bluuebunny/crossref_metadata_2025 huggingface dataset:
Differences in seed germination responses may promote coexistence of four sympatric Piper species
Authors: Daws M. I. , Burslem D. F. R. P. , Crabtree L. M. , Kirkman P. , Mullins C. E. , Dalling J. W.
Published: March 2003

Species‐specific responses to the range of microsites resulting from canopy gap formation may contribute to coexistence in tropical forests. We investigated the effects of four factors affected by canopy gap formation (red : far‐red light, soil nitrate concentrations, soil temperature fluctuations and soil water potential) on the germination response of four pioneer Piper species (P. dilatatum, P. hispidum, P. marginatum and P. peltatum) that are typically found in canopy gaps and clearings.

 All four Piper species required light for maximum germination. However, the ratio of red : far‐red light (R : FR) resulting in maximum germination varied between species. Piper peltatum will germinate in simulated understorey light conditions; P. dilatatum and P. hispidum require conditions typically found in small to medium gaps; while P. marginatum requires the conditions appropriate to large gaps.

 Only P. marginatum was affected by nitrate concentration: elevated concentrations increased the germination rate. This suggests that this species could detect canopy gaps using a combination of high R : FR and elevated soil nitrate concentrations.

 The germination rate of P. marginatum was least sensitive to low water potentials and high daytime temperatures characteristic of large gaps. Piper peltatum was most sensitive to these treatments, while P. dilatatum and P. hispidum were intermediate in response.

 A principal components analysis of the ratios of germination in understorey to large gap conditions, for four variables, generated a significant axis that explained 88·5% of the variance in germination response between species. Differential species distribution along this axis, based on species‐specific responses, may allow germination to occur in the most suitable microsite for onward growth of the seedling and contribute to species coexistence by reducing interspecific competition.
Modelling unimodal flowering phenology with exponential sine equations
Authors: Malo J. E.
Published: March 2003

1. This paper presents a method for modelling unimodal flowering phenology based on fitting data to an exponential sine function with a minimum number of parameters.

2. Such parameters have the advantage of being direct surrogates for the most relevant features of any phenology (start, duration, intensity, skewness and length of the tails).

3. The use of the proposed function is exemplified using flowering data collected from populations of five Cistaceae species (four from the genus Cistus and one from Halimium).

4. The fitted models account for a large part (> 90%) of the variance in the data, and their parameters are easily interpreted in ecological terms.
Mussel valve discrimination and strategies used in valve discrimination by the Oystercatcher,Haematopus ostralegus
Authors: Nagarajan R. , Lea S. E. G. , Goss‐Custard J. D.
Published: March 2003

1. In 72% of a sample of common musselsMytilus edulisL. that had been attacked by ventrally hammering Oystercatchers,Haematopus ostralegusL., the mussel had been opened through the right valve rather than the left.

2. In a matched sample of unopened shells, 56·5% of mussels were ventrally thinner on the right. Mussel thickness differences were unrelated to overall mussel thickness, so this proportion should apply also to opened shells even though they are on average thinner than unopened shells.

3. Since Oystercatchers attack a higher proportion of mussels by the right valve than are actually thinner on the right, their preference cannot depend solely on detecting measurable differences in thickness.

4. The pattern of preference can best be explained if Oystercatchers detect and attack the thinner valve in the mussel when the thickness difference between the two valves is more than a threshold of 0·036 mm, and otherwise always attack the right valve. This strategy would save 15·5% of the blows the birds would make if they attacked shells without regard to differences in valve thickness.

5. The improvement in the overall net energy intake rate that could be achieved by such a valve thickness discrimination strategy was 3·6%.
A trade‐off between clutch size and incubation efficiency in the Barn Swallow Hirundo rustica
Authors: Engstrand S. M. , Bryant D. M.
Published: March 2003

To investigate reproductive costs associated specifically with incubation for a single‐sex intermittent incubator, clutches of the Barn Swallow (Hirundo rustica Linnaeus) were manipulated in successive trios, involving reduced (n − 2), control (n) and enlarged (n + 2) clutch sizes (n is the natural clutch). All manipulated clutches were within the natural range.

Manipulations were made shortly after clutch completion and reversed immediately prior to hatching, so the costs of incubation were examined in isolation while the demands of egg‐laying and nestling rearing were maintained at natural levels.

The efficiency of incubation was dependent on clutch size: the period from laying to hatch was shortest for those incubating reduced (R), and longest for birds incubating enlarged (E) clutches (R: 14·8 ± 0·1 days, n = 28; control (C): 15·3 ± 0·3, n = 24; E: 15·6 ± 0·2 days, n = 28). Also, hatching success was higher amongst R (92%) than either C (85%) or E (81%) clutches.

There was no evidence of an incubation cost due to clutch size in terms of parental body condition, provisioning of nestlings, or in the time interval before a second clutch.

No difference was found in female nest attendance behaviour or in the decline in egg density (which could reflect water loss) according to our clutch manipulations, indicating that other factors probably underlie the effects observed.

It was shown that the number of eggs being incubated affects components of fitness in Barn Swallows, independently of laying and chick‐rearing costs. Intraseasonal costs of incubation included prolonged egg development and reduced hatching success among larger clutches but did not affect nestling growth or interclutch intervals. We suggest that an inability to maintain optimal temperature for egg development is likely to underlie the observed responses to clutch size manipulations.
Influence of venom availability on a spider's prey‐choice behaviour
Authors: Wullschleger B. , Nentwig W.
Published: March 2003

A neotropical hunting spider, Cupiennius salei, was studied to test whether spiders optimize venom use.

