bluuebunny/crossref_metadata_2025 huggingface dataset:
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

Background

Bone 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.

Results

The 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.

Conclusions

These 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

Background

Multiprotein 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.

Results

The 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.

Conclusions

Structural 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

Background

The 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.

Results

We 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.

Conclusions

Although 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

Background

Synapse‐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.

Results

N‐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.

Conclusion

SAPAP 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

Background

Transcriptional 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.

Results

Using 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.

Conclusions

These 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

Background

The 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).

Results

To 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.

Conclusions

Greatly 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

Background

Several 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.

Results

In 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.

Conclusions

Since 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

Background

Id 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.

Results

We 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.

Conclusions

We 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.
Somatic mutations of synaptic cadherin (CNR family) transcripts in the nervous system
Authors: Hirayama Teruyoshi , Sugino Hidehiko , Yagi Takeshi
Published: March 2003

Background

Cadherin‐related neuronal receptor (CNR) family genes have been identified in the nervous system by screening molecules bound to Fyn‐tyrosine kinase. The CNR family is comprised of diverse synaptic cadherins. The genomic organization of the CNR genes, composed of variable and constant regions, is similar to that of the immunoglobulin gene cluster. The nervous system is characterized by the acquisition of diverse function. This feature is similar to the immune system. In the immune system, the generation and selection of immunoglobulin gene mutants is the underlying basis for acquired immunity. We therefore examined somatic regulation of the CNR family genes in the nervous system to determine whether a similar mechanism controls nervous system development.

Results

We first demonstrated that approximately 10% of the CNR3 transcripts were trans‐transcripts between the variable and constant exons at the adult stage. Furthermore, somatic mutations of CNR3 transcripts accumulated during brain development, with a marked bias for A‐to‐G and U‐to‐C transitions. Approximately 70% of the CNR3 transcripts exhibited a mutation characterized by the addition of a CU‐repeat; these transcripts contained seven continuous CU‐repeats in the 3′ untranslated region at the adult stage, whereas the CNR genomic DNA contained six continuous CU‐repeats. Interestingly, a high rate of replacement mutations was detected in the first cadherin domain that functions in acquisition of specificity for cell adhesion in cadherins, while in the other regions the occurrence of silent mutations increased during development.

Conclusion

The present report is the first description of the generation and possible selection of somatic mutations of synaptic cadherin transcripts. The somatic alterations of CNR family transcripts and their synaptic localization have interesting implications for the molecular basis underlying the establishment of somatic rearrangement of neuronal networks.
TBP‐interacting protein TIP120A is a new global transcription activator with bipartite functional domains
Authors: Kayukawa Kentaro , Kitajima Yoko , Tamura Taka‐aki
Published: March 2003

Background

We previously identified a TBP (TATA‐binding protein)‐interacting protein 120A (TIP120A) from rat liver nuclear extracts. TIP120A is thought to be a unique global transcription factor that can interact with TBP and can stimulate all classes of eukaryotic transcription.

Results

We produced various truncation proteins of TIP120A to delineate its functional domains. TIP120A binds to TBP in the acidic amino acid‐rich N‐terminal region and in the leucine‐rich C‐terminal region. These regions exhibited an ability to stimulate basal transcription in vitro. In addition, these two regions overlap with domains that facilitate nonspecific DNA‐binding of RNA polymerase II. The sequences of these two regions are significantly conserved among TIP120A homologues of eukaryotes.

Conclusions

TIP120A is a bipartite transcription factor, and both N‐terminal and C‐terminal regions exhibit TBP‐binding activity and stimulate the basal transcription ability.
Defective development of secretory neurones in the hypothalamus of Arnt2‐knockout mice
Authors: Hosoya Tomonori , Oda Yoshihito , Takahashi Satoru , Morita Masanobu , Kawauchi Shimako , Ema Masatsugu , Yamamoto Masayuki , Fujii‐Kuriyama Yoshiaki
Published: March 2003

Background

Within the basic region‐helix‐loop‐helix (bHLH)‐PAS family of transcription factors, Arnt and Arnt2 play unique roles; these two factors not only heterodimerize with themselves, but also with other members of this family and they act as transcription regulators which bind to specific DNA elements. Whereas Arnt is broadly expressed in various tissues, the expression of Arnt2 is known to be limited to the neural tissues.

