bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of engrailed promoter elements essential for interactions with a stripe enhancer in Drosophila embryos
Authors: Orihara Minako , Hosono Chie , Kojima Tetsuya , Saigo Kaoru
Published: March 2003

Background

The structures and functions of promoter sequences of most genes have been analysed using in vitro transcription and/or cultured cell systems, neither possessing tissue‐specific enhancers. Promoter–enhancer interactions in vivo, in particular, during ontogeny, are still poorly understood.

Results

We have established a new method for the assessment of promoter activity in cells that participate in fly body formation, using the UAS/GAL4 system. A functional analysis was then conducted on the promoter sequence of the engrailed gene in Drosophila embryos. A 38‐bp‐long sequence, terminating with an initiator or RNA start site and a downstream promoter element, was found to be capable of receiving activation signals from the engrailed stripe enhancer. Transcriptional efficiency was improved significantly by the presence of upstream promoting elements, most functionally replaceable with synthetic GAGA factor binding sites.

Conclusions

We identified the in vivo minimum promoter of engrailed and demonstrated that the GAGA factor binding sites serve primarily as quantitative elements which augment transcriptional efficiency. Evidence was also obtained that indicated that not only enhancer but also promoter sequences were involved in the determination of the tissue‐specificity of gene expression.
Negative regulation of theptsoperon by Mlc: mechanism underlying glucose induction inEscherichia coli
Authors: Tanaka Yuya , Kimata Keiko , Inada Toshifumi , Tagami Hideaki , Aiba Hiroji
Published: March 2003

Background

Theptsoperon ofEscherichia coliconsists of three genesptsH,ptsIandcrr, each encoding for central components of the phosphoenolpyruvate: carbohydrate phosphotransferase system, HPr, enzyme I and IIAGlc, respectively. Transcription of theptsoperon is stimulated when glucose is present in the culture medium. One of the two major promoters, P0, is responsible for this glucose induction. However, no regulatory protein responsible for the glucose induction of theptsoperon has been identified yet and molecular mechanism by which glucose stimulates theptstranscription is not known.

Results

We found by Northern blotting that theptsmRNA levels in cells lacking Mlc, a new global repressor of carbohydrate metabolism, were increased without external glucose and that the addition of glucose had no effect on theptsmRNA levels in the mutant cells. Western blotting revealed that the enzyme I level in themlc−cells was also elevated without glucose and no further increase in the enzyme I level was observed in the presence of glucose. S1 analysis revealed that transcription of the glucose‐sensitive promoter, P0, occurs constitutively in themlc−cells independently from the external glucose.In vitrotranscription studies indicated that Mlc strongly inhibited P0 transcription. DNase I footprinting experiment revealed that Mlc bound to P0 promoter region to prevent RNA polymerase binding at P0.

Conclusion

We conclude that Mlc is a repressor for theptstranscription acting as a major regulatory protein involved in the glucose induction ofptsoperon. We propose that glucose induces theptstranscription by modulating the Mlc activity. The mechanism by which glucose modulates the Mlc action remains to be studied.
Similar and differential behaviour between the nectin‐afadin‐ponsin and cadherin‐catenin systems during the formation and disruption of the polarized junctional alignment in epithelial cells
Authors: Asakura Takeshi , Nakanishi Hiroyuki , Sakisaka Toshiaki , Takahashi Kenichi , Mandai Kenji , Nishimura Miyuki , Sasaki Takuya , Takai Yoshimi
Published: March 2003

Background

We have recently identified a novel cell‐cell adhesion system, named NAP system, which is localized at cadherin‐based cell‐cell adherens junctions (AJs). The NAP system is composed of at least nectin, afadin and ponsin. Nectin is an immunoglobulin‐like cell adhesion molecule. Afadin is an actin filament‐binding protein which associates nectin with the actin cytoskeleton. Ponsin is an afadin‐binding protein which furthermore binds to vinculin and provides a possible linkage of nectin‐afadin to cadherin‐catenin through vinculin. We compared here the behaviour of the NAP and cadherin‐catenin systems during the formation and disruption of the polarized junctional alignment in epithelial cells.

Results

At the early stage of the formation of the polarized junctional alignment in MTD‐1 A cells, primordial spot‐like junctions were formed at the tips of thin cellular protrusions radiating from adjacent cells. Nectin, afadin, ponsin, cadherin and catenin were simultaneously recruited to these junctions. As the cell polarization proceeded, the spot‐like junctions were gradually fused to form belt‐like AJs where all these proteins were concentrated. The disruption of cell‐cell AJs in MDCK cells by culturing at a low Ca2+ concentration caused rapid endocytosis of cadherin, but not that of nectin or afadin. Addition of 12‐O‐tetradecanoylphorbol‐13‐acetate to the cells formed a tight junction‐like structure where nectin and afadin, but not cadherin, accumulated.

Conclusion

These results indicate that the NAP and cadherin‐catenin systems show similar and differential behaviour during the formation and disruption of the polarized junctional alignment in epithelial cells.
Long range interaction of cis‐DNA elements mediated by architectural transcription factor Bach1
Authors: Yoshida Chikashi , Tokumasu Fuyuki , Hohmura Ken I. , Bungert Jörg , Hayashi Norio , Nagasawa Toshiro , Engel James Douglas , Yamamoto Masayuki , Takeyasu Kunio , Igarashi Kazuhiko
Published: March 2003

Background

A central question in vertebrate transcriptional regulation is how cis‐regulatory modules, including enhancers, silencers and promoters, communicate with each other over long distances to mandate proper gene expression. In order to address this question we analysed protein/DNA interactions in the human β‐globin locus control region (LCR). One of the many proteins that are potentially implicated in LCR function is Bach1. Bach1 possesses a basic leucine zipper (bZip) domain, as well as a BTB/POZ domain that has been shown to be involved in the regulation of chromatin structure. Bach1 forms heterodimers with small Maf proteins through its leucine zipper and binds to
Maf
recognition
elements (MARE).