A prey‐choice experiment was performed with several cockroach species. These easily accepted prey species are especially suitable for such an experiment because the LD50 varies over a range of 40. Cupiennius salei spiders had either full venom glands (control) or their venom glands were experimentally emptied by electrical milking or by allowing them to bite into a series of crickets.

Two pairs of cockroaches were used: Blatta orientalis (LD50: 0·4 nl mg−1) vs Nauphoeta cinerea (LD50: 17·5 nl mg−1) and Periplaneta australasiae (LD50: 0·75 nl mg−1) vs Periplaneta americana (LD50: 10 nl mg−1). These pairs provide a difference of 40‐fold and 10‐fold, respectively, in venom sensitivity. Cupiennius salei preferred B. orientalis significantly over N. cinerea when the venom glands were empty, but not when the full amount of venom was available. No effect was evident for the other pair, indicating that a factor of approximately 10 is too low.

We conclude that the spiders know how much venom is available in their venom glands and that they select prey items that are appropriate for the amount of venom available.
Root longevity and phenology differences between two co‐occurring savanna bunchgrasses with different leaf habits
Authors: West J. B. , Espeleta J. F. , Donovan L. A.
Published: March 2003

Aristida strictaandSchizachyrium scopariumare C4perennial bunchgrass species native toPinus palustrissavannas in the south‐eastern USA. Species differences in growth rate, tissue nutrient content and distribution suggest thatA. strictais more stress‐tolerant (sensuGrime) thanS. scoparium. In addition,A. strictaretains leaves all year, whereasS. scopariumis winter deciduous. Based on these observations, we wanted to determine (1) whether root longevity was higher forA. strictaas compared toS. scoparium; and (2) whether the leaf habits of these species had consequences for seasonal patterns of root production and death.

Usingin siturhizotrons, we recorded root number and length of these species over the course of 2·5 years on flat, transparent 50 × 50 cm windows. Kaplan–Meier (product‐limit) analysis was used to produce survival functions and to estimate median lifespans for the right‐censored root longevity data. These survival functions were compared using log‐rank χ2statistics, and a proportional hazards model was used to examine the effects of season and species on the survival of each cohort. Final demographic variables were analysed usinganova.

The median lifespan ofA. strictawas 777 days whereas that ofS. scopariumwas 374 days. This significant difference in median lifespan was consistent between species for all cohorts in both years. Also, coincident with winter leaf senescence, root production nearly stopped forS. scopariumwhereasA. strictaproduced roots all year. This is probably due to the winter photosynthetic capacity ofA. stricta. Root death did not exhibit any seasonal pattern in either species. A pulse of roots produced in apparent response to the 2000 drought was evident forS. scopariumbut not forA. stricta. This cohort, however, rapidly died during the dormant season, probably due to the lack of winter photosynthesis in that species.

Aristida strictaandS. scopariumexhibited root production, death and longevity patterns that were predicted on the basis of ecological and leaf habit differences between them. These findings suggest species‐specific differences in performance and element cycling in these nutrient‐poor soils.
Leaf‐size divergence along rainfall and soil‐nutrient gradients: is the method of size reduction common among clades?
Authors: MDonald P. G. , Fonseca C. R. , Overton J. McC. , Westoby M.
Published: March 2003

Ecologists have long recognized that plants occurring in areas of low rainfall or soil nutrients tend to have smaller leaves than those in more favourable regions.

Working with a large data set (690 species at 47 sites spread widely through south‐east Australia) for which this reduction has been described previously, we investigated the morphology of leaf size reduction, asking whether any patterns observed were consistent across evolutionary lineages or between environmental gradients.

Leaf length, width and surface areas were measured; leaf traits such as pubescence or lobing were also scored qualitatively. There was no correlation between soil phosphorus and rainfall across sites. Further, there was no evidence that pubescence, lobing or other traits assessed served as alternatives to reduction of leaf size at the low ends of either environmental gradient.

Leaf size reduction occurred through many combinations of change in leaf width and length, even within lineages. Thus consistent patterns in the method of leaf size reduction were not found, although broad similarities between rainfall and soil P gradients were apparent.
Sample size and the estimation of physiological parameters in the field
Authors: McNab B. K.
Published: March 2003

Estimates of basal rate of metabolism in fruit pigeons and flying foxes, derived from the mean of individual means, were compared to the mean basal rate obtained by pooling all data into a single estimate.

Pooled estimates of basal rate differ by ≤1% of the basal rates obtained from the mean of individual means in 7 of 10 species of pigeons and 4 of 11 species of flying foxes.

The coefficient of variation of the mean of individual mean basal rates decreased with an increase in the mean number of measurements per individual, which, given that the number of measurements per individual was 8·4 in pigeons and 3·8 in flying foxes, partially accounts for the smaller coefficients in pigeons.

The maximal absolute difference between individual mean basal rates and the mean of individual means within a species decreased with an increase in the number of measurements per individual to 8, beyond which the difference remained constant.

Repeated measurements on a single individual usually gave an estimate of basal rate within 2–8% of the species’ mean when eight or more measurements were made.

