bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular assembly of RNA polymerase II from the fission yeast Schizosaccharomyces pombe: subunit–subunit contact network involving Rpb5
Authors: Miyao Takenori , Yasui Kiyoshi , Sakurai Hitomi , Yamagishi Masahiro , Ishihama Akira 
Published: November 2003

Background: Eukaryotic RNA polymerase II is composed of more than 10 polypeptide chains. The minimum and essential subunits for RNA synthesis have not yet been identified. Toward this ultimate goal, we analysed the topological arrangement of the putative subunits. Here we report a subunit–subunit contact network involving subunit 5 of the fission yeast Schizosaccharomyces pombe RNA polymerase II. Results: The rpb5+ gene encoding subunit 5 of RNA polymerase II was cloned from the fission yeast Schizosaccharomyces pombe. The polypeptide predicted from DNA sequence of the rpb5+ gene consists of  210 amino acids with a calculated molecular weight of 23 914. The homology of the amino acid sequence is 55% and 43% with Saccharomyces cerevisiae RPB5 and human hRPB25, respectively. Far‐Western blot analysis of S. pombe RNA polymerase II using 32P‐labelled recombinant Rpb5 fused to glutathione S‐transferase (GST) as a probe, indicated that Rpb5 binds strongly to membrane‐immobilized Rpb1, Rpb2 and Rpb3 and weakly to Rpb5 and a 15‐kDa subunit (Rpb8 or Rpb11). In agreement with this result, the 32P‐labelled Rpb3 probe showed a strong binding signal against Rpb5 in addition to Rpb1 and Rpb2. The existence of Rpb5–Rpb3 contact was supported by detection of complexes formed between these two proteins synthesized in vitro using protein‐immobilized beads. Conclusion: Rpb3 and Rpb5, the putative subunits of RNA polymerase II, associate each other to form binary complexes. These two subunits also bind to the two large subunits, Rpb1 and Rpb2, independently.
Regulation of IFN‐α/β genes: evidence for a dual function of the transcription factor complex ISGF3 in the production and action of IFN‐α/β
Authors: Harada Hisashi , Matsumoto Masahito , Sato Mitsuharu , Kashiwazaki Yasuo , Kimura Tohru , Kitagawa Motoo , Yokochi Taeko , Sok‐Pin Tan Rosemary , Takasugi Tomohiro , Kadokawa Yuzo , Schindler Chris , Schreiber Robert D. , Noguchi Shigeru , Taniguchi Tadatsugu 
Published: November 2003

Background: Efficient production of interferons (IFNs) in virally infected cells is an essential aspect of the host defence. The transcription factor complex ISGF3 (IFN‐stimulated gene factor 3) was originally identified as a critical mediator of the IFN signal; it is formed upon IFN receptor (IFNR) stimulation and binds to ISREs (IFN‐stimulated response elements) to activate IFN‐inducible genes. It has recently been shown that the DNA binding component of ISGF3, p48 (ISGF3γ) also binds to virus‐inducible elements in the IFN‐α/β genes, suggesting a potential new role of p48 in IFN production. Results: Primary cells from mice with a targeted disruption of the p48 gene show severe defects in virus‐induced IFN‐α/β gene expression. A similar defect was also observed in cells lacking type I IFNR or Stat1, further demonstrating the role of IFN signalling in the induction of these IFN genes. ISGF3 in fact binds to the virus‐inducible elements within the IFN‐α/β promoters. We also provide evidence showing that these elements are additionally controlled by an unidentified factor(s) which presumably triggers the primary phase of IFN gene induction. Conclusions: Our results demonstrate that the IFN signal transducing complex ISGF3 plays a crucial role in IFN production and suggest that ISGF3 may participate directly in the activation of IFN‐α/β promoters. This dual function of ISGF3 may insure the efficient operation of this cytokine system in the host defence.
Roles of cysteine residues of DsbB in its activity to reoxidize DsbA, the protein disulphide bond catalyst of Escherichia coli
Authors: Kishigami Satoshi , Ito Koreaki 
Published: November 2003

