bluuebunny/crossref_metadata_2025 huggingface dataset:
Passage through bird guts increases germination rate and seedling growth inSorbus aucuparia
Authors: Paulsen T. R. , Högstedt G.
Published: March 2003

On the west coast of Norway,Turdusspp. (Thrushes) are important dispersal agents ofSorbus aucupariaL. (Rowan) seeds, and avian and mammalian gut treatment often alters seed germination characteristics. In the present study the effects of avian gut treatment onS. aucupariaseeds are described, with emphasis on subsequent seedling growth.

Seeds ingested byTurdusspp. and non‐ingested control seeds were sown singly or multiply in soil, in pomes, or in bird droppings.

Defecated seeds were ≈9% heavier than control seeds, and seedling growth was faster from defecated seeds than controls. In addition, differences in the rate of seedling emergence were found, with seedlings from ingested seeds appearing first.

The increased growth may be due to seedlings emerging earlier from defecated seeds, giving them an extended growth period at a time of increasing day length.

We argue that factors such as seedling growth, rate of seedling emergence and seed mass, in addition to percentage seed germination, are important in determining seedling survival.
Energy requirements of female Macaroni Penguins breeding at South Georgia
Authors: Green J. A. , Butler P. J. , Woakes A. J. , Boyd I. L.
Published: March 2003

Breeding female Macaroni Penguins (Eudyptes chrysolophus) were implanted with heart rate, temperature and depth data loggers in order to estimate their rate of energy expenditure from heart rate.

The estimated average daily metabolic rates (ADMR) of birds during the brood and crèche phases of the breeding season were 8·17 ± 0·44 W kg−1 and 8·24 ± 0·41 W kg−1, respectively. There was no significant difference between these estimates and the pooled ADMR for all birds was 8·22 ± 0·38 W kg−1.

Metabolic rate while the penguins were at‐sea was 9·03 ± 0·39 W kg−1 and this was significantly greater than the metabolic rate of 6·27 ± 0·38 W kg−1 while they were on‐shore.

Females undertook shorter foraging trips during the crèche phase than during the brood phase.

When considered as a breeding pair, Macaroni Penguins expend more energy in raising their chick during the crèche phase of the breeding season as the male assists in provisioning the chick only at this time.
A new mammalian period gene predominantly expressed in the suprachiasmatic nucleus
Authors: Takumi Toru , Matsubara Chiaki , Shigeyoshi Yasufumi , Taguchi Kouji , Yagita Kazuhiro , Maebayashi Yoshiro , Sakakida Yoko , Okumura Ko , Takashima Naoyuki , Okamura Hitoshi
Published: March 2003

Background

In mammals, two possible clock genes (Clock, Per1) have very recently been reported. mPer1 (the first identified mouse period gene), in particular, shows a circadian expression in suprachiasmatic nuclei (SCN), the mammalian circadian centre. However, only mPer1 and Clock as clock components may not be sufficient to understand all the events in circadian oscillation and entrainment.

Results

A mammalian period complementary DNA, mPer2, has been isolated from the mouse brain. The amino acid sequence of mPer2 is similar to mPer1 and Drosophila Period (dPer), indicating that mPer2 is a member of the family which contains mPer1, itself a homologue of dPer. mPer2 mRNA is predominantly expressed in SCN. A robust circadian rhythmic expression in the SCN supports the view that mPer2 is a clock gene. mPer2 is strongly expressed at the subjective afternoon in constant darkness, distinct from a morning‐phase clock mPer1. Our precise quantitative in situ hybridizations have revealed that the peak expression of mPer2 transcripts is delayed by 8 h in LD (light–dark) or 4 h in DD (dark–dark) conditions when compared to mPer1. A short brief light exposure at the early subjective night, prompting a phase‐shift in locomotor rhythms, induces a transient increase of mPer2 transcripts with delayed onset, as compared to mPer1 mRNA induction. Furthermore, mPer2 is co‐expressed with mPer1 in single SCN cells.

Conclusions

Mammalian period genes show molecular heterogeneity, each of which is composed of a different oscillator, and may serve to establish stable circadian rhythms in mammalian oscillating cells.
Fas‐induced DNA fragmentation and proteolysis of nuclear proteins
Authors: Kawahara Atsuo , Enari Masato , Talanian Robert V. , Wong Winnie W. , Nagata Shigekazu
Published: March 2003

Background

Fas is a member of the tumour necrosis factor (TNF) receptor family. Activation of Fas by its ligand or an agonistic anti‐Fas antibody causes apoptosis in Fas‐bearing cells, by activating various members of the caspase family.

Results

Specific fluorogenic substrates (MCA‐DEVDAPK[dnp] and MCA‐VEVDAPK[dnp]) for caspases 3 and 6 were prepared. Using these substrates, a gradual increase of the caspase 3‐ and 6‐like proteases were detected during the Fas engagement in human Jurkat. This activation of caspases correlated well with the cleavage of poly(ADP‐ribose) polymerase and lamin B1, as well as with DNA fragmentation. When the recombinant caspases were added to the extracts from Jurkat cells, caspase 3 produced active caspase 6‐like protease, while caspase 6 activated the caspase 3 protease, suggesting that these proteases can activate each other. The caspase‐treated cell extracts, as well as the extracts from the Fas‐activated cells, caused the proteolysis of nuclear proteins and DNA degradation. The cleavage of nuclear proteins was inhibited by caspase inhibitors, while the same inhibitors had no effect on DNA degradation.

