bluuebunny/crossref_metadata_2025_split huggingface dataset:
Nik1: a Nim1‐like protein kinase of S. cerevisiae interacts with the Cdc28 complex and regulates cell cycle progression
Authors: Tanaka Seiji , Nojima Hiroshi 
Published: November 2003

Background: The eukaryotic cell cycle is driven by CDK‐cyclin complexes. A number of proteins interact either with CDK or the CDK complex to regulate CDK activity. A search for novel cell cycle regulators in the budding yeast Saccharomyces cerevisiae yielded multicopy suppressors of the cdc2‐L7mutation of the fission yeast, Schizosaccharomyces pombe. Results: One of the isolated genes was found to encode a putative protein kinase similar to Nim1 of S. pombe and was termed NIK1 (Nim1‐like kinase 1). Transcription of NIK1‐was periodic and peaked at the G1/S boundary. Although NIK1 is not essential, Δnik1 cells showed G2 delay and hydroxyurea (HU) sensitivity. Anomalously elongated buds were observed in the stationary phase or in the presence of HU. Moreover, DNA was aberrantly distributed in the Δnik1 cdc28 double mutant. Genetical and biochemical evidence suggests that Nik1 interacts with the Cdc28 complex. Conclusions: Nik is a structural and functional homo‐logue of Nim1. Nik1 interacts with the Cdc28 complex and functions not only at the G2/M transition but also at other points of the cell cycle.
A cell‐free system of Tn3 transposition and transposition immunity
Authors: Maekawa Takafumi , Yanagihara Katsuhiko , Ohtsubo Eiichi 
Published: November 2003

Background: Tn3 is a bacterial transposon, which encodes transposase required for its transposition. Tn3 has terminal inverted repeat (IR) sequences of 38 bp in length, whose inner region, called the B domain, is bound by transposase. Tn3 confers transposition immunity, a phenomenon in which Tn3 transposes to a target replicon with Tn3 much less frequently than to a target replicon with no Tn3. Results: To understand transposition and transposition immunity at the molecular level, we constructed a cell‐free system using a plasmid as the target. Transpositional recombination occurred in a cell extract containing transposase between the target and a donor plasmid carrying mini‐Tn3 at a high frequency. The reaction required ATP, Mg2+, dNTPs and 2% polyvinyl alcohol, and was inhibited by inhibitors for DNA synthesis and DNA gyrase. In this system, when a plasmid with the IR sequence was used as the target, the frequency of transposition was significantly decreased, demonstrating that the transposition immunity conferred by Tn3 is reproduced in vitro. Preincubation of the target in the cell extract increased the level of transposition immunity. On the other hand, mutations within the B domain in the IR sequence of the target abolished transposition immunity. Conclusions: Transposition of Tn3 and transposition immunity could be reproduced in vitro. The results demonstrate that the binding of transposase to domain B of the IR sequence in the target replicon is responsible for transposition immunity. We propose that the transposition immunity results from conversion of the normal synaptic complex formed between the donor and target molecules to another complex which is inactive for transposition, due to the interaction between transposases binding to the IR sequences in the donor and target molecules.
Specific nicking at the 3′ ends of the terminal inverted repeat sequences in transposon Tn3 by transposase and an E. coli protein ACP
Authors: Maekawa Takafumi , Yanagihara Katsuhiko , Ohtsubo Eiichi 
Published: November 2003

Background: Tn3, a bacterial transposon, carries tnpA gene encoding transposase which is essential for its transposition. The transposition of Tn3 has been reproduced in vitro in a cell extract containing transposase by using a plasmid carrying mini‐Tn3 as the donor and another plasmid as the target. Transposase has the ability to bind to the 38‐bp terminal inverted repeats (IRs) of Tn3. The molecular mechanism of the initiation step of the Tn3 transposition reaction promoted by the transposase has, however, not been understood. Results: We found that nicking occurred efficiently in the cell‐free system at each of the 3′ ends of the IRs of mini‐Tn3 in the closed circular or linear donor molecules. The nicking reaction required transposase and Mg2+, but did not require ATP, an ATP‐regenerating system, dNTPs and polyvinyl alcohol, which were the requirements for the transposition reaction. By using the nicking assay employed here, transposase was purified almost to homogeneity. Gel filtration and sedimentation analyses indicate that transposase forms a dimer in a solution containing 0.5 m NaCl. The nicking activity of the purified transposase was weak and was found to be stimulated by a host factor. The nicking stimulation factor was subsequently purified and found to be ACP, an Escherichia coli acyl carrier protein. Conclusions: Nicking occurred efficiently at the 3′ ends of mini‐Tn3 in the reaction mixture containing transposase and ACP. ACP is known to act as a factor which modulates enzymes that are involved in several biological processes either in the acylated or unacylated form. ACP may also modulate transposase to initiate the transposition reaction with nicking at the 3′ ends of Tn3.
Purification and characterization of XRad51.1 protein, Xenopus RAD51 homologue: recombinant XRad51.1 promotes strand exchange reaction
Authors: Maeshima Kazuhiro , Morimatsu Katsumi , Horii Toshihiro 
Published: November 2003

