bluuebunny/crossref_metadata_2025 huggingface dataset:
Maternal body volume constrains water uptake by lizard eggs in utero
Authors: Qualls C. P. , Andrews R. M.
Published: March 2003

1. Flexible shelled eggs of squamate reptiles normally take up substantial amounts of water, and swell accordingly, during development. However, most water uptake occurs after oviposition, beginning immediately or soon after oviposition, and water uptake is severely restricted in eggs that are retained in utero past the normal time of oviposition.

2. These observations suggest that some aspect of retention in the oviductal environment limits the amount of water eggs can absorb prior to oviposition.

3. This paper presents evidence, from two species of Sceloporus lizard, supporting the hypothesis that limited space within the mother’s body cavity physically constrains the ability of eggs to expand, and thereby their ability to absorb water. When adjusted for maternal body size, the water content of a female’s clutch of eggs is negatively correlated with the dry mass of her clutch (the space available in the body cavity is finite, and thus a greater dry mass content leaves less volume for water).

4. If such a constraint on water uptake is widespread, it can have important implications for our understanding of the evolution of viviparity, the costs of reproduction, and the determinants of reproductive output in squamate reptiles.
Allocation of nitrogen and carbon in leaves of Metrosideros polymorpha regulates carboxylation capacity and δ13C along an altitudinal gradient
Authors: Cordell S. , Goldstein G. , Meinzer F. C. , Handley L. L.
Published: March 2003

1.
Metrosideros polymorpha (O’hia), the dominant tree species in Hawaiian forest ecosystems, grows from sea level to treeline (2500 m). Consistent changes in its morphology and anatomy occur along this altitudinal/temperature gradient. Patterns of variation in photosynthetic gas exchange, leaf nitrogen content, nitrogen‐use efficiency, δ13C, and morphological and anatomical characteristics were determined across the elevational gradient. In addition, on‐line carbon isotope discrimination studies of high and low elevation M. polymorpha were performed.

2. Observed trends with increasing altitude were: (1) progressively higher carboxylation efficiency, leaf N content on an area basis, leaf mass per unit area (LMA), less negative foliar δ13C and (2) progressively smaller leaf size. Net CO2 assimilation (A) expressed on an area basis, leaf dry mass and N content per leaf remained relatively constant along the gradient.

3. Foliar δ13C became less negative with increasing elevation (– 30‰ at low elevation to – 24‰ at high elevation) and was strongly correlated with foliar N and LMA. Foliar δ13C was also correlated with variations in the ratio of intercellular to ambient partial pressure of CO2 (pi/pa), as determined by field gas‐exchange studies.

4. Results from on‐line fractionation experiments suggested that the relatively large internal resistance to CO2 diffusion did not differ between high and low elevation populations, despite differences in LMA. Less negative values of δ13C at high elevations and corresponding lower values of pi/pa were associated with increased carboxylation efficiency and N content on a unit leaf area basis.

5. Two major homeostatic responses in M. polymorpha plants along elevational/temperature gradients were observed: (1) maintenance of similar photosynthetic rates per unit leaf surface area despite suboptimal conditions for CO2 assimilation at high elevation and (2) similar N content per leaf despite lower soil N availability at high elevations. These homeostatic mechanisms allow M. polymorpha to maintain a relatively high level of growth‐related activities at high elevation, despite limiting environmental conditions.
Selfing rates in natural populations of Echium vulgare: a combined empirical and model approach
Authors: Rademaker M. C. J. , De Jong T. J. , Van Der Meijden E.
Published: March 2003

1. We quantified geitonogamous selfing in Echium vulgare, a self‐compatible, bumble‐bee pollinated plant. A maximum estimate of selfing was determined using a paternity analysis with RAPDs. In the first experiment, bumble‐bees visited a sequence of virgin flowers. The percentage selfing increased rapidly from 12% in the first flower visited, up to 50% in the 15th flower visited in the sequence. In the second experiment, when bees visited plants in a natural population, the average selfing of plants increased with the number of open flowers from 0% to maximally 33%.

2. The results obtained in both experiments are consistently lower than predicted by our model on pollen dynamics (Rademaker, de Jong & Klinkhamer 1997). We modified the model on pollen dynamics to link it more to the field situation with observations on flower stage, flower opening and bumble‐bee preference, so that the bumble‐bees encounter a variable number of pollen grains per flower. We also adjusted the parameters. If less pollen adheres to the bee (25% instead of 50%) after removal from the anthers, or if bees arrive at a plant with more pollen grains (6000 instead of 4448), the predictions of the model in regard to selfing could be improved but were still high compared with the observed selfing rates measured with RAPDs.

3. We suggest that the model is consistent with pollen dynamics in the field. However, post‐pollination processes like selective abortion could play a role in E. vulgare.
Changing fitness consequences of hsp70 copy number in transgenic Drosophila larvae undergoing natural thermal stress
Authors: Roberts S. P. , Feder M. E.
Published: March 2003

1. Transgenic manipulation of the gene copy number of hsp70, which encodes the major inducible heat‐shock protein of Drosophila melanogaster (Hsp70), affects both Hsp70 levels and inducible thermotolerance in the laboratory; here parallel effects in transgenic Drosophila larvae undergoing natural or simulated natural thermal stress are demonstrated.

2. Necrotic fruit was infested with larvae of either of two transgenic strains, one transformed with 12 extra copies of the hsp70 gene (extra‐copy strain) and a sister strain possessing only the wild‐type number (10) of hsp70 genes (excision strain), and then allowed to heat to variable extents.