Results

To elucidate the function of Arnt2 in detail, we cloned the mouse Arnt2 gene and its gene structure was determined. We subsequently generated germ line Arnt2 mutant mice by gene targeting technology. Heterozygous Arnt2 mice were viable, but homozygous Arnt2 gene knockout mice died shortly after birth. Histological and immunological analyses revealed that the supraoptic nuclei (SON) and the paraventricular nuclei (PVN) are hypocellular. Moreover, secretory neurones identified by the expression of neurosecretory hormone such as arginine vasopressin, oxytocin, corticotrophin‐releasing hormone and somatostatin are completely absent in SON and PVN in the mutant Arnt2 mice. Consistent with these observations, prospective SON and PVN neurones which express Brn2 appeared around E13.5 in the mantle zone, but no neurones which expressed the neurosecretory hormones were found in the SON and PVN regions.

Conclusions

These data show that the transcription factor Arnt2 controls the development of the secretory neurones at the later or final stages of differentiation rather than at the beginning stage. Strikingly similar observations have been reported with the Sim1 deficient mice. Taken together, our results demonstrate that Arnt2 is an indispensable transcription factor for the development of the hypothalamus, and suggest that Arnt2 is an obligatory partner molecule of Sim1 in the developmental process of the neuroendocrinological cell lineages.
Requirement of Syk‐phospholipase C‐γ2 pathway for phorbol ester‐induced phospholipase D activation in DT40 cells
Authors: Hitomi Tomohiro , Yanagi Shigeru , Inatome Ryoko , Ding Junyi , Takano Tomoko , Yamamura Hirohei
Published: March 2003

Background

Treatment of many cell types with phorbol esters stimulates phospholipase D (PLD) activity implying regulation of the enzyme by protein kinase C. Studies of the effects of several protein‐tyrosine kinase (PTK) inhibitors have suggested that PTK(s) play some roles in the phorbol ester‐induced PLD activation, but it remains unclear how and which PTK(s) is involved in this pathway. In this study, we investigated the roles of Syk and other PTKs for the phorbol esters, 12‐O‐tetradecanoylphorbol 13‐acetate (TPA)‐induced PLD activation in K562 and DT40 cells.

Results

TPA‐induced PLD activation was remarkably reduced in both Syk dominant negative mutant K562 cells and Syk deficient DT40 B cells. Mutational analysis further indicated that two major autophosphorylation sites (Tyr‐518 and Tyr‐519) of Syk are critical for PLD activation. Similarly, TPA‐induced PLD activation was reduced in Btk deficient cells, but unaffected in Lyn deficient cells. Finally, in cells deficient in the PLC‐γ2, one of the phosphorylated substrates regulated by Syk and Btk, TPA‐induced PLD activation, as well as phosphatidylinositol 4,5‐bisphosphate (PIP2) hydrolysis was remarkably reduced.

Conclusions

We demonstrated that the Syk, Btk and PLC‐γ2 pathways are required for TPA‐induced PLD activation in DT40 cells.
PAR‐6 regulates aPKC activity in a novel way and mediates cell‐cell contact‐induced formation of the epithelial junctional complex
Authors: Yamanaka Tomoyuki , Horikoshi Yosuke , Suzuki Atsushi , Sugiyama Yuki , Kitamura Koichi , Maniwa Rika , Nagai Yoko , Yamashita Akio , Hirose Tomonori , Ishikawa Hiroko , Ohno Shigeo
Published: March 2003

Background PAR‐6, aPKC and PAR‐3 are polarity proteins that co‐operate in the establishment of cell polarity in Caenorhabditis elegans and Drosophila embryos. We have recently shown that mammalian aPKC is required for the formation of the epithelia‐specific cell‐cell junctional structure. We have also revealed that a mammalian PAR‐6 forms a ternary complex with aPKC and ASIP/PAR‐3, and localizes at the most apical end of the junctional complex in epithelial cells.