Background: DsbA, a periplasmic protein, catalyses the disulphide bond formation of other cell surface proteins in E. coli. Reoxidation of DsbA for catalytic turn over is assured by DsbB, a membrane protein with four essential cysteine residues facing the periplasm. We and others previously reported that the reactive Cys30 residue of DsbA forms a mixed disulphide with DsbB in the absence of its partner Cys33 residue.Results: Under the medium condition in which the DsbA mutant lacking Cys33 forms a mixed disulphide only with DsbB, we examined cysteine mutants of epitope‐tagged DsbB for their ability to form the complex. It was shown that Cys104 of DsbB is absolutely required while other three cysteines are also required for maximum interaction. Examination of the redox states of cysteines in wild‐type and mutant DsbB suggested that Cys104 and Cys130 form a disulphide bond which will be transferred to DsbA. In agreement with this notion, DsbB mutants lacking one of the N‐terminally located cysteines retain weak DsbB activity in vivo. The primary role of the N‐terminally located thioredoxin‐like motif of DsbB is probably to reoxidize Cys104 and Cys130Conclusions: We propose the following reaction cycle. DsbB is initially oxidized (State A in Summary Figure). Disulphide interaction between Cys30 of DsbA and Cys104 of DsbB should then trigger the recycling reaction of DsbA (State B), allowing over all electron transfer from newly secreted protein via DsbA (Cys30/Cys33) to DsbB in which intrachain electron flow from Cys104/Cys130 (State C) to Cys41/Cys44 (State D) may occur.
Localization of mouse Rad51 and Lim15 proteins on meiotic chromosomes at late stages of prophase 1
Authors: Ikeya Tomoatsu , Shinohara Akira , Sato Shusei , Tabata Satoshi , Ogawa Tomoko 
Published: November 2003

Background: In meiosis, eukaryotic chromosomes show a series of morphological changes, during which chromosomes synapse and recombine. To understand the mechanisms of the morphological changes and recombination of chromosomes, we examined stage‐specific localization of the Rad51 and Lim15 proteins on the chromosomes in meiotic prophase 1. These proteins are homologous with the RecA protein and have general properties of searching and pairing of homologous DNA sequences. We used mouse chromosomes whose small sizes allow us to identify the locations of these proteins on the entire structures of the chromosomes. Results: In the leptotene and zygotene stages, the Rad51 protein was present on chromatin loops of mouse testis chromosomes then the protein left the loops. In the pachytene stage, the Rad51 protein was present almost exclusively along the core of the synaptonemal complexes (SC). When the stage proceeded to diplotene, the protein was present in the synaptic regions of chromosomes, in particular, in the chiasma regions. The protein was not present on separated homologous SC cores. On the other hand, the Lim15 protein that was found on chromatin loops in early prophase 1, was present almost exclusively at both ends of the SC cores throughout the late stages of prophase 1. Conclusion: The Rad51 and Lim15 proteins are present in chromatin loops when chromosomes form SC. The proteins may promote pairing of homologous DNA sequences that would lead formation of SC. The Rad51 protein in the SC cores may be involved in chiasma formation in late stages. The Lim15 protein, instead, may be involved in recombination in the telomeric region or in cohesion of sister chromatids for segregation.
The directionality of RuvAB‐mediated branch migration: in vitro studies with three‐armed junctions
Authors: Hiom Kevin , Tsaneva Irina R. , West Stephen C. 
Published: November 2003

Background:  The Escherichia coli RuvA and RuvB proteins promote the branch migration of 4‐way (Holliday) junctions during genetic recombination. The active complex is a tripartite structure in which RuvA protein is bound to the crossover and is sandwiched between two hexameric rings of RuvB. Branch migration requires ATP hydrolysis and occurs as the DNA passes through each RuvB ring. Results:  In this work, we have investigated the mechanism by which RuvAB catalyses the branch migration of a three‐armed (Y) junction. Using synthetic DNA structures, we observed the formation of DNA products, a partial duplex DNA molecule and a single‐stranded oligonucleotide, indicative of a branch migration reaction that occurred with unique polarity. Analysis of the RuvAB‐junction complex by DNase footprinting showed that RuvA bound asymmetrically to the junction and targeted a single hexameric RuvB ring to one arm of DNA. Conclusion:  Branch migration of a three‐armed junction occurs in a unidirectional manner that is determined by the assembly of a single RuvB ring onto one arm of the DNA. The asymmetry of the complex and observed directionality of branch migration indicate that strand passage occurs as the DNA is pulled into the RuvB ring structure, a reaction likely to be coupled with DNA unwinding.
Architectural flexibility in lambda site‐specific recombination: three alternate conformations channel the attL site into three distinct pathways
Authors: Segall Anca M. , Nash Howard A. 
Published: November 2003