. The analysis of limited samples most often occurs in the study of elusive or endangered species, when the availability of individuals for study is greatly limited.
Large scale isolation of osteoclast‐specific genes by an improved method involving the preparation of a subtracted cDNA library
Authors: Kobori Masato , Ikeda Yasushi , Nara Hiromi , Kato Miyuki , Kumegawa Masayoshi , Nojima Hiroshi , Kawashima Hiroyuki
Published: March 2003

Background

Osteoclasts play crucial roles in bone resorption, which triggers bone remodeling. Molecular mechanisms underlying these osteoclast‐specific biological functions remain elusive because only a limited number of osteoclast‐specific genes have been identified. To circumvent this, we isolated a large number of osteoclast‐specific genes by preparing a subtracted cDNA library of high quality.

Results

We first constructed a plasmid expression vector (pAP3neo) that allowed an efficient subtraction. Then, we improved the standard protocols for preparation of the cDNA library and the subsequent subtraction procedure. Using our protocol, we prepared a rabbit osteoclast cDNA library of high complexity. Subsequently, we prepared an osteoclast‐specific cDNA library of high complexity by subtracting it with biotin‐labelled mRNA, derived from rabbit spleen through the biotin–avidin method. The resulting library included a high proportion of full‐length cDNA inserts. Using DNA dot blot analysis, we found that the osteoclast‐specific cDNA clones were highly enriched in this subtracted cDNA library, i.e. nearly 70% of the analysed clones were primarily detected in osteoclasts but not in spleen. Multiple‐tissue Northern analysis also showed that many of these clones were expressed almost exclusively in osteoclasts. DNA sequencing of randomly selected clones showed that 424 cDNA species out of 1136 analysed were novel. DNA sequencing also showed that our subtracted cDNA library was almost equalized, suggesting that the library may contain almost all of the osteoclast‐specific genes.

Conclusion

From these data, we conclude that our subtraction protocols, and the subsequent procedure for the analysis of the isolated clones developed here, are useful for the comprehensive isolation and identification of transcriptionally up‐ or down‐regulated genes in general.
Transcription factor CP2 is essential for lens‐specific expression of the chicken αA‐crystallin gene
Authors: Murata Takuya , Nitta Masahiro , Yasuda Kunio
Published: March 2003

Background:

Lens‐specific transcriptional activation of the chicken αA‐crystallin gene is controlled by the distal and proximal enhancers, αCE1 and αCE2, respectively. Analysis using specific monoclonal antibodies against purified αCE1‐binding factor αCEF1 revealed that αCEF1 is composed of two distinct subunits.

Results:

We have demonstrated that one of the subunits of αCEF1 is encoded by chicken ubiquitous transcription factor CP2 (cCP2), which is homologous to mouse CP2, and human CP2/LBP‐1/LSF‐1. Electrophoretic mobility shift assays and cross‐linking experiments showed that αCEF1 and bacterially expressed cCP2 form a tetramer. Overexpression of cCP2 activates transcription through αCE1, but a mutant cCP2 lacking the DNA‐binding domain reduced the transcription to basal levels. Although cCP2 binds to the CP2 template from the mouse α‐globin promoter, it fails to promote transcription through this template. Element substitution experiments between αCE1 and the CP2 template revealed that the lens‐specific enhancer activity of αCE1 is due to the 6 bp sequence (−139/−134; lens‐specific element (LSE)) adjacent to the 3′ of the cCP2 binding site within αCE1.

Conclusion:

We have shown that the tetrameric transcription factor cCP2 is essential for lens‐specific transcription of the chicken αA‐crystallin gene, although it is ubiquitously expressed. We propose a model where cCP2 cooperates with a putative lens‐specific factor which binds to LSE. Fig. 6.
Modulation of the nucleoid, the transcription apparatus, and the translation machinery in bacteria for stationary phase survival
Authors: Ishihama Akira
Published: March 2003

Upon sensing an impending saturation level of their population density, Escherichia coli cells enter into the stationary phase. We have identified structural and functional modulations of the nucleoid, the transcription apparatus and the translation machinery occurring during the transition from exponential growth to stationary phase. The major DNA‐binding proteins, Fis, HU and Hfq, in the exponential‐phase nucleoid are replaced by a single stationary‐phase protein Dps, thereby compacting the nucleoid and ultimately leading to silencing of the DNA functions. The transcription apparatus is modified by replacing the major promoter recognition subunit, σ70, with σS. A stationary‐phase protein, Rsd (Regulator of Sigma D), with the binding activity of σ70 is involved in the efficient replacement of σ and/or the storage of unused σ70. Changes in cytoplasmic composition also differentially influence the activity of Eσ70 and EσS holoenzymes. Together, these effects may result in the preferential transcription of stationary‐phase specific genes. The translation machinery is also modulated in stationary phase, by the formation of translationally incompetent 100S ribosomes. A small stationary‐phase protein, RMF (Ribosome Modulation Factor), is involved in the dimerization of 70S ribosome monomers.
Molecular cloning and characterization of a novel member of the MAP kinase superfamily
Authors: Miyata Yoshihiko , Akashi Makoto , Nishida Eisuke
Published: March 2003

Background

Members of the MAP kinase superfamily play important roles in a wide variety of signal transduction pathways, and several members have been identified. However, the diversity and complexity of cellular responses in mammalian systems may imply existence of hitherto unidentified members of the MAP kinase superfamily.

