bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hydraulic differentiation of Ponderosa pine populations along a climate gradient is not associated with ecotypic divergence
Authors: Maherali H. , Williams B. L. , Paige K. N. , Delucia E. H. 
Published: March 2003

Summary 1.
Pinus ponderosa occurs in a range of contrasting environments in the western USA. Xeric populations typically have lower leaf : sapwood area ratio (AL/AS) and higher whole‐tree leaf specific hydraulic conductance (KL) than mesic populations. These climate‐driven shifts in hydraulic architecture are considered adaptive because they maintain minimum leaf water potential above levels that cause xylem cavitation. 2. Using a common garden study, we examined whether differences in biomass allocation and hydraulic architecture between P. ponderosa populations originating from isolated outcrops in the Great Basin desert and Sierran montane environments were caused by ecotypic differentiation or phenotypic plasticity. To determine if populations were genetically differentiated and if phenotypic and genetic differentiation coincided, we also characterized the genetic structure of these populations using DNA microsatellites. 3. Phenotypic differentiation in growth, biomass allocation and hydraulic architecture was variable among populations in the common garden. There were no systematic differences between desert and montane climate groups that were consistent with adaptive expectations. Drought had no effect on the root : shoot and needle : stem ratio, but reduced seedling biomass accumulation, leaf area ratio, AL/AS and KL. Stem hydraulic conductance (KH) was strongly size‐dependent, and was lower in droughted plants, primarily because of lower growth. 4. Although microsatellites were able to detect significant non‐zero (P < 0·001) levels of differentiation between populations, these differences were small and were not correlated with geographic separation or climate group. Estimates of genetic differentiation among populations were low (<5%), and almost all the genetic variation (>95%) resided within populations, suggesting that gene flow was the dominant factor shaping genetic structure. 5. These results indicate that biomass allocation and hydraulic differences between desert and montane populations are not the result of ecotypic differentiation. Significant drought effects on leaf : sapwood allocation and KL suggest that phenotypic differentiation between desert and montane climates could be the result of phenotypic plasticity.
Characterization of RecA1332 in vivo and in vitro. A role for α‐helix E as a liaison between the subunit–subunit interface and the DNA and ATP binding domains of RecA protein
Authors: Bianco Piero R. , Weinstock George M. 
Published: March 2003

Background:The RecA protein of Escherichia coli is essential for homologous recombination and induction of the SOS response. RecA has three cysteines located at positions 90, 116 and 129. Chemical modification of these residues abolishes ATP hydrolysis and repressor cleavage, and causes a reduction in the DNA strand exchange and DNA strand annealing activities. Several mutants at each of these positions were isolated and partially characterized. One of these, recA1332, replaces cysteine 129 with methionine. Although this is a relatively conservative mutation based on hydrophobicity, recA1332 was completely defective for DNA repair but the purified protein was active for ATPase in vitro.Results: In vivo, strains containing this mutant allele were shown to be defective when assayed for all RecA‐dependent activities. In vitro, RecA1332 protein possessed DNA‐dependent ATP hydrolysis activity that showed an increased sensitivity to inhibition by monovalent cations, and whose kcat was reduced 3‐ to 12‐fold. In addition, RecA1332 was unable to use oligodeoxyribonulceotides as ssDNA cofactors in the ATPase reaction. RecA1332 showed altered binding to single‐ and double‐stranded DNA and, although it was able to perform DNA strand exchange, it was slowed in its ability to both form joint molecule intermediates and to convert these species to product.Conclusions:Our results are consistent with a defect in intermolecular interactions between RecA monomers. We propose that α‐helix E (which includes C129M) is a liaison that connects the subunit–subunit interactions to DNA and ATP binding, thereby creating filament stability and cooperativity.
Abortive recombination in Escherichia coli ruv mutants blocks chromosome partitioning
Authors: Ishioka Ken , Fukuoh Atsushi , Iwasaki Hiroshi , Nakata Atsuo , Shinagawa Hideo 
Published: March 2003