Background:  In the phage lambda life cycle, the Integrase (Int) protein carries out recombination between two different sets of DNA substrates: attP and attB in integration, attL and attR in excision. In each case, the partners are very different in structure from each other and the recombination reaction between them is effectively irreversible. For comparison, we have studied the recombination mediated by Int between two identical attL sites. Both in vitro and in vivo, recombination between two attL sites can be mediated inefficiently by Int alone. But, while IHF can stimulate recombination 5–10‐fold in vivo (to the level of excision and integration), this stimulation is not observed under standard conditions in vitro Results:  We find that IHF can stimulate the in vitro recombination between two attLs that are modified to be defective in one of the high affinity binding sites for Int, P′1. With such substrates, the efficiency of IHF‐stimulated recombination is comparable to that seen in vivo. The requirements for this reaction distinguish it from other lambda recombination pathways, as does the performance of several mutant Int proteins. Recombination of attL sites on intracellular plasmids suggests that this pathway is effective in vivo, but that some unknown factor or condition permits it to operate on wild‐type as well as mutated attL sites. Conclusions:  The recombination pathway described in this work apparently uses a unique attL architecture, one which requires bending by IHF and is inhibited by Int bound at the P′1 site. In addition to demonstrating the architectural flexibity of the lambda system, this pathway should be a valuable resource for separating the basic requirements of strand exchange chemistry from the features which impart directionality.
Subcellular localization of proteins governing the proteolytic activation of a developmental transcription factor in Bacillus subtilis
Authors: Resnekov Orna , Alper Scott , Losick Richard 
Published: November 2003

Background: Spore formation in Bacillus subtilis takes place in a sporangium consisting of two compartments called the forespore and the mother cell. Late in development, when the forespore is wholly contained within the mother cell, gene transcription is coordinated between the compartments by an intercellular signal transduction pathway. This pathway operates at the level of proteolytic processing of the proprotein precursor (pro‐σK) to the mother‐cell transcription factor σK. The conversion of pro‐σK to σK is governed by the putative processing enzyme SpoIVFB and its negative regulator SpoIVFA, which are produced in the mother cell. Results: We used fluorescence microscopy in conjunction with antibodies against SpoIVFA and SpoIVFB and a fusion of SpoIVFB to the Green Fluorescent Protein from Aquorea victoria to visualize these proteins in the sporangium. Both proteins were found to co‐localize with the forespore region of the sporangium, a finding consistent with the idea that SpoIVFA and SpoIVFB, which are inferred to be integral membrane proteins, are located in the mother cell membrane that surrounds the forespore. Conclusions:  We conclude that SpoIVFA and SpoIVFB are situated at the boundary between the forespore and the mother cell, at which location SpoIVFB could be activated by a signalling protein produced in the forespore.
Proto‐oncogene of int‐3, a mouse Notch homologue, is expressed in endothelial cells during early embryogenesis
Authors: Shirayoshi Yasuaki , Yuasa Yoshihiro , Suzuki Takashi , Sugaya Kimihiko , Kawase Eihachiro , Ikemura Toshimichi , Nakatsuji Norio 
Published: November 2003

Background: Notch and its homologues are key regulatory receptors of the cell fate decision in various developmental processes. The int‐3 oncogene was originally identified as a frequent target in Mouse Mammary Tumour Virus (MMTV)‐induced mammary tumours and has been regarded as a Notch homologue, based on its similarity to the intracellular domain of Notch. Studies with int‐3 transgenic mice have suggested that the int‐3 transgene affects the differentiation capacity of stem cells and leads to neoplastic proliferation in epithelial cells. However, the exact nature and the in vivo expression pattern of the int‐3 proto‐oncogene are unknown. The function of gene products in embryogenesis is also not clear. Results: We isolated cDNA clones corresponding to the proto‐oncogene of int‐3 and analysed its overall structure. The predicted amino acid sequence of the int‐3 proto‐oncogene contains the conserved motif found in Notch family receptors. Therefore, we name Notch‐4 for the int‐3 proto‐oncogene. However, Notch‐4 has fewer EGF repeats and shows less similarity to Notch, compared with other mammalian Notch homologues. In embryogenesis, the expression of Notch‐4 was detected in endothelial cells of blood vessels forming tissues such as the dorsal aorta, intersegmental vessels, yolk sac vessels, cephalic vessels, heart, vessels in branchial arches, and capillary plexuses. In these tissues, Notch‐4 expression coincided with flk‐1, the major regulatory gene of vasculogenesis and angiogenesis. We also found that Notch‐4 expression was up‐regulated in vitro during the differentiation of endothelial cells from embryonic stem cells (ES cells). Conclusion: The endothelial cell specific expression pattern of Notch‐4, as well as its structural similarity of Notch, suggest that Notch‐4 is an endothelial cell specific homologue of Notch and it may play a crucial role in vasculogenesis and angiogenesis.
Caenorhabditis elegans RBX1 is essential for meiosis, mitotic chromosomal condensation and segregation, and cytokinesis
Authors: Sasagawa Yohei , Urano Takeshi , Kohara Yuji , Takahashi Hideyuki , Higashitani Atsushi 
Published: November 2003