Results

We report the molecular cloning and characterization of a novel member of the MAP kinase superfamily. We isolated full‐length mouse and human cDNAs that encode complete open reading frames of a novel protein kinase, termed MOK. MOK consists of 419 (human) and 420 (mouse) amino acids, with a calculated molecular weight of 48kDa. MOK contains all of the protein serine/threonine kinase consensus motifs and shows a modest similarity to members of the MAP kinase superfamily and MAK and MAK‐related kinase (MRK). In addition, MOK possesses a Thr‐Glu‐Tyr (TEY) motif in the activation loop domain, as do classical MAP kinases. MOK is widely expressed in normal tissues and organs and localizes to the cytoplasm. MOK is able to phosphorylate several known MAP kinase substrates and to undergo autophosphorylation. A mutation in the TEY motif to AEF abolished the kinase activity of MOK, and the treatment of cells with a phosphatase inhibitor, okadaic acid, enhanced the kinase activity of MOK, suggesting the existence of an upstream kinase. Phorbol ester TPA was found to stimulate the kinase activity of MOK, whereas serum stimulation, osmotic shock, or anisomycin treatment did not significantly activate MOK.

Conclusion

These results indicate that MOK is distantly related to members of known subfamilies of the MAP kinase superfamily and can therefore be classified as a novel member.
Hyperosmotic stress‐induced reorganization of actin bundles in Dictyostelium cells over‐expressing cofilin
Authors: Aizawa Hiroyuki , Katadae Maiko , Maruya Mikako , Sameshima Masazumi , Murakami‐Murofushi Kimiko , Yahara Ichiro
Published: March 2003

Background

Cofilin is a low‐molecular weight actin‐modulating protein, which binds to, severs, and depolymerizes actin filaments in vitro. Aip1, an actin‐interacting protein, was recently identified as a product of a gene on a multicopy plasmid which suppresses the temperature‐sensitive phenotype of a cofilin mutant in Saccharomyces cerevisiae. Actin cytoskeleton plays an essential role in resistance to hyperosmotic stress in Dictyostelium discoideum. The roles of cofilin and Aip1 in this resistance are not known.

Results

In response to hyperosmotic stress, D. discoideum cells round up. This stress‐induced morphological change involves the redistribution of cofilin, together with actin filaments, into cortical contractile portions of the cells, followed by their contraction. Over‐expression of cofilin increases and thickens cortical actin bundles in cells. The bundles become tight and are reorganized into a ring‐shaped structure in response to hyperosmotic stress. The ring structure of actin bundles had two characteristic bands across them; bright and dark bands, heavily stained and not stained with phalloidin. In the bundles, straight filaments with a diameter of 5.3‐nm were aligned parallel by cross‐bridge structures. In cells lacking the myosin‐II heavy chain, the bundles, which were induced by an over‐expression of cofilin, shortened and became straight following hyperosmotic stress, forming a polygonal structure. D. discoideum Aip1/Wrp2 enhanced the severing of actin filaments by cofilin in vitro and colocalized with cofilin in cells, including those that were over‐expressing cofilin before and after exposure to hyperosmotic stress.

Conclusions

Cofilin plays a pivotal role in concert with Aip1/Wrp2 in the reorganization of actin architectures into bundles that contract in a myosin‐II‐independent manner, in response to hyperosmotic stress.
Multiple mammalian proteasomal ATPases, but not proteasome itself, are associated with TATA‐binding protein and a novel transcriptional activator, TIP120
Authors: Makino Yasutaka , Yoshida Tatsushi , Yogosawa Shingo , Tanaka Keiji , Muramatsu Masami , Tamura Taka‐aki
Published: March 2003

Background

SUG1 belongs to proteasomal ATPase. Previous studies have demonstrated that SUG1 is associated with TBP. It is assumed to be involved in transcriptional regulation in addition to proteolysis. In this study, we investigated the association of mammalian SUG1 with TBP in more detail.

Results

Pull‐down experiments with TBP revealed multiple TBP‐interacting proteins (TIPs) that were recovered dependent upon the presence of C‐terminal conserved domain of TBP. By 2‐D electrophoresis, we identified SUG1 in TIPs. By using far‐Western analysis, we identified two proteins that could directly bind to TBP: SUG1 and another proteasomal ATPase (S4). Protein microsequencing and Western blotting identified all the remaining proteasomal ATPases (MSS1, TBP1, TBP7, and SUG2) in the TIP preparations. We present evidence that TBP and at least SUG1, MSS1, and S4 form a complex in the cell. However, no evidence of association of TBP with the 26S proteasome or its 19S regulatory unit was obtained. The molecular mass of the TBP/ATPases‐complex, which also included a novel transcription regulatory factor, TIP120, was estimated to be ≈800 kDa.

Conclusion

These results suggest that there is a novel multisubunit complex containing TBP and proteasomal ATPases. Based on our findings, we hypothesize that proteasomal ATPases are involved in transcriptional regulation in addition to proteolysis.
Rho‐dependence of Schizosaccharomyces pombe Pck2
Authors: Sayers Lee G. , Katayama Satoshi , Nakano Kentaro , Mellor Harry , Mabuchi Issei , Toda Takashi , Parker Peter J.
Published: March 2003

Background

In metazoans, the HR1 domain, a motif found in a number of proteins including the protein kinase C‐related PRKs, is responsible for an interaction with Rho‐GTPases. The structural similarity between the Schizosaccaromyces pombe Pck proteins and the mammalian Rho‐dependent protein kinase C‐related family, has led us to investigate the relationship between the function of Rho and that of Pck1/2.