Background:All the ruvA, ruvB and ruvC mutants of Escherichia coli are sensitive to treatments that damage DNA, and are mildly defective in homologous recombination. It has been reported that the ruv mutants form nonseptate, multinuclear filaments after low doses of UV irradiation, dependent on the sfiA gene product. In vitro, the RuvAB complex promotes the branch migration of Holliday junctions, and RuvC resolves the junctions endonucleolytically.Results:After a low UV dose (5 J/m2), both ΔruvAB and ΔruvC mutant cells became filamentous, with their chromosomes aggregated in the central region. This corresponded to an increase in nonmigrating DNA on pulsed field gel electrophoresis of the XbaI digested chromosome. Upon further incubation, they produced a large number of anucleoid cells of normal size. A recA mutation, but not a recB mutation, suppressed these phenotypes of the ruv mutants. The ruv polA12(Ts) double mutants were inviable at the nonpermissive temperature and mimicked the morphological phenotypes of the UV irradiated ruv mutants.Conclusion: ruvA, B and C mutations block chromosome partitioning in UV irradiated cells because the abortive homologous recombination covalently links chromosomes together. There is a recBCD independent pathway for the recA dependent formation of recombination intermediates. An Ruv‐mediated resolution of recombination intermediates is required for the repair of strand breaks produced in UV irradiated cells and in the polA mutant cells.
Smad6 functions as an intracellular antagonist of some TGF‐β family members during Xenopus embryogenesis
Authors: Nakayama Takuya , Gardner Holly , Berg Linnea K. , Christian Jan L. 
Published: March 2003

Background:Bone morphogenetic proteins (BMPs) transmit signals via the intracellular protein Smad1, which is phosphorylated by ligand bound receptors, translocates to the nucleus, and functions to activate BMP target genes. Recently, a subclass of Smad proteins has been shown to inhibit, rather than transduce, BMP signalling, either by binding to the intracellular domain of BMP receptors, thereby preventing phosphorylation‐mediated activation of Smad1, or by binding directly to Smad1, thereby inhibiting its ability to activate gene transcription.Results:We have identified a Xenopus Smad (Smad6) that is 52% identical to mammalian Smad6, an inhibitory Smad. The spatial pattern of expression of Smad6 changes dynamically during embryogenesis and is similar to that of BMP‐4 at the tailbud stage. Overexpression of Smad6 in Xenopus embryos phenocopies the effect of blocking BMP‐4 signalling, leading to dorsalization of mesoderm and neuralization of ectoderm. Xenopus Smad6 completely blocks the activity of exogenous BMP‐4, and, unlike human Smad6, partially blocks the activity of activin, in a mesoderm induction assay. We also find that Smad6 protein accumulates at the membrane in some cells but is partially or completely restricted to nuclei of most overexpressing cells.Conclusions:We have identified an inhibitory Xenopus Smad, Smad6, that functions as an intracellular antagonist of activin and BMP‐4 signalling. Our finding that Smad6 protein is partially or completely restricted to nuclei of most overexpressing cells suggests that it may employ a novel or additional mechanism of action to antagonize TGF‐β family signalling other than that reported for other inhibitory Smads.
BMP regulates vegetal pole induction centres in early Xenopus development
Authors: Nachaliel Nira , Re'em‐Kalma Yael , Eshed Orna , Elias Sarah , Frank Dale 
Published: March 2003