Results

Using atomic force microscopy we visualized large looped DNA structures between MAREs located in different regulatory elements within the human β‐globin LCR that were mediated by Bach1/MafK heterodimers. The formation of these DNA loops required the Bach1 BTB/POZ protein interaction domain. Furthermore, in transfection studies we found that Bach1 repressed the enhancer activity of the LCR in a BTB/POZ domain‐dependent manner.

Conclusion

Our results suggest that Bach1 and other BTB/POZ transcription factors may represent a class of nuclear architectural proteins that mediate long range interactions between cis‐regulatory elements in order to regulate gene expression.
Region between α‐helices 3 and 4 of the Mad homology 2 domain of Smad4: functional roles in oligomer formation and transcriptional activation
Authors: Tada Keiichiro , Inoue Hirofumi , Ebisawa Takanori , Makuuchi Masatoshi , Kawabata Masahiro , Imamura Takeshi , Miyazono Kohei
Published: March 2003

Background

Smad4 has a unique region of 35 amino acids between α‐helices 3 and 4 (termed H3/4 loop) of the Mad homology (MH) 2 domain. In order to elucidate the functional importance of the H3/4 loop, we prepared chimeric constructs of Smad4 containing the region corresponding to the α‐helix 3, H3/4 loop and α‐helix 4 of different Smads, including a chimera containing that of Smad2 (Smad4‐HL2).

Results

Smad4‐HL2 constitutively induced the transcriptional activation of p3TP‐Lux, a TGF‐β‐responsive reporter construct. However, co‐transfection of Smad2 with Smad4‐HL2 did not induce a further increase in the activation of p3TP‐Lux. Smad4‐HL2 did not induce the activation of pAR3‐Lux, which contains FAST1‐binding sites and is activated by a complex composed of FAST1, Smad2 and Smad4. Smad4‐HL2 formed a homo‐oligomer more efficiently than wild‐type Smad4 in mammalian cells. Moreover, Smad4‐HL2 bound to DNA containing the Smad‐binding sites with a gretaer affinity than the wild‐type Smad4.

Conclusion

Smad4‐HL2 spontaneously forms a homo‐oligomer, which may bind to DNA with relatively high affinity and induce transcriptional activation of p3TP‐Lux. The H3/4 loop of Smad4 may thus play a role in precluding the spontaneous oligomer formation of Smad4.
Mechanism of catabolite repression in the bgl operon of Escherichia coli: involvement of the anti‐terminator BglG, CRP‐cAMP and EIIAGlc in mediating glucose effect downstream of transcription initiation
Authors: Gulati Abhilasha , Mahadevan Subramony
Published: March 2003

Background

Expression of the bgl operon of Escherichia coli, involved in the regulated uptake and utilization of aromatic β‐glucosides, is extremely sensitive to the presence of glucose in the growth medium. We have analysed the mechanism by which glucose exerts its inhibitory effect on bgl expression.

Results

Our studies show that initiation of transcription from the bgl promoter is only marginally sensitive to glucose. Instead, glucose exerts a more significant inhibition on the elongation of transcription beyond the rho‐independent terminator present within the leader sequence. Transcriptional analyses using plasmids that carry mutations in bglG or within the terminator, suggest that the target for glucose‐mediated repression is the anti‐terminator protein, BglG. Introduction of multiple copies of bglG or the presence of mutations that inhibit its phosphorylation by Enzyme IIBgl (BglF), result in loss of glucose repression. Studies using crp, cya and crr strains show that both CRP‐cAMP and the Enzyme IIAGlc (EIIAGlc) are involved in the regulation. Although transcription initiation is normal in a crp, cya double mutant, no detectable transcription is seen downstream of the terminator, which is restored by a mutation within the terminator. Transcription past the terminator is also partly restored by the addition of exogenous cAMP to glucose‐grown cultures of a crp+ strain. Glucose repression is lost in the crr mutant strain.

Conclusions

The results summarized above indicate that glucose repression in the bgl operon is mediated at the level of transcription anti‐termination, and glucose affects the activity of BglG by altering its phosphorylation by BglF. The CRP‐cAMP complex is also involved in this regulation. The results using the crr mutant suggest a negative role for EIIAGlc in the catabolite repression of the bgl genes.
Schnurri interacts with Mad in a Dpp‐dependent manner
Authors: Udagawa Yoshiyuki , Hanai Jun‐ichi , Tada Kei‐ichiro , Grieder Nicole C. , Momoeda Mikio , Taketani Yuji , Affolter Markus , Kawabata Masahiro , Miyazono Kohei
Published: March 2003

Background

Decapentaplegic (Dpp) is a member of the transforming growth factor‐β superfamily. Dpp governs various developmental processes in Drosophila through the transcriptional regulation of a variety of genes. Signals of Dpp are transmitted from the cell membrane to the nucleus by Medea and Mad, both belonging to the Smad protein family. Mad was shown to bind to the Dpp‐responsive element in genes such as vestigial, labial, and Ultrabithorax. The DNA binding affinity of Smad proteins is relatively low, and requires other nuclear factor(s) to form stable DNA binding complexes. schnurri (shn) was identified as a candidate gene acting downstream of Dpp receptors, but its relevance to Mad has remained unknown.