Conclusions

At one stage of the caspase cascade, caspases activate each other, and amplify the apoptotic signal. Caspases downstream of the cascade then cause the proteolysis of nuclear proteins and DNA degradation.
Stress‐activated signalling pathways in yeast
Authors: Toone W. Mark , Jones Nic
Published: March 2003

Eukaryotic cells have developed response mechanisms to combat the harmful effects of a variety of stress conditions. In the majority of cases, such responses involve changes in the gene expression pattern of the cell, leading to increased levels and activities of proteins that have stress‐protective functions. Over the last few years, considerable progress has been made in understanding how stress‐dependent transcriptional changes are brought about, and it transpires that the underlying mechanisms are highly conserved, being similar in organisms ranging from yeast to man. Many of the stress signals derive from the extracellular environment and accordingly these signals require transduction from the cell surface to the nucleus. This is accomplished through stress‐activated signalling pathways, key amongst which are the highly conserved stress‐activated MAP kinase pathways. Stimulation of these pathways leads to the increased activity of specific transcription factors and consequently the increased expression of certain stress‐related genes. In this review, we focus on the progress that has been made in understanding these stress responses in yeast.
Meiotic behaviours of chromosomes and microtubules in budding yeast: relocalization of centromeres and telomeres during meiotic prophase
Authors: Hayashi Aki , Ogawa Hideyuki , Kohno Kenji , Gasser Susan M. , Hiraoka Yasushi
Published: March 2003

Background

" Meiosis is a process of universal importance in eukaryotic organisms, generating variation in the heritable haploid genome by recombination and re‐assortment of chromosomes. The intranuclear movement of chromosomes is expected to achieve pairing and recombination of homologous chromosomes during meiosis. Meiosis in the budding yeast Saccharomyces cerevisiae has been extensively studied, both genetically and by molecular biology; here we report cytological observations of meiotic chromosomal events in this organism.

Results

Using fluorescence microscopy, we have examined the behaviour of chromosomes and microtubules during meiosis in S. cerevisiae. We first observed the dynamic behaviour of nuclei in living cells using jellyfish green fluorescent protein (GFP) fused with nucleoplasmin, a Xenopus oocyte nuclear protein. The characterization of nuclear movement in living cells was extended by an analysis of chromosomes and microtubules in fixed specimens. In addition, the nuclear localization of centromeres and telomeres was determined by indirect immunofluorescence microscopy in synchronous populations of meiotic cells. While telomeres remain in clusters of 5–8 throughout meiosis, centromeres change their nuclear localization dramatically during the progression of meiosis: centromeres are first clustered at a single site near the spindle‐pole body before the induction of meiosis, and become scattered during the meiotic prophase.

Conclusions

Our observations have demonstrated that nuclear and cytoskeletal reorganization take place with meiosis in S. cerevisiae. In particular, the distinct relocalization of centromeres during meiosis indicates a considerable movement of chromosomes within the meiotic prophase nucleus.
Function in vivo of separate segments of the β subunit of Escherichia coli RNA polymerase
Authors: Trigwell Susan , Glass Robert E.
Published: March 2003

Background:

Transcription of genetic material is catalysed by the enzyme DNA‐dependent, RNA polymerase. The multimeric RNA polymerases consist of between 4 and 16 different subunits, of which the two largest, termed β and β′, are conserved throughout nature. The β subunit has been implicated in all of the stages of transcription that are catalysed by the complete enzyme. Several lines of evidence have suggested that the function of the β subunit is not dependent upon the contiguity of the sequence blocks. In this report, a complementary immunological and genetic approach was adopted in order to investigate the individual regions of the β subunit of RNA polymerase. To this end, the β structural gene rpoB was separated into four near‐equal, non‐overlapping segments (as well as ‘half’ genes) on the basis of ‘split’ genes in nature, known functional organization and sequence conservation. These segments were used to prepare sequence‐specific antibodies against the four individual regions, as well as being expressed in vivo from a tight, lac‐controlled high‐copy number vector.

Results:

Immunological probing of the holoenzyme in vitro suggested that the amino‐terminal half of the β polypeptide is buried within the enzyme complex. Of the four segments expressed in vivo, the extreme C‐terminal segment was trans‐dominant lethal (of the effect of large N‐terminal amber fragments on cellular growth; Nene & Glass 1982) and this isolated region was shown to bind the translational elongation factor EF‐Tu in vivo.

Conclusions:

These in vivo and in vitro studies, in conjunction with recent in vitro work (Severinov et al. 1995), unambiguously demonstrate that individual regions of β may adopt structurally and functionally competent forms, and underline the possibility of in vivo investigation of separate regions of this massive polypeptide chain. A model is presented for the role of EF‐Tu in stringent control.
Two F‐box/WD‐repeat proteins Pop1 and Pop2 form hetero‐ and homo‐complexes together with cullin‐1 in the fission yeast SCF (Skp1‐Cullin‐1‐F‐box) ubiquitin ligase
Authors: Kominami Kin‐ichiro , Ochotorena Itziar , Toda Takashi
Published: March 2003

Background:

In the ubiquitin‐dependent proteolysis pathway, a ubiquitin ligase (E3) is responsible for substrate selectivity and timing of degradation. A novel E3, SCF (Skp1‐Cullin‐1/Cdc53‐F‐box) plays a pivotal role in cell cycle progression. In fission yeast, F‐box/WD‐repeat protein Pop1 regulates the level of the CDK (cyclin‐dependent kinase) inhibitor Rum1 and the S phase regulator Cdc18.

Results:

We have cloned and characterized the pop2+ gene which encodes the Pop1‐related F‐box/WD‐repeat protein. Pop2 plays a role which overlaps with Pop1 in the degradation of Rum1 and Cdc18. However, these two proteins are not functional homologues. Pop1 and Pop2 form hetero‐ as well as homo‐dimers in the cell. We have analysed two fission yeast cullin members and found that cullin‐1 functions as a component of SCFPop1,2, whilst cullin‐3 is involved in the distinct stress‐response pathway.

