced gad7 gene product was a bZIP protein of 566 amino acids, which could bind to the CRE (cAMP response element) sequence in vitro. Disruption of gad7 resulted in the same phenotypes as gad7‐1. Expression of ste11, which encodes a key transcription factor for sexual development, was not inducible in the disruptant. Gad7 was co‐immunoprecipitated with another bZIP protein Pcr1, suggesting that the two proteins form a heterodimer in vivo. Gad7 was phosphorylated, and the state of its phosphorylation appeared to be modified in pka1Δ or wis1Δ cells. Conclusions: Gad7, a CRE‐binding protein that cooperates with Pcr1, is required for proper G1 arrest and gene expression under nitrogen starvation. Gad7 is a phosphoprotein, whose activity may be regulated by protein kinases including the cAMP‐dependent protein kinase (Pka1) and Wis1 osmosensory MAP kinase kinase.
A yeast gene product, Fob1 protein, required for both replication fork blocking and recombinational hotspot activities
Authors: Kobayashi Takehiko , Horiuchi Takashi 
Published: November 2003

Background:  In the rRNA gene cluster on the Saccharomyces cerevisiae chromosome XII, a unique site that blocks progression of the replication fork exists in a single unit of the repeats. The site RFB (replication fork blocking) is located within one of two cis‐elements present in a nontranscribed region of each repeated unit, which are required for obtaining maximal activity of a hotspot (HOT1) dependent recombination. Results:  To investigate the correlation between replication fork blocking at RFB and homologous recombination at HOT1, we have isolated Hot1‐defective mutants and examined their ability for fork blocking at RFB. Amongst 23 isolated mutants, four were found to be defective in both abilities. Genetic analysis of the mutants reveals that a single mutation, named fob1 (fork blocking less), is responsible for the defects in both abilities. The FOB1 gene is located on chromosome IV and has no homology with any other genes listed in DNA data banks. Conclusion:  The pleiotropic effect of the fob1 mutation suggests that homologous recombination at HOT1 is closely linked with DNA replication fork blocking event at RFB.
Subunit assembly in vivo of Escherichia coli RNA polymerase: role of the amino‐terminal assembly domain of alpha subunit
Authors: Kimura Makoto , Ishihama Akira 
Published: November 2003

Background: The RNA polymerase core enzyme of Escherichia coli is assembled in the sequence α→α2→α2β→α2 ββ′. The amino‐terminal domain down to residue 235 of the Escherichia coli RNA polymerase α subunit plays a key role in enzyme assembly. In vitro reconstitution studies from mutant α subunits have indicated the involvement of multiple sites for α dimerization, two regions (one near residue 45 and the other near residue 80) for β association, and two regions (one around residue 80 and the other between residues 180 and 200) for β′ association. The mechanism of RNA polymerase assembly in vivo, however, remains largely unknown. Results: RNA polymerase assembly in vivo was analysed for E. coli strains carrying expression plasmids for four amino‐terminal deletion and 11 Ala‐Ser (AS) dipeptide‐insertion mutant α subunits. For detection of RNA polymerase complexes, a hexa‐histidine (H6) tag was added to all these mutant α at their carboxy‐termini, and subunit complexes containing the H6‐tagged α were isolated by passing cell extracts through Ni2+‐affinity columns. The assembly properties of most α mutants were consistent with those observed in in vitro reconstitution studies. Some mutants defective in β′ association in vitro such as those carrying mutations at residues 80 and 200 were, however, assembled in vivo, suggesting that a specific condition(s) or factor(s) supports RNA polymerase assembly in vivo. One possible candidate supporting the RNA polymerase assembly is the molecular chaperon(s), because DnaK (hsp70) was always associated with assembly‐defective RNA polymerase mutants. Most assembly competent mutants complemented two temperature‐sensitive mutant alleles of rpoA, but two assembly competent mutants, one (αΔN20) carrying a deletion at the extreme amino‐terminal region and the other (αI‐60) with AS insertion at residue 60, failed to complement these ts mutants. The failure suggests that these assembly competent but complementation‐negative α mutants lack an as yet unidentified function(s). In the case of these two mutants, DnaK was associated, with apparently assembled RNA polymerase. Conclusion: The α–α, α–β and α–β′ contact sites on the RNA polymerase α subunit identified in in vitro reconstitution studies also participate in the subunit assembly in vivo. Some α mutants defective in assembly in vitro are, however, assembled in vivo. A factor(s) such as the molecular chaperon DnaK or a specific intracellular condition(s) may affect RNA polymerase assembly in vivo.
Initiation of Sendai virus multiplication from transfected cDNA or RNA with negative or positive sense
Authors: Kato Atsushi , Sakai Yuko , Shioda Tatsuo , Kondo Toru , Nakanishi Mahito , Nagai Yoshiyuki 
Published: November 2003