Results

We characterized the biochemical functions of Shn. Shn forms homo‐oligomers. Shn is localized in the nucleus, and is likely to have multiple nuclear localizing signals. Finally, we found that Shn interacts with Mad in a Dpp‐dependent manner.

Conclusions

The present results argue that Shn may act as a nuclear component of the Dpp signalling pathway through direct interaction with Mad.
Molecular beacon aptamer fluoresces in the presence of Tat protein of HIV‐1
Authors: Yamamoto Rika , Kumar Penmetcha K. R.
Published: March 2003

Background

We reported an aptamer, RNATat that binds to the Tat protein of HIV with two orders of magnitude greater (133‐fold) affinity over the TAR RNA of HIV‐1 and specifically inhibits the Tat‐dependent trans‐activation of transcription, both in vitro and in vivo (demonstrated in the accompanying article, Yamamoto et al., this issue pp. 371–388). We now report the use of aptamer‐derived oligomers to analyze the Tat of HIV and the possible applications of such constructs in the field of biosensors.

Results

To make new molecular beacon, we constructed two RNA oligomers that derived from RNATat. To one of the split RNA oligomers that forms a hairpin structure, the fluorophore and quencher were attached at the 5′‐ and 3′‐ends, respectively. Specifically in the presence of Tat or its peptides, but not in the presence of other RNA binding proteins, the two oligomers undergo a conformational change to form a duplex that leads to relieving of fluorophore from the quencher, and thus a significant enhancement of the fluorescence of fluorescein was observed.

Conclusion

A novel strategy for exploiting aptamers in the analysis of Tat (analyte) has been described. A similar strategy could be used to study other analytes such as proteins and small molecules. In addition, the molecular beacon aptamer requires half the length of target sequence (eight nucleotides) in comparison with molecular beacons. Thus, it is conceivable that we could insert an analyte‐binding site into molecular beacons to convert them to signalling beacons.
Interaction between neurone and microglia mediated by platelet‐activating factor
Authors: Aihara Makoto , Ishii Satoshi , Kume Kazuhiko , Shimizu Takao
Published: March 2003

Background

Platelet‐activating factor (PAF) is a potent phospholipid mediator that plays various roles in neuronal function and brain development. The production and release of PAF in the brain has also been reported under various pathological conditions. However, neither the cell types and mechanism responsible for the synthesis of PAF nor its target cells have been fully identified.

Results

Using primary culture cells derived from rat brain and a very sensitive assay method for PAF, we found that PAF was synthesized in neurones following stimulation with glutamic acid. PAF synthesis required activation of NMDA receptors and subsequent elevation of intracellular calcium ions. Microglia, which express functional PAF receptors to a high level, showed a marked chemotactic response to PAF. This chemotaxis is a receptor‐mediated process, as microglia from PAF‐receptor‐deficient mice did not show such a response. The activation of a pertussis‐toxin‐sensitive G‐protein and mitogen‐activated protein kinase presumably plays a role in intracellular signalling leading to chemotaxis.

Conclusions

Considering the cytoprotective and cytotoxic roles of microglia, PAF functions as a key messenger in neurone–microglial interactions.
Multiple downstream signalling pathways from ROCK, a target molecule of Rho small G protein, in reorganization of the actin cytoskeleton in Madin–Darby canine kidney cells
Authors: Takaishi Kenji , Matozaki Takashi , Nakano Katsutoshi , Takai Yoshimi
Published: March 2003

In Madin–Darby canine kidney cells, Rho small G protein regulates formation of stress fibres, focal adhesions, and peripheral bundles through reorganization of the actin cytoskeleton. There are two morphologically distinguishable types of Rho‐regulated stress fibres: parallel and stellate. Of these, effects of Rho small G protein, mDia1 regulates the formation of parallel stress fibres, whereas ROCK regulates the formation of stellate stress fibres, peripheral bundles and focal adhesions. Both mDia1 and ROCK are direct downstream targets of Rho small G protein.

The ROCK‐induced formation of stellate stress fibres is regulated mainly through the myosin light chain kinase‐dependent phosphorylation of myosin light chain and the LIM‐kinase‐dependent phosphorylation of cofilin. The ROCK‐induced formation of focal adhesions is mainly regulated through a downstream pathway of ROCK other than myosin light chain and cofilin. The ROCK‐induced formation of peripheral bundles is regulated at least through ERM proteins, but not through the myosin light chain or cofilin.

Our present and previous findings suggest the presence of multiple downstream signalling pathways from ROCK to reorganization of the actin cytoskeleton in Madin–Darby canine kidney cells.
Mouse Peg9/Dlk1 and human PEG9/DLK1 are paternally expressed imprinted genes closely located to the maternally expressed imprinted genes: mouse Meg3/Gtl2 and human MEG3
Authors: Kobayashi Shin , Wagatsuma Hirotaka , Ono Ryuichi , Ichikawa Hitoshi , Yamazaki Masaaki , Tashiro Hiroyuki , Aisaka Kohzo , Miyoshi Naoki , Kohda Takashi , Ogura Atsuo , Ohki Misao , Kaneko‐Ishino Tomoko , Ishino Fumitoshi
Published: March 2003

Genomic imprinting significantly influences development, growth and behaviour in mammals. Systematic screening of imprinted genes has been extensively carried out to identify the genes responsible for imprinted phenotypes and to elucidate the biological significance of this phenomenon. In this study, we applied DNA chip technology for isolating paternally expressed imprinted genes (Pegs). We compared the resulting expression profiles of parthenogenetic and fertilized control embryos to identify novel imprinted genes.