3. As the intensity of thermal stress increased, the consequences of extra hsp70 copies reversed. After no or moderate thermal stress, excision larvae survived better than did extra copy larvae. By contrast, extra copy larvae tolerated intense hyperthermia better than did excision larvae.

4. These results establish that the Hsp70‐mediated enhancement of stress tolerance, previously demonstrated only for artificial stress regimes in the laboratory, extends to natural stress regimes.

5. Mortality due to overexpression of Hsp70, however, also increases under mild natural stress regimes, buttressing the ecological relevance of a hypothesized evolutionary trade‐off of the benefits and adverse consequences of Hsp70 expression.
Ecomorphological correlates of habitat partitioning in Corsican lacertid lizards
Authors: Vanhooydonck B. , Van Damme R. , Aerts P.
Published: March 2003

1. Morphological features (i.e. snout–vent length, SVL, mass and limb parameters), locomotor performance (sprint speed, climbing speed, manoeuvrability and endurance) and microhabitat use were measured in three lacertid lizard species. Additionally, microhabitat preference was tested in the laboratory with species kept apart and in combination with each other to assess mutual interference.

2. It was predicted that each species possesses morphological adaptations that allow it to excel in those performance measures relevant in its particular microhabitat.

3. The three species differed in microhabitat use. Lacerta bedriagae was mostly seen on boulders, while Podarcis sicula occurred among vegetation. Podarcis tiliguerta seemed more general in its microhabitat use. The results from the microhabitat preference in the laboratory corresponded well to these field observations.

4. Besides differing in SVL, the three species differed in limb length and diameter, and body mass (all relative to SVL). L. bedriagae has long forelimbs, short hindlimbs and a high body mass, while P. sicula has short forelimbs, long hindlimbs and a low body mass. P. tiliguerta is morphologically intermediate.

5. The differences found in morphology translated partially into performance differences. Only climbing speed and endurance differed among species. L. bedriagae excelled in both cases. Considering its microhabitat use, this seemed adaptive.

6. Unexpectedly, the interference experiments suggested that L. bedriagae is the least competitive of the three species.
Elongated chambers for field studies across atmospheric CO2 gradients
Authors: Johnson H. B. , Polley H. W. , Whitis R. P.
Published: March 2003

1. We describe a field facility that controls CO2 along continuous gradients from superambient to subambient concentrations. The facility consists of two transparent, tunnel‐shaped chambers, each 1‐m wide and tall and 60‐m long. Pure CO2 is injected into one chamber during daylight to initiate a superambient CO2 gradient (550–350 µmol mol−1). Ambient air is introduced to the second chamber to initiate a subambient CO2 gradient (350–200 µmol mol−1). CO2 concentrations at night are regulated at 150 µmol mol−1 above daytime values along each gradient. The CO2 gradients are maintained by varying the rate and direction (day/night) of air flow.

2. Air temperature and vapour pressure deficit are regulated near ambient values by cooling and dehumidifying air at 5‐m intervals along chambers.

3. Desired CO2 gradients were regulated on grassland for virtually the entire 9‐month growing season in 1998, including a 6‐month drought. Consistent CO2 concentrations were maintained along gradients despite seasonal variation in species composition, leaf area, and temperature.

4. Daytime temperatures in chambers tracked the seasonal pattern in 1998. The polyethylene covering on chambers transmitted 90% of incident light, but usually increased the ratio of diffuse to direct light.

5. By enabling the study of trends in plant and ecosystem responses to CO2 over both subambient and superambient concentrations, elongated chambers fill a void in CO2 research facilities.
Photosynthetic refixation in branches of Western White Pine
Authors: L. A. Cernusak , J. D. Marshall
Published: March 2003

1. We investigated the functional controls on light‐dependent refixation of respired CO2 in photosynthetic bark of Western White Pine (Pinus monticola Dougl. ex D. Don) branches.

2. Gross photosynthesis increased with increasing intensity of photosynthetically active radiation (PAR) to > 2000 µmol m−2 s−1 and with increasing bark surface temperature from 15 to > 45 °C. Maximum observed refixation rates were 76 ± 3% (mean ± SE) of dark respiration.

3. At a given temperature and irradiance, dark respiration rate was the physiological parameter that correlated most strongly with gross photosynthesis (R = 0·89, P < 0·0001, n = 43). Dark respiration rates varied 20‐fold among branches. Over this range, refixation averaged 55 ± 2% of dark respiration at 25 °C and 1000 µmol PAR m−2 s−1. However, refixation was not correlated with internal CO2 concentrations in illuminated branches; these concentrations remained fairly constant.

4. Specific bark area, bark conductance to water vapour, bark chlorophyll a/b ratio, and bark nitrogen concentration varied markedly with canopy depth in mature trees.

5. Results are summarized in a simple model of refixation driven by light, temperature and dark respiration rate. The model may prove useful in assessing the quantitative significance of refixation in the carbon balance of forest trees.
Physical apertures as constraints on egg size and shape in the Common Musk Turtle, Sternotherus odoratus
Authors: Clark P. J. , Ewert M. A. , Nelson C. E.
Published: March 2003

Egg size in turtles often increases with female size, contrary to expectations of optimality. Functional constraints on egg width imposed by the pelvic aperture or the gap between the carapace and plastron (the caudal gap) have been inferred for a few populations but appear inapplicable in others.

 For Sternotherus odoratus (the Common Musk Turtle), the pelvic aperture was always wider than the width of the female’s largest egg by at least 3·7 mm. The caudal gap was narrower than the widest egg for 25·7% of the females.

 Egg width increased, and elongation (length/width) decreased, as female size and clutch size increased.