BackgroundBone morphogenetic protein (BMP) plays an important role in mesoderm patterning in Xenopus. The ectopic expression of BMP‐4 protein hyperventralizes embryos, whereas embryos expressing a BMP‐2/4 dominant‐negative receptor (DNR) are hyperdorsalized. Mesoderm is initially induced in the marginal zone by cells in the underlying vegetal pole. While much is known about BMP's expression and role in patterning the marginal zone, little is known about its early role in regulating vegetal mesoderm induction centre formation.ResultsThe role of BMP in regulating formation of vegetal mesoderm inducing centres during early Xenopus development was examined. Ectopic BMP‐4 expression in vegetal pole cells inhibited dorsal mesoderm induction but increased ventral mesoderm induction when recombined with animal cap ectoderm in Nieuwkoop explants. 32‐cell embryos injected with BMP‐4 RNA in the most vegetal blastomere tier were not hyperdorsalized by LiCl treatment. The ectopic expression of Smad or Mix.1 proteins in the vegetal pole also inhibited dorsal mesoderm induction in explants and embryos. Expression of the BMP 2/4 DNR in the vegetal pole increased dorsal mesoderm induction and inhibited ventral mesoderm induction in explants and embryos.ConclusionsThese results support a role for BMP signalling in regulating ventral vegetal and dorsal vegetal mesoderm induction centre formation during early Xenopus development.
Identification of the core domain and the secondary structure of the transcriptional coactivator MBF1
Authors: Ozaki Jun , Takemaru Ken‐ichi , Ikegami Takahisa , Mishima Masaki , Ueda Hitoshi , Hirose Susumu , Kabe Yasuaki , Handa Hiroshi , Shirakawa Masahiro 
Published: March 2003

BackgroundMultiprotein bridging factor 1 (MBF1) is a transcriptional coactivator necessary for transcriptional activation caused by DNA binding activators, such as FTZ‐F1 and GCN4. MBF1 bridges the DNA‐binding regions of these activators and the TATA‐box binding protein (TBP), suggesting that MBF1 functions by recruiting TBP to promoters where the activators are bound. In addition, MBF1 stimulates DNA binding activities of the activators to their recognition sites. To date, little is known about structures of coactivators that bind to TBP.ResultsThe two‐dimensional (2D) 1H‐15N correlation spectrum of 15N labeled MBF1 indicated that MBF1 consists of both flexible and well structured parts. Limited digestion of MBF1 by α‐chymotrypsin yielded a ~9 kDa fragment. N‐terminal sequence analysis and NMR measurements revealed that this fragment originates from the C‐terminal 80 residues of MBF1 and form a well structured C‐terminal domain of MBF1, MBF1CTD. As previous deletion analyses have shown that MBF1CTD is capable of binding to TBP, it is suggested that MBF1CTD is the TBP binding domain of MBF1. Sequential assignments have been obtained by means of three‐dimensional (3D) and four dimensional (4D) heteronuclear correlation spectroscopies, and then the secondary structure of MBF1CTD was determined. As a result, MBF1CTD was shown to contain four amphipathic helices and a conserved C‐terminal region. Asp106 which is assumed to be responsible for the binding to TBP is located at the hydrophilic side of the third helix.ConclusionsStructural analyses revealed that MBF1 consists of two structurally different domains. A N‐terminal region is indispensable for the binding to activators, and does not form a well defined structure. In contrast, the C‐terminal 80 residues, which is capable of binding to TBP by itself, form a well‐structured domain, MBF1CTD. MBF1CTD is made up of four amphipathic helices and a conserved C‐terminal tail. A putative TBP binding residue is located on the hydrophilic surface of the third helix.
Requirement of cooperative functions of two repeated death effector domains in caspase‐8 and in MC159 for induction and inhibition of apoptosis, respectively
Authors: Tsukumo Shin‐ichi , Yonehara Shin 
Published: March 2003