A novel paternally expressed mouse imprinted gene, Peg9/Dlk1, was identified. Consistent with this finding, the paternal expression of its human homologue, PEG9/DLK1, was also confirmed. These two genes form imprinted gene clusters with the reciprocally imprinted mouse Meg3/Gtl2 and human MEG3 genes that we first identified on distal chromosome 12 and chromosome 14q32, respectively.

As DNA chip technology allows us to quickly screen a large number of genes, using this technology to search for imprinted genes could accelerate the identification of genes responsible for human and mouse genetic diseases. Dlk1 and DLK1, which encode transmembrane proteins, have six EGF‐like repeats and show homology to the Delta gene in Drosophila melanogaster. Because of its homology to mammalian Delta homologues, PEG9/DLK1 may contribute to the scoliosis phenotype observed in maternal uniparental disomy 14 (mUPD14) patients.
Roles of partly unfolded conformations in macromolecular self‐assembly
Authors: Namba Keiichi
Published: March 2003

From genes to cells there are many steps of hierarchical increments in building up complex frameworks that provide intricate networks of macromolecular interactions, through which cellular activities such as gene expression, signal processing, energy transduction and material conversion are dynamically organized and regulated. The self‐assembly of macromolecules into large complexes is one such important step, but this process is by no means a simple aggregation of macromolecules with predefined, rigid complementary structures. In many cases the component molecules undergo either domain rearrangements or folding of disordered portions, which occurs only following binding to their correct partners. The partial disorder is used in some cases to prevent spontaneous assembly at inappropriate times or locations. It is also often used for finely tuning the equilibrium and activation energy of reversible binding. In other cases, such as protein translocation across membranes, an unfolded terminus appears to be the prerequisite for the process as an initiation signal, as well as the physical necessity to be taken into narrow channels. Self‐assembly processes of viruses and bacterial flagella are typical examples where the induced folding of disordered chains plays a key role in regulating the addition of new components to a growing assembly. Various aspects of mechanistic roles of natively unfolded conformations of proteins are overviewed and discussed in this short review.
Identification and expression of a novel gene in odour‐taste associative learning in the terrestrial slug
Authors: Nakaya Tadashi , Kawahara Shigenori , Watanabe Satoshi , Lee Dong‐Seok , Suzuki Toshiharu , Kirino Yutaka
Published: March 2003

Odour‐taste associative learning in the terrestrial slug offers a useful model for long‐term memory formation and retention. The genes which are expressed over 2 h after a learning event are thought to include those related to memory formation and/or consolidation. It is very important to examine what kinds of genes are expressed following associative learning.

We identified a novel slug gene, the expression of which was regulated by associative learning and mostly restricted to the procerebrum (PC), a place that olfactory information is believed to be processed in slug. This gene encodes a 121 amino acid, 18 kDa secretory protein which we term LAPS18. Expression of the LAPS18 gene was induced in somata and the protein spread to neurites in the PC of slugs subjected to paired conditioning. Recombinant LAPS18 promoted the aggregation and movement of PC neurones in culture and they were blocked by the anti‐LAPS18 antibody. Beads coupled with LAPS18 protein attached to PC neurones and the beads aggregated through PC cells but not by themselves, suggesting that LAPS18 may require a counterpart molecule for PC neurone aggregation.

An increased expression and translocation of LAPS18 protein after paired conditioning may be needed for long‐term memory formation and retention in the slug. Since genes homologous to LAPS18 genes in the land slug Limax are found from vertebrates including human, analysing the expression and function of LAPS18 may be important in understanding the molecular mechanism of memory formation and retention.
Influenza virus RNA polymerase PA subunit is a novel serine protease with Ser624 at the active site
Authors: Hara Koyu , Shiota Mayumi , Kido Hiroshi , Ohtsu Yasushi , Kashiwagi Takahito , Iwahashi Jun , Hamada Nobuyuki , Mizoue Kazutoshi , Tsumura Naoki , Kato Hirohisa , Toyoda Tetsuya
Published: March 2003

Background

Influenza virus RNA polymerase is a multifunctional enzyme that catalyses both transcription and replication of the RNA genome. The function of the influenza virus RNA polymerase PA subunit in viral replication is poorly understood, although the enzyme is known to be required for cRNA → vRNA synthesis. The protease related activity of PA has been discussed ever since protease‐inducing activity was demonstrated in transfection experiments.

Results

PA protein was highly purified from insect cells infected with the recombinant baculovirus carrying PA cDNA, and a novel chymotrypsin‐type serine protease activity was identified with the synthetic peptide, Suc‐LLVY‐MCA, in the PA protein. [3H]DFP was crosslinked with PA and a mutational analysis revealed that serine624 was as an active site for the protease activity.

Conclusions

These results constitute the demonstration of protease activity in PA subunit of the influenza virus RNA polymerase complexes.
Selenocysteine codons decrease polysome association on endogenous selenoprotein mRNAs
Authors: Martin III Glover W. , Berry Marla J.
Published: March 2003

Background

Selenocysteine incorporation has been reported to be inefficient in all systems studied, including Escherichia coli, baculovirus‐insect cell systems, rabbit reticulocyte in vitro translation systems, transiently transfected mammalian cells, and intact animals. Nonetheless, full‐length selenoproteins containing up to 17 selenocysteine residues are produced in animals, indicating that the efficiency observed in manipulated systems might not accurately reflect the true efficiency of this process in nature.