 Females at three ecologically contrasting sites differed appreciably in size but produced eggs of the same mean shape and size, despite the strong within‐site changes in both egg size and shape with female size. As the younger females at all sites were of similar age and produced eggs of similar size and shape (again, despite differences in body size), egg size and shape may be age‐specific.

 No optimal egg size prevailed but the scaled residuals of egg size with female mass were less variable than were those for clutch size.
Allometric and ecological relationships of ventricle and liver mass in anuran amphibians
Authors: Withers P. C. , Hillman S. S.
Published: March 2003

There is an isometric relationship for allometry of ventricle mass (slope = 0·952) and liver mass (slope = 1·027) and phylogenetic correlations of ventricle and liver mass for museum specimens of 42 species of Australian frogs.

 There was a strong relationship for ventricle mass with body mass, phylogeny and dehydration tolerance, but not habit, body shape or environmental variables. The relatively large ventricle of more dehydration‐tolerant species may also be associated with a higher metabolic scope for activity. There was a strong relationship for liver mass with body mass, phylogeny, habit (burrowing, terrestrial, arboreal), body shape and some environmental variables, but not dehydration tolerance. For burrowing frogs, a relatively large liver is presumably important because it is an energy store during long periods of dormancy and is a source of amino acids for cocoon formation or urea synthesis. For arboreal frogs, a small liver might reduce the cost of transport by limiting body mass, and allow a more elongate shape.

 Relative ventricle mass of museum specimens is representative of values for field‐collected frogs. Relative liver mass is higher for museum specimens; this might be an artefact of museum preservation and/or reflect seasonal or geographic variation.
Soil and atmospheric water deficits and the distribution of New Zealand’s indigenous tree species
Authors: J. R. Leathwick , Whitehead D.
Published: March 2003

1. An extensive data set describing the composition of New Zealand’s remaining indigenous forests was used to estimate the degree of correlation between measures of both soil and atmospheric water deficit and the distribution of common tree species.

2. For most species, regression models incorporating measures of air saturation deficit in early autumn, as well as an annual integral of root zone water deficit, provided the best explanation of spatial distribution. This accords strongly with the mechanistic effects of air saturation deficits on transpiration from trees, and the hydraulic risks experienced by trees under high evaporative demand.

3. Adjustment of root zone water deficits to account for reductions in rainfall in dry years substantially improved model predictions. This suggests that extreme climatic events, such as the El Niño phase of the Southern Oscillation, are likely to have strongly influenced the historic composition of forests in New Zealand’s drier eastern lowlands.
Relative importance of microhabitat, plant form and photosynthetic physiology to carbon gain in two alpine herbs
Authors: Germino M. J. , Smith W. K.
Published: March 2003

1. The effects of microhabitat and plant form on sunlight interception, leaf temperatures, frost occurrence and photosynthesis were evaluated for Caltha leptosepala DC and Erythronium grandiflorum Pursh. Both plants are perennials that commonly emerge from alpine snowbanks where the combination of cool temperatures and strong sunlight is among the most extreme for vascular plants.

2.  Caltha leptosepala occurred in microsites where colder air accumulates, and has larger, less inclined and more densely clustered leaves compared to E. grandiflorum (which has two steeply inclined leaves).

3. These differences in microsite and plant form led to leaf temperatures below 0 °C on 70% of nights during the summer growth season in C. leptosepala, compared to only 38% in E. grandiflorum. Leaves of C. leptosepala warmed more slowly on mornings following frosts compared to E. grandiflorum, due to less aerodynamic coupling between leaf and air temperature, and also a 45% smaller ratio of sunlit to total leaf area due to mutual shading among leaves.

4. As a result, night frost did not affect subsequent CO2 assimilation (A) in E. grandiflorum, while frostless nights and warmer mornings led to 35% greater A in C. leptosepala in the early morning.

5. There were no appreciable differences in the temperature and light response of photosynthesis between the two species. The apparent quantum yield of A declined only ≈8% in both species following frost and exposure to strong sunlight, indicating little adjustment of photosynthetic physiology.

6. Greater daily carbon gain probably occurs for E. grandiflorum because of its plant form and microclimate, rather than differences in photosynthetic physiology.
Species‐level versus community‐level patterns of mycorrhizal dependence on phosphorus: an example of Simpson’s paradox
Authors: Allison V. J. , Goldberg D. E.
Published: March 2003

1. Ecological studies commonly assume that it is possible to extrapolate from a response shown by a fixed set of species to the response when the species composition is allowed to change. However, as described by Simpson’s paradox, this is not necessarily a reasonable expectation.

2. The impact of Simpson’s paradox on an ecological question was tested using a meta‐analysis of data on plant responses to arbuscular mycorrhizas. Although species‐level response commonly declines as phosphorus availability increases, we hypothesized that the community‐level response could either decline or remain constant.

3. As expected, mycorrhizal response of individual species declined significantly as P supply increased. The response averaged across multiple species was negative but not robust, so we cannot distinguish clearly between the hypotheses.

4. It is impossible to assume that community‐level responses to environmental gradients are the same as those found at species level. We recommend that experimental tests of hypotheses should allow species identity to change with the environment.
Survival of terrestrial soil‐dwelling arthropods on and in seawater: implications for trans‐oceanic dispersal
Authors: Coulson S. J. , Hodkinson I. D. , Webb N. R. , Harrison J. A.
Published: March 2003

1. The ability of six species of high Arctic soil arthropod to survive oceanic transport in or on sea water was tested experimentally.

