bluuebunny/crossref_metadata_2025 huggingface dataset:
On the measurement of growth with applications to the modelling and analysis of plant growth
Authors: M. L. Roderick
Published: March 2003

1. In this paper, a theoretical framework for the analysis of growth is described. Growth is equated with change in volume (V) and the growth rate is given by the equation; dV/dt = (dm/dt)(1/ρ) − (dρ/dt)(m/ρ2) wheremis the mass andρthe density. The volume is inclusive of internal air spaces.

2. The second term of the growth equation (see above) can be ignored if density is constant over time. Data for humans (and presumably other large animals) show that while composition changes over time, the density is approximately constant at about that of water. In that case, the growth rate can be estimated from measures of the rate of change of mass. However, the density of plants is variable (c. 0·4–1·2 g cm−3) and measures of mass and density are necessary to analyse plant growth.

3. To use the theory as the basis of plant growth models, it is necessary to develop simple methods for estimating the surface area of roots, stems and leaves assuming that the mass and volume are known. A literature review found that the surface area to volume ratios of leaves and roots generally increase with the mass concentration of water. Theoretical arguments are used to predict that in woody stems, the situation should be reversed such that the surface area to volume ratio increases with the mass concentration of dry matter. Those relationships should be very useful in the development of plant growth models.

4. Measures of plant dry mass and estimates of the rate of change in dry mass are shown to be very difficult to interpret because of differences in the mass concentration of dry matter between individuals and over time.

5. It is concluded that measures of mass and density will be necessary before plant growth analysis can achieve its full potential. A framework for extending the theory to include the forces necessary for growth to occur is described.
Seed bank dynamics: the role of fungal pathogens and climate change
Authors: Leishman M. R. , Masters G. J. , Clarke I. P. , Brown V. K.
Published: March 2003

1. One of the climate change scenarios predicted for the UK is warmer winters and additional summer rainfall, which may favour growth and survival of fungal pathogens. We tested several hypotheses on the fate of persistent seeds in the soil and the role of fungal pathogens under this predicted climate change.

2. We buried seed bags containing fungicide‐treated and non‐fungicide‐treated seeds of four species with persistent seed banks (Convolvulus arvensis L., Lotus corniculatus L., Medicago lupulina L. and Rubus fruticosus L.) under control and simulated climate change (winter warming plus supplemented summer rain) conditions, and monitored seed survival over 1 to 2 years.

3. Fungicide treatment resulted in a significant increase in the percentage of intact seeds recovered for only two of the four species, M. lupulina and R. fruticosus. Seeds of M. lupulina that were treated with fungicide remained viable in the soil for longer than non‐treated seeds. Thus, the effect of fungal pathogens on seed persistence in the soil appears to be species specific.

4. There was no significant effect of the simulated climate (winter warming plus supplemented summer rain) on seed persistence in the soil, for any of the four species. Neither was a significant climate × fungicide treatment interaction found for any of the four species. Thus, it does not appear that the conditions provided in the simulated climate plots favoured the growth and survival of fungal pathogens affecting the soil seed banks of the four species studied here.

5. The use of fungicides in manipulative experiments and the importance of field experiments that simulate predicted climate change are discussed.
Locomotor performance and dominance in male Tree Lizards, Urosaurus ornatus
Authors: Robson M. A. , Miles D. B.
Published: March 2003

1. The importance of locomotor performance to the social status of male lizards may lie in its effect on the outcome of agonistic interactions and territorial contests. Presumably, dominant males are able to acquire and defend larger territories, encounter more potential mates and have greater reproductive success.

2. Therefore, the hypothesis that dominance status should covary with locomotor performance was tested using the Tree Lizard, Urosaurus ornatus.

3. Two components of locomotor performance, maximum sprint speed and stamina, in males were measured. The former variable was estimated as the fastest speed on a 0·25‐m length along a 2‐m long racetrack. The second variable was measured as the elapsed time a lizard could sustain a walking pace on a canvas belt moving at 0·50 km h−1.

4. The relative social status of size‐matched males was independently determined by staging paired encounters in a novel arena. Dominance was measured by behavioural scores of aggression and submission.

5. The dyadic encounters resulted in the unambiguous determination of dominant individuals for 48 of 50 trials. Dominant males were significantly faster than submissive males. In addition, endurance capacity of dominant males exceeded the submissive males.

6. Dominance behaviour and locomotor performance are strongly associated. Although the exact underlying mechanism is unknown, the results suggest a link between hormones, performance and behaviour in territorial lizards.
Constraints on the plasticity of Daphnia magna influenced by fish‐kairomones
Authors: Stibor H. , Navarra D. Müller
Published: March 2003

1.  Daphnia magna reproduced at a smaller size (body mass) when exposed to fish‐kairomone than D. magna growing up without this cue.

2. The total egg mass for first reproduction was the same in both groups, but the proportion of clutch mass to total body mass was about 11% higher in daphniids exposed to fish‐kairomone. This difference was about the same for low and high food concentrations (0·5 or 1·5 mgC l−1).

3. Although egg mass was the same, triglyceride (TAG) content of the egg material was significantly lower when daphniids were exposed to fish‐kairomones for both food concentrations.

4. The TAG content of the clutch was lowered to the extent that the TAG proportion in the clutch in relation to the somatic mass of the animal was the same for animals treated with fish‐kairomone and controls (5% of body mass at first reproduction).