Conclusions:

Fission yeast SCF is composed of Pop1 and Pop2, two structurally related but functionally independent F‐box/WD‐repeat proteins. By forming three distinct complexes, SCFPop1/Pop1, SCFPop1/Pop2 and SCFPop2/Pop2, SCF has evolved a sophisticated mechanism to control the level of Rum1 and Cdc18. Fission yeast SCF also contains cullin‐1 as a universal scaffold and each cullin member plays a distinct biological role.
Impaired extrapyramidal function caused by the targeted disruption of Retinoid X receptor RXRγ1 isoform
Authors: Saga Yumiko , Kobayashi Masahiko , Ohta Hisashi , Murai Naomi , Nakai Nami , Oshima Masanobu , Taketo Makoto M.
Published: March 2003

Background

Retinoid X receptors RXRα, β and γ exert multiple functions in the genetic regulation of mammalian signalling systems by forming heterodimeric complexes with several nuclear ligand receptors. In contrast to the widespread expression of RXRα and RXRβ, the expression of RXRγ is restricted to particular tissues in which RXRγ1 is the major isoform expressed in the mouse corpus striatum.

Results

To investigate the function of this particular isoform RXRγ1, we generated RXRγ1 gene‐knockout mice by homologous recombination in ES cells. Both heterozygous and homozygous mice showed severe runting after birth, which often resulted in the early death of mice of the 129/C57BL‐6 genetic background. Independent of genetic background, however, the expression of choline acetyltransferase (ChAT) in the cholinergic interneurones in the striatum (caudal putamen) was markedly reduced in the RXRγ1 gene‐null mice. Furthermore, the mutant exhibited an altered response to the administration of dopamine receptor antagonists, haloperidol and chlorpromazine, which normally induce catalepsy in mice.

Conclusions

These results strongly suggest that RXRγ1 plays an important role in either the development or activation of cholinergic neurones in nigrostriatal extrapyramidal pathways.
The G10BP‐1 gene encoding a GC box binding protein, is a target of Myc and Jun/Fos
Authors: Shirasuna Kenna , Takeuchi Arata , Bando Takayoshi , Nakajima Takuma , Oda Kinichiro
Published: March 2003

Background

G10BP, a serum‐inducible factor, represses the transcription of the fibronectin gene through binding to the G‐rich sequences in the promoter excluding Sp1 from binding to these sequences.

Results

The 5′ flanking sequence of the G10BP‐1 gene was isolated by polymerase chain reaction of the adaptor‐ligated genomic DNA library using the adaptor primer and the G10BP‐1 cDNA primer. The elements required for activation of the G10BP‐1 promoter following serum stimulation were analysed by transfection of quiescent rat 3Y1 cells with G10BP‐1 promoter‐luciferase cDNA constructs containing 5′ sequential deletions or base substitutions. The results showed that the promoter was activated by Myc and Jun through the E box and AP1 sites. The formation of DNA–protein complexes with 32P‐labelled oligonucleotides containing the E box or AP1 site with cell extracts prepared during G1 progression was correlated with the promoter activation and greatly reduced by immunodepletion of Myc or c‐Jun from the extracts.

Conclusion

These results indicate that the G10BP‐1 gene is a target of Myc and Jun/Fos and that these factors repress the fibronectin gene expression through induction of G10BP‐1 during G1‐to‐S phase progression.
Amber (UAG) suppressors affected in UGA/UAA‐specific polypeptide release factor 2 of bacteria: genetic prediction of initial binding to ribosome preceding stop codon recognition
Authors: Yoshimura Kuniyasu , Ito Koichi , Nakamura Yoshikazu
Published: March 2003

Background

Prokaryotic translational release factors, RF1 and RF2, catalyse protein release at UAG/UAA and UGA/UAA stop codons, respectively. Mutations in RF1 and RF2 are known to cause non‐sense suppression for UAG (amber) and UGA (opal) codons, respectively, and they do not exert a reciprocal (‘cross’) suppression phenotype. We aimed to isolate RF mutants of such cross‐suppression activity, which we designated ‘Csu’ phenotype in this paper.

Results

Using a lacZ (UAG) reporter, we selected amber suppressor alleles occurring in the plasmid‐bearing RF2 gene of Salmonella typhimurium. Of nine such RF2 csu alleles, five were mis‐sense mutations and four were non‐sense mutations. The former mis‐sense mutants retained the RF2 activity and catalysed UGA termination both in vivo and in vitro. RF2 C‐terminal deletions equivalent to the non‐sense alleles exerted amber suppression as well as opal suppression activity. Moreover, the equivalent RF1 segments also showed both the suppression phenotypes.

Conclusions

All the csu mutations were mapped at the C‐terminal half of RF2 and are strikingly coincident with the highly conservative amino acids, suggesting that they affect the conserved function of bacterial RFs. We propose here that there should be an ‘initial binding’ step of RFs to the ribosome, preceding stop codon recognition (‘initial binding’ hypothesis) and that the N‐terminal RF domain(s), that are truncated or affected by the csu mutations, are responsible for this step and interfere with the proper functioning of cognate release factors on the ribosome.
Critical role of the membrane‐proximal, proline‐rich motif of the interleukin‐2 receptor γc chain in the Jak3‐independent signal transduction
Authors: Tsujino Shiho , Miyazaki Tadaaki , Kawahara Atsuo , Maeda Michiyuki , Taniguchi Tadatsugu , Fujii Hodaka
Published: March 2003

Background

The interleukin‐2 receptor (IL‐2R) consists of three subunits, the IL‐2Rα, IL‐2Rβc, and IL‐2Rγc chains. The essential role of the IL‐2Rγc cytoplasmic domain, consisting of 86 amino acids, in signal transmission has been well documented. Particularly, the carboxyl ter‐minal region containing 48 amino acids. is essential for the association with and activation of the Jak3 protein tyrosine kinase. On the other hand, little is known about the role of the rest of the IL‐2Rγc cytoplasmic region consisting of the membrane‐proximal 38 amino acids.