Results

Rho1 is shown to interact with the conserved N‐terminal HR1 domain of Pck1/2 in vitro and in vivo. Lethal overproduction of Rho1 is neutralized by co‐expression of the Pck2 HR1 domain, which by itself compromises growth when overproduced. The Pck2–Rho1 interaction has a profound effect on the steady state expression of Pck2 and this is shown to parallel the immunoprecipitated activity and phosphorylation of Pck2 at its activation loop site. It is further shown that Pck2 becomes localized at the septum, where Rho1 is also located.

Conclusions

The results demonstrate that the Pck proteins are Rho1 effectors in fission yeast and that the HR1 domain is a universal motif for the Rho‐GTPase interaction. Furthermore, the evidence supports the contention that the yeast Pck1 and Pck2 proteins are primitive protein kinases, which in vertebrates have evolved into the two distinct PKC and PRK families.
Regulation of interaction of the acetyltransferase region of p300 and the DNA‐binding domain of Sp1 on and through DNA binding
Authors: Suzuki Toru , Kimura Akatsuki , Nagai Ryozo , Horikoshi Masami
Published: March 2003

Background

The coactivator p300 acts as a transcriptional adaptor for many DNA‐binding activators. The finding that p300 possesses intrinsic acetyltransferase activity which, by chemically modifying histone tails affects the nucleosomal environment and transcription, has greatly advanced our understanding of its function. Subsequent recent studies have shown that non‐histone proteins are also acetylated. However, one central question which has remained unanswered is how the coactivator/acetyltransferase interacts with DNA‐binding activators to modulate their actions.

Results

Here we have demonstrated physical and functional interaction between the acetyltransferase region of p300 and the DNA‐binding domain (DBD) of the transcription factor Sp1. This interaction stimulates DNA binding by the DBD of Sp1, which is mediated primarily by physical interaction rather than acetylation, despite acetylation of the DBD of Sp1 by the acetyltransferase region of p300. Furthermore, DNA binding by the DBD of Sp1 inhibits both its association and acetylation by the acetyltransferase region of p300.

Conclusions

These findings suggest a new role for p300 in regulating promoter access by DNA‐binding activators through multiple regulatory interactions.
Cell cycle mechanisms of sister chromatid separation; Roles of Cut1/separin and Cut2/securin
Authors: Yanagida Mitsuhiro
Published: March 2003

The correct transmission of chromosomes from mother to daughter cells is fundamental for genetic inheritance. Separation and segregation of sister chromatids in growing cells occurs in the cell cycle stage called ‘anaphase’. The basic process of sister chromatid separation is similar in all eukaryotes: many gene products required are conserved. In this review, the roles of two proteins essential for the onset of anaphase in fission yeast, Cut2/securin and Cut1/separin, are discussed with regard to cell cycle regulation, and compared with the postulated roles of homologous proteins in other organisms. Securin, like mitotic cyclins, is the target of the anaphase promoting complex (APC)/cyclosome and is polyubiquitinated before destruction in a manner dependent upon the destruction sequence. The anaphase never occurs properly in the absence of securin destruction. In human cells, securin is an oncogene. Separin is a large protein (MW ≈180 kDa), the C‐terminus of which is conserved, and is thought to be inhibited by association with securin at the nonconserved N‐terminus. In the budding yeast, Esp1/separin is thought to be a component of proteolysis against Scc1, an essential subunit of cohesin which is thought to link duplicated sister chromatids up to the anaphase. Whether fission yeast Cut1/separin is also implicated in proteolysis of cohesin is discussed.
Temporal and selective association of multiple sigma factors with RNA polymerase during sporulation in Bacillus subtilis
Authors: Fujita Masaya
Published: March 2003

Background

During sporulation in Bacillus subtilis, an asymmetric division produces two cells, a forespore and mother cell, with which follow different developmental paths. The highly ordered programme of temporal and spatial gene activation during sporulation is governed by the principal RNA polymerase holoenzyme (EσA) and alternative holoenzyme forms containing the developmental sigma factors σH, σF, σE, σG and σK, which appear successively during development. The control mechanism(s) of temporal and selective association of multiple sigma factors with core RNA polymerase is unclear. As a first step to addressing these issues, this report quantifies the amount of each subunit of RNA polymerase that is present in the sporangium during sporulation, and analyses in vitro the relative affinities of each sigma subunit for core RNA polymerase.

Results

Using quantitative immunoblot analysis, the amounts of EσA, EσH, EσE and EσK in relation to the total amount of RNA polymerase at appropriate time‐points were found to be 15%, 1%, 6% and 2%, respectively. Therefore, the core RNA polymerase is predicted to be in excess. The level of core RNA polymerase and σA remained constant during the transition from vegetative growth to sporulation, whereas the sporulation‐specific sigma factors appeared successively, in the order σH, σE and σK. Competition experiments between sigma factors in an in vitro transcription system revealed the dominance of σA over σH and σE for open promoter complex formation. These results are inconsistent with the idea that late appearing sigma factors can displace earlier appearing sigmas from the core enzyme.

Conclusions

As the core RNA polymerase is in excess, the results suggest that successive sigma factors can bind to core RNA polymerase without having to displace earlier appearing sigma factors. Thus, the programme of gene expression during sporulation might not require mechanisms for the substitution of one sigma factor by another on the core RNA polymerase.
MSSP promotes ras/myc cooperative cell transforming activity by binding to c‐Myc
Authors: Niki Takeshi , Izumi Sachiyo , Saëgusa Yumiko , Taira Takahiro , Takai Toshiki , Iguchi‐Ariga Sanae M. M. , Ariga Hiroyoshi
Published: March 2003

Background

MSSPs, mycsingle
strand binding
proteins, were originally identified as proteins recognizing a putative replication origin/transcriptional enhancer in the human c‐Myc gene. The cDNAs encoding four of the family proteins, MSSP‐1, MSSP‐2, Scr2 and Scr3, were cloned. These proteins carry two copies of the putative RNA binding domains, RNP‐A and RNP‐B, and have been suggested to participate in DNA replication and cell cycle progression from the G1 to the S phase.