Results

To begin to address this apparent discrepancy, we have examined the polysome profiles of endogenously expressed selenoprotein mRNAs in a mammalian cell line, and compared them with nonselenoprotein mRNAs. We report that three selenoprotein mRNAs, type 1 deiodinase, glutathione peroxidase and selenoprotein P, are under‐loaded with ribosomes, based on their predicted open reading frame sizes. The average numbers of ribosomes per mRNA correspond to the sizes predicted by termination at the UGA selenocysteine codons. Appropriate loading on the type 1 deiodinase mRNA is seen following substitution of a cysteine codon for the selenocysteine codon, indicating that the UGA codon confers a translational penalty on the mRNA. Surprisingly, ribosomal loading is also increased by the expression of eukaryotic release factors eRF1 and eRF3.

Conclusions

These results suggest that the presence of a selenocysteine codon confers a translational penalty on selenoprotein mRNAs, and that increased levels of release factors may alter the kinetics of termination.
Crk family adaptor proteins trans‐activate c‐Abl kinase
Authors: Shishido Tomoyuki , Akagi Tsuyoshi , Chalmers Alistair , Maeda Makiko , Terada Toshie , Georgescu Maria‐Magdalena , Hanafusa Hidesaburo
Published: March 2003

Background

c‐Abl kinase is activated in response to a variety of biological stimuli. Crk family adaptor proteins can interact physically with c‐Abl and be involved in the activation of c‐Abl kinase.

Results

We report that the Crk family of adaptor proteins act as trans‐acting activators of c‐Abl kinase. The interaction of the amino‐terminal Src‐homology (SH) 3 domain of c‐Crk and the proline‐rich motifs of c‐Abl is an essential step for the phosphorylation of c‐Crk by c‐Abl, as well as the activation of c‐Abl by c‐Crk. The activation of c‐Abl by c‐Crk is negatively regulated by phosphorylation of the tyrosine 221 of c‐Crk. Our data suggest that, in the absence of phosphorylation of the tyrosine Y221, the SH2 domain of c‐Crk becomes free to bind to target molecules while the carboxyl‐terminal SH3 domain of c‐Crk binds to the proline‐rich region of c‐Abl, inducing the activation of c‐Abl by c‐Crk.

Conclusion

This study suggests that the Crk family functions as trans‐acting activators of c‐Abl kinase. The phosphorylation of c‐Crk may regulate c‐Abl kinase.
Self‐splicing of the Tetrahymena group I ribozyme without conserved base‐triples
Authors: Ikawa Yoshiya , Yoshioka Wataru , Ohki Yasushi , Shiraishi Hideaki , Inoue Tan
Published: March 2003

Background

Group I introns share a conserved core region consisting of two domains, P8‐P3‐P7 and P4‐P6, joined by four base‐triples. We showed previously that the T4 td intron can perform phosphoester transfer reactions at two splice sites in the absence of both P4‐P6 and the conserved base‐triples, whereas it is barely able to perform the intact splicing reaction due to the difficulty of conducting the sequential reactions.

Results

Based on previous findings, we constructed a bimolecular ribozyme lacking a large portion of P4‐P6 and the base‐triples from the Tetrahymena intron, on the assumption that the long–range interactions of the peripheral regions in the two RNAs can compensate for the deteriorated core. The bimolecular ribozyme performed the intact splicing reaction.

Conclusion

The present analysis indicates that the base‐triples are nonessential, but that L4 and the distal part of P4 in P4‐P6 are important for conducting the splicing reaction. The reconstituted self‐splicing ribozyme provides an amenable system for analysing the role(s) of elements in the core region in the self‐splicing reaction mechanism.
Yeast Saccharomyces cerevisiae has two cis‐prenyltransferases with different properties and localizations. Implication for their distinct physiological roles in dolichol synthesis
Authors: Sato Miyuki , Fujisaki Shingo , Sato Ken , Nishimura Yukinobu , Nakano Akihiko
Published: March 2003

Background Dolichol is a family of long‐chain polyprenols, which is utilized as a sugar carrier in protein glycosylation in the endoplasmic reticulum (ER). We have identified a key enzyme of the dolichol synthesis, cis‐prenyltransferase, as Rer2p from Saccharomyces cerevisiae. We have also isolated a multicopy suppressor of an rer2 mutant and named it SRT1. It encodes a protein similar to Rer2p but its function has not been established.

Results The cis‐prenyltransferase activity of Srt1p has been proved biochemically in the lysate of yeast cells lacking Rer2p. The polyprenol product of Srt1p is longer in chain length than that of Rer2p and is not sufficiently converted to dolichol and dolichyl phosphate, unlike that of Rer2p. The subcellular localization of these two isozymes has been examined by immunofluorescence microscopy and by the use of GFP fusion proteins. Whereas GFP‐Rer2p is localized to the continuous ER and some dots associated with the ER, GFP‐Srt1p shows only punctate localization patterns. Immunofluorescence double staining with Erg6p, a marker of lipid particles in yeast, indicates that Srt1p is mainly localized to lipid particles (lipid bodies). RER2 is mainly expressed in the early logarithmic phase, while the expression of SRT1 is induced in the stationary phase.