5. The lower TAG content per clutch resulted in a lower quality of the individual egg in daphniids exposed to fish‐kairomone. The resulting higher susceptibility to starvation under low food conditions is a substantial cost of the plastic response to fish‐kairomones.

6. This cost may set a limit on the plastic response in resource allocation of Daphnia magna exposed to fish‐kairomones. However, daphniids living in a system with fish predation are more likely to be eaten by fish than to starve to death.
Effect of drought on the growth of Lolium perenne genotypes with and without fungal endophytes
Authors: Cheplick G. P. , Perera A. , Koulouris K.
Published: March 2003

Grass leaves are often inhabited by fungal endophytes that can enhance host growth. In some forage species, endophytes improve host resistance to, and recovery from, drought.

Our objective was to determine if the growth of genotypes of Lolium perenne L. was improved by endophytes during recovery from drought.

Thirteen infected genotypes were cloned into ramets. Half were treated with a systemic fungicide to eliminate the endophyte (E−); half were untreated and retained high endophyte levels (E+). In a glasshouse, half of all E− and E+ ramets were watered regularly, whilst half were exposed to a 2 week drought on two occasions, each followed by a 3 week recovery period.

After the first drought and recovery period, endophytes significantly reduced tiller production in the drought‐stressed group.

After the second drought and recovery period, effects of drought on live leaf area and dry mass were highly dependent on host genotype, but not endophytes. The mean tiller mass of E+ ramets after drought was significantly less than that of watered E+ ramets, but this was not true in E− ramets. For six genotypes there was greater mass allocation to storage in the tiller bases of E− ramets after drought.

This perennial ryegrass population showed marked genotypic variation in the ability to recover from drought stress, but endophytes played little or no role in this ability. For some host genotypes there may be a metabolic cost of harbouring endophytes during environmentally stressful conditions.
Effects of temperature on the development of an arctic Collembola (Hypogastrura tullbergi)
Authors: Birkemoe T. , Leinaas H. P.
Published: March 2003

Embryonic development, juvenile moulting and growth rates, and maximum size of the arctic collembolanHypogastrura tullbergiwere investigated at 5, 10 and 15 °C in laboratory experiments. The embryonic development was also investigated at 21 °C.

The lower temperature threshold of the embryonic development (t0) was −1·3 °C, possibly reflecting a slight cold adaptation. The temperature of maximum development rate exceeded 21 °C.

Instar duration rate was linearly related to temperature. Growth per instar, however, was thermally dependent, giving an overall non‐linear correlation between growth and temperature. This emphasizes the importance of studying growth and moulting as separate processes in Collembola.

Development of genital area (number of hairs) over succeeding instars was affected by the temperature, suggesting that a higher proportion of individuals reached the adult stage in an earlier instar at 15 than at 10 °C.

Contrary to the general pattern in ectotherms of increasing size at lower temperatures, adultH. tullbergireached a larger maximum size at 15 than at 10 °C.

No aspects of postembryonic development inH. tullbergishowed any signs of cold adaptation, probably because it is more important to be able to efficiently utilize high temperatures that frequently occur in the microhabitat during sunny periods in summer.
Seasonal nitrogen storage and remobilization in the forb Rumex acetosa
Authors: Bausenwein U. , Millard P. , Thornton B. , Raven J. A.
Published: March 2003

The contribution of N storage and remobilization to the vegetative and reproductive growth of the forb Rumex acetosa was quantified using 15N labelling techniques with plants derived from semi‐natural grasslands in Scotland.

 The contribution of remobilized N to the total N in the new above‐ground tissues was highest at the beginning of the growing season at 58%. New leaves and reproductive organs contained equal amounts of remobilized N.

 During early vegetative growth, the taproot was the main source of remobilized N, whereas during reproductive growth, N was additionally remobilized from fine roots and leaves.

 Free amino acids (mainly arginine and glutamine) and proteins were identified as the main storage compounds in the taproots. The protein pool did not show any seasonal variations that indicated the existence of a vegetative storage protein, indicating that such proteins are not a necessary component of N storage/remobilization in all species.

 The ability to store and remobilize N provides a mechanism for growth in the spring when the availability of soil N is low, and means that growth depends upon environmental conditions during more than one year.
Leaf respiration in two tropical rainforests: constraints on physiology by phosphorus, nitrogen and temperature
Authors: Meir P. , Grace J. , Miranda A. C.
Published: March 2003

Leaf respiration is a major component of the terrestrial carbon cycle, but is poorly quantified for tropical forests. We measured dark respiration, R, and nutrient concentration (nitrogen, N and phosphorus, P) of leaves within two forest canopies: in Reserva Jarú, south‐west Brazil; and Mbalmayo Reserve, central Cameroon. The data, expressed on a leaf area basis (Ra, Na, Pa) and a leaf mass basis (Rm, Nm, Pm), were used to quantify the temperature sensitivity of R and to test the hypothesis that leaf metabolism is more strongly constrained by phosphorus than by nitrogen in these lowland rainforests.