Results

We report that MSSP‐1 and MSSP‐2 bound directly to the C‐terminal portion of c‐Myc, along with Max, side by side. MSSP, c‐Myc and Max formed a ternary complex in vivo, although MSSP did not directly associate with Max. The MSSP/Myc/Max ternary complex lost the binding activity to the E‐box sequence—the recognition sequence of c‐Myc/Max complex—thereby abrogating the E‐box‐dependent transcription activity of c‐Myc. MSSP specifically stimulated the cooperative transforming activity of c‐myc with ras, in a manner dependent upon the RNP sequences, while mssp itself showed no transforming activity in mouse NIH3T3 cells. The NIH3T3 transformants, together with ras, myc and mssp, grew to form very large colonies in soft agar, as compared to those with ras plus myc or ras alone.

Conclusions

MSSP is a modulator of c‐Myc and the c‐Myc/MSSP complex may deregulate cell cycle controls and lead cells towards transforming pathways.
Bidirectional migration of SeqA‐bound hemimethylated DNA clusters and pairing of oriC copies in Escherichia coli
Authors: Hiraga Sota , Ichinose Chiyome , Onogi Toshinari , Niki Hironori , Yamazoe Mitsuyoshi
Published: March 2003

Background

We previously found that SeqA protein, which binds preferentially to newly replicated hemimethylated DNA, is localized as discrete fluorescent foci in Escherichia coli cells. A single SeqA focus, localized at midcell, separates into two foci and these foci migrate abruptly in opposite directions.

Results

The present study shows that (i) appearance of SeqA foci depends on continuous DNA replication, suggesting that the SeqA foci represent clusters consisting of SeqA and newly replicated hemimethylated DNA, (ii) in a synchronous round of replication, a single SeqA focus at midcell separates into two foci and these foci abruptly migrate in opposite directions midway through replication from oriC to the terminus, and (iii) oriC is replicated at midcell but replicated oriC copies remain linked with each other at midcell for 40 min after replication at 30 °C. Subsequently, the linked oriC copies separate and migrate gradually towards both borders of the nucleoid before cell division.

Conclusions

A single cluster of SeqA‐bound hemimethylated DNA segment separates into two clusters and these clusters migrate abruptly in a bipolar fashion during progress of replication and prior to separation of linked sister oriC copies.
Targeted disruption of the homeobox transcription factor Bapx1 results in lethal skeletal dysplasia with asplenia and gastroduodenal malformation
Authors: Akazawa Hiroshi , Komuro Issei , Sugitani Yoshinobu , Yazaki Yoshio , Nagai Ryozo , Noda Tetsuo
Published: March 2003

Background

NK homeobox genes have been shown to play important roles in cell‐type specification and organogenesis. Murine Bapx1, a member of NK homeobox gene family, is expressed in all the cartilageous tissues that undergo endochondral bone formation, and in gut mesentery during embryogenesis, suggesting that Bapx1 may be a key transcription factor ragulating the development of these organs.

Results

We generated Bapx1‐deficient mice by gene targeting. Bapx1−/− mice exhibited lethal skeletal dysplasia, with abnormal development of the vertebral column and some craniofacial bones, accompanied with asplenia and gastroduodenal malformation. We showed that the proliferative activity of the sclerotome cells, forming the vertebral column, was significantly reduced in Bapx1−/− embryos. The sclerotome cells of the mutants appeared to migrate and condense normally, but subsequent differentiation into the mature vertebral bodies and intervertebral discs were affected. The sclerotome cells in the vertebral bodies failed to differentiate into hypertrophic chondrocytes, as revealed by the undetected expression of Col10a1 and Osteopontin, and the sclerotome cells in the intervertebral discs failed to express the typical extracellular matrix proteins Col2a1, Col9a2 and aggrecan. Furthermore, we investigated the effect of loss of Bapx1 on the expression of some transcription factors, identified to be expressed in the developing sclerotome and be required for normal development of the vertebral column. Among them, we found that the expression of MFH‐1 (mesenchyme forkhead‐1), which was reported to regulate the proliferation and differentiation of sclerotome cells, was significantly reduced in ventromedial sclerotome cells in Bapx1−/− mice.

Conclusion

Our analysis provided evidence that Bapx1 was indispensable for normal development of ventromedial structure of vertebral column and some of craniofacial bones, splenogenesis and morphogenesis of gastroduodenal tract.
Transactivation mechanisms of mouse clock transcription factors, mClock and mArnt3
Authors: Takahata Sho , Ozaki Takahiro , Mimura Junsei , Kikuchi Yasuo , Sogawa Kazuhiro , Fujii‐Kuriyama Yoshiaki
Published: March 2003

Background

The Arnt3 (also termed as BMAL1 or MOP3)/Clock heterodimer is a positive regulator of circadian rhythm and activates the transcription of target genes such as per1 and vasopressin.