BackgroundThe death effector domain (DED), which functions as a domain for a homophilic protein interaction, plays a role in death receptor‐mediated apoptosis. Two tandemly repeated DEDs in the prodomain of caspase‐8 (Casp8NC‐DED) and those in MC159 (viral FLIP) have been shown to positively and negatively regulate apoptosis, respectively, by binding to caspase‐8 and/or Fas‐
associated
death
domain (FADD). However, characteristics of each DED in Casp8NC‐DED and those in MC159 have not been well examined.ResultsWe analysed deletion and chimera mutants of DEDs derived from Casp8NC‐DED and MC159, and found that MC159 and Casp8NC‐DED require the combined effects of the two repeated DEDs to exert their binding and biological activities. The carboxy‐terminal DED of Casp8NC‐DED (Casp8C‐DED) has the potential to induce apoptosis, and the amino‐terminal DED of MC159 showed a dominant inhibitory effect on apoptosis when combined with Casp8C‐DED. In addition, the two repeated DEDs in Casp8NC‐DED and MC159 were shown to regulate the activities of caspase differently from the caspase recruitment domain (CARD) in the prodomains of caspase‐2, ‐9 and Apaf‐1.ConclusionsAlthough each of the DEDs in Casp8NC‐DED and MC159 has the potential to stimulate or inhibit apoptosis, the combination of the two‐repeated DEDs is necessary for the DED‐containing proteins to stimulate or inhibit apoptosis.
Association of synapse‐associated protein 90/ postsynaptic density‐95‐associated protein (SAPAP) with neurofilaments
Authors: Hirao Kazuyo , Hata Yutaka , Deguchi Maki , Yao Ikuko , Ogura Misa , Rokukawa Chiaki , Kawabe Hiroshi , Mizoguchi Akira , Takai Yoshimi 
Published: March 2003

BackgroundSynapse‐associated protein (SAP) 90/Postsynaptic density (PSD)‐95‐associated protein (SAPAP) (also called Guanylate kinase‐associated protein/hDLG‐associated protein) interacts with the guanylate kinase domains of PSD‐95 and synaptic scaffolding molecule (S‐SCAM) via the middle region containing 5 repeats of 14 amino acids. SAPAP also binds the recently identified proteins, nArgBP2 and synamon (also called Shank 1a), via the proline‐rich region and the C‐terminus, respectively. SAPAP is highly enriched in the Triton X‐100‐insoluble PSD fraction, and recruits PSD‐95 into the Triton X‐100‐insoluble fraction in transfected cells. We have further characterized here the Triton X‐100‐insolubility of SAPAP and tried to identify the Triton X‐100‐insoluble structures which SAPAP interacts with.ResultsN‐Methyl‐ d‐aspartate receptors were recruited into the Triton X‐100‐insoluble fraction with PSD‐95 by SAPAP. The N‐terminal region of SAPAP was Triton X‐100‐insoluble, whereas the middle and C‐terminal regions were Triton X‐100‐soluble. We identified proteins interacting with 35S‐methionine‐labelled SAPAP in the overlay assay, determined their amino acid sequences, and found them to be neurofilaments. SAPAP interacted with neurofilaments via the N‐terminal region, was co‐immunoprecipitated with neurofilaments from the rat brain, and co‐localized with neurofilaments in transfected cells.ConclusionSAPAP associates with neurofilaments via the N‐terminal region and may link various components of the PSD to neurofilaments.
Functional interaction of general transcription initiation factor TFIIE with general chromatin factor SPT16/CDC68
Authors: Kang Seung‐Woo , Kuzuhara Takashi , Horikoshi Masami 
Published: March 2003