 Leaf respiration rate (Ra, at 25 °C) at Jarú was nearly half that at Mbalmayo (the range in Ra from near the ground to the upper canopy was 0·11–0·78 µmol m−2 s−1 at Jarú versus 0·22–1·19 µmol m−2 s−1 at Mbalmayo), and the mean Q10 for respiration at each site was 2·3 ± 0·9 (1 SD) and 2·0 ± 0·5 (1 SD), respectively. There were significant differences (P < 0·01) between sites in leaf phosphorus concentration, but not in leaf nitrogen concentration: Pm was very low at Jarú (0·2–0·7 mg g−1) but higher at Mbalmayo (0·5–2·4 mg g−1), whilst Nm was similar at both forests (10–45 mg g−1).

 Rm was not significantly associated with canopy position or specific leaf area (SLA, m2 g−1) in either forest, but a significant relationship between SLA and Nm was found for both sites (P < 0·05), consistent with existing data. At Jarú, Rm was strongly related to Pm (P < 0·001) and less strongly related to Nm (P < 0·05), but at Mbalmayo, Rm was not significantly related to either Pm or Nm.

 Ra was linearly related to Na and Pa at both sites (P < 0·01), principally because of changes in leaf mass per area (LMA, g m−2) associated with canopy position. At Mbalmayo, LMA explained 70% of the variation in Ra, but only 20% at Jarú. For Jarú, the strongest relationship with Ra was obtained by combining LMA with Pm in a multiple regression (r2 = 0·53); further inclusion of Nm did not improve the regression. At Mbalmayo neither Nm or Pm improved the regression of Ra on LMA.

 These results indicate a strong influence of LMA on the relative rates of Ra within the vertical gradient of each canopy. They also suggest that at JarúPm constrains respiration more strongly than Nm, and further, that the very low Pm at Jarú may explain the lower absolute values of respiration there relative to Mbalmayo, where Pm was higher. The leaves at both sites are typical of lowland tropical rainforests in not having particularly low Nm, and consistent with this, Nm was a weaker predictor of respiration than Pm or LMA.
Active brood care in Cancer setosus (Crustacea: Decapoda): the relationship between female behaviour, embryo oxygen consumption and the cost of brooding
Authors: Baeza J. A. , Fernández M.
Published: March 2003

Previous studies have shown that oxygen is limiting in embryo masses of marine invertebrates. It has been suggested that several behaviours found in brooding females of brachyuran crabs are used to ventilate and provide oxygen to the embryo masses.

 The relationship between female brooding behaviour, oxygen consumption of embryos and oxygen provision to the brood mass for embryos at different developmental stages was studied, using the marine crab Cancer setosus. The changes in oxygen consumption of brooding females associated with changes in oxygen provision to the brood were also estimated.

Brooding females of C. setosus behaved differently from non‐brooding females. Abdominal flapping was associated with an increase in oxygen availability in the centre of the brood mass; the frequency of abdominal flapping increased with embryonic development, as oxygen demand of crab embryos increased. Oxygen consumption of brooding females also increased throughout embryonic development. The difference in oxygen consumption between brooding and non‐brooding females was used as an indicator of the cost of oxygen provision (brooding).

 These results provide the first evidence – among crabs and other marine invertebrates – of a direct link between active brood care and oxygen provision. It is possible that parental care in marine invertebrates is strongly linked to oxygen provision, since oxygen limitation has been reported for several brooding taxa. The simple physiological constraint of oxygen provision in marine invertebrates may have important ecological and evolutionary consequences.
The balanced‐growth hypothesis and the allometry of leaf and root biomass allocation
Authors: Shipley B. , Meziane D.
Published: March 2003

1. Many ecological models of plant growth assume balanced growth: that biomass is allocated preferentially to leaves or roots to increase capture of the limiting external resource. An alternative explanation is based on nonlinear (allometric) allocation as a function of plant size. The objective of this study was to test between these two alternative explanations.

2. A total of 1150 plants from 22 different herbaceous species were grown in hydroponic sand culture in factorial combinations of high (1100 µmol m−2 s−1) and low (200 µmol m−2 s−1 PAR) irradiance crossed with a full‐strength and a 1/6 dilution of Hoagland’s hydroponic solution. Plants were harvested at 15, 20, 25, 30 and 35 days postgermination, and dry mass was determined for leaf and root components. These data were used to test the hypotheses of balanced growth and of allometric allocation.

3. Both irradiance and nutrient supply affected the slope and intercept of the root : shoot allometry, contrary to the allometric hypothesis but in agreement with the hypothesis of balanced growth; decreased nutrient supply increased allocation to roots; and decreased irradiance increased allocation to leaves.

4. Plants allocated relatively more biomass to roots than to leaves as plants grew larger. In order for the balanced‐growth hypothesis to be correct, the net rate of nutrient uptake per unit root mass must have been decreasing relative to the net rate of carbon gain per unit leaf mass.

5. We suggest two reasons why this might be the case: (i) older roots decreased their efficiency of nutrient uptake; and (ii) larger root systems more rapidly decreased the available nutrients between flushes of hydroponic solution.

6. These results support the notion of balanced growth that is found in many ecological models of plant growth.
Decreased heat‐shock resistance and down‐regulation of Hsp70 expression with increasing age in adult Drosophila melanogaster
Authors: Sørensen J. G. , Loeschcke V.
Published: March 2003

1. Adult Drosophila melanogaster Meigen were examined for Hsp70 expression level and heat‐shock resistance from 0 to 8 days of age – the latter being close to the mean life span in natural D. melanogaster populations.

2. Over the 8 days, both induced Hsp70 expression level and induced heat‐shock resistance showed a linear decrease. Heat‐shock resistance without prior hardening also decreased, but at a lower rate than both induced Hsp70 expression level and heat‐shock resistance after hardening.