2. Five species of Collembola,Hypogastrura tullbergi(Schäffer),Onychiurus groenlandicus(Tullberg),Onychiurus arcticus(Tullberg),Folsomia quadrioculata(Tullberg) andTetracanthella arcticaSchött were exposed on the surface film of seawater in chambers agitated within a shaking water bath.

3. All five species survived for over 14 days, at the end of which survival among species varied between 27 and 100%.

4. In a separate experiment the cryptostigmatic miteCamisia anomiaColloff and the ‘wettable’ collembolanT. arcticasurvived submersion in sea water for longer than 2 weeks. Survival ofC. anomiaexceeded 75% but that ofT. arctica, when submerged, fell to 12%.

5. Survival times are sufficient to permit ocean current transport from northern Norway to Svalbard in the high Arctic, a distance of around 700 km. The implications of the experiments for long‐distancetrans‐oceanic dispersal of non‐specialized soil arthropods are discussed.
Effects of low calcium concentrations on two common freshwater crustaceans, Gammarus lacustris and Astacus astacus
Authors: Rukke N. A.
Published: March 2003

1. Field studies indicate that low Ca concentrations limit the distribution and success of Ca‐demanding freshwater crustaceans in soft‐water localities. A long‐lasting depletion of Ca caused by reversed acidification could thus be detrimental to such species.

2. Experimental studies of Gammarus lacustris and Astacus astacus revealed decreased survival and retarded growth at ambient Ca concentrations <5 mg l−1, compared with animals reared at 10 mg l−1.

3. A significant proportion of the total body Ca content was lost during the moult, but moulting frequencies were not affected by ambient Ca concentration. Complete postmoult calcification of the carapace could not be achieved at ambient Ca concentrations <5 mg l−1, and individuals reared at such conditions had a lower specific Ca content than individuals reared at sufficient Ca levels.

4. A juvenile bottleneck regarding the susceptibility to Ca deficiency was shown in G. lacustris, as neonates had a relatively higher mass‐specific Ca content and a higher Ca threshold for survival than adults.

5. Because of a short postmoult calcification period of less than 2 days, G. lacustris had limited ability to compensate for low water Ca levels by obtaining Ca from food. However, food might be an important source of Ca for A. astacus living at low ambient Ca, because eating could be resumed during the relatively long calcification period (>15 days).
Soil organic matter distribution and below‐ground competition between Calluna vulgaris and Nardus stricta
Authors: Genney D. R. , Alexander I. J. , Hartley S. E.
Published: March 2003

We conducted an experiment to investigate the effect of organic matter distribution on below‐ground competition between the dwarf shrub Calluna vulgaris (L.) Hull and the coarse upland grass Nardus stricta (L.).

The two species were grown alone, or in competition in pots where the substrate was either a sand layer overlain by a peat layer (layered), or the same volume of peat evenly mixed with sand throughout the pot (mixed).

Root length and allocation in the top, middle and bottom of each pot was measured, along with plant biomass.

Calluna allocated the greatest proportion of root length to the top of the pot, irrespective of the peat distribution. Nardus allocated a large proportion of root length to the bottom of the pot. Root allocation by Nardus to the top and middle of the pot was positively related to the distribution of peat.

Nardus grown alone attained greater shoot mass in the layered than in the mixed substrate, but this was not the case when Calluna was present. Calluna was the superior competitor in both layered and mixed‐substrate pots, but more so in the latter.

This was attributed to (i) the ability of Calluna to exclude Nardus roots from the upper organic material in the layered substrate, and (ii) the greater plasticity of root allocation of Nardus, enabling it to avoid competition with Calluna.
Phosphorus uptake, not carbon transfer, explains arbuscular mycorrhizal enhancement of Centaurea maculosa in the presence of native grassland species
Authors: Zabinski C. A. , Quinn L. , Callaway R. M.
Published: March 2003

Previous studies have shown that arbuscular mycorrhizas (AM) enhance the growth of the invasive forb Centaurea maculosa when growing with native grass species. Using 13CO2, we tested the hypothesis that this enhancement is explained by carbon transfer from native species to C. maculosa via mycorrhizal hyphal linkages.

A C. maculosa plant was paired with one of five native species – three grasses (Festuca idahoensis, Koeleria cristata and Pseudoroegneria spicata) and two forbs (Achillea millefolium and Gaillardia aristata) – in pots that separated the plants with either a mesh barrier (28 µm, excludes fine roots but not hyphae) or a membrane barrier (0·45 µm, excludes roots and hyphae).

13CO2 was added to the atmosphere of either Centaurea or the native species after 20 weeks’ growth. A 25 min pulse application was followed by 7 days’ growth and subsequent harvest.

The biomass response of C. maculosa was consistent with previous experiments: C. maculosa was larger when growing in mesh barrier pots, when hyphae were able to access the opposite side of the pot; in mesh barrier pots only, biomass varied with neighbouring species. Native plant biomass did not vary between mesh‐ vs membrane‐barrier pots.

There was no evidence of carbon transfer, either from the native plant to C. maculosa or in the reverse direction.

Centaurea maculosa contained significantly more phosphorus in mesh‐divided pots, but this depended on the neighbouring plant. The P concentration in C. maculosa was significantly higher in mesh‐divided pots when growing with a grass and not a forb. Native species contained more P in mesh‐divided pots than membrane‐divided pots, and P concentration differed between species (higher in forbs than grasses), but did not vary between mesh‐ and membrane‐divided pots.

Our study suggests that C. maculosa is able to exploit its mycorrhizal symbiosis more effectively than the native grassland species. The mechanism for this appears to be luxury consumption of P through efficient utilization of extra‐radical hyphae, but that effect is dependent on neighbouring species, and occurs when growing with a grass neighbour.