Background:
The RAD51 gene of Saccharomyces cerevisiae is homologous to the Escherichia coli recA gene and plays a key role in genetic recombination and DNA double‐strand break repair. To construct an improved experimental system of homologous recombination in higher eukaryotes, we have chosen the South African clawed frog, Xenopus laevis, whose egg extracts might be useful for the in vitro studies. We identified and characterized a Xenopus homologue of RAD51 gene, the XRAD51.1.
Results:
Recombinant XRad51.1 was expressed in E. coli. The purified XRad51.1 protein showed ssDNA‐dependent ATPase activity and promoted the DNA strand exchange reaction between two 55‐mer oligonucleotides. The binding stoichiometry of XRad51.1 to ssDNA was determined by fluorescence of poly(dεA), a chemically modified poly(dA), and was found to be about six bases/XRad51.1 monomer in a nucleoprotein filament, a similar value to E. coli RecA protein. The kinetics of the fluorescence change of poly(dεA) after XRad51.1 binding in the presence of ATP was significantly different from that observed with RecA protein. The affinity of XRad51.1 to ssDNA in the presence of ATP was higher than that of RecA protein, and the dissociation of the XRad51.1‐ssDNA complex was slower than the RecA‐ssDNA complex.
Conclusions:
Purified recombinant XRad51.1 protein promoted the strand exchange between short DNA molecules. While the binding stoichiometry of XRad51.1 protein to ssDNA was identical to that of the RecA protein, XRad51.1 has a significantly higher affinity and binding stability to ssDNA than did the RecA protein in the presence of ATP.
A hamster temperature‐sensitive G1 mutant, tsBN250 has a single point mutation in histidyl‐tRNA synthetase that inhibits an accumulation of cyclin D1
Authors: Motomura Seiichi , Fukushima Kohtaro , Nishitani Hideo , Nawata Hajime , Nishimoto Takeharu 
Published: November 2003

Background:
We have previously isolated a series of temperature‐sensitive mutants for cell‐proliferation from the BHK21 cell line, derived from the golden hamster. These mutants proliferate at 33.5 °C, the permissive temperature, but not at 39.5 °C the restrictive temperature. Using DNA‐mediated gene transfer, human genes complementing these ts mutants were cloned.
Results:
At 39.5 °C the tsBN250 cell line, a temperature‐sensitive mutant of the BHK21 cell line, had a defect in the G1 phase, but not in the S phase. The human gene complementing tsBN250 cells was found to encode histidyl‐tRNA synthetase. Indeed, the tsBN250 cell line had a single base change—guanine to adenine at the second position of the 362nd codon of hamster histidyl‐tRNA‐synthetase, converting arginine to histidine. Following release from serum starvation, cyclin E, but not cyclin D1, was accumulated, while, at 39.5 °C, the mRNA of cyclin D1 was normally expressed in tsBN250 cells. A similar inhibition of cyclin D1 accumulation was observed in another ts mutant, tsBN269, which has a single point mutation in lysyl‐tRNA synthetase. Overexpression of cyclin D1 enabled tsBN250 cells to enter the S phase.
Conclusion:
tsBN250 cells have a single point mutation in histidyl tRNA synthetase that causes a loss of histidyl‐tRNA synthetase activity which in turn reduces the content of cyclin D1, but not of cyclin E, thereby resulting in G1 arrest.
Dissociation kinetics of RepA dimers: implications for mechanisms of activation of DNA binding by chaperones
Authors: Chattoraj Dhruba K. , Ghirlando Rodolfo , Park Kyusung , Dibbens Justin A. , Lewis Marc S. 
Published: November 2003