3. The results suggest that the decrease in induced Hsp70 expression level in D. melanogaster with age is regulatory and not due to a reduced ability to express Hsp70 as a result of senescence.

4. The changes in induced Hsp70 expression level with age are likely to be determined by natural selection and are evolutionarily adaptive. The decrease with age in stress resistance after hardening may then be considered a direct consequence of age‐dependent reduction in expression levels of induced Hsp70.
Roads as barriers to movement for hedgehogs
Authors: Rondinini C. , Doncaster C. P.
Published: March 2003

1. Movements of hedgehogs,Erinaceus europaeusL., were analysed in relation to roads by recording their nocturnal foraging trajectories in urban areas adjacent to major road systems. Four male and four female hedgehogs at each of two sites were each tracked over five replicate trajectories of more than 3 h, using radio‐telemetry and direct observation.

2. Frequency of road crossings and use of habitat were compared to simulated random trajectories. For each observed trajectory, 100 trajectories were simulated with the same distribution of speeds as observed, but with random direction.

3. Observed trajectories had lower rates of crossing large but not small roads than simulated, indicating that hedgehogs treat large roads as barriers during nightly foraging activities. Most hedgehogs were never observed to cross roads, whereas almost all simulated trajectories did cross both large and small roads.

4. Crossing behaviour differed by site, but not by sex.

5. Roads and road verges had the lowest rank in a habitat preference analysis at both sites, while playing fields, gardens and urban areas were preferred habitats. These results are discussed in relation to the regional scale preference for urban areas, and the attraction to road verges while dispersing, shown by hedgehogs.
Predicting changes in community composition and ecosystem functioning from plant traits: revisiting the Holy Grail
Authors: Lavorel S. , Garnier E.
Published: March 2003

The concept of plant functional type proposes that species can be grouped according to common responses to the environment and/or common effects on ecosystem processes. However, the knowledge of relationships between traits associated with the response of plants to environmental factors such as resources and disturbances (response traits), and traits that determine effects of plants on ecosystem functions (effect traits), such as biogeochemical cycling or propensity to disturbance, remains rudimentary.

We present a framework using concepts and results from community ecology, ecosystem ecology and evolutionary biology to provide this linkage. Ecosystem functioning is the end result of the operation of multiple environmental filters in a hierarchy of scales which, by selecting individuals with appropriate responses, result in assemblages with varying trait composition. Functional linkages and trade‐offs among traits, each of which relates to one or several processes, determine whether or not filtering by different factors gives a match, and whether ecosystem effects can be easily deduced from the knowledge of the filters.

To illustrate this framework we analyse a set of key environmental factors and ecosystem processes. While traits associated with response to nutrient gradients strongly overlapped with those determining net primary production, little direct overlap was found between response to fire and flammability.

We hypothesize that these patterns reflect general trends. Responses to resource availability would be determined by traits that are also involved in biogeochemical cycling, because both these responses and effects are driven by the trade‐off between acquisition and conservation. On the other hand, regeneration and demographic traits associated with response to disturbance, which are known to have little connection with adult traits involved in plant ecophysiology, would be of little relevance to ecosystem processes.

This framework is likely to be broadly applicable, although caution must be exercised to use trait linkages and trade‐offs appropriate to the scale, environmental conditions and evolutionary context. It may direct the selection of plant functional types for vegetation models at a range of scales, and help with the design of experimental studies of relationships between plant diversity and ecosystem properties.
Nutrient concentration, resorption and lifespan: leaf traits of Australian sclerophyll species
Authors: Wright I. J. , Westoby M.
Published: March 2003

Most plants withdraw nutrients from leaves as they age, and redeploy them elsewhere in the plant. The proportion of nutrients resorbed and the residual nutrient concentration in senesced leaves are different but complementary indices of nutrient conservation via this process. A major spectrum of strategic variation runs from plant species with typically long leaf lifespan (LL), high leaf mass per area (LMA), low leaf nutrient concentrations, and low photosynthetic capacity, to species with the opposite characteristics. It is unknown to what extent either facet of resorption covaries with the LL–LMA spectrum.

Green‐leaf and senesced‐leaf N and P concentrations were quantified for 73 evergreen species from four sites in eastern Australia (nutrient‐rich and nutrient‐poor sites in each of two rainfall zones). Leaf nutrient concentrations in green and senesced leaves were negatively correlated with LL across all species and at most sites, especially if N2‐fixing species were excluded from analyses involving leaf N.

Proportional resorption did not differ with soil nutrients, as has been found elsewhere, nor was it correlated with LL. Green‐leaf and senesced‐leaf nutrient concentrations were lower for species on poorer soils. A simple model was described in which the proportion of resorbed vs soil‐derived nutrients deployed in new leaves is set by the relative cost of nutrients from the two sources. The model provides a prospective explanation for the observed differences between species from nutrient‐rich and nutrient‐poor habitats.