Background: The mononegavirus superfamily (Mononegavirales) comprises three families, Rhabdoviridae, Paramyxoviridae and Filoviridae. These viruses possess a single stranded negative sense RNA as the genome. Recent success in the recovery of infectious virus from a transfected cDNA of mononegaviruses including Sendai virus, a prototypic paramyxovirus, is opening the possibility of their genetic engineering. However, infectious viruses have been recovered only by initiating the infectious cycle with cDNA directing the synthesis of antigenomic positive sense (+) RNA. Starting with genomic negative sense (−) RNA has been unsuccessful. Furthermore, the recovery efficiency has often been extremely low. Results: We describe here an analogous system that allows recovery of Sendai virus at a high rate, from cells in which the transfected cDNA and plasmids to support the synthesis of viral nucleocapsid protein and RNA polymerases are coexpressed by vaccinia virus‐driven bacteriophage T7 polymerase. Our system was able to recover the virus from cDNA directing not only (+)RNA but also (−)RNA. Moreover, using this system, we succeeded in recovery of the virus by transfection of in vitro synthesized (+)RNA or (−)RNA. This improved virus recovery appeared to be accomplished by supplying the supporting plasmids at an optimal ratio and by minimizing the cytopathic effect of the vaccinia virus by specific inhibitors. In addition, it was probably critical that our cDNAs were constructed to generate viral authentic RNAs without adding T7 promoter‐specific nucleotides to the 5′ ends. An immediate application of the system was demonstrated by the creation of a candidate vaccine strain with a predetermined attenuating mutation in the cleavage‐activation site of the viral fusion glycoprotein. Conclusion: We have established methods which greatly improve the recovery of Sendai virus from cDNA. There is essentially no absolute obstacle to recovery of the virus from the (−)RNA template. Even the complete full length RNA chain in the naked form appears to be properly encapsidated to become a functional template.
Signalling from endoplasmic reticulum to nucleus: transcription factor with a basic‐leucine zipper motif is required for the unfolded protein‐response pathway
Authors: Mori Kazutoshi , Kawahara Tetsushi , Yoshida Hiderou , Yanagi Hideki , Yura Takashi 
Published: November 2003

Background: Accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers the transcriptional induction of molecular chaperones and folding enzymes localized in the ER. Thus, eukaryotic cells possess an intracellular signalling pathway from the ER to the nucleus, called the unfolded protein‐response (UPR) pathway. In Saccharomyces cerevisiae, such induction is mediated by the cis‐acting unfolded protein‐response element (UPRE) which has been thought to be recognized by one or more transcription factor(s). Results: Extensive mutational analysis revealed that UPRE contains a partial palindrome with a spacer of one nucleotide (CAGCGTG) that is essential for its function. We then cloned the ERN4 (presumably identical with HAC1) gene using yeast one‐hybrid screening, in which the GAL4‐ERN4 fusion gene constitutively activates the UPR pathway. The ERN4 gene encodes a basic‐leucine zipper protein (Ern4p) that specifically binds to UPRE in vitro and activates transcription in vivo. Cells lacking Ern4p are unable to induce transcription of any of the five target genes tested and exhibit sensitivity to ER stress and inositol requirement for growth. Conclusion: We concluded that Ern4p represents a major component of the putative transcription factor (UPRF) responsible for the UPR leading to the induction of ER‐localized stress proteins.
Essential role of active nuclear transport in apoptosis
Authors: Yasuhara Noriko , Eguchi Yutaka , Tachibana Taro , Imamoto Naoko , Yoneda Yoshihiro , Tsujimoto Yoshihide 
Published: November 2003

Background
: Apoptosis is defined by chromatin condensation, nuclear fragmentation and the formation of apoptotic bodies. Because apoptotic signals are transmitted through a common pathway that includes the target steps of death‐driving ICE‐family proteases and anti‐cell death protein Bcl‐2 in the cytoplasm, the signals must be transferred from the cytoplasm to the nucleus, at least to induce the apoptotic manifestation of the nucleus. Small signal molecules might diffuse across nuclear pores, but larger molecules are transported by active mechanisms requiring ATP and GTP hydrolysis. It is not known whether apoptotic signals are transmitted into the nucleus by the mechanisms of active nuclear transport.
Results
: To test the possibility that active nuclear transport is involved in apoptotic signal transmission, we have analysed the effects of molecules that inhibit active nuclear transport on apoptosis. Wheat germ agglutinin (WGA), excess amounts of p10 protein, Ran‐GTPγS complex, and anti‐PTAC58 antibody, which all inhibit active nuclear transport when exogenously microinjected, prevent Fas‐induced apoptotic nuclear manifestation. WGA also prevents apoptotic nuclear change promoted by microinjected active CPP32β/Yama protease (an ICE family member), which plays an essential role in most apoptosis.
Conclusions
: The results presented here strongly suggest that active nuclear transport is essential for apoptotic signal transduction.
Transcriptional activation through interaction of MBF2 with TFIIA
Authors: Li Feng‐Qian , Takemaru Ken‐ichi , Goto Masahide , Ueda Hitoshi , Handa Hiroshi , Hirose Susumu 
Published: November 2003