Results

We show that a truncated mutant form of IL‐2Rγc which lacks the membrane‐distal 48 amino acids is still capable of inducing the activation of Jak1 and Stat3/Stat5 in the absence of Jak3 activation. This membrane‐proximal region can also mediate the IL‐2‐induced tyrosine phosphorylation of the p85 subunit of phosphatidylinositol‐3‐kinase (PI3K). Furthermore, these signalling events are completely abrogated when mutations are introduced into the proline‐rich motif in this region.

Conclusions

In this study, we identified a Jak3‐independent signalling pathway(s) from the membrane‐proximal region of IL‐2Rγc. Our results indicate that the proline‐rich motif in this region plays a critical role in this signalling pathway(s). The present study may provide further insight into the mechanism of cellular responses mediated by IL‐2 and other cytokines which utilize the IL‐2Rγc for their signal transmission.
Identification and characterization of E‐APC, a novel Drosophila homologue of the tumour suppressor APC
Authors: Hamada Fumihiko , Murata Yoji , Nishida Ayumu , Fujita Fumitaka , Tomoyasu Yoshinori , Nakamura Makoto , Toyoshima Kumao , Tabata Tetsuya , Ueno Naoto , Akiyama Tetsu
Published: March 2003

Background

Mutations in the adenomatous polyposis coli (APC) tumour suppressor gene are implicated in the genesis of colorectal cancers. The product of the APC gene forms a complex with β‐catenin, glycogen synthase kinase 3β (GSK‐3β) and Axin/conductin, and induces the degradation of β‐catenin.

Results

We have identified a novel Drosophila homologue of APC, E‐APC, which is similar to but differs in several respects from D‐APC. The E‐APC cDNA encodes a protein of predicted 1067 amino acids, with seven armadillo repeats, two copies of the 15‐amino acid repeat, five copies of the 20‐amino acid repeat, and one Axin/conductin binding site. E‐APC directly interacts with D‐Axin and Armadillo (Arm, the Drosophila homologue of β‐catenin) in vitro, destabilizes intracellular β‐catenin, and suppresses β‐catenin/TCF‐regulated transcription in APC−/− colon cancer cells. The E‐APC mRNA is ubiquitously expressed throughout all developmental stages in Drosophila.

Conclusion

Our findings suggest that E‐APC may be universally involved in the regulation of the Wingless signalling pathway by down‐regulating the level of Arm in Drosophila.
Intragenic suppression of trans‐dominant lethal substitutions in the conserved GEME motif of the β subunit of RNA polymerase: evidence for functional cooperativity within the C‐terminus
Authors: Malik Talat , Ahmad Khalid , Buyukuslu Nihal , Cromie Karen , Glass Robert E.
Published: March 2003

Background

The ubiquitous multimeric RNA polymerases contain two large, conserved subunits, of which the β subunit has been implicated in all three stages of transcription. We have previously described a genetic system involving random, PCR‐mediated mutagenesis of the region of rpoB encoding the C‐terminal 116 amino acids of the β subunit of Escherichia coli RNA polymerase and the characterization of dominant‐negative mutations. This study identified the invariant motif GEME (residues 1271→1274;
Cromie et al. 1999
). Starting with three of these GEME‐motif lethal mutations (G1271E, G1271V, M1273V), we have selected for
intragenic suppressors, located within the same 3′‐region, that prevent expression of the trans‐dominant phenotype.

Results

We isolated a total of 24 missense mutants and a further 14 frameshift alleles (the latter generating a nested set of C‐terminal deletions of the β subunit) and studied the effect of the missense suppressors in vivo and in vitro. The majority of the second‐site substitutions pinpoint highly conserved residues and were allele‐specific. In contrast, one particular missense substitution (S1332P) acted on all three primary site mutations whilst not appreciably affecting assembly proficiency, suggesting motif‐specific suppression. Two missense substitutions were found to perturb assembly of the β subunit (M1232T and L1233P) and define a small conserved region (1228→1233) adjacent to one of the active‐site residues identified by affinity‐labelling, H1237. The majority of primary mutations were located in three main clusters within the 116 amino acid region.

Conclusions

The importance and functional co‐operativity of the three main clusters pinpointed is supported by the present isolation of suppressors of three different GEME primary mutations in the same three regions (whereas the suppressors of G1271V and M1273V are located in all three clusters, those for G1271E are all C‐terminal of this residue). Moreover, the location of the suppressors suggests that the GEME and HLVDDK regions are present as α‐helices in holoenzyme, and that functional co‐operativity is through one particular face of each helix.
Hsc70/Hsp40 chaperone system mediates the Hsp90‐dependent refolding of firefly luciferase
Authors: Minami Yasufumi , Minami Michiko
Published: March 2003

Background

The 90‐kDa heat shock protein, Hsp90, was previously shown to capture firefly luciferase during thermal inactivation, thereby preventing its irreversible off‐pathway aggregation and maintaining it in a folding‐competent state. However, subsequent refolding of the luciferase required addition of rabbit reticulocyte lysate.

Results

Here we demonstrate that Hsc70 (cytosolic Hsp70) and Hsp40/Hdj1 (cytosolic DnaJ homologue) are the effective components in a reticulocyte lysate, while other unidentified factor in the lysate is also required for the refolding of Hsp90‐captured luciferase. Though another cytosolic DnaJ homologue, Hdj2/HSDJ, was more efficient than Hsp40 in suppressing the aggregation of rhodanese, Hdj2 was less effective for the refolding of luciferase than Hsp40. In the absence of the third factor, Hsp40 could bind to the luciferase captured by Hsp90, which suggested that Hsp40 on its own was able to bind the substrate protein, but Hsc70 could not.