The results from this study provide support for the argument that selection to minimize nutrient losses has affected the residual nutrient concentration in senesced leaves, rather than proportional resorption per se. Further, variation among species in residual nutrient concentration was correlated with one of the key spectra of strategic variation between plant species, the leaf lifespan–LMA axis of variation.
BRAM1, a BMP receptor‐associated molecule involved in BMP signalling
Authors: Kurozumi Kimikazu , Nishita Michiru , Yamaguchi Kyoko , Fujita Takashi , Ueno Naoto , Shibuya Hiroshi
Published: March 2003

Background

TGF‐β superfamily members elicit signals through the stimulation of serine/threonine‐kinase receptors. Recently, molecules associated with several TGF‐β family receptors have been cloned. One such molecule, the immunophilin FKBP12, has been reported to interact with TGF‐β family type I receptors. However, the identity of signalling specific molecules interacting with the receptor was unknown.

Results

To clarify the factors mediating bone morphogenetic protein (BMP) receptor signalling, a cytoplasmic molecule associated with the BMP type IA receptor (BMPR‐IA) was isolated using the yeast two‐hybrid system. We designated the molecule BMP receptor associated molecule 1 (BRAM1). BRAM1 is an alternatively spliced form of BS69, a factor previously identified as an adenovirus E1A‐associated protein. BRAM1 was localized to the cytoplasmic region in mammalian cells, whereas BS69 is localized to the nucleus. BRAM1 bound specifically to BMPR‐IA in mammalian cells. The C‐terminal half of BRAM1 was found to be sufficient for binding to BMPR‐IA.

Conclusions

BRAM1, a BMPR‐IA associated molecule, was isolated using the yeast two‐hybrid system, and found to associate specifically with BMPR‐IA. BRAM1 may thus serve as an interacting protein in the BMP signal pathway.
How protein reads the stop codon and terminates translation
Authors: Nakamura Yoshikazu , Ito Koichi
Published: March 2003

Translation termination requires two codon‐specific protein‐release factors in prokaryotes and one factor in eukaryotes. The underlying mechanism for stop codon recognition, as well as the biological meaning of the conservation of one or two release factors in the evolutionary kingdoms, are not known. The recent discovery of release factor genes and the molecular mimicry between translational factors and tRNA provide us with clues to the mechanisms of how proteins read the stop codon and terminate translation, shedding some light on the evolutionary aspect of release factors.
The proteasome: a protein‐destroying machine
Authors: Tanaka Keiji , Chiba Tomoki
Published: March 2003

Most cellular proteins are targeted for degradation by the proteasome, a eukaryotic ATP‐dependent protease, after they have been covalently attached to ubiquitin (Ub) in the form of a poly Ub chain functioning as a degradation signal. The proteasome is an unusually large multisubunit proteolytic complex, consisting of a central catalytic machine (called the 20S proteasome) and two terminal regulatory subcomplexes, termed PA700 or PA28, that are attached to both ends of the central portion in opposite orientations, to form enzymatically active proteasomes. The large assembled proteasome acts as a protein‐destroying machine responsible for the selective breakdown of numerous ubiquitinylated cellular proteins and certain nonubiquitinylated proteins. To date, proteolysis mediated by the Ub‐proteasome pathway has been shown to be involved in a wide variety of biologically important processes, such as the cell cycle, apoptosis, metabolism, signal transduction, immune response and protein quality control, implying that it functions as a previously unrecognized regulatory system for determining the final fate of protein factors involved in these biological reactions.
Involvement of CRM1, a nuclear export receptor, in mRNA export in mammalian cells and fission yeast
Authors: Watanabe Mina , Fukuda Makoto , Yoshida Minoru , Yanagida Mitsuhiro , Nishida Eisuke
Published: March 2003

Background

CRM1, an evolutionarily conserved protein, was shown to be a receptor for leucine‐rich nuclear export signal (NES)‐dependent protein transport. In lower eukaryotes CRM1 is reported to be required for the export of mRNA, however, involvement of the NES‐dependent transport pathway in mRNA export in higher eukaryotes has not been established.

Results

We have found that treatment of mammalian cells with leptomycin B (LMB), a specific inhibitor of CRM1, induces the nuclear accumulation of endogenous mRNA, probably due to the inhibition of its export. In fission yeasts, the nuclear accumulation of mRNA also occurred in cells treated with LMB or in a temperature‐sensitive crm1 mutant at a restrictive temperature. A synthetic mRNA that was injected into the nucleus of mammalian cultured cells was exported from the nucleus within 5 h. This export was inhibited by both wheat germ agglutinin and a temperature of 4 °C. Importantly, this mRNA export was inhibited by LMB or by an excess amount of the NES peptide‐conjugates. LMB treatment, on the other hand, rapidly induced the nuclear entry of RanBP1, a factor involved in the active nucleocytoplasmic transport, although the treatment did not interfere with a nuclear localization signal‐dependent transport system within 7 h.

Conclusion

These results suggest that CRM1 is involved in mRNA export in eukaryotic cells.
Orientation specificity of the Lactococcus lactis Chi site
Authors: El Karoui Meriem , Schaeffer Matthieu , Biaudet Véronique , Bolotin Alexandre , Sorokin Alexeï , Gruss Alexandra
Published: March 2003

Background

In Escherichia coli, the Chi sequence modulates the activity of RecBCD, a powerful double‐stranded (ds) DNA exonuclease/helicase. Chi attenuates RecBCD exonuclease activity and stimulates homologous recombination in an orientation‐dependent manner. ChiEc is frequent and over‐represented on its genome, which is thought to be related to its role in dsDNA break repair. We previously identified a Chi‐like sequence (referred to as ChiLl) and an exonuclease/helicase in the Gram‐positive bacterium Lactococcus lactis. ChiLl and RexAB are functional analogues of ChiEc and RecBCD.