Background: Transcriptional activation of the Drosopohila melanogaster fushi tarzu gene by FTZ‐F1 or its silkworm counterpart BmFTZ‐F1 requires two cofactors MBF1 and MBF2 which do not directly bind to DNA. MBF1 is a bridging molecule that connects FTZ‐F1 (or BmFTZ‐ F1), MBF2 and TATA binding protein TBP. MBF2 is a positive cofactor that activates transcription. Results: To elucidate the mechanism of transcriptional activation by MBF2, we isolated a cDNA coding for the factor. Northern blot analyses showed temporally restricted expression of MBF2 mRNA similar to that of BmFTZ‐F1 mRNA. The cDNA sequence predicts a polypeptide of 10 kDa whereas natural MBF2 is a glycoprotein of 22 kDa. The deduced amino acid sequence of the factor showed no homology with proteins in the databases. Farwestern analyses and glutathione S–transferase interaction assays demonstrated that MBF2 makes a direct contact with the β‐subunit of TFIIA. In a HeLa cell nuclear extract, bacterially expressed recombinant MBF2 activated transcription from various promoters as natural MBF2 did. This activation requires the MBF2–TFIIA interaction. When recombinant MBF2 was added to the HeLa cell nuclear extract in the presence of MBF1 and FTZ622 bearing the DNA‐binding region of FTZ‐F1, it selectively activated transcription of the fushi tarazu gene. This selective activation also requires the MBF2–TFIIA interaction. Conclusion: MBF2 activates transcription through its interaction with TFIIA. Selective transcriptional activation occurs when MBF2 is recruited to a promoter carrying the FTZ‐F1 binding site by FTZ‐F1 and MBF1.
Microtubule‐associated coiled‐coil protein Ssm4 is involved in the meiotic development in fission yeast
Authors: Yamashita Akira , Watanabe Yoshinori , Yamamoto Masayuki 
Published: November 2003

Background: In fission yeast, an RNA species named meiRNA is specifically required for the promotion of the first meiotic division. To dissect the function of this RNA and its partner RNA‐binding protein Mei2, we screened for high‐copy‐number suppressors of the arrest prior to the first meiotic division caused by loss of meiRNA. Results: Analysis of one of the suppressors, named ssm4, suggested that it encodes a coiled‐coil protein carrying a microtubule‐binding motif at its N‐terminus. Expression of ssm4 was restricted to cells undergoing meiosis. Disruption of ssm4 affected neither vegetative growth nor conjugation, but resulted in frequent generation of asci carrying less than four spores. Tagged Ssm4 could colocalize with microtubules in mitotic cells, and was seen to localize at spindles during both the first and the second meiotic division. The microtubule‐binding motif was essential for the association of Ssm4 with microtubules and for its function during meiosis, but not for the suppression of loss of meiRNA. Ssm4 appeared to possess a potential to migrate to the nucleus. Conclusions: Ssm4 is a microtubule‐colocalizing protein that plays a role specifically in meiosis. Ssm4 appears to modify the structure or the function of nuclear microtubules in order to promote the meiotic nuclear division.
Identification of an extended half‐site motif required for the function of peroxisome proliferator‐activated receptor α
Authors: Osada Shiho , Tsukamoto Toshiro , Takiguchi Masaki , Mori Masataka , Osumi Takashi 
Published: November 2003

Background:Peroxisome proliferator‐activated receptors (PPARs) belong to the nuclear hormone receptor superfamily and regulate many genes of the proteins involved in lipid metabolism, including peroxisomal acyl‐CoA oxidase (AOX). Through heterodimerization with retinoid X receptors (RXRs), PPAR was believed to recognize the sequence elements consisting of two directly repeating 6‐bp half‐sites spaced by one nucleotide (DR‐1), located in the regulatory regions of these genes.Results:Employing the peroxisome proliferator‐responsive enhancer of the rat AOX gene, we analysed the minimal sequence requirements for enhancer activity and PPARα/RXRα binding. We found that the sequence just downstream of the DR‐1 motif is indispensable for both functions. By a direct selection procedure of high‐affinity binding sites from a random sequence pool, we identified a consensus sequence at the four positions next to DR‐1. We also suggest that PPARα binds to the downstream half‐site, whereas RXRα binds to the upstream half‐site of the AOX DR‐1.Conclusions:An extended half‐site of 10‐bp, but not a simple 6‐bp half‐site, is required for the PPARα binding, upon heterodimer formation with RXRα. The binding polarity of PPARα/RXRα seems to be opposite to that of other RXR‐involving heterodimers.