Background: The replication initiator of plasmid P1, RepA, binds DNA as monomer. The binding is stimulated by the chaperones DnaJ, DnaK and GrpE of Escherichia coli. Two models of chaperone action have been proposed. (i) Chaperones dissociate RepA dimers, which are inactive in DNA binding, into active monomers. (ii) The dissociation occurs spontaneously but the monomeric products require the chaperones for refolding into the active form. The latter model was based on the observation that RepA diluted 1000‐fold below the KD for dimer dissociation, still required the chaperones for DNA binding.Results: We have confirmed that under the condition of DNA binding experiments, the RepA dimers dissociate reversibly into monomers with a KD value of 1.1 ± 0.1 μm. In the vicinity of this concentration, the sedimentation coefficient of RepA was concentration dependent, allowing estimation of s20,w coefficients for the RepA monomer (2.95 S) and dimer (4.01 S). Dynamic light scattering experiments indicated an increase of the monomer fraction within 5 min of RepA dilution. Circular dichroism (CD) measurements were consistent with these results.Conclusion: RepA monomerization is efficient without the mediation of chaperones. They are required to activate RepA most likely because they are needed to re‐fold RepA monomers.
Histone‐like protein HU as a specific transcriptional regulator: co‐factor role in repression of gal transcription by GAL repressor
Authors: Aki Tsunehiro , Choy Hyon E. , Adhya Sankar 
Published: November 2003

Background: Transcription initiation from the two overlapping promoters of the gal operon in Escherichia coli is negatively regulated by binding of Gal repressor (GalR) to bipartite operators, which encompass the promoters. Coordinated repression of the two promoters requires GalR binding to both operators. In a purified system, GalR, nevertheless, fails to show the coordinated repression, predicting the participation of an additional factor(s) in the regulation in vivoResults: We have purified a protein that restored the expected GalR‐mediated repression for the in vitro system and have identified this factor to be the bacterial histone‐like protein HU. In vitro transcription assays in the presence of GalR and HU show that, just as in vivo, the coordinated repression of the two gal promoters requires GalR binding to both operators and is sensitive to the inducer, d‐galactose. The GalR and HU dependent repression also requires supercoiled DNA template and prevents open complex formation.Conclusion: We propose that HU, acting as a co‐factor, brings about the GalR‐mediated repression by forming a distinct nucleoprotein complex of higher order structure. Although how HU participates in the assembly process is unknown, there may be a cooperative effect in the formation of the repression complex.
A membrane cofactor protein transgenic mouse model for the study of discordant xenograft rejection
Authors: Yannoutsos Nikos , IJzermans Jan N. M. , Harkes Clara , Bonthuis Fred , Zhou Chun‐Yan , White David , Marquet Richard L. M. , Grosveld Frank 
Published: November 2003

Background: In recent years, interest has been revived in the possibility of transplanting organs into humans from a phylogenetically disparate species such as the pig (xenotransplantation). Such discordant xenografts, however, are subject to hyperacute rejection (HAR) and activation of host complement plays a major role in this rejection. This problem may be solved through the use of transgenic technology by providing the grafted tissue with molecules that down‐regulate the action of host complement. Results: Transgenesis with a yeast artificial chromosome (YAC) was used to produce transgenic mice with the complete genomic gene of the human complement regulator membrane cofactor protein (MCP). Transgenic mice were obtained that exhibit full regulation of MCP as normally observed in humans. Hearts from these mice were shown to be significantly protected from HAR caused by human serum in an in vivo experimental procedure. Conclusions: We conclude that MCP can protect discordant xenografts from HAR caused by human serum and that transgenic mice can be used effectively as in vivo models for the study of the role of human complement regulatory molecules in xenotransplantation.
A novel nuclease activity from Xenopus laevis releases short oligomers from 5′‐ends of double‐and single‐stranded DNA
Authors: Reichenberger Susanne , Brüll Nicole , Feldmann Elke , Göttlich Bernd , Vielmetter Walter , Pfeiffer Petra 
Published: November 2003