Results

We report that ChiLl attenuates RexAB exonuclease activity and stimulates homologous recombination in an orientation‐dependent manner. Analysis of ChiLl distribution on the L. lactis chromosome reveals that ChiLl is frequent, highly over‐represented, and oriented with respect to the direction of replication.

Conclusion

Our results show that a single orientation of ChiLl interacts with RexAB. The active orientation is preferentially found on the replication leading strand of the L. lactis genome, consistent with a primary role of ChiLl in repair of dsDNA breaks at the replication fork. We propose that orientation‐dependence of Chi activity and over‐representation of Chi sequences on bacterial genomes may be conserved properties of exonuclease/helicase–Chi couples. Other properties of the Chi sequence distribution on the genomes might reflect more specific characteristics of each couple and of the host.
The paternal methylation imprint of the mouse H19 locus is acquired in the gonocyte stage during foetal testis development
Authors: Ueda Takayuki , Abe Kuniya , Miura Asuka , Yuzuriha Misako , Zubair Mohamad , Noguchi Motoko , Niwa Katsutoshi , Kawase Yosuke , Kono Tomohiro , Matsuda Yoichi , Fujimoto Hirokazu , Shibata Hideo , Hayashizaki Yoshihide , Sasaki Hiroyuki
Published: March 2003

Background

Germline‐specific differential DNA methylation that persists through fertilization and embryonic development is thought to be the ‘imprint’ distinguishing the parental alleles of imprinted genes. If such methylation is to work as the imprinting mechanism, however, it has to be reprogrammed following each passage through the germline. Previous studies on maternally methylated genes have shown that their methylation imprints are first erased in primordial germ cells (PGCs) and then re‐established during oocyte growth.

Results

We have examined the timing of the reprogramming of the paternal methylation imprint of the mouse H19 gene during germ cell development. In both male and female PGCs, the paternal allele is partially methylated whereas the maternal allele is unmethylated. This partial methylation is completely erased in the female germline by entry into meiosis, establishing the oocyte methylation pattern. In the male germline, both alleles become methylated, mainly during the gonocyte stage, establishing the sperm methylation pattern.

Conclusion

The paternal methylation imprint of H19 is established in the male germline and erased in the female germline at specific developmental stages. The identification of the timings of the methylation and demethylation should help to identify and characterize the biochemical basis of the reprogramming of imprinting.
Ribonuclease H attack of leukaemic fused transcripts AML1‐MTG8(ETO) by DNA/RNA chimeric hammerhead ribozymes
Authors: Kozu Tomoko , Sugio Yasuhiro , Fukuyama Tomofusa , Sueoka Eisaburo , Otsuka Teruhisa , Akagi Kiwamu
Published: March 2003

Background

Catalytic anti‐sense oligonucleotides might be useful tools for controlling specific gene expression. However, to obtain effective oligonucleotides of the desired function in vivo is still a difficult task.

Results

To evaluate the usefulness of synthesized DNA/RNA hammerhead ribozymes targeting AML1‐MTG8 (ETO) leukaemic fusion transcripts in vivo, we analysed their effects on cell growth and the mechanism of action using isolated cell nuclei. These ribozymes inhibited the growth of leukaemic cell lines expressing the AML1 ‐MTG8 and degraded AML1‐MTG8 mRNA in isolated nuclei of these cells. However, the reactions gave rise to additional cleavage products. Systematic cleavage analyses using an anti‐sense oligonucleotide array revealed that the cleavage was induced by endogenous RNase H at specific sites, in accordance with their calculated melting temperature (Tm) values. With suppression of RNase H by sulfhydryl agents, the DNA/RNA ribozyme had a ribozyme catalytic activity. In addition, the ribozymes and anti‐sense oligonucleotides suppressed the AML1‐MTG8 protein in the leukaemic cells.

Conclusions

The DNA/RNA ribozymes inhibited cell growth primarily via anti‐sense effects, the main role of which was the activation of RNase H‐digestion by their DNA arms. In addition, the isolated nuclei provided a useful assay system for modelling in vivo conditions for the quantitative evaluation of anti‐sense/ribozyme activity.
The conserved nuclear receptor Ftz‐F1 is required for embryogenesis, moulting and reproduction in Caenorhabditis elegans
Authors: Asahina Masako , Ishihara Takeshi , Jindra Marek , Kohara Yuji , Katsura Isao , Hirose Susumu
Published: March 2003

Background

Nuclear receptors are essential players in the development of all metazoans. The nematode Caenorhabditis elegans possesses more than 200 putative nuclear receptor genes, several times more than the number known in any other organism. Very few of these transcription factors are conserved with components of the steroid response pathways in vertebrates and arthropods. Ftz‐F1, one of the evolutionarily oldest nuclear receptor types, is required for steroidogenesis and sexual differentiation in mice and for segmentation and metamorphosis in Drosophila.

Results

We employed two complementary approaches, direct mutagenesis and RNA interference, to explore the role of nhr‐25, a C. elegans ortholog of Ftz‐F1. Deletion mutants show that nhr‐25 is essential for embryogenesis. RNA interference reveals additional requirements throughout the postembryonic life, namely in moulting and differentiation of the gonad and vulva. All these defects are consistent with the nhr‐25 expression pattern, determined by in situ hybridization and GFP reporter activity.