Results The ternary complex formation and junctional co‐localization of PAR‐6 with aPKC and ASIP/PAR‐3 are observed during the early stage of epithelial cell polarization. In addition, over‐expression of the PAR‐6 mutant with CRIB/PDZ domain in MDCK cells disturbs the cell‐cell contact‐induced junctional localization of tight junction proteins, as well as inhibiting TER development. Furthermore, the binding of Cdc42:GTP to the CRIB/PDZ domain of PAR‐6 enhances the kinase activity of PAR‐6‐bound aPKC. Detailed analyses suggest that the binding of PAR‐6 to aPKC has the intrinsic potential to activate aPKC, which is only released when Cdc42:GTP binds to the CRIB/PDZ domain.

Conclusion The results indicate the involvement of PAR‐6 in the aPKC function which is required for the cell‐cell adhesion‐induced formation of epithelial junctional structures, possibly through the cooperative regulation of aPKC activity with Cdc42.
Reconstitution of recombinant TFIIH that can mediate activator‐dependent transcription
Authors: Fukuda Aya , Yamauchi Jun , Wu Shwu‐Yuan , Chiang Cheng‐Ming , Muramatsu Masami , Hisatake Koji
Published: March 2003

Background TFIIH is one of the general transcription factors required for accurate transcription of protein‐coding genes by RNA polymerase II. TFIIH has helicase and kinase activities, plays a role in promoter opening and promoter escape, and is also implicated in efficient activator‐dependent transcription.

Results We have established a reconstitution system of recombinant TFIIH using a three‐virus baculovirus expression system. The recombinant TFIIH was active in CTD kinase and DNA helicase assays, and showed both basal and activator‐dependent transcriptional activities that were indistinguishable from those of HeLa cell‐derived TFIIH. Further analyses using recombinant TFIIH confirmed a critical role of TFIIH in activator‐dependent transcription. The dose response of TFIIH in activator‐dependent transcription suggested that mere recruitment of TFIIH is not sufficient for transcriptional activation. The sensitivity of activator‐dependent transcription to nonhydrolysable ATP analogues indicated the importance of the enzymatic activities of TFIIH in transcriptional activation.

Conclusions Our results raise a possibility that transcriptional activation by GAL4‐VP16 requires enzymatic activities. Recombinant TFIIH reconstituted from this baculovirus system should be useful for analysis of the mechanisms of activation by GAL4‐VP16.
The mouse Snrpn minimal promoter and its human orthologue: activity and imprinting
Authors: Hershko Alon Y. , Finberg Yael , Kantor Boris , Shemer Ruth , Razin Aharon
Published: March 2003

Background Microdeletions in chromosome 15q13‐15 of Prader‐Willi (PWS) and Angelman Syndrome (AS) patients suggested that SNRPN promoter/exon 1, together with a short sequence located approximately 35 kb upstream, constitute an imprinting control centre that regulates the entire 2 Mb PWS/AS imprinted domain. We have recently shown that a minitransgene composed of the human upstream sequence and mouse Snrpn promoter/exon 1 harbours all the elements necessary for establishing and maintaining an imprinted state.

Results Here we describe, using transfection experiments, the Snrpn minimal promoter (SMP), being composed of the entire 76 bp exon 1 and 84 bp of upstream sequence. A 7 bp element (SBE) within SMP that, in its unmethylated state binds a specific protein, is absolutely required for promoter activity. The orthologous human sequence, in spite of the fact that it possesses an identical SBE, failed to display promoter activity in transfection experiments and failed to create a methylated state of the maternal allele. Transgenic experiments reveal that a mutation in SBE of the mouse sequence did not completely abolish methylation of the maternal allele, indicating that sequences outside SBE participate in this process. Replacement of human exon 1 with the mouse orthologue replenished promoter activity, but left the maternal allele in the transgenic experiment unmethylated. The reciprocal chimera, in which mouse exon 1 was replaced by the human orthologue resulted in loss of promoter activity and did not support differential methylation.