AbstractBackground:  The RING‐H2 finger protein RBX1 (ROC1/HRT1) is a common subunit of SKP1‐CDC53/CUL1‐F‐box (SCF), other cullins and von Hippel‐Lindau (VHL) tumour suppressor E3 ubiquitin ligase complexes. RBX1 protein sequences are highly conserved in various species, including yeasts, Drosophila melanogaster, mice and humans. In Saccharo‐myces cerevisiae, RBX1 is essential for the G1/S transition.Results:  Caenorhabditis elegans RBX1 is strongly expressed in early embryos and in the gonad, including meiotic cells. Depletion of RBX1 by RNA‐mediated interference (RNAi) caused pronounced defects in the first meiotic division. Several irregular phenotypes were identified in embryos that escaped from meiotic arrest: defects in mitotic chromosomal condensation and segregation, abnormal chromosome bridges, giant nuclei, abnormal cortical protrusion, multinucleate cells and defects in germ cell proliferation. Moreover, histone H3 phosphorylation at Ser10 and Ser28 was significantly reduced in these embryos. The histone H3 phosphorylation defect of embryos was rescued by the additional depletion of protein phosphatase 1 (GLC7α/β) by RNAi.Conclusion:  These results indicate that the RBX1 protein participates in diverse functions relevant to chromosome metabolism and cell cycle control.
Double‐stranded DNA binding properties of Saccharomyces cerevisiae DNA polymerase ɛ and of the Dpb3p‐Dpb4p subassembly
Authors: Tsubota Toshiaki , Maki Satoko , Kubota Hajime , Sugino Akio , Maki Hisaji 
Published: November 2003

AbstractBackground:  DNA polymerase ɛ (Pol ɛ) of Saccharomyces cerevisiae participates in many aspects of DNA replication, as well as in DNA repair. In order to clarify molecular mechanisms employed in the multiple tasks of Pol ɛ, we have been characterizing the interaction between Pol ɛ and DNA.Results:  Analysis of the four‐subunit Pol ɛ complex by gel mobility shift assay revealed that the complex binds not only to single‐stranded (ss) DNA but also equally well to double‐stranded (ds) DNA. A truncated polypeptide consisting of the N‐terminal domain of Pol2p catalytic subunit binds to ssDNA but not to dsDNA, indicating that the Pol2p C‐terminal domain and/or the auxiliary subunits are involved in the dsDNA‐binding. The dsDNA‐binding by Pol ɛ does not require DNA ends or specific DNA sequences. Further analysis by competition experiments indicated that Pol ɛ contains at least two distinct DNA‐binding sites, one of which binds exclusively to ssDNA and the other to dsDNA. The dsDNA‐binding site, however, is suggested to also bind ssDNA. The DNA polymerase activity of Pol ɛ is inhibited by ssDNA but not by dsDNA. Furthermore, purification of the Pol ɛ auxiliary subunits Dpb3p and Dpb4p revealed that these proteins form a heterodimer and associate with dsDNA.Conclusions:  Pol ɛ has multiple sites at which it interacts with DNA. One of these sites has a strong affinity for dsDNA, a feature that is not generally associated with DNA polymerases. Involvement of the Dpb3p‐Dpb4p complex in the dsDNA‐binding of Pol ɛ is inferred.
A comparison of diploid and tetraploid perennial ryegrass and tetraploid ryegrass/white clover swards under continuous sheep stocking at controlled sward heights. 3. Sward characteristics and animal output, years 4–8
Authors: Vipond J. E. , Swift G. , Cleland A. T. , Fitzsimons J. , Hunter E. A. 
Published: November 2003