Conclusions

Our data link the C. elegans Ftz‐F1 ortholog with a number of developmental processes. Significantly, its role in the periodical replacement of cuticle (moulting) appears to be evolutionarily shared with insects and thus supports the monophyletic origin of moulting.
In vitro analysis of STAT5 activation by granulocyte‐macrophage colony‐stimulating factor
Authors: Sakurai Yoshiko , Arai Ken‐ichi , Watanabe Sumiko
Published: March 2003

The granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) receptor activates multiple and complex signalling pathways in response to GM‐CSF stimulation. Biochemical studies suggested that signalling pathways are transmitted through protein/protein interactions, but how these biochemical cascades are initiated and transmitted in response to cytokine stimulation is largely unknown.

To investigate these events biochemically, we established an in vitro system leading to the GM‐CSF‐dependent activation of Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 5 in cell homogenates prepared from BA/F3 cells expressing the GM‐CSF receptor. Activation of STAT5 DNA binding ability requires both membrane and cytoplasmic fractions while phosphorylation of JAK2 requires only the membrane fraction. Since the addition of anti‐βc or phosphotyrosine antibodies inhibited GM‐CSF induced STAT5 DNA binding activity, we examined the role of tyrosine residues of βc for in vitro activation of STAT5. Addition of synthetic tyrosine‐phosphorylated peptides derived from βc cytoplasmic tyrosines prior to GM‐CSF stimulation inhibited the in vitro activation of STAT5. The association between these tyrosine‐phosphorylated peptides and STAT5 was observed by using peptide‐coupling beads and BA/F3 lysates.

We established a GM‐CSF‐dependent in vitro system. In cases of STAT5 activation, each phosphorylated tyrosine residue of βc can act as a docking site and enhance STAT5 activation.
Induction of IRF‐3/‐7 kinase and NF‐κB in response to double‐stranded RNA and virus infection: common and unique pathways
Authors: Iwamura Tomokatsu , Yoneyama Mitsutoshi , Yamaguchi Kazumi , Suhara Wakako , Mori Wakana , Shiota Kazutaka , Okabe Yasutaka , Namiki Hideo , Fujita Takashi
Published: March 2003

Backgrounds

Infection by virus or treatment with double‐stranded RNA (dsRNA) results in the activation of transcription factors including IRF‐3, IRF‐7 and a pleiotropic regulator NF‐κB by specific phosphorylation. These factors are important in triggering a cascade of antiviral responses. A protein kinase that is yet to be identified is responsible for the activation of these factors and plays a key role in the responses.

Results

The signal cascade was analysed using sensitive assays for the activation of IRF‐3 and NF‐κB, and various inhibitors. We found that the activation of IRF‐3 and NF‐κB by dsRNA or virus involves a process that is sensitive to Geldanamycin. Although the induction of NF‐κB by dsRNA/virus and TNF‐α involves common downstream pathways including IKK activation, the upstream, Geldanamycin‐sensitive process was unique to the dsRNA/virus‐induced signal. By an in vitro assay using cell extract, we found an inducible protein kinase activity with physiological specificity of IRF‐3 phosphorylation. Furthermore, the same extract specifically phosphorylated IRF‐7 in a similar manner.

Conclusions

Double‐stranded RNA or virus triggers a specific signal cascade that results in the activation of the IRF‐3/‐7 kinase we detected, which corresponds to the long‐sought signalling machinery that is responsible for triggering the early phase of innate response. The signal branches to a common NF‐κB activation cascade, thus resulting in the activation of a set of critical transcription factors for the response.
Identification of the cis‐acting region in the NF2 gene promoter as a potential target for mutation and methylation‐dependent silencing in schwannoma
Authors: Kino Takeshi , Takeshima Hideo , Nakao Mitsuyoshi , Nishi Toru , Yamamoto Keizo , Kimura Takahiro , Saito Yoshiki , Kochi Masato , Kuratsu Jun‐ichi , Saya Hideyuki , Ushio Yukitaka
Published: March 2003

Background

Although mutational inactivation and allelic loss in the NF2 gene appear to be causal events in the majority of vestibular schwannomas, involvement of another potentially important mechanism, transcriptional inactivation, has not been investigated.

Results

We cloned and functionally characterized the 5′‐flanking region of the human NF2 gene and identified the molecular mechanisms that regulate NF2 expression. Luciferase assay and site‐directed mutagenesis demonstrated that a 70‐base pair (bp) region (−591 to −522 bp from the translation start site) was essential for the basic expression of the NF2 gene. A gel mobility shift assay indicated recognition by nuclear protein of the unusually long (≈66 bp) sequences in this region. Recognition was inhibited by either mutation of the binding core sequence or by methylation of three CpG sites. Point mutations at these CpG sites significantly decreased promoter activity, suggesting the importance of these sites. In 14 of 23 vestibular schwannomas, these three CpG sites were methylated in a site‐specific manner and the methylation status was consistent with the expression of NF2 mRNA.

Conclusion

Suppressed expression by aberrant methylation or mutation of the promoter elements could be an alternative mechanism for inactivation of the NF2 gene.
Detection of tmRNA‐mediated trans‐translation products in Bacillus subtilis
Authors: Fujihara Ai , Tomatsu Hisashi , Inagaki Sachi , Tadaki Toshimasa , Ushida Chisato , Himeno Hyouta , Muto Akira
Published: March 2003

Background:

Bacterial tmRNA (10Sa RNA) is involved in a trans‐translation reaction, which contributes to the degradation of incompletely synthesized peptides and the recycling of stalled ribosomes. To investigate the physiological roles of this reaction in Bacillus subtilis, we devised a system for detecting the proteins that are subject to in vivo trans‐translation.