Conclusions

Hsc70, Hsp40 and at least another additional component in the reticulocyte lysate are necessary for full accomplishment of the refolding of Hsp90‐captured luciferase. The third factor may be required for the loading of Hsc70 on to the substrate protein bound to Hsp90.
The loose coupling mechanism in molecular machines of living cells
Authors: Oosawa Fumio
Published: March 2003

Living cells have molecular machines for free energy conversion, for example, sliding machines in muscle and other cells, flagellar motors in bacteria, and various ion pumps in cell membranes. They are constructed from protein molecules and work in the nm (nanometer), pN (piconewton) and ms (millisecond) ranges, without inertia. In 1980s, a question was raised of whether the input–output or influx–efflux coupling in these molecular machines is tight or loose, and an idea of loose coupling was proposed. Recently, the long‐distance multistep sliding of a single myosin head on an actin filament, coupled with the hydrolysis of one ATP molecule, was observed by Yanagida's group using highly developed techniques of optical microscopy and micromanipulation. This gave direct evidence for the loose coupling between the chemical reaction and the mechanical event in the sliding machine. In this review, I will briefly describe a historical overview of the input–output problem in the molecular machines of living cells.
A novel RNA motif that binds efficiently and specifically to the Tat protein of HIV and inhibits the trans‐activation by Tat of transcription in vitro and in vivo
Authors: Yamamoto Rika , Katahira Masato , Nishikawa Satoshi , Baba Tadashi , Taira Kazunari , Kumar Penmetcha K. R.
Published: March 2003

Background

To find a novel RNA that would bind efficiently and specifically to Tat protein but not to other cellular factors, we used an in vitro selection method and isolated a novel aptamer RNATat, a 37‐mer RNA oligomer, that binds efficiently to the Tat protein of HIV‐1. In the present study, we analysed various properties of aptamer RNATat, including binding kinetics, identification of functional groups for Tat binding, and inhibition of Tat function.

Results

The binding affinity of the isolated aptamer RNATat to Tat‐1 was 133 times higher than that of authentic TAR‐1 RNA. RNATat is composed of inverted repeats of two TAR‐like motifs, and even though RNATat had two Tat‐binding core elements, the interaction with Tat took place at a molar ratio of 1 : 1. Several functional groups of aptamer RNATat responsible for Tat binding were identified. The selected aptamer RNATat competed effectively for binding to Tat even in the presence of a large excess of TAR‐1 or TAR‐2 RNA in vitro, and specifically prevented Tat‐dependent trans‐activation both in vitro and in vivo.

Conclusions

Our results indicate that a novel aptamer, RNATat, retained strong affinity for Tat even in the presence of a large excess of HIV TAR. RNATat binds efficiently to Tat proteins or peptides derived from either HIV‐1 or HIV‐2. Unlike TAR RNA, RNATat affinity does not depend upon cellular proteins such as cyclin T1, thus RNATat has the potential for use as a molecular recognition element in biosensors.
Ste20‐like kinase (SLK), a regulatory kinase for polo‐like kinase (Plk) during the G2/M transition in somatic cells
Authors: Ellinger‐Ziegelbauer Heidrun , Karasuyama Hajime , Yamada Eitaro , Tsujikawa Kazutake , Todokoro Kazuo , Nishida Eisuke
Published: March 2003

Background

Activation of the cyclin‐dependent kinase cdc2‐cyclin B1 at the G2/M transition of the cell cycle requires dephosphorylation of threonine‐14 and tyrosine‐15 in cdc2, which in higher eukaryotes is brought about by the Cdc25C phosphatase. In Xenopus, there is evidence that a kinase cascade comprised of xPlkk1 and Plx1, the Xenopus polo‐like kinase 1, plays a key role in the activation of Cdc25C during oocyte maturation. In the mammalian somatic cell cycle, a polo‐like kinase homologue (Plk1) also functions during mitosis, but a kinase upstream of Plk is still unknown.

Results

We show here that human Ste20‐like kinase (SLK), which is a ubiquitously expressed mammalian protein related to xPlkk1, can phosphorylate and activate murine Plk1. During progression through the G2 phase of the mammalian cell cycle, the activity of endogenous SLK is increased. The amount of SLK protein is decreased in quiescent and differentiating cells. Treatment with okadaic acid induces a phosphorylation‐dependent enhancement of SLK activity.

Conclusions

We propose that SLK has a role in the regulation of Plk1 activity in actively dividing cells during the somatic cell cycle. SLK itself is suggested to be regulated by phosphorylation.
Ebp2p, yeast homologue of a human protein that interacts with Epstein–Barr virus Nuclear Antigen 1, is required for pre‐rRNA processing and ribosomal subunit assembly
Authors: Tsujii Rota , Miyoshi Keita , Tsuno Akiko , Matsui Yasushi , Toh‐e Akio , Miyakawa Tokichi , Mizuta Keiko
Published: March 2003

Background

A defect in the secretory pathway causes the transcriptional repression of both rRNA and ribosomal protein genes in Saccharomyces cerevisiae, suggesting a coupling of ribosome synthesis and plasma membrane synthesis. Rrs1p, an essential nuclear protein, is required for the secretory response.