BackgroundTranscriptional initiation of class II genes is one of the major targets for the regulation of gene expression and is carried out by RNA polymerase II and many auxiliary factors, which include general transcription initiation factors (GTFs). TFIIE, one of the GTFs, functions at the later stage of transcription initiation. As recent studies indicated the possibility that TFIIE may have a role in chromatin transcriptional regulation, we isolated TFIIE‐interacting factors which have chromatin‐related functions.ResultsUsing the yeast two‐hybrid screening system, we isolated the C‐terminal part of the human homologue of Saccharomyces cerevisiae (y) Spt16p/Cdc68p, a general chromatin factor. The C‐terminal part of human SPT16/CDC68 directly interacts with TFIIE, and ySpt16p/Cdc68p also interacts with yTFIIE (Tfa1p/Tfa2p), thus indicating the existence of an evolutionarily conserved interaction between TFIIE and SPT16/CDC68. Functional interaction of yTFIIE and ySpt16p/Cdc68p was examined using a conditional yTFIIE‐α mutant strain. Over‐expression of ySpt16p/Cdc68p suppressed the phenotype of cold sensitivity of the yTFIIE‐α‐cs mutant strain, and in vitro binding assays revealed that yTFIIE‐α‐cs mutant protein showed diminished binding affinity to ySpt16p/Cdc68p.ConclusionsThese observations indicate that general transcription initiation factor TFIIE functionally interacts with general chromatin factor SPT16/CDC68, a finding which provides new insight into the involvement of TFIIE in chromatin transcription. This may well lead to a breakthrough in relationships between the transcription initiation process and structural changes in chromatin.
Direct imaging of phosphorylation‐dependent conformational change and DNA binding of CREB by electron microscopy
Authors: Usukura Jiro , Nishizawa Yuji , Shimomura Atsushi , Kobayashi Kazuto , Nagatsu Toshiharu , Hagiwara Masatoshi 
Published: March 2003

BackgroundThe second messenger cAMP stimulates the expression of numerous genes through the PKA‐dependent phosphorylation of CREB. The cAMP‐regulated transcription factor CREB undergoes conformational change in response to phosphorylation by PKA at Ser 133. The phosphorylation enables interaction between the kinase‐inducible domain (KID) of CREB and KIX domain of CREB binding protein (CBP).ResultsTo understand the activation mechanism of CREB‐mediated gene expression, we performed the electron‐microscope imaging of the transcription machinery. We improved the metal shadowing techniques to achieve higher resolution to detect phosphorylation‐induced conformation change of the protein. Homodimer formation of CREB and the complex formation of phosphorylated CREB with CBP were observed under the electron microscope. The binding of the CREB dimer to CREs on the somatostatin and tyrosine hydroxylase promoters were also visualized directly and stereoscopically.ConclusionsGreatly improved resolution achieved by our modified metal shadowing techniques makes it possible to visualize that the shape of CREB homodimer was changed in phosphorylation‐dependent manner and that the promoter DNA strands containing CREs appeared to be bent and twisted slightly by the holding in the crevice of the CREB homodimer. This method may be applicable to visualize transcriptional activation process of nuclear receptors or general transcription machinery.
Significant activity of a modified ribozyme with N7‐deazaguanine at G10.1: the double‐metal‐ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes
Authors: Nakamatsu Yuka , Warashina Masaki , Kuwabara Tomoko , Tanaka Yoshiyuki , Yoshinari Koichi , Taira Kazunari 
Published: March 2003