Although no single study can disprove the carbon‐transfer hypothesis, our work suggests that AM‐mediated neighbour effects are the result of mycorrhizal networks that increase species’ access to P. Whether the synergistic effects of neighbours are due to complementarity of AM fungal symbionts utilized by different plant species, or have to do with the structure of AM networks that develop more extensively with multiple host plants, remains to be investigated.
Human ubiquitin‐protein ligase Nedd4: expression, subcellular localization and selective interaction with ubiquitin‐conjugating enzymes
Authors: Anan Tadashi , Nagata Yoichi , Koga Hisashi , Honda Yoshiomi , Yabuki Nami , Miyamoto Chikara , Kuwano Akira , Matsuda Ichiro , Endo Fumio , Saya Hideyuki , Nakao Mitsuyoshi
Published: March 2003

Background

Nedd4 is a ubiquitin‐protein ligase containing a calcium/lipid‐binding domain, multiple WW domains and a C‐terminal Hect domain, which is required for both the ubiquitin transfer and the association with E2 ubiquitin‐conjugating enzymes. Nedd4 has been reported to be involved in the selective ubiquitination of some regulatory proteins in transcription and membrane transport.

Results

Three mRNA species for human Nedd4 were found to be 6.4‐, 7.8‐ and 9.5‐kb in size, and their expression patterns varied among normal tissues and cancer cell lines, indicating the tissue‐ and cell‐specificities of Nedd4 expression. The Nedd4 protein, ≈120 kDa in weight, was found in the cytoplasm, mainly in the perinuclear region and cytoplasmic periphery, of human cultured cells. Neural differentiation induced not only the down‐regulation of Nedd4 but also the localization of the protein to both the cytoplasm and neurites. To identify the ubiquitination pathway that is linked to Nedd4, we demonstrated that specific E2 enzymes, including human Ubc4, UbcH5B, UbcH5C, UbcH6 and UbcH7, could transfer ubiquitin molecules to Nedd4 at the active cysteine residue, whereas E6AP accepted ubiquitins from Ubc4, UbcH5B, UbcH5C and UbcH7. Furthermore, nuclear localization of N‐terminal deletion mutant Nedd4 enabled us to investigate the interaction between Nedd4 and E2 enzyme (Ubc4 or UbcH7) in the cell. The simultaneous expression of the full‐length Nedd4 and E2 enzyme revealed that both proteins mostly colocalized in the cytoplasmic periphery, while the N‐terminal deleted Nedd4 induced the nuclear and perinuclear colocalization with E2 enzyme.

Conclusion

Our findings suggested that Nedd4 plays an important role in the cell regulation, including neural differentiation through cooperation with specific E2 ubiquitination pathways.
Incorporation of Drosophila TAF110 into the yeast TFIID complex does not permit the Sp1 glutamine‐rich activation domain to function in vivo
Authors: Keaveney Marie , Struhl Kevin
Published: March 2003

Background

Acidic activation domains function across eukaryotic species, and hence stimulate transcription by a conserved molecular mechanism. In contrast, glutamine‐rich activation domains function in flies, mammals, and fission yeasts but not in the budding yeast Saccharomyces cerevisiae. The glutamine‐rich activation domain of Sp1 interacts with TAF110, and it has been suggested that this interaction is important for transcriptional activation. S. cerevisiae does not contain a homologue of TAF110, suggesting a potential mechanism to account for the failure of glutamine‐rich activation domains to stimulate transcription.

Results

Here, we have artificially recruited Drosophila TAF110 into the yeast TFIID complex by fusing it to yeast TBP. The resulting TFIID complex supports normal cell growth, but it is unable to mediate Sp1‐dependent activation.

Conclusions

Thus, the interaction of glutamine‐rich activation domains with TAF110 is insufficient for transcriptional activation in vivo, indicating that other targets within the PolII machinery are necessary.
Smad8B, a Smad8 splice variant lacking the SSXS site that inhibits Smad8‐mediated signalling
Authors: Nishita Michiru , Ueno Naoto , Shibuya Hiroshi
Published: March 2003

Background

Members of the TGF‐β superfamily of ligands bind to and activate surface serine/threonine‐kinase receptors. Transduction of these signals requires the Smad proteins, which transiently interact with the activated receptor complex and are phosphorylated on their C terminus, SSXS site, by the type I receptor. Smad8 is a downstream signalling mediator of ALK2/ActRIA.

Results

We have cloned a splice variant of Smad8, designated Smad8B. The Smad8 and Smad8B cDNAs are identical in sequence, except that Smad8B lacks a portion encoding 47 amino acids, including the SSXS phosphorylation site, in the C‐terminal MH2 region. Both Smad8 and Smad8B were expressed in many of the same cell types. Smad8B was capable of specific complex formation with either Smad8 or Smad4 in mammalian cells. In cells expressing constitutively activated ALK2, Smad8B was localized to the cytoplasmic region, whereas Smad8 was translocated into the nucleus. In mammalian cells, Smad8B acted as a dominant inhibitor of BMP signalling.