Results

EBP2, encoding the yeast homologue of a human protein that interacts with Epstein–Barr virus Nuclear Antigen 1, was cloned in a two‐hybrid screen using RRS1 as a bait. The rrs1‐1 mutation, which produces Rrs1p without the C‐terminal half and causes a defect in the secretory response, almost abolished the interaction with Ebp2p. Ebp2p is essential for growth and is mainly localized in the nucleolus. The effects of Ebp2p depletion on ribosome biogenesis is quite similar to that of Rrs1p depletion; in the Ebp2p‐depleted cells, the rate of pre‐rRNA processing is slower, and significantly less mature 25S rRNA is produced compared to those in wild‐type cells. The polysome pattern indicates that Ebp2p‐depletion causes a decrease of 80S monosomes and polysomes, an accumulation of 40S subunits, and the appearance of half‐mer polysomes.

Conclusions

Ebp2p is required for the maturation of 25S rRNA and 60S subunit assembly. Ebp2p may be one of the target proteins of Rrs1p for executing the signal to regulate ribosome biogenesis.
Phosphorylation of ERM proteins at filopodia induced by Cdc42
Authors: Nakamura Nao , Oshiro Noriko , Fukata Yuko , Amano Mutsuki , Fukata Masaki , Kuroda Shinya , Matsuura Yoshiharu , Leung Thomas , Lim Louis , Kaibuchi Kozo
Published: March 2003

Background

ERM (ezrin, radixin, and moesin) proteins function as membrane‐cytoskeletal linkers, and are known to be localized at filopodia and microvilli‐like structures. We have shown that Rho‐associated kinase (Rho‐kinase)/ROKα/ROCK II phosphorylates moesin at Thr‐558 at the lower stream of Rho, and the phosphorylation is crucial to the formation of microvilli‐like structures (Oshiro, N., Fukata, Y. & Kaibuchi, K. (1998) Phosphorylation of moesin by Rho‐associated kinase (Rho‐kinase) plays a crucial role in the formation of microvilli‐like structures. J. Biol. Chem. 273, 34663– 34666). However, the role of ERM proteins in the formation of filopodia is less well characterized.

Results

Here we examined the phosphorylation state of ERM during filopodia formation induced by Cdc42 using the antibody recognizing ERM proteins phosphorylated at COOH (C)‐terminal threonine. When NIH 3T3 cells were transfected with constitutively active Cdc42 (Cdc42V12), filopodia formation was induced and phosphorylation of ERM at C‐terminal threonine was observed at the tip of filopodia, while the phosphorylation levels of ERM were lower and phosphorylated ERM was distributed throughout the cytoplasm in the control cells. We also showed that
Myotonic dystrophy kinase‐
related Cdc42‐binding
kinase (MRCK) which has been identified as an effector of Cdc42, phosphorylated moesin at C‐terminal threonine in a cell‐free system. Coexpression of the dominant negative form of MRCK inhibited both the formation of filopodia and accumulation of C‐terminal threonine‐phosphorylated ERM proteins at filopodia induced by Cdc42V12.

Conclusion

The formation of filopodia induced by Cdc42 is accompanied by phosphorylation of ERM proteins, and MRCK is a candidate for the kinase that phosphorylates ERM proteins at filopodia.
MBD2‐MBD3 complex binds to hemi‐methylated DNA and forms a complex containing DNMT1 at the replication foci in late S phase
Authors: Tatematsu Ken‐ichiro , Yamazaki Tetsu , Ishikawa Fuyuki
Published: March 2003

Background

In vertebrates and plants, DNA methylation is one of the major mechanisms regulating gene expression. Recently, a family of methyl‐CpG‐binding proteins has been identified, and some members, such as MeCP2 and MBD2, were shown to mediate gene repression by recruiting histone deacetylase complexes to methylated genes. However, the function of another member of this family, MBD3, remained elusive.

Results

It was shown that MBD2 and MBD3 form homo‐ and hetero‐dimers (or multimers) in vitro and in vivo. Significantly, the MBD2‐MBD3 complex showed an affinity to hemi‐methylated DNAs, a property that has never been reported with any member of the family proteins. MBD2 and MBD3 were co‐localized with DNMT1 at replication foci in 293 cell nuclei at late S phase. Moreover, by a co‐immunoprecipitation experiment, DNMT1 was shown to form a complex with MBD2 and MBD3. Finally, the abundance of MBD3 was highest in the late S phase when the DNMT1 is also most abundant, whereas the MBD2 level was largely constant throughout the cell cycle.

Conclusions

The results suggest that MBD3 may play an important role in the S phase. We hypothesize that the MBD2‐MBD3 complex recognizes hemi‐methylated DNA concurrent with DNA replication and recruits histone deacetylase complexes, as well as DNMT1, to establish and/or maintain the transcriptionally repressed chromatin.
Identification and characterization of human Wee1B, a new member of the Wee1 family of Cdk‐inhibitory kinases
Authors: Nakanishi Makoto , Ando Hitomi , Watanabe Nobumoto , Kitamura Kenzo , Ito Kenji , Okayama Hiroto , Miyamoto Tomomi , Agui Takashi , Sasaki Makoto
Published: March 2003

Background

In eukaryotic cells, the kinase activity of the mitosis‐promoting complex composed of cyclin B and Cdc2 (Cdk1) is negatively regulated by the phosphorylation of Cdk1 on threonine or tyrosine residues within its ATP binding domain.

Results

We identified human Wee1B by searching a sequence database. The predicted human Wee1B protein comprises 561 amino acids. Northern blot analysis revealed that human Wee1B mRNA is particularly abundant in testis. Interestingly, RT‐PCR using early embryos revealed that the Wee1B product was readily detectable at the mature oocyte, but abruptly disappeared at embryonic day 2.5, suggesting that the amount of Wee1B mRNA is dependent on the maternal expression. GFP‐Wee1B showed a predominantly nuclear localization in HeLa cells. Human Wee1B was able to rescue the lethal phenotype of the fission yeast wee1‐50Δmik1 mutant, and over‐expression of the human protein in these cells resulted in cell elongation as a result of arrest of the cell cycle at the G2‐M transition. Recombinant Wee1B effectively phosphorylated cyclin B‐associated Cdk1 on tyrosine‐15, resulting in an inactivation of the kinase activity of Cdk1.