Results

We investigated the transcriptional mechanism of mArnt3/mClock heterodimer. While mClock did not possess any distinct activation domain, mArnt3 contained a transcriptional activation domain at the most C‐terminal end, the activity of which was not expressed, even in the one hybrid system, until it was bound by mClock. It has been suggested that mClock plays a regulatory or structural role in exerting a transcription enhancing effect of the mArnt3/mClock heterodimer. Deletion proceeding from amino acids 559–492 of mClock markedly reduced the transactivation activity of mArnt3/mClock heterodimer, in consistence with the results of the Clock‐Δ19 mutant. Yeast and mammalian two‐hybrid systems revealed that CBP and p300 interacted with mArnt3 via the CREB binding domain. The In vivo interaction between mArnt3 and CBP was confirmed by the GST pull down assay.

Conclusion

Taken together, these results suggest that the mArnt3/mClock heterodimer exerted its transactivation activity via CBP or p300 interacting with mArnt3 in the heterodimer with mClock playing a structural or regulatory role in the transactivation process.
Extracellular matrix tenascin‐X in combination with vascular endothelial growth factor B enhances endothelial cell proliferation
Authors: Ikuta Tomoki , Ariga Hiroyoshi , Matsumoto Ken‐ichi
Published: March 2003

An extracellular matrix tenascin‐X (TNX) is highly expressed in muscular tissues, especially heart and skeletal muscle, and is also prominent around blood vessels. The precise in vivo role of TNX remains to be elucidated. To identify proteins that interact with TNX in the extracellular environment, we searched for TNX‐binding proteins using a yeast two‐hybrid system.

We used mouse TNX‐specific fibronectin type III repeats (mTNX/FNIII13‐25) as a bait for the screening. We found that vascular endothelial growth factor B (VEGF‐B) binds to mTNX/FNIII13‐25. This interaction was confirmed by pull‐down assays and co‐immunoprecipitation assays. The full‐length mTNX, as well as mTNX/FNIII13‐25, interacted with both alternative splice isoforms VEGF‐B186 and VEGF‐B167. Furthermore, the full‐length mTNX also bound to VEGF‐A. The minimal region of TNX that interacts with VEGF‐B was mapped to the FNIII repeats (FNIII13‐25) but not to the other characteristic domains of TNX. The TNX‐binding site of VEGF‐B was located in the N‐terminal 115‐amino acid region. mTNX/FNIII13‐25 did not prevent the interaction of VEGF‐B with VEGFR‐1 (VEGF receptor 1), and VEGF‐B could simultaneously bind to both mTNX/FNIII13‐25 and VEGFR‐1. A conditioned medium from transfected 293T cells coexpressing full‐length TNX and VEGF‐B could promote DNA synthesis in bovine endothelial cells in which VEGFR‐1 were expressed. VEGFR‐1 phosphorylation triggered by VEGF‐B186 were increased in cells plated with mTNX/FNIII13‐25 or full‐length mTNX, compared with cells plated with VEGF‐B186 alone.

TNX interacts with VEGF‐B and enhances the ability of VEGF‐B to stimulate cell proliferation. This enhanced mitogenecity is caused by increased signals mediated by the VEGFR‐1 receptor. This finding suggests a role for TNX in the regulation of the development of blood vessels such as vasculogenesis and angiogenesis.
Requirement of Runx1/AML1/PEBP2αB for the generation of haematopoietic cells from endothelial cells
Authors: Yokomizo Tomomasa , Ogawa Minetaro , Osato Motomi , Kanno Tomohiko , Yoshida Hisahiro , Fujimoto Tetsuhiro , Fraser Stuart , Nishikawa Satomi , Okada Hitoshi , Satake Masanobu , Noda Tetsuo , Nishikawa Shin‐Ichi , Ito Yoshiaki
Published: March 2003

Recent studies revealing that endothelial cells derived from E8.5‐E10.5 mouse embryos give rise to haematopoietic cells appear to correspond to previous histological observations that haematopoietic cell clusters are attached to the ventral aspect of dorsal aorta in such a way as if they were budding from the endothelial cell layer. Gene disruption studies have revealed that Runx1/AML1 is required for definitive haematopoiesis but not for primitive haematopoiesis, but the precise stage of gene function is not yet known.

We found that mice deficient in Runx1/AML1 (an α subunit of the transcription factor PEBP2/CBF) lack c‐Kit+ haematopoietic cell clusters in the dorsal aorta, omphalomesenteric and umbilical arteries, as well as yolk sac vessels. Moreover, endothelial cells sorted from the embryo proper and the yolk sac of AML1–/– embryos are unable to differentiate into haematopoietic cells on OP9 stromal cells, whereas colonies of AML1–/– endothelial cells can be formed in culture.

These results strongly suggest that the emergence of haematopoietic cells from endothelial cells represents a major pathway of definitive haematopoiesis and is an event that also occurs in the yolk sac in vivo, as suggested by earlier in vitro experiments.
Sequence‐specific termination by T7 RNA polymerase requires formation of paused conformation prior to the point of RNA release
Authors: Song Hoseok , Kang Changwon
Published: March 2003

Background

The sequence‐specific, hairpin‐independent termination signal for the bacteriophage RNA polymerases in Escherichia coli rrnB t1 terminator consists of two modules. The upstream module includes the conserved sequence and the downstream one is U‐rich.