Conclusions We have shown that yeast has two active cis‐prenyltransferases with different properties. This result implies that the two isozymes have different physiological roles during the life cycle of the yeast.
Roles of nodal‐lefty regulatory loops in embryonic patterning of vertebrates
Authors: Juan Hou , Hamada Hiroshi
Published: March 2003

Nodal is a signalling molecule that belongs to the transforming growth factor–β superfamily of proteins, and Lefty proteins are antagonists of Nodal signalling. The nodal and lefty genes form positive and negative regulatory loops that resemble the reaction‐diffusion system. As a pair, these genes control various events of vertebrate embryonic patterning, including left‐right specification and mesoderm formation. In this review, we will focus on recent studies that have addressed the roles of nodal and lefty in mouse development.
Inhibition of BMP2‐induced, TAK1 kinase‐mediated neurite outgrowth by Smad6 and Smad7
Authors: Yanagisawa Makoto , Nakashima Kinichi , Takeda Kohsuke , Ochiai Wataru , Takizawa Takumi , Ueno Masaya , Takizawa Makiko , Shibuya Hiroshi , Taga Tetsuya
Published: March 2003

Background BMP2 is known to play a wide variety of roles, including some in the development of the nervous system. This cytokine has been reported to induce neurite outgrowth in rat pheochromocytoma PC12 cells via the activation of a p38 MAP kinase, although its regulatory mechanism remains largely to be elucidated.

Results BMP2‐induced neurite outgrowth in PC12 cells was inhibited by the introduction of a kinase‐negative form of a MAP kinase kinase kinase, TAK1, an upstream regulatory kinase for p38 kinase. Following BMP2 stimulation, the expression of Smad6 and Smad7, inhibitory Smad species that are known to inhibit the BMP2‐restricted Smad species, Smad1, Smad5 and Smad8, was up‐regulated. Unexpectedly, over‐expression of either Smad6 or Smad7 in PC12 cells repressed the BMP2‐induced neurite outgrowth and severely impeded the p38 kinase pathway. Both of these inhibitory Smads were found to interact physically with TAK1‐binding protein, a molecule required for TAK1 activation.

Conclusions This study demonstrates that BMP2‐induced neurite outgrowth in PC12 cells involves activation of the TAK1‐p38 kinase pathway which is inhibited by Smad6 and Smad7.
Step‐wise divergence of primitive and definitive haematopoietic and endothelial cell lineages during embryonic stem cell differentiation
Authors: Fujimoto Tetsuhiro , Ogawa Minetaro , Minegishi Naoko , Yoshida Hisahiro , Yokomizo Tomomasa , Yamamoto Masayuki , Nishikawa Shin‐ichi
Published: March 2003

Background The developmental processes leading from the mesoderm to primitive and definitive haematopoietic and endothelial lineages, although of great importance, are still poorly defined. Recent studies have suggested a model in which common precursors give rise to endothelial progenitors and haematopoietic progenitors, the latter subsequently generating both primitive and definitive haematopoietic lineages. However, this model is contradicted by findings that suggest the emergence of haematopoietic cells from the endothelial lineage.

Results We found sequential steps in the differentiation of FLK1+ mesoderm into haematopoietic and endothelial lineages in an in vitro differentiation system of embryonic stem (ES) cells: (i) the GATA‐1+ subset of FLK1+ mesodermal cells loses the capacity to give rise to endothelial cells and is restricted to primitive erythroid, macrophage and definitive erythroid progenitors; (ii) the remaining GATA‐1− cells give rise to VE‐cadherin+ endothelial cells; and subsequently (iii) multiple definitive haematopoietic progenitors and endothelial cells branch off from a subset of VE‐cadherin+ cells.

Conclusions These observations strongly suggest that the divergence of primitive and multilineage definitive haematopoietic/endothelial lineages occurs first, and then multilineage definitive haematopoietic progenitors arise from VE‐cadherin+ endothelial cells in the development of haematopoietic and endothelial cells.
Characterization, expression and complex formation of the murine Fanconi anaemia gene product Fancg
Authors: Van De Vrugt Henri J. , Koomen Mireille , Berns Mariska A. D. , De Vries Yne , Rooimans Martin A. , Van Der Weel Laura , Blom Eric , De Groot Jan , Schepers Rik J. , Stone Stacie , Hoatlin Maureen E. , Cheng Ngan Ching , Joenje Hans , Arwert Fré
Published: March 2003

Background: Fanconi anaemia (FA) is an autosomal recessive chromosomal instability disorder. Six distinct FA disease genes have been identified, the products of which function in an integrated pathway that is thought to support a nuclear caretaker function. Comparison of FA gene characteristics in different species may help to unravel the molecular function of the FA pathway.

Results: We have cloned the murine homologue of the Fanconi anaemia complementation group G gene, FANCG/XRCC9. The murine Fancg protein shows an 83% similarity to the human protein sequence, and has a predicted molecular weight of 68.5 kDa. Expression of mouse Fancg in human FA‐G lymphoblasts fully corrects their cross‐linker hypersensitivity. At mRNA and protein levels we detected the co‐expression of Fancg and Fanca in murine tissues. In addition, mouse Fancg and Fanca proteins co‐purify by immunoprecipitation. Upon transfection into Fanca‐deficient mouse embryonic fibroblasts EGFP‐Fancg chimeric protein was detectable in the nucleus.

Conclusions: We identified a murine cDNA, Fancg, which cross‐complements the cellular defect of human FA‐G cells and thus represents a true homologue of human FANCG. Spleen, thymus and testis showed the highest Fancg expression levels. Although Fancg and Fanca are able to form a complex, this interaction is not required for Fancg to accumulate in the nuclear compartment.
Tropomyosin is required for the cell fusion process during conjugation in fission yeast
Authors: Kurahashi Hiroshi , Imai Yoshiyuki , Yamamoto Masayuki
Published: March 2003

Background:  Tropomyosin is an actin‐binding protein, which is thought to stabilize actin filaments and influence many aspects of F‐actin. In fission yeast, the cdc8 gene encodes tropomyosin, and the gene product Cdc8p is known to be essential for the formation of the F‐actin contractile ring and hence for cytokinesis in the mitotic cell cycle.