Conclusions

Smad8B, a splice variant of Smad8, was isolated and found to specifically associate with both Smad8 and Smad4. Smad8B inhibited BMP signalling. Smad8 and Smad8B thus represent novel signal transduction proteins that may regulate the BMP signalling pathway.
The Pho85 kinase, a member of the yeast cyclin‐ dependent kinase (Cdk) family, has a regulation mechanism different from Cdks functioning throughout the cell cycle
Authors: Nishizawa Masafumi , Suzuki Kohji , Fujino Marie , Oguchi Tomoko , Toh‐e Akio
Published: March 2003

Background

The PHO85 gene is a negative regulator of the PHO system in the yeast Saccharomyces cerevisiae and encodes a protein kinase (Pho85p) which is highly homologous to the Cdc28 kinase (Cdc28p). Although the two kinases share a 51% identity and their functional domains are well conserved, PHO85 fails to replace CDC28. Pho85p forms complexes with G1‐cyclin homologues, including Pcl1p, Pcl2p and Pcl9p, and is thought to be involved in the cell‐cycle regulation at G1 and the end of M. By analysing the genetic and biochemical properties of Pho85p, we studied whether the regulation of Pho85p activity is similar to other cyclin‐dependent kinases (Cdks) directly involved in cell cycle regulation.

Results

A functional analysis of various Pho85 mutants revealed that E53 in the PSTAIRE sequence was important for Pho85p function. On the other hand, residues in the T‐loop including S166, S167 and E168, appeared dispensable for Pho85p function, suggesting that the phosphorylation of S166, corresponding to T161 of Cdc2p and T169 of Cdc28p, was not required for the kinase activity of Pho85p. Instead, we found that phosphorylation of Y18, corresponding to Y15 of Cdc2p and Y19 of Cdc28p, may be important for the binding of Pho80p but not of Pcl1p, suggesting that tyrosine phosphorylation may function as a signal which discriminates various Pho85‐cyclins.

Conclusion

In Cdks functioning throughout the cell cycle, tyrosine phosphorylation is inhibitory to the activation of kinase, whereas the phosphorylation of threonine in the T‐loop is essential for activation. Our finding indicates that the regulation mechanism of Pho85p activation appears to be distinct from these Cdks.
Heterogeneous nuclear RNA‐ribonucleoprotein F binds to DNA via an oligo(dG)‐motif and is associated with RNA polymerase II
Authors: Yoshida Tatsushi , Kokura Kenji , Makino Yasutaka , Ossipow Vincent , Tamura Taka‐aki
Published: March 2003

Background

The heterogeneous nuclear ribonucleoprotein F (hnRNP‐F) is one of the constituents of the splicing‐related hnRNP complex. Recent studies suggest that pre‐mRNA modification and splicing factors are associated with transcriptional initiation factors and RNA polymerase II (RNA pol II) at a promoter, implying that pre‐mRNA‐engaged factors might be associated with a promoter.

Results

We isolated a cDNA of rat hnRNP‐F and expressed the recombinant protein. HnRNP‐F selectively bound to oligo(dG) in constructions with other oligohomonucleotides. The ssDNA of an SV40 promoter sequence having the GC‐boxes was bound to hnRNP‐F, while hnRNP‐F bound to the (G/C)‐stretch as dsDNA. Consequently, hnRNP‐F was designated as an oligo(dG)‐binding protein. None of the RNA‐binding domains (RBDs) in hnRNP‐F were critical for the oligo(dG)‐binding. In contrast, the GY‐rich region that exists between RBD‐II and ‐III exhibited an oligo(dG)‐binding ability. We suggest that the GY‐rich region is a novel DNA‐binding motif. HnRNP‐F was shown to contain two types of binding motifs for RNA and DNA. Additionally, we found that hnRNP‐F was co‐precipitated with RNA pol II. Interestingly, the RNA pol II holoenzyme also contained hnRNP‐F. The tissue distribution profile of hnRNP‐F was similar to that of both RNA pol II and TBP.

Conclusion

The above results suggest that hnRNP‐F is associated with a transcriptional initiation apparatus that includes RNA pol II. The DNA‐binding ability of hnRNP‐F might facilitate the entry of pre‐mRNA modification/splicing factors at a promoter.
ORC1 interacts with c‐Myc to inhibit E‐box‐dependent transcription by abrogating c‐Myc–SNF5/INI1 interaction
Authors: Takayama Masa‐aki , Taira Takahiro , Tamai Katsuyuki , Iguchi‐Ariga Sanae M. M. , Ariga Hiroyoshi
Published: March 2003

Background

The c‐myc oncogene product (c‐Myc) is a transcription factor that forms a complex with Max and recognizes the E‐box sequence. c‐Myc plays key functions in cell proliferation, differentiation and apoptosis. As for its activity towards cell proliferation, it is generally thought that c‐Myc transactivates the E‐box‐containing genes that encode proteins essential to cell‐cycle progression. Despite the characterization of candidate genes regulated by c‐Myc in culture cells, these have still not been firmly recognized as real target genes for c‐Myc.

Results

We found that c‐Myc directly bound to the N‐terminal region of origin recognition complex‐1 (ORC1), a region that is responsible for gene silencing, in a state of complex containing other ORC subunits and Max in vivo and in vitro. Furthermore, ORC1 inhibited E‐box‐dependent transcription activity of c‐Myc by competitive binding to the C‐terminal region of c‐Myc with SNF5, a component of chromatin remodelling complex SNF/Swi1.

Conclusions

These results suggest that ORC1 suppresses the transcription activity of c‐Myc by its recruitment into an inactive form of chromatin during some stage of the cell cycle.
The Schizosaccharomyces pombe spo6+ gene encoding a nuclear protein with sequence similarity to budding yeast Dbf4 is required for meiotic second division and sporulation
Authors: Nakamura Tomohiro , Kishida Masao , Shimoda Chikashi
Published: March 2003

Background

Sporulation of the fission yeast Schizosaccharomyces pombe is a cell differentiation process which accompanies meiosis. The spo6+ gene was identified as a sporulation‐specific gene, whose transcription was regulated by the forkhead family transcription factor Mei4.