Results for years 4–8 of a long‐term grazing experiment on swards of a diploid perennial ryegrass (Lolium perenne), var. Contender (D swards), a tetraploid ryegrass, var. Condesa (T swards) and Condesa with S184 white clover (Trifolium repens) (TC swards), direct sown in May 1987, are presented. The swards were continuously stocked with sheep from 1988 to 1990, as previously reported, and for a further 5 years, 1991–95, at a target sward surface height (SSH) of 4–6 cm. Control of sward height was successfully achieved by variable stocking, except in 1993 when paddocks were set stocked and the resulting mean SSH was 9·3 cm. Grass swards received on average 160 kg N ha−1 year−1; grass/clover swards were mainly not fertilized with N with the exception that they were given 30 kg N ha−1 as a remedial mid‐summer application during a period of low herbage mass on offer in 1994 and 1995.Mean white clover content of the swards fell from 18·2% of herbage dry‐matter (DM) in 1992 to 8·5% in 1993, whereas stolon lengths fell from 120 to 58 m m−2. A return to lower sward heights in 1994–95 resulted in an increase in white clover content to 12·8% by the final sampling in August 1995. Perennial ryegrass content of the grass swards remained high throughout (mean 96·7% in 1995). Perennial ryegrass tiller densities recorded in August 1991, 1993 and 1994 showed consistently significant (P < 0·001) sward differences (3‐year mean 16 600, 13 700 and 10 100 perennial ryegrass tillers m−2 for the D, T and TC swards). In 1994, the year after lax grazing, a low perennial ryegrass tiller density (9100 m−2) and low white clover content (mean 4·3%) in the TC swards resulted in a much lower herbage bulk density than in the grass swards (April–July means 72, 94 and 44 kg OM ha−1 cm−1 for the D, T and TC swards). There was a consistent 40 g d−1 increase in lamb liveweight gain on the TC swards over the T swards, except in 1994. In that year there was a reduction in lamb liveweight gain of 33 g d−1 on the TC swards and a significant increase in ewe liveweight loss (117 g d−1) associated with low herbage bulk density despite optimal sward height. Lamb output (kg liveweight ha−1) on TC swards reflected white clover content, falling from a similar output to that produced from grass given 160 kg N ha−1, at 18% white clover DM content, down to 60% of grass + N swards with around 5% clover. A 6% greater output from the T than the D swards was achieved mainly through higher stocking rate. The experiment demonstrated a rapid, loss in white clover under lax grazing, and showed that the relationship between performance and sward height is also dependent on herbage density. High lamb output from a grass/clover sward was only achieved when the clover content was maintained at 15–20% of the herbage DM.
Is it worth taking a biopsy of the normal‐looking endometrium?
Authors: Badawy A. , Ash A. , Nagele F. , O'Connor H. , Davis A. , Magos A. L. 
Published: November 2003

Objectives To determine whether it is necessary to supplement the finding of a normal hysteroscopic appearance at diagnostic hysteroscopy with endometrial biopsy in the investigation of women with abnormal uterine bleeding.
Design Observational study.
Setting An undergraduate teaching hospital.
Subjects The endometrial biopsy reports of 756 patients aged 19–77 years old with abnormal uterine bleeding, who were found to have a normal hysteroscopy, were used. A normal hysteroscopic appearance was defined as: (a) normally shaped uterine cavity, (b) normal‐looking, homogeneous‐appearing endometrium, and (c) absence of focal lesions such as submucous myoma and polyps. An enlarged endometrial cavity, atrophic endometrium, and polypoid secretory‐appearing endometrium were all considered normal.
Results Endometrial histology was normal in 97.5% of cases (n = 737). Abnormalities found included chronic endometritis (n = 17) and low‐risk hyperplasia (n = 2). No age‐ or symptom‐related differences were seen any of the histological findings, and no cases of malignancy or atypical hyperplasia were detected.
Conclusion The visual diagnosis at panoramic hysteroscopy is reliable when performed properly and according to strict criteria of what is normal. It seem unnecessary to obtain tissue for pathological examination when the hysteroscopic view is within normal limits, but biopsy should still be obtained if the uterine cavity appears in any way abnormal.