Results: We isolated fission yeast mutants that were defective in cell fusion during conjugation. One of them turned out to carry a point mutation in cdc8. We found that the original temperature‐sensitive cdc8 mutant frequently failed to undergo cell fusion when mated at a semi‐permissive temperature. Additional cdc8 mutants isolated by targeted mutagenesis also showed defects in both cell fusion and cytokinesis. A decrease in the amount of intracellular Cdc8p also affected both, but cell growth was more severely blocked than cell fusion in this case. Immunostaining revealed that Cdc8p was localized as a spot at the cell‐to‐cell attachment site during conjugation, without overlapping with F‐actin patches.

Conclusions: Tropomyosin Cdc8p is indispensable for cell fusion during conjugation in fission yeast. However, cell fusion appears to require fewer tropomyosin molecules than cytokinesis. We speculate that tropomyosin may organize a small F‐actin‐containing organelle at the cell‐to‐cell contact site in each mating cell, which plays a key role in cell fusion.
Hyper‐processive and slower DNA chain elongation catalysed by DNA polymerase III holoenzyme purified from the dnaE173 mutator mutant of Escherichia coli
Authors: Sugaya Yutaka , Ihara Keiichi , Masuda Yuji , Ohtsubo Eiichi , Maki Hisaji
Published: March 2003

Background: A strong mutator mutation, dnaE173, leads to a Glu612 → Lys amino acid change in the α subunit of Escherichia coli DNA polymerase III (PolIII) holoenzyme and abolishes the proofreading function of the replicative enzyme without affecting the 3′ → 5′ exonuclease activity of the ɛ subunit. The dnaE173 mutator is unique in its ability to induce sequence‐substitution mutations, suggesting that an unknown function of the α subunit is hampered by the dnaE173 mutation.

Results: A PolIII holoenzyme reconstituted from dnaE173 PolIII* (DNA polymerase III holoenzyme lacking the β clamp subunit) and the β subunit showed a strong resistance to replication‐pausing on the template DNA and readily promoted strand‐displacement DNA synthesis. Unlike wild‐type PolIII*, dnaE173 PolIII* was able to catalyse highly processive DNA synthesis without the aid of the β‐clamp subunit. The rate of chain elongation by the dnaE173 holoenzyme was reduced to one‐third of that determined for the wild‐type enzyme. In contrast, an exonuclease‐deficient PolIII holoenzyme was vastly prone to pausing, but had the same rate of chain elongation as the wild‐type.

Conclusions: The hyper‐processivity and slower DNA chain elongation rate of the dnaE173 holoenzyme are distinct effects caused by the dnaE173 mutation and are likely to be involved in the sequence‐substitution mutagenesis. A link between the proofreading and chain elongation processes was suggested.
Carboxyl‐terminal basic amino acids in the X domain are essential for the nuclear import of phospholipase C δ1
Authors: Okada Masashi , Fujii Makoto , Yamaga Masaki , Sugimoto Hiroaki , Sadano Hiroyuki , Osumi Takashi , Kamata Hideaki , Hirata Hajime , Yagisawa Hitoshi
Published: March 2003

Background: Although phospholipase C (PLC)δ1 containing a functional nuclear export signal (NES) is normally localized at the plasma membrane and in the cytoplasm, it shuttles between the nucleus and the cytoplasm. Since nucleocytoplasmic shuttling of a molecule is generally regulated by a balance between its NES and the nuclear localization signal (NLS), we examined whether PLCδ1 contains an NLS sequence.

Results: A region corresponding to the C terminus of the X domain and the XY‐linker, which contains clusters of basic amino acid residues, was essential for the nuclear import of PLCδ1 in Madin–Darby canine kidney cells. A series of point mutations on lysine residues in this region revealed that K432 and K434 in combination were important for the nuclear import. A short synthetic peptide corresponding to residues 429–442, however, was not able to function as an NLS sequence when they were injected into the cytoplasm in a carrier‐conjugated form. Neither a longer peptide equivalent to PLCδ1 412–498 fused to a protein tag consisting of glutathione S‐transferase and green fluorescent protein was imported to the nucleus after microinjection into the cytoplasm.

Conclusion: The nuclear import of PLCδ1 requires the C‐terminus of the X domain, particularly the amino acid residues K432 and K434, and the XY‐linker. The region alone, however, cannot serve as a functional NLS. The machinery for nuclear transport may require additional structural component(s) of the enzyme.
Calcineurin is implicated in the regulation of the septation initiation network in fission yeast
Authors: Lu Yabin , Sugiura Reiko , Yada Tomoko , Cheng Hong , Sio Susie O. , Shuntoh Hisato , Kuno Takayoshi
Published: March 2003

Background: In fission yeast, calcineurin has been implicated in cytokinesis because calcineurin‐deleted cells form multiple septa and cell separation is impeded. However, this mechanism remains unclear.

Results: We screened for mutations that confer syn‐thetic lethality with calcineurin deletion and isolated a mutant, its10‐1/cdc7‐i10, a novel allele of the cdc7+ gene involved in the septation initiation network (SIN). The mutation created a termination codon, resulting in the truncation of Cdc7 by 162 amino acids, which is not localized in the spindle pole body. Following treatment with the immune suppressive drug FK506, cdc7‐i10 and the original cdc7‐24 mutant cells showed highly elongated multinuclear morphology with few visible septa, closely resembling the phenotype at the restrictive temperature. Other SIN mutants, cdc11, spg1, sid2 and mob1 showed similar phenotypes following FK506 treatment. Consistent with this, expression of the constitutively active calcineurin suppressed the growth defects and septum initiation deficiency of these SIN mutants at the restrictive temperature. Moreover, electron microscopy revealed that calcineurin‐deleted cells had very thick multiple septa which were partially and ectopically formed.