BackgroundSeveral reports have appeared recently of experimental evidence for a double‐metal‐ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes. In one case, hammerhead ribozyme‐mediated cleavage was analysed as a function of the concentration of La3+ ions in the presence of a fixed concentration of Mg2+ ions so that the role of metal ions that are directly involved in the cleavage reaction could be monitored. The resultant bell‐shaped curve for activation of cleavage was used to support the proposed double‐metal‐ion mechanism of catalysis. However, other studies have demonstrated that the binding of a metal ion (the most conserved P9 metal ion) to the pro‐Rp oxygen (P9 oxygen) of the phosphate moiety of nucleotide A9 and to the N7 of nucleotide G10.1 is critical for efficient catalysis, despite the large distance (≈20 Å) between the P9 metal ion and the labile phosphodiester group in the ground state. In fact, it was demonstrated that an added Cd2+ ion binds first to the pro‐Rp phosphoryl P9 oxygen but not with the pro‐Rp phosphoryl oxygen at the cleavage site.ResultsIn earlier discussions, it was difficult to completely exclude the possibility that La3+ ions might have replaced the P9 metal ion and, as a result, created conditions represented by the bell‐shaped curve. In order to clarify this situation, we examined a chemically synthesized hammerhead ribozyme (7‐deaza‐R34) that included a minimal modification, namely, an N7‐deazaguanine residue in place of G10.1. We compared the kinetic properties of this ribozyme with those of the parental ribozyme (R34). Kinetic analysis revealed that, unlike the cases of added Cd2+ ions, the added La3+ ions did not replace the pre‐existing P9 metal ion, and that the replacement of N7 by C7 at G10.1 reduced the catalytic activity to a limited extent. This result indicates that the binding of a Mg2+ ion to N7 at G10.1 is catalytically important but not indispensable. Most importantly, 7‐deaza‐R34 also yielded a bell‐shaped curve upon addition of La3+ ions to the reaction mixture.ConclusionsSince the data based on our experiments with 7‐deaza‐R34 are completely free from potential artefacts, due to the binding of a La3+ ion to N7 at G10.1, our results, that 7‐deaza‐R34 yielded a bell‐shaped curve following the addition of La3+ ions to the Mg2+‐background reaction mixture, strongly supports the proposal that a double‐metal‐ion mechanism is operative in the cleavage reaction which is catalysed by hammerhead ribozymes.
MIDA1 is a sequence specific DNA binding protein with novel DNA binding properties
Authors: Inoue Toshiaki , Shoji Wataru , Obinata Masuo 
Published: March 2003

BackgroundId proteins not only regulate cell differentiation negatively, but they also promote growth and apoptosis. To know the mechanism of how Id regulates cell fate, we previously isolated an Id‐associating protein, MIDA1, which positively regulates cell growth. Its predicted amino acid sequence contains tryptophan‐mediated repeats (Tryp‐med repeats) similar to the DNA binding region of the c‐Myb oncoprotein. We determined whether MIDA1 can bind to DNA in a sequence specific manner by PCR‐assisted binding site selection.ResultsWe identified a 7‐base sequence (GTCAAGC) surrounded by a 1–3 bp palindromic sequence as the DNA sequence recognized by the Tryp‐med repeats of MIDA1. This motif is located within the 5′‐flanking sequence of several growth regulating genes. Gel shift assays revealed that this sequence and a certain length of flanking DNA are necessary for MIDA1 to bind DNA in a stable manner. Methylation interference and DNase I footprint analysis suggested that the DNA binding of MIDA1 is resistant to DNA methylation and that MIDA1 does not specifically localize on this particular motif.ConclusionsWe concluded that MIDA1 is a novel sequence‐specific DNA binding protein with some different properties from the usual transcription factors and that MIDA1 may act as a mediator of Id‐mediated growth‐promoting function through its DNA binding activity.
The hDLG‐associated protein DAP interacts with dynein light chain and neuronal nitric oxide synthase
Authors: Haraguchi Keiko , Satoh Kiyotoshi , Yanai Hiroyuki , Hamada Fumihiko , Kawabuchi Masahiro , Akiyama Tetsu 
Published: March 2003

Postsynaptic density (PSD)‐95 interacts with and mediates clustering of the N‐methyl‐ d‐aspartate‐receptors (NMDA‐R). PSD‐95 also interacts with the hDLG‐associated protein DAP, which is also called Synapse‐associated protein 90‐associated protein (SAPAP), and Guanylate kinase‐associated protein (GKAP).DAP interacted directly with the dynein light chain (DLC) family of proteins. DLC was contained in the NMDA‐R‐PSD‐95‐DAP‐neuronal nitric oxide synthase (nNOS) complex. Furthermore, DAP interacted with nNOS and recruited it into the Triton X‐100‐insoluble fraction of transfected cells.DAP interacts directly with DLC and nNOS, and links these proteins to the NMDA‐R‐PSD‐95 complex.