Results: The wild‐type tmRNA gene (ssrA) in the genome was replaced by a variant ssrA encoding a tag‐peptide sequence containing six histidine residues (His‐tag) and two aspartic acids at the C‐terminus. The His‐tagged proteins that accumulated in the cells without degradation were fractionated by Ni2+‐NTA column and gel electrophoresis and were detected by Western blotting with an anti‐His‐tag antibody. The results showed that the trans‐translation occurred more frequently at a high temperature (50 °C) than at a low temperature (37 °C). Two‐dimensional (2D) gel electrophoresis of the products revealed many distinct spots, which represent specific target proteins for the trans‐translation reaction. Furthermore, the 2D gel patterns of the products from cells cultured at high and low temperatures were apparently different. Several tagged proteins were identified by the N‐terminal amino acid sequences of the products.

Conclusion: Trans‐translation occurs more frequently at high temperature than at low temperature, and different proteins are tagged at different temperatures.
The γ‐tubulin complex protein Alp4 provides a link between the metaphase checkpoint and cytokinesis in fission yeast
Authors: Vardy Leah , Fujita Akiko , Toda Takashi
Published: March 2003

Background:  The progression of cytokinesis requires cyclin B destruction by the anaphase promoting complex (APC/C) and, in fission yeast, activation of the septation initiation network (SIN) is also essential. The γ‐tubulin complex (γ‐TuC) localizes to the centrosome throughout the cell cycle and is directly involved in the organization of the mitotic spindle.

Results:  We have previously shown that the mutant defective in alp4+ (Spc97/GCP2) displays bipolar spindle defects due to a failure in the recruitment of the γ‐TuC on to the spindle pole body (SPB, the centrosome equivalent). Here we show that in these mutants the Mad2 checkpoint is activated, yet septation proceeds due to the untimely activation of the SIN. The Sid1 kinase, the downstream effector of the SIN, is recruited prematurely to both, instead of only one, of the SPBs, which triggers septation despite the presence of monopolar spindles. Remarkably, cyclin B levels, which would normally have declined, remain high at the SPB in septated mutant cells.

Conclusions:  We propose a novel role of the γ‐TuC in inhibiting activation of the SIN until cyclin B is destroyed. Given the ubiquitous existence of the γ‐TuC, this mechanism may be conserved throughout evolution and function to couple cytokinesis to mitotic exit.
Gene products encoded in the ninR region of phage λ participate in Red‐mediated recombination
Authors: Tarkowski Trudee A. , Mooney Duane , Thomason Lynn C. , Stahl Franklin W.
Published: March 2003

Background:  The ninR region of phage λ contains two recombination genes, orf (ninB) and rap (ninG), that were previously shown to have roles when the RecF and RecBCD recombination pathways of E. coli, respectively, operate on phage λ.

Results:  When λ DNA replication is blocked, recombination is focused at the termini of the virion chromosome. Deletion of the ninR region of λ decreases the sharpness of the focusing without diminishing the overall rate of recombination. The phenotype is accounted for in large part by the deletion of rap and of orf. Mutation of the recJ gene of the host partially suppresses the Rap– phenotype.

Conclusion: ninR functions Orf and Rap participate in Red recombination, the primary pathway operating when wild‐type λ grows lytically in rec+ cells. The ability of recJ mutation to suppress the Rap– phenotype indicates that RecJ exonuclease can participate in Red‐mediated recombination, at least in the absence of Rap function. A model is presented for Red‐mediated RecA‐dependent recombination that includes these newly identified participants.
Cleavage reaction of HDV ribozymes in the presence of Mg2+ is accompanied by a conformational change
Authors: Tanaka Yoichiro , Tagaya Mitsuhiro , Hori Tamaki , Sakamoto Taiichi , Kurihara Yasuyuki , Katahira Masato , Uesugi Seiichi
Published: March 2003

Background: Hepatitis delta virus (HDV) ribozymes cleave RNA in the presence of divalent metal ions. We have previously elucidated the solution conformation of a minimized trans‐acting HDV ribozyme and obtained evidence by NMR study that an Mg2+ ion binds to a site close to the cleavage site.

Results: We examined two ribozyme systems: a pre‐cleavage complex with a non‐cleavable substrate analogue (mS8) and a post‐cleavage complex with a 3′ cleavage product (P7). Upon titration with MgCl2, the complex with P7 showed a profound spectral change, while that with mS8 showed broadening of the signals. Analysis of the NOESY spectra of the P7 complex at high Mg2+ concentration revealed that a G:U pair is formed within the L3 loop, and the P1 and P4 stems are stabilized with respect to those of the pre‐cleavage complex.

Conclusion: The present analysis indicates that the cleavage reaction of the HDV ribozyme produces a big conformational change. Furthermore, presence of the 5′‐terminal cytidine residue prevents this conformational change and its absence stabilizes the product–ribozyme complex in the presence of Mg2+. The structure of the Mg2+‐bound P7 complex is similar to the crystal structure found for a product–ribozyme complex but is different from the pre‐cleavage structure.