Conclusion

We identified human Wee1B as a novel Cdk1‐inhibitory kinase. The identification of this new member of the Wee1 family suggests that inhibition of Cdk1 is mediated at multiple levels in mammals.
Membrane‐associated guanylate kinase with inverted orientation (MAGI)‐1/brain angiogenesis inhibitor 1‐associated protein (BAP1) as a scaffolding molecule for Rap small G protein GDP/GTP exchange protein at tight junctions
Authors: Mino Akihisa , Ohtsuka Toshihisa , Inoue Eiji , Takai Yoshimi
Published: March 2003

Membrane‐associated guanylate kinase (MAGUK) with inverted orientation (MAGI)‐1/brain angiogenesis inhibitor 1‐associated protein (BAP1), is a member of the MAGUK family that has multiple PDZ domains and interacts with many transmembrane proteins, including receptors and channels, through these domains. MAGI‐1/BAP1 is ubiquitously expressed and localized at tight junctions in epithelial cells. It is an isoform of the neurone‐specific synaptic scaffolding molecule (S‐SCAM), which is known to interact with NMDA receptors and neuroligins. We have recently found that S‐SCAM also interacts with a signalling molecule, a GDP/GTP exchange protein (GEP) that is specific for Rap1 small G protein, Rap GEP, which has also recently been referred to as RA‐GEF/PDZ‐GEFI/CNras‐GEF. In this study, we have examined whether MAGI‐1/BAP1 also interacts with and serves as a scaffolding molecule for Rap GEP at tight junctions in epithelial cells.

MAGI‐1/BAP1 similarly interacted with Rap GEP in cell‐free and intact cell systems. A Northern blot analysis revealed that Rap GEP was expressed in most tissues examined. However, neither postsynaptic density (PSD)‐95/synapse‐associated protein (SAP) 90 (another member of the MAGUK family) nor SAP97/human discs‐large tumour suppressor gene product (another ubiquitously expressed MAGUK localizing to adherens junctions in epithelial cells and the isoform of PSD‐95/SAP90) interacted with Rap GEP.

These results indicate that MAGI‐1/BAP1 serves as a scaffolding molecule for Rap GEP at tight junctions in epithelial cells.
E2F regulates growth‐dependent transcription of genes encoding both catalytic and regulatory subunits of mouse primase
Authors: Nishikawa Naoko S. , Izumi Masako , Yokoi Masayuki , Miyazawa Hiroshi , Hanaoka Fumio
Published: March 2003

DNA polymerase α‐primase is one of the principal enzymes involved in eukaryotic chromosomal DNA replication. Mouse DNA polymerase α‐primase consists of four subunits with molecular masses of 180, 68, 54 and 46 kDa. Protein and mRNA expression levels of the four subunits are up‐regulated in a coordinated manner in response to growth stimulation. We have previously analysed the transcription of the 180 kDa (p180) and 68 kDa (p68) subunits, which form the DNA polymerase catalytic complex, and found that growth‐dependent regulation of transcription of the mouse p180 and p68 genes is mediated by a common factor, E2F, while the basal transcription of the genes is regulated by different transcription factors.

We characterized the transcriptional regulation of the 54 kDa (p54) and 46 kDa (p46) subunits, which form the DNA primase catalytic complex. We isolated genomic clones spanning the 5′‐flanking regions of the p54 and p46 genes and showed, using transient expression and gel mobility shift assays, that the basal transcription of p54 is controlled by Sp1 and GA‐binding protein, as is the basal transcription of the p180 gene. The basal transcription of p46 is controlled by unknown factor(s) which were bound to the upstream sequence. The variant E2F sites close to the transcription initiation sites of the p54 and p46 genes had no basal promoter activity, but were essential for the growth‐dependent transcription of both genes.

The promoter regions of the four subunits of mouse DNA polymerase d‐primase complex share several common features. The coordinated transcription of all four subunits in response to growth stimulation appears to be controlled by E2F.
Involvement of RNase G in in vivo mRNA metabolism in Escherichia coli
Authors: Umitsuki Genryou , Wachi Masaaki , Takada Ayako , Hikichi Takafusa , Nagai Kazuo
Published: March 2003

Background

Escherichia coli rng gene (previously called cafA) encodes a novel RNase, named RNase G, which is involved in the 5′ end‐processing of 16S rRNA. In rng mutant cells, a precursor form of 16S rRNA, 16.3S rRNA, is accumulated. Here we report a role of RNase G in the in vivo mRNA metabolism.

Results

We found that rng::cat mutant strains overproduced a protein of about 100 kDa. N‐terminal amino acid sequencing of this protein showed that it was identical to the fermentative alcohol dehydrogenase, the product of the adhE gene located at 28 min on the E. coli genetic map. The level of adhE mRNA was significantly higher in the rng::cat mutant strain than that in its parental strain, while such differences were not seen in other genes we examined. A rifampicin‐chase experiment revealed that the half‐life of adhE mRNA was 2.5‐fold longer in the rng::cat disruptant than in the wild‐type.

Conclusion

These results indicate that, in addition to rRNA processing, RNase G is involved in in vivo mRNA degradation in E. coli.
Conformational switching of Escherichia coli RNA polymerase‐promoter binary complex is facilitated by elongation factor GreA and GreB
Authors: Sen Ranjan , Nagai Hiroki , Shimamoto Nobuo
Published: March 2003

Background

The initiation arrest at a modified λPR promoter is caused by irreversible divergence of the reaction pathway into productive and arrested branches. Escherichia coli GreA and GreB induce cleavage of the nascent transcript and relieve arrest in elongation. They also reduce abortive synthesis at several promoters and relieve initiation arrest. Their mechanism of action during initiation, and its relationship to the branched initiation pathway are unknown.