Conclusions The observations made by in vitro and in vivo experiments suggest that several cis elements which are involved in Snrpn promoter activity and the imprinting process are present in the mouse promoter and absent in the human orthologous sequence.
Herpes simplex virus encodes a virion‐associated protein which promotes long cellular processes in over‐expressing cells
Authors: Takakuwa Hiroki , Goshima Fumi , Koshizuka Tetsuo , Murata Takayuki , Daikoku Tohru , Nishiyama Yukihiro
Published: March 2003

Background Herpes simplex virus (HSV) possesses a number of accessory genes which are dispensable for replication in cell culture. A previous study showed that the UL21 gene product of HSV type 1 is a virion component that is not necessary for viral replication. The function of the gene product remains unknown.

Results We found that the HSV‐1 UL21 gene product, a capsid‐associated tegument protein with an apparent molecular mass of 62 kDa, promotes the outgrowth of long cellular processes when it is over‐expressed in non‐neural cells. The UL21 protein co‐localizes and physically associates with microtubules in the long processes. Analysis using mutant proteins implicates a proline‐rich region in promotion of the processes.

Conclusions The results suggest that the UL21 protein, like tau and other MAPs, promotes the process by directly or indirectly interacting with microtubules and facilitates the intracellular transport of the virus.
Transgenic over‐expression of MafK suppresses T cell proliferation and function in vivo
Authors: Yoh Keigyou , Sugawara Takehiko , Motohashi Hozumi , Takahama Yousuke , Koyama Akio , Yamamoto Masayuki , Takahashi Satoru
Published: March 2003

Background The small Maf proteins regulate gene transcription from Maf recognition elements (MARE). These proteins do not contain a canonical transactivation domain. Depending upon the ratio of small Maf proteins to their partner proteins, which either possess a transactivation domain or not, transcription can be switched on or off.

Results Transgenic mice were generated which over‐express the small Maf family member MafK, specifically in the T cell lineage. It was our expectation that the high level of MafK would shift the balance to the formation of MafK homodimer and thereby repress MARE‐dependent transcription. The transgenic mice had a shortened life span because of Pneumocystis carinii pneumonia and displayed a decrease in thymocytes and lower IL‐2 and IL‐4 mRNA expression levels. Analyses by electrophoretic gel mobility shift assay revealed that over‐expressed MafK could interact with the proximal AP‐1 sequence of IL‐2 and the MARE in the IL‐4 promoter region.

Conclusion These results indicate that when over‐expressed, MafK binds to a MARE‐like sequence and represses MARE‐dependent transcription. Consequently, T cell proliferation and cytokine secretion are affected. The MafK homodimer serves as an important molecular probe for evaluating the role played by cis‐acting MAREs in the proliferation and function of T cells.
Fission yeast chk1 mutants show distinct responses to different types of DNA damaging treatments
Authors: Francesconi Stefania , Smeets Monique , Grenon Muriel , Tillit Jeanne , Blaisonneau Joël , Baldacci Giuseppe
Published: March 2003

Background: Chk1 kinase is activated by phosphorylation at serine‐345 by Rad3 checkpoint kinase and is required for DNA damage checkpoint in late S and G2 phase of S. pombe cell cycle. We studied the ability of two chk1 mutants, chk1‐1 and chk1‐2, to undergo phosphorylation and to delay cell cycle progression in response to different types of DNA lesions.

Results: Both the Chk1‐1 and Chk1‐2 mutant proteins are phosphorylated to various extents when DNA is damaged in early G2 phase of cell cycle by either UV irradiation or gamma irradiation. However, chk1‐2 mutant does not delay cell cycle progression in a dose dependent manner specifically upon gamma irradiations. This defect is not associated with an important loss of survival. Furthermore, both chk1 mutants survive to Camptothecin treatment despite undetectable Chk1‐1 or Chk1‐2 phosphorylated forms. We show that both mutant proteins are not phosphorylated in cds1 devoid cells treated with ribonucleotide reductase inhibitor hydroxyurea or when the replisome is affected by a thermosensitive mutation in DNA polymerase δ. This inability is associated with the loss of checkpoint function. We found that an increased level of Crb2/Rhp9 protein specifically complements the defect of the chk1‐1 mutant allowing Chk1‐1 phosphorylation upon treatment with hydroxyurea of dcds1 cells.