Results

Elongation complexes of T7 RNA polymerase paused 2 bp before reaching the termination site at a 500 µm concentration of NTP. At 5–50 µm NTP, however, they paused and terminated there or resumed elongation beyond the termination site. Only at higher concentrations of NTP (500 µm), the pause complex proceeded slowly to and became incompetent at the termination site. At 4 bp or more before the termination site, the unprotected single‐stranded region of transcription bubble shrank at the trailing edge to 4–5 bp from ≈10 bp, resulting from duplex formation of the conserved sequence. The pause and bubble collapse were not observed with an inactive mutant of the termination signal.

Conclusion

Sequence‐specific termination requires the slow elongation mode of paused conformation, working only at high concentrations of NTP for a few bp prior to the RNA release site. The collapse of bubble that was observed several base pairs before the termination site and/or the resulting duplex might subsequently lead to the paused conformation of T7 elongation complexes.
Developmental roles and molecular characterization of a Drosophila homologue of Arabidopsis Argonaute1, the founder of a novel gene superfamily
Authors: Kataoka Youhei , Takeichi Masatoshi , Uemura Tadashi
Published: March 2003

Background

Arabidopsis Argonaute1 (AGO1) is the founder of a novel gene superfamily that is conserved from fission yeasts to humans. AGO1, and several other members of this superfamily are necessary for stem cell renewal or RNA interference. However, little has been reported about their roles in animal development or about the molecular activities of any of the members.

Results

We have isolated a Drosophila homologue of AGO1, dAGO1, in our attempt to search genetically for regulators of Wingless (Wg) signal transduction. dAGO1 is broadly expressed in the embryo and the imaginal disc. dAGO1 over‐expression at wing margins suggested that it behaves as a positive regulator in the genetic background employed. Loss‐of‐function mutations of dAGO1, unexpectedly, did not give typical segment polarity phenotypes of the wg class; instead, dAGO1 maternal and zygotic mutant embryos showed developmental defects, with malformation of the nervous system being the most prominent. The mutant decreased in the numbers of several types of neurones and glia examined. The dAGO1 protein was distributed in the cytoplasm and co‐sedimented with poly(U)‐ or poly(A)‐conjugated beads.

Conclusion

Our results suggest that the dAGO1 protein exerts its developmental functions by binding to RNA either directly or indirectly.
Werner helicase relocates into nuclear foci in response to DNA damaging agents and co‐localizes with RPA and Rad51
Authors: Sakamoto Shuichi , Nishikawa Kaori , Heo Seok‐Jin , Goto Makoto , Furuichi Yasuhiro , Shimamoto Akira
Published: March 2003

Background

Werner syndrome (WS) is an autosomal recessive disorder with many features of premature ageing. Cells derived from WS patients show genomic instability, aberrations in the S‐phase and sensitivity to genotoxic agents. The gene responsible for WS (WRN) encodes a DNA helicase belonging to the RecQ helicase family. Although biochemical studies showed that the gene product of WRN (WRNp) interacts with proteins that participate in DNA metabolism, its precise biological function remains unclear.

Results

Using immunocytochemistry, we found that WRNp forms distinct nuclear foci in response to DNA damaging agents, including camptothecin (CPT), etoposide, 4‐nitroquinolin‐N‐oxide and bleomycin. The presence of aphidicolin inhibited CPT‐induced WRNp foci strongly but not bleomycin‐induced foci. These WRNp foci overlapped with the foci of replication protein A (RPA) almost entirely and with the foci of Rad51 partially, implicating cooperative functions of these proteins in response to DNA damage. We also found that WRNp foci partially co‐localize with sites of 5‐bromo‐2′‐deoxy‐uridine incorporation.

Conclusions

These findings suggest that WRNp form nuclear foci in response to aberrant DNA structures, including DNA double‐strand breaks and stalled replication forks. We propose that WRNp takes part in the homologous recombinational repair and in the processing of stalled replication forks.
Identification and characterization of DAlk: a novel Drosophila melanogaster RTK which drives ERK activation in vivo
Authors: Lorén Christina E. , Scully Audra , Grabbe Caroline , Edeen Philip T. , Thomas John , McKeown Michael , Hunter Tony , Palmer Ruth H.
Published: March 2003

Background
The mammalian receptor protein tyrosine kinase (RTK), Anaplastic Lymphoma Kinase (ALK), was first described as the product of the t(2;5) chromosomal translocation found in non‐Hodgkin's lymphoma. While the mechanism of ALK activation in non‐Hodgkin's lymphoma has been examined, to date, no in vivo role for this orphan insulin receptor family RTK has been described.

Results
We describe here a novel Drosophila melanogaster RTK, DAlk, which we have mapped to band 53 on the right arm of the second chromosome. Full‐length DAlk cDNA encodes a phosphoprotein of 200 kDa, which shares homology not only with mammalian ALK but also with the orphan RTK LTK. Analysis of both mammalian and Drosophila ALK reveals that the ALK family of RTKs contains a newly identified MAM domain within their extracellular domains. Like its mammalian counterpart, DAlk appears to be expressed in the developing CNS by in situ analysis. However, in addition to expression of DAlk in the Drosophila brain, careful analysis reveals an additional early role for DAlk in the developing visceral mesoderm where its expression is coincident with activated ERK.

Conclusion
In this paper we describe a Drosophila melanogaster Alk RTK which is expressed in the developing embryonic mesoderm and CNS. Our data provide evidence for the existence of a DAlk RTK pathway in Drosophila. We show that ERK participates in this pathway, and that it is activated by DAlk in vivo. Expression patterns of dALK, together with activated ERK, suggest that DAlk fulfils the criteria of the missing RTK pathway, leading to ERK activation in the developing visceral mesoderm.