Background: Double‐strand breaks in chromosomal DNA of eucaryotic cells are assumed to be repaired by mechanisms of illegitimate recombination capable of direct rejoining of the broken ends. Cell‐free extracts of Xenopus laevis eggs efficiently perform these end joining reactions with any pair of noncomplementary DNA termini whose single‐stranded 5′‐ or 3′‐overhangs do not exceed a length of ≈ 10 nt. Results: Using hairpin‐shaped oligonucleotides that allow the construction of double‐strand break termini with 5′‐ or 3′‐overhangs of defined length and sequence we show that 5′‐overhangs of more than 9–10 nt are exonucleolytically resected in the extract to produce shorter 5′‐overhangs that can be metabolized in the end joining reaction. 5′‐recessed ends in double‐stranded DNA with 3′‐overhangs of more than 2 nt as well as the 5′‐ends of single‐stranded DNA also serve as substrates for the exonuclease activity. In all cases, oligomers of about 10 nt are released from the 5′‐ends. Conclusions: We describe here a novel 5′‐exonuclease activity present in eggs from Xenopus laevis that reproducibly removes decameric oligonucleotides from 5′‐ends of double‐ and single‐stranded DNA. A possible function of this unusual activity is discussed in the context of homologous and illegitimate genetic recombination processes.
Implications of the zinc‐finger motif found in the DNA‐binding domain of the human XPA protein
Authors: Morita Eugene H. , Ohkubo Tadayasu , Kuraoka Isao , Shirakawa Masahiro , Tanaka Kiyoji , Morikawa Kosuke 
Published: November 2003

Background: The XPA (xeroderma pigmentosum group A) protein specifically recognizes the UV‐ or chemically damaged DNA lesions, and triggers the nucleotide excision repair process. This XPA protein contains the functional domain which is crucial to the recognition of damaged DNA. Its primary structure suggests that this DNA binding domain may contain a zinc‐finger motif. To gain a more detailed insight into this zinc‐finger motif, we have measured the 113Cd‐NMR spectra of the DNA binding domains derived from the wild‐type and mutant XPA proteins. Results: 113Cd‐NMR analysis, combined with atomic absorption and site‐directed mutagenesis, revealed that the DNA binding domain contains one zinc ion, coordinated with four cysteine residues (Cys105, Cys108, Cys126, and Cys129), that is essential for correct protein folding in vivo and in vitro Conclusions: The four ligand cysteine residues form a Cys‐X2‐Cys‐X17‐Cys‐X2‐Cys motif, which is reminiscent of the (Cys)4 type zinc‐finger motif found in numerous transcriptional regulatory proteins. However, the secondary structure prediction and the 3D‐1D compatibility analysis demonstrate that there is no structural similarity in the vicinity of the zinc‐finger motif between the XPA protein and other zinc‐finger containing proteins. We conclude that the XPA protein contains a new type of zinc‐finger motif.
Developmental expression of the Bombyx Antennapedia homologue and homeotic changes in the Nc mutant
Authors: Nagata Toshifumi , Suzuki Yoshiaki , Ueno Kohji , Kokubo Hiroki , Xu Xin , Hui Chi‐chung , Hara Wajiro , Fukuta Masakazu 
Published: November 2003

Background: Abundant availability of homeotic mutants in Bombyx mori provides us with clues to study the body plan of the silkworm. Previous studies indicated that the ECa/ECa and EN/EN embryos, which reveal drastic morphological changes (Itikawa 1943, 1952) lack the homologue of abd‐A and the homologues of abd‐A and Ubx, respectively (Ueno et al. 1992). It will be interesting to characterize a mutant named Nc, the locus of which was mapped about 1.4 cm apart from the E loci (Itikawa 1944, 1952). Results: In the present study we cloned and identified the Bombyx Antennapedia cDNA from a library of larval middle silk gland. The developmental expression patterns of the Bombyx Antennapedia were analysed in embryos and larval middle silk glands. We found the Bombyx Antennapedia mRNA of the Nc mutant lacks the homeobox region and its downstream region. This defect comes from a corresponding deletion on the Nc chromosome. Development of the Nc/Nc embryos terminates at stage 26, revealing a transformation of the prothoracic legs into antennae, milder changes in the mesothoracic legs, compression between the prothoracic and mesothoracic segments as well as in the gnathocephalon region, and a severe growth suppression of the silk glands. Conclusions: These results give a molecular basis and detailed morphological abnormalities of the Nc mutant known since 1944.
Recognition mechanisms of the minus‐strand origin of phage f1 by Escherichia coli RNA polymerase
Authors: Higashitani Nahoko , Higashitani Atsushi , Guan Zhi‐Wen , Horiuchi Kensuke 
Published: November 2003