Results

spo6
+ encodes a protein with sequence similarity to Saccharomyces cerevisiae Dbf4p, which is required for the initiation of DNA replication. However, doubling time and cell morphology of spo6 deletion mutants and spo6‐cDNA over‐expressing cells were indistinguishable from wild‐type cells. Spliced mature mRNAs of spo6+ appeared when diploid cells committed to meiosis. Spo6p fused to green fluorescent protein (GFP) preferentially localized in a nucleus. Although spo6Δ diploids normally underwent premeiotic DNA replication and meiosis‐I, approximately 80% of cells were blocked at the binucleate stage during meiosis and virtually no asci were formed. Anti‐tubulin staining revealed that only 25% of the binucleate cells assembled spindle microtubules for meiosis‐II. In a small number of tetranucleate cells, sister nuclei insufficiently separated and spindles were frequently fragmented. The meiosis‐II arrest phenotype was exaggerated at low temperature and in the presence of caffeine.

Conclusions

These results indicate that Spo6p is a novel Dbf4‐related nuclear protein, which is expressed during meiosis and is indispensable for normal progression of meiosis‐II and sporulation.
Interactions between Mcm10p and other replication factors are required for proper initiation and elongation of chromosomal DNA replication in Saccharomyces cerevisiae
Authors: Kawasaki Yasuo , Hiraga Shin‐ichiro , Sugino Akio
Published: March 2003

MCM10 is essential for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae. Previous work showed that Mcm10p interacts with the Mcm2–7 protein complex that may be functioning as the replication‐licensing factor. In addition, Mcm10p is required during origin activation and disassembly of the prereplicative complex, which allows smooth passage of replication forks.

We show that an mcm10 mutation causes a slow progression of DNA synthesis and a loss of chromosome integrity during the S phase and prevents entry into mitosis, despite apparent completion of chromosomal DNA replication at nonpermissive temperatures. Furthermore, Mcm10p interacts genetically with the origin recognition complex (ORC) and various replication elongation factors, including a subunit of DNA polymerases ε and δ. Mcm10p is an abundant protein (approximately 4 × 104 copies per haploid cell) that is almost exclusively localized in the chromatin and/or nuclear matrix fractions during all phases of the cell cycle. When it is visualized by the chromosome‐spreading method followed by immunostaining, Mcm10p forms punctate foci on chromatin throughout the cell cycle and these foci mostly overlap with those of Orc1p, a component of ORC.

These results suggest that Mcm10p, like the Mcm2–7 proteins, is a critical component of the prereplication chromatin and acts together with ORC during the initiation of chromosomal DNA replication; in addition, Mcm10p plays an important role during the elongation of DNA replication.
Identification of Tetrahymena hsp60 as a 14‐nm filament protein/citrate synthase‐binding protein and its possible involvement in the oral apparatus formation
Authors: Takeda Tetsuya , Yoshihama Isao , Numata Osamu
Published: March 2003

Background

Tetrahymena 14‐nm filament protein (14FP) is bifunctional, with roles as a citrate synthase in mitochondria and as a cytoskeletal protein in nuclear events during fertilization and in oral morphogenesis. In this study, to further our understanding of the bifunctional property of 14FP, we attempted to screen 14FP‐binding proteins using affinity column chromatography.

Results

Through the screening of 14FP‐binding proteins using 14FP‐affinity chromatography, we detected 65 kDa and 70 kDa proteins that bound to 14FP in an ATP dependent manner. From the N‐terminal amino acid sequence, these proteins were identified as the Tetrahymena mitochondrial chaperones, hsp60 and mthsp70, respectively. Tetrahymena hsp60 was recognized with a monoclonal antibody raised against human hsp60. Immunofluorescence and immunoelectron microscopy using the monoclonal antibody showed that Tetrahymena hsp60 was localized to mitochondria. Moreover, Tetrahymena hsp60 was also present at extramitochondrial sites including basal bodies of cilia and oral apparatus, and particularly at the developing oral apparatus during cell division.

Conclusion

These results suggest that Tetrahymena hsp60 is localized in basal bodies and is involved in cortical patterning such as the formation of the oral apparatus as well as having a role in the folding of mitochondrial proteins in mitochondria.
Tumour suppressor activity of human imprinted gene PEG3 in a glioma cell line
Authors: Kohda Takashi , Asai Akio , Kuroiwa Yoshimi , Kobayashi Shin , Aisaka Kohzoh , Nagashima Goro , Yoshida Michihiro C. , Kondo Yasumitsu , Kagiyama Naoto , Kirino Takaaki , Kaneko‐Ishino Tomoko , Ishino Fumitoshi
Published: March 2003

Background

Mouse imprinted gene Peg3 encodes a large C2H2 type zinc finger protein with unique characteristics. Peg3 knockout mice were found to show an impairment in maternal behaviour of the adult female. Mouse Peg3 is located on the proximal region of chromosome 7 which is syntenic to the long arm of human chromosome 19. It has been reported that a loss of heterozygosity (LOH) of chromosome 19q occurs frequently in several glioma types.

Results

We isolated human PEG3 cDNA. Both human and mouse PEG3 were strongly expressed in the adult brain and the Peg3 protein was localized in the nuclei of both neurones and glial cells. A significant decrease in PEG3 expression was more commonly observed in glioma cell lines as compared with that in primary cultures of astrocytes. Transfection of PEG3 cDNA in a glioma cell line resulted in a loss of tumorigenicity in nude mice.