Conclusion: These results suggest that calcineurin is involved in the regulation of the SIN pathway, and is required for the proper formation and maturation of the septum in fission yeast.
Isolation and expression of a novel mitochondrial septin that interacts with CRMP/CRAM in the developing neurones
Authors: Takahashi Shusuke , Inatome Ryoko , Yamamura Hirohei , Yanagi Shigeru
Published: March 2003

Background: Collapsin response mediator proteins (CRMPs) and CRAM belong to the unc‐33 gene family which is implicated in axon guidance and outgrowth during neural development. However, their exact roles remain largely unknown. To understand the molecular basis of CRMP/CRAM function, we have undertaken to identify CRMP/CRAM interacting proteins.

Results: We have identified a novel mitochondrial septin (M‐septin) as one of the CRMP/CRAM interacting proteins from the developing rat brain. M‐septin is a major, alternatively spliced variant of the H5 gene in developing mouse brain and its expression is up‐regulated during the neuronal differentiation of embryonal carcinoma P19 cells. In COS‐7 cells, M‐septin is specifically localized to mitochondria whereas H5 is diffusely distributed to the perinuclear cytoplasm and plasma membranes. In contrast to H5, M‐septin induces the mitochondrial translocation of CRAM but not CRMP2. Finally, M‐Septin is found to be transiently translocated to mitochondria before the induction of the neurites and then dissociates from the mitochondria after neurite extension in P19 cells.

Conclusions: Our results suggest that M‐septin has a role which is distinct from H5, and together with CRMP/CRAM, may play an important role in the neuronal differentiation and axon guidance through the control of mitochondrial function.
MDG1/ERdj4, an ER‐resident DnaJ family member, suppresses cell death induced by ER stress
Authors: Kurisu Junko , Honma Asuka , Miyajima Hiroshi , Kondo Shinichi , Okumura Masayo , Imaizumi Kazunori
Published: March 2003

Background: Alterations in homeostasis after various cellular stresses, which prevent protein folding and cause an accumulation of misfolding or malfolding proteins in the endoplasmic reticulum (ER), have the potential to induce cellular damage, and are therefore a type of ‘ER stress.’ To understand the molecular events or cascades underlying the ER stress response regulated by gene transcription and mediated by stress transducers, it is crucial to identify the molecules induced during ER stress and to analyse the roles of these genes.

Results: We identified MDG1/ERdj4, a member of the DnaJ protein family, as an inducible gene during ER stress. MDG1/ERdj4 contains the J domain, which is essential for interacting with Hsp70s, at the N‐terminal portion and just at the back of the transmembrane domain. Its trypsin digestion and glycosylation of a chimeric protein composed of MDG1/ERdj4 fused with the extracellular domain of the amyloid precursor protein at its C‐terminus, showed that its C‐terminal portion containing the J domain could be orientated to the ER lumen. Over‐expression of it inhibited the cell death induced by ER stress. In contrast, its mutants with the J domain deleted showed no protective effects against cell death.

Conclusions: MDG1/ERdj4 may play roles in stabilizing GRP78/BiP binding to unfolded substrate proteins in a J domain‐dependent manner and prevent the accumulation of unfolded proteins in the ER, consequently protecting cells from ER stress.
Volcanoes and peat bogs
Authors: Holmes Jane , Hall Valerie , Wilson Peter
Published: March 2003

Peat bogs in the British Isles contain tephra from volcanic eruptions in Iceland; tephra in the Falkland Islands most likely derives from Andean volcanoes. Study of these far‐travelled volcanic products is making a significant contribution to our understanding of past environmental changes.
Crystal Geyser – Utah's cold one
Authors: Waltham Tony
Published: March 2003

A cold geyser appears to be a contradiction in terms. But a combination of carbon dioxide, effervescing groundwater and a fortuitous oil exploration well can create a very spectacular water fountain.
The magma‐to‐mud cycle
Authors: Merriman Dick
Published: March 2003

For more than 3700 million years of Earth history, the magma‐to‐mud cycle has steadily generated new crustal rock. Mud formed by rock weathering coats the Earth's surface and helps to sustain life. Reactive clay minerals are the key components of mud, and control the absorption and release of chemicals and fluids that are critical for the recycling of crustal rocks.
An experimental comparison of three direct methods of marine source signature estimation
Authors: Laws Robert , Landrø Martin , Amundsen Lasse
Published: March 2003

Three methods for estimation of the pressure wavefield generated by a marine airgun array are tested experimentally and compared. In the trial a variety of radiation angles and array configurations were used and some large synchronization errors were deliberately introduced. The source was equipped with near‐field hydrophones and a subsource ministreamer. A tethered far‐field hydrophone was used so that the three estimated far‐field signatures could be compared with an independent measurement.

The knowledge of the source signature is important for on‐board source array QC, deconvolution, multiple attenuation, stratigraphic trap prediction, modelling and inversion, AVO analysis and reservoir monitoring.

The methods perform very well and give estimates whose frequency‐domain spectra match the measured spectra to within a few dB and within a few tens of degrees of phase over the tested bandwidth of 3.5–110 Hz. The time‐domain error‐energy is typically only a few per cent of the signal energy for radiation angles within about 30° of the vertical. The third method proved to be sensitive to an experimental shortcoming leading to overloading of the ministreamer and meaningful comparison was not possible for some test configurations.