Results

The Gre factors mitigated initiation arrest only when they were added to the binary complex of the holoenzyme bound to the λPR promoter, prior to RNA synthesis. They exerted little effect when they were added to ternary initiation complexes. They accelerated the exchange of the binary complex with its free components by 6–9‐fold. When they are present, a high concentration of the initiating nucleotide increased yield of the full‐length transcript, whereas a low concentration did not.

Conclusions

All the results presented above can be explained by a model where the productive and arrested pathways diverge at the binary complex stage. The Gre factors relieve the initiation arrest by introducing reversibility between subspecies of the binary complex that are precursors of the two pathways. RNA cleavage is unlikely to cause relief of initiation arrest.
Terminal deoxynucleotidyltransferase directly interacts with a novel nuclear protein that is homologous to p65
Authors: Yamashita Nobuyuki , Shimazaki Noriko , Ibe Shiro , Kaneko Reo , Tanabe Akiko , Toyomoto Tsuguri , Fujita Kiyoko , Hasegawa Tomohiko , Toji Shingo , Tamai Katsuyuki , Yamamoto Hiroko , Koiwai Osamu
Published: March 2003

Background Terminal deoxynucleotidyltransferase (TdT) is a DNA polymerase that enhances Ig and TcR gene diversity in the N region in B‐ and T‐cells. TdT is found as a member of a large protein complex in the lysate of the thymocytes. To elucidate the molecular mechanism of the synthesis of the N region, we first attempted to isolate the genes with products that are interacting directly with TdT.

Results Using a yeast two‐hybrid system, we isolated a cDNA clone encoding a novel nuclear protein that interacts with TdT. This protein was designated as TdT interacting factor 1 (TdIF1). TdIF1 has a high degree of homology to the transcription factor p65, which belongs to the nuclear receptor superfamily. TdIF1 contains HMG‐I and HMG‐Y DNA binding domains (AT‐hooks) and can bind to single‐ and double‐stranded DNA. TdT and TdIF1 were co‐eluted at position 232 kDa by gel filtration of MOLT4 lysate. TdIF1 can enhance TdT activity fourfold in vitro assay system using oligo(dT)16 as primers.

Conclusions TdIF1 binds directly to TdT, both in vitro and in vivo. TdIF1 and TdT exist as the members of a 232 kDa protein complex. TdIF1 can enhance TdT activity maximum fourfold in vitro assay system, suggesting that it positively regulates the synthesis of the N region during V(D)J recombination in the Ig and TcR genes.
Pathways for repair of topoisomerase I covalent complexes in Saccharomyces cerevisiae
Authors: Pouliot Jeffrey J. , Robertson Carol A. , Nash Howard A.
Published: March 2003

Background The covalent linkage between DNA and the active site tyrosine of topoisomerase I can be stabilized by chemotherapeutic agents, adjacent DNA lesions, or mutational defects in the topoisomerase itself. Following collision with a replication fork, the covalent complex can be converted to a double‐strand break. Tdp1, an enzyme that can hydrolyse the bond between topoisomerase I and DNA, is thought to be involved in the repair of these lesions, but little is known about how such repair is accomplished.

Results Reaction kinetics with model substrates reveal that the catalytic efficiency of Saccharomyces cerevisiae Tdp1 is relatively poor when the scissile bond is located in the middle of a duplex, but much better when it is located at the end of a structure. Survival of yeast after induction of a toxic topoisomerase is substantially reduced by inactivation of the TDP1 gene. Comparison of survival of single and double mutants places TDP1 and RAD52 in the same epistasis group but TDP1 and RAD9 in different epistasis groups. In the absence of RAD9, inactivation of TDP1 has a significant effect on the survival of cells following exposure to camptothecin but is without consequence for the survival of agents that do not target topoisomerase I.

Conclusions Tdp1 acts as a specific repair enzyme for topoisomerase I lesions. Rather than working at their earliest occurrence, the enzyme acts after covalent complexes have been converted to DSBs. A second repair pathway also exists that functions independently of Tdp1 but requires RAD9 function to efficiently repair topoisomerase I‐linked DSBs. The efficiency of these pathways differs for complexes induced with the chemotherapeutic agent camptothecin vs. those accumulated by mutant forms of topoisomerase I.
A deubiquitinating enzyme, UCH/CeUBP130, has an essential role in the formation of a functional microtubule‐organizing centre (MTOC) during early cleavage in C. elegans
Authors: Lee Jungsoo , Jee Changhoon , Lee Jin Il , Lee Myon Hee , Lee Moon Hee , Koo Hyeon‐Sook , Chung Chin Ha , Ahnn Joohong
Published: March 2003

Background Deubiquitinating enzymes generate monomeric ubiquitin in protein degradation pathways and are known to be important for the early development in many organisms.

Results RNA interference experiments targeted for a UBP homologue, UCH/CeUBP130, in C. elegans resulted in cell division defective embryos. Immunostaining localized UCH/CeUBP130 in the sperm and at the microtubule‐organizing centre (MTOC) during early cleavage. Furthermore, the embryonic lethal phenotype was rescued by mating with wild‐type males.

Conclusions Since it is known that the MTOC in the fertilized embryo is contributed by sperm asters in C. elegans, we suggest that UCH/CeUBP130 and ubiquitin protein degradation pathways may be involved in microtubule‐based sperm aster formation. Therefore UCH/CeUBP130 is necessary for the formation of a functional MTOC in the fertilized embryo of C. elegans.