Conclusions: Mutants chk1‐1 and chk1‐2 behave differently according to the type of lesion generated on DNA.
The SH3, HOOK and guanylate kinase‐like domains of hDLG are important for its cytoplasmic localization
Authors: Kohu Kazuyoshi , Ogawa Fumiaki , Akiyama Tetsu
Published: March 2003

Background: hDLG, the human homologue of the Drosophila tumour suppressor dlg, functions as a scaffolding protein that facilitates the transmission of diverse downstream signals. hDLG possesses multiple protein‐binding domains, including three PDZ domains, an SH3 domain, a HOOK domain and a guanylate kinase‐like (GK) domain.

Results: We studied the significance of the PDZ, SH3, HOOK and GK domains in the cytoplasmic localization of hDLG. We found that mutation of the SH3 or GK domain, but not the PDZ domain, resulted in a re‐localization of hDLG to the nucleus. Furthermore, hDLG was found to possess a potential nuclear localization signal in the HOOK domain.

Conclusion: These results suggest that the SH3, HOOK and GK domains of hDLG are important for its cytoplasmic localization.
Diving deep on a Pleistocene reef in eastern Jamaica
Authors: DONOVAN STEPHEN K. , HARPER DAVID A. T.
Published: March 2003

Exhumed early Pleistocene reefs along the eastern coast of Jamaica expose deep‐water environments downslope from the more familiar, shallow‐water reef frameworks. Such environments have quite different faunas from the shallow parts of the reefs, including common ‘Palaeozoic‐type’ biotas like the brachiopods and crinoids. These animals are found in deeper‐water settings around the Caribbean today; however, these biotas are not readily available nor easily studied. The slightly older Pleistocene faunas are much more accessible and informative. Here the past may be the key to the present.
Stegosaur tracks and the persistence of facies — the Lower Cretaceous of Western Australia
Authors: PAGE DAVID
Published: March 2003

Sometimes between January and June 1996, the only known stegosaur tracks were stolen from Broome in north‐western Australia (Fig. 1). Vandalism or loss of any type material is always to be lamented, yet there is good cause to suspect that the trackmaker at Broome was not a stegosaur. The reasons why this should be so relate to some of the factors involved in the formation of dinosaur trace fossils and the difficulties encountered in their interpretation. The stratigraphy at Broome is notable for preserving evidence of potential Wealden‐type deposition in Australia.
Joint estimation of AVO and kinematic parameters
Authors: Grion S. , Mazzotti A. , Spagnolini U.
Published: March 2003

Conventional amplitude‐versus‐offset (AVO) analysis such as linear fitting of isotime samples after NMO correction does not give reliable results in the presence of interfering reflections or velocity errors. For this reason we propose a new method that is able to remove interference effects on the AVO of the target reflection and minimize the effects of residual moveout. The method is based on the minimization of the difference between observed data and a model that includes theoretical descriptions of the AVO and traveltimes. This minimization is carried out jointly with respect to AVO and kinematic parameters (velocities and traveltimes) and requires the a priori knowledge of the propagating wavelet. The kinematic parameters are given by the NMO equation extended to the fourth‐order term and the AVO is described as a linear combination of a set of orthogonal functions. The AVO functions are derived from a statistical model of reflection amplitude in the presence of velocity error.

Applications to synthetic and real data demonstrate the ability of the method to attenuate distortion effects on the AVO of the primary reflection by interfering coherent noise. The real data example pertains to a marine case where the primary event is contaminated by multiple reflections generated in the water layer and by another event, reflected from an interface deeper than the target, that merges with the examined reflections at the far‐offsets. This causes a subtle tuning that distorts the AVO of the target. Our method attenuates the effects of the multiples and discriminates the ‘double event’ nature of the target reflection at the far‐offsets.
Improvement of MT data processing using stationary and coherence tests
Authors: Lamarque
Published: March 2003