Background: The primer RNA for the synthesis of the minus strand of filamentous coliphages is produced by host RNA polymerase at a specific site on the plus strand template. The mechanism used by the enzyme in recognizing the origin is unknown, but minus strand replication requires the holoenzyme form of RNA polymerase. The origin contains two inverted repeats which can form hairpins. Results: When the origin sequence is drawn arranging the two hairpins horizontally, it resembles a stretch of a mostly double‐stranded molecule. The nucleotides protected from nucleases by the holoenzyme were found to be located in two regions on this drawing: one around the 35th nucleotide upstream of the RNA start site, and the other ranging from the 10th nucleotide upstream to the 10th downstream, of the start site. The core enzyme did not show any protection. Mutational analyses of the origin indicated that the base‐paired structure in the former region was important for origin activity. In the region around the 10th nucleotide upstream of the start site, specific base(s) in the non‐template strand were required for origin activity, while the base sequence of the template strand was irrelevant. Conclusions: It is likely that the recognition mechanism of the origin by RNA polymerase shares common features with that of transcriptional promoters.
Chi‐activated RecBCD enzyme possesses 5′→3′ nucleolytic activity, but RecBC enzyme does not: evidence suggesting that the alteration induced by Chi is not simply ejection of the RecD subunit
Authors: Anderson Daniel G. , Churchill Jason J. , Kowalczykowski Stephen C. 
Published: November 2003

Background: Homologous recombination in Escherichia coli is initiated by the RecBCD enzyme, and is stimulated by DNA elements known as Chi (χ) sites. The RecBCD enzyme is both a helicase and a nuclease. Recognition of χ causes both attenuation of the 3′→5′ exonuclease activity of the RecBCD enzyme, and activation of an exonuclease activity with 5′→3′ polarity, while leaving the helicase activity unaffected. A variety of evidence suggests that χ‐recognition by RecBCD enzyme is accompanied by ejection of the RecD subunit. Results:Through examination of RecBCD exonuclease activity under a variety of conditions, we have shown that recognition of χ by the RecBCD enzyme results in a net reduction of nuclease activity. In addition, the exact location of the first cleavage event elicited by χ‐activation of the 5′→3′ nuclease is dependent upon the concentration of free magnesium ions. Finally, we have demonstrated that purified RecBC enzyme (i.e. without the RecD subunit) possesses no significant exonuclease activity under conditions where the χ‐modified RecBCD enzyme is an active 5′→3′ exonuclease. Conclusions: We have shown that, despite the activation of a 5′→3′ exonuclease, recognition of χ by the RecBCD enzyme results in a net preservation of DNA. This new χ‐activated nucleolytic action shows surprising variability in the exact location of its initial cleavage. We have demonstrated that purified RecBC enzyme is not an exact analogue of the χ‐activated RecBCD enzyme, suggesting that the biochemical basis of χ‐activation is not simply ejection of the RecD subunit.
Xenopus cyclin A1 can associate with Cdc28 in budding yeast, causing cell‐cycle arrest with an abnormal distribution of nuclear DNA
Authors: Funakoshi Minoru , Sikder Hashmat , Ebihara Hiroko , Irie Kenji , Sugimoto Katsunori , Matsumoto Kunihiro , Hunt Tim , Nishimoto Takeharu , Kobayashi Hideki 
Published: November 2003

Background:Cyclins play a regulatory role in cell cycle progression, associated with cyclin‐dependent kinases. We have investigated the structure–function relationships of cyclin A, mainly using Xenopus egg extracts in vitro. To further analyse the function and structure of cyclin A in vivo, we expressed Xenopus cyclin A1 in the budding yeast Saccharomyces cerevisiae.Results:We herein show that vertebrate cyclin A1 can associate with endogenous Cdc28 to form histone H1 kinase. The growth of the yeast was inhibited by the expression of indestructible cyclin A1, but not by a non‐Cdk binding cyclin A1 mutant. The induction of cyclin A1 expression in yeast caused cell cycle arrest with an abnormal distribution of nuclear DNA to the daughter bud. Suppressors of the cyclin A1‐mediated growth arrest were identified as new alleles of the cdc28 mutation that reduced the binding of cyclin A1 and possessed different affinities for the cyclin–Cdc28 complexes. The temperature‐sensitivity of the cdc28 mutation was thus preferentially suppressed by the endogenous cyclins CLN2 and CLB2.Conclusions:These results suggest that the Cdc28 protein kinase activity mediated by vertebrate cyclin A1 may be involved in the process of nuclear movement in the yeast, and thereby affect the dependence of the M phase on the completion of the S phase through a preferential binding affinity of the cyclin–Cdc28 complex.