Conclusions

The human PEG3 gene is a paternally expressed imprinted gene. Introduction of PEG3 cDNA into the glioma cells suggests that human PEG3 protein functions as a tumour suppressor. Human PEG3 is located on 19q13.4 and is one of the candidates for tumour suppressor genes that are predicted to be sited in gliomas.
Cofilin‐2, a novel type of cofilin, is expressed specifically at aggregation stage of Dictyostelium discoideum development
Authors: Aizawa Hiroyuki , Kishi Yoshiro , Iida Kazuko , Sameshima Masazumi , Yahara Ichiro
Published: March 2003

Background A conventional cofilin, cofilin‐1 in Dictyostelium discoideum plays significant roles in cell proliferation, phagocytosis, chemotactic movement and macropinocytosis.

Results We identified a new member of the cofilin family, named cofilin‐2 in D. discoideum. Cofilin‐2 shows significant homology to a conventional Dictyostelium cofilin, cofilin‐1, through its entire sequence, and contains residues conserved among the cofilin family that are responsible for actin‐binding. On the other hand, several residues that are conserved among the cofilin family are missing from cofilin‐2. Purified cofilin‐2 depolymerized actin filaments in a dose‐ and pH‐dependent manner and reduced the apparent viscosity of an actin solution, although they did not co‐sediment with actin filaments at all. Cofilin‐2 was not expressed in vegetative cells, but was transiently induced during the aggregation stage of development, whereas cofilin‐1 was predominantly expressed in vegetative cells. Immunocytochemistry revealed that cofilin‐2 localizes at substrate adhesion sites, where cofilin‐1 is almost completely excluded. Disruption of the cofilin‐2 gene caused an increase in actin accumulation at the substrate adhesion sites. We also found that cofilin‐2 did not rescue Δcof1 yeast cells, whereas cofilin‐1 did.

Conclusions Cofilin‐2 may play a distinct role from that of cofilin‐1 in destabilization of the actin cytoskeleton during Dictyostelium development.
Level of the RNA polymerase II in the fission yeast stays constant but phosphorylation of its carboxyl terminal domain varies depending on the phase and rate of cell growth
Authors: Sakurai Hitomi , Ishihama Akira
Published: March 2003

Background: The RNA polymerase II of the fission yeast Schizosaccharomyces pombe consists of 12 Rpb subunits, of which four (Rpb1, Rpb2, Rpb3 and Rpb11) form the assembly and catalytic core and five (Rpb5, Rpb6, Rpb8, Rpb10 and Rpb12) are shared among RNA polymerases I, II and III. The intracellular levels of three RNA polymerase forms should be interrelated, but the control of RNA polymerase formation remains mostly unknown.

Results: To reveal the physiological role and the synthesis control of each Rpb subunit, the intracellular levels of the Rpb proteins were examined in S. pombe growing at various phases under various conditions. Results indicate that the intracellular concentrations of the Rpb proteins stay constant at levels characteristic of the rate and phase of cell growth, and the relative level between the 12 subunits also remains constant, together implying that the intracellular concentration of RNA polymerase II stays constant, as in the case of prokaryotes. As an attempt to gain insights into the activity control of RNA polymerase II, we also analysed the phosphorylation level of the carboxyl‐terminal domain (CTD) of the largest subunit Rpb1. Phosphorylated forms of Tyr1 and Thr4 within 29 repeats of the YSPTSPS heptapeptide were detected in both slow‐migrating IIo and fast‐migrating IIa forms of Rpb1 on SDS‐PAGE (polyacrylamide gel electrophoresis). However, phosphorylated Ser2 and Ser5 were identified only in the IIo form, indicating that Ser phosphorylation contributes to the conformational change in CTD. The phosphorylation levels of Ser, Thr and Tyr all vary depending on the cell culture conditions.

Conclusion: The intracellular level of RNA polymerase II stays constant, but the amount engaged in transcription cycle varies depending on the culture conditions, as estimated from the sites and levels of phosphorylation of Rpb1 CTD.
Transcription termination and anti‐termination in E. coli
Authors: Nudler Evgeny , Gottesman Max E.
Published: March 2003

Transcription termination in Escherichia coli is controlled by many factors. The sequence of the DNA template, the structure of the transcript, and the actions of auxiliary proteins all play a role in determining the efficiency of the process. Termination is regulated and can be enhanced or suppressed by host and phage proteins. This complex reaction is rapidly yielding to biochemical and structural analysis of the interacting factors. Below we review and attempt to unify into basic principles the remarkable recent progress in understanding transcription termination and anti‐termination.
Mispaired P3 region in the hierarchical folding pathway of the Tetrahymena ribozyme
Authors: Ohki Yasushi , Ikawa Yoshiya , Shiraishi Hideaki , Inoue Tan
Published: March 2003

Background: The Tetrahymena group I ribozyme folds into a complex three‐dimensional structure for performing catalytic reactions. The catalysis depends on its catalytic core consisting of two helical domains, P4–P6 and P3–P7, connected by single stranded regions. In the folding process, most of this ribozyme folds in a hierarchical manner in which a kinetically stable intermediate determines the overall folding rate.

Results: Although the nature of this intermediate has not yet been elucidated, a mispaired P3 stem (alt‐P3) appears a likely candidate. To examine the effects of the alt‐P3 structure on the kinetic and thermodynamic properties of the active structure of the ribozyme or its P3–P7 domain formation, we prepared and analysed variant ribozymes in which relative stabilities of the original P3 and alt‐P3 structure were altered systematically.

Conclusion: The results indicate that the alt‐P3 structure is not the major rate‐limiting factor in the folding process.