The great majority of data processing methods for magnetotelluric measurements are based on an average Fourier spectral analysis to estimate either the transfer function or the coherence function. This assumes that the time‐series data are stationary. The aim of this paper is to present a preprocessing method able to extract the stationary and coherent part of the original signals. The practitioner can then apply the magnetotelluric method of his choice to these new data. This preliminary data sorting is done in four steps: (i) slow drifts are eliminated using a high‐pass filter whose cut‐off frequency is determined by an iterative procedure. Based on run tests, this procedure is also able to remove segments with non‐independent samples in the time series; (ii) non‐stationary segments are eliminated after band‐pass filtering; (iii) non‐coherent segments are eliminated before spectral analysis; (iv) the impedance tensor value is then retained, at a given frequency, only if the signals are coherent. This preprocessing method was tested on the simplest, but still used, magnetotelluric method which uses only two field components, and it was found that the average resistivity standard deviation decreased significantly from 14.6 Ωm without sorting to 8.6 Ωm after sorting.
Practical aspects of wavefield separation of two‐component surface seismic data based on polarization and slowness estimates
Authors: Richwalski Sandra , Roy‐Chowdhury Kabir , Mondt Jaap C.
Published: March 2003

The processing of multicomponent seismic data, carried out individually on the different wavetypes (P‐, S‐ and converted waves), should result in an improved image of the subsurface. We examine the wavefield‐separation method proposed by Cho and Spencer. We discuss practical aspects related to the separation of interfering waves on two‐component surface seismic data and illustrate these using synthetic data. A sliding spatial window is used for analysis. The choice of its width represents a trade‐off between stabilizing the method in the presence of random noise and ensuring a good spatial resolution. No a priori knowledge of the subsurface is required, but locally, the characteristic parameters of the waves, i.e. horizontal slowness and polarization, are assumed to be constant inside the analysis window. These parameters are estimated at each frequency, but a statistical analysis provides a more robust estimate, especially in the presence of random noise. This approach also solves the problem of eigenvalue sharing and switching. Additional smoothing of the estimates according to a model may further improve the results. The width of the analysis window may be decreased if the waves inside the data window differ significantly in amplitude. The dominant wave in each case is separated from the lower‐amplitude waves and subtracted from the data. This novel iterative approach thereby allows for the isolation of low‐amplitude events.
An approximate analytical approach to compute geoelectric dipole–dipole responses due to a small buried cube
Authors: Szalai Sándor , Szarka László
Published: March 2003

A simple analytical solution is presented for computing direct current (DC) electric field distortion due to a small cube in a homogeneous half‐space, measured with a dipole–dipole array on the surface. Both the transmitter and the receiver may have any orientation; furthermore their position on the horizontal surface and the depth of the cube can be freely selected. It is shown that a simple approximate analytical method may replace more complicated 3D numerical modelling algorithms.

The approximation lies in the linearization of the problem: the secondary source (i.e. the cube) is considered as a system of three perpendicular electric dipoles. In spite of this first‐order approximation, in the case of realistic depths z (zR≈0.1–0.5, where R is the transmitter–receiver distance), this approximate solution fits very well with true 3D numerical modelling results, and with analogue modelling results if aR≤0.1, where a is the length of the side of the cube. Due to its simplicity, this method could be used for computing DC field distortion effects, estimating parameter‐sensitivities, or even determining some initial models for further inversions.
Distortions of EM transients in coincident loops at short time‐delays
Authors: Kamenetsky F. , Oelsner ChR.
Published: March 2003

Transient electromagnetic measurements with short time‐delays of transients are used for solving different problems within the upper part of a geoelectric section. However, it is necessary to take into consideration distortions connected with self‐transients within the transmitter–receiver system. From the practical point of view, it is important to estimate the minimum time‐delay after which these distortions may be neglected. We present such an estimation which uses a simple approximation method for a single‐loop (or coincident‐loop) configuration. For common values of the loop size (10 m × 10 m to 40 m × 40 m) and of the resistivity of a homogeneous half‐space (1–100 Ωm), the minimum time‐delay beyond which we can use a standard interpretation is in the range of 2–10 µs. This is equivalent to a minimum depth of investigation in the range of 1–30 m.