bluuebunny/crossref_metadata_2025 huggingface dataset:
Sons and daughters: age‐specific differences in parental rearing capacities
Authors: Daunt F. , Monaghan P. , Wanless S. , Harris M. P. , Griffiths R.
Published: March 2003

1. Numerous studies of iteroparous breeders have demonstrated an increase in average breeding performance with parental age.

2. One of the most widely suggested mechanisms to explain this pattern is that the foraging performance of young breeders is relatively poor. Where this is coupled to differences in the costs of rearing male and female offspring, for example because of sexual size dimorphism, young breeders may have difficulties rearing the more expensive sex.

3. This was investigated in the Shag, a monogamous seabird in which adult males are on average 20% heavier than adult females. Young pairs breed later in the season than older pairs, and lay smaller eggs. Differences in timing of breeding and egg quality were controlled for in a cross‐fostering experiment in which older and young individuals reared chicks simultaneously from eggs of similar quality.

4. Male chicks reared by young parents grew more slowly and attained a lower peak mass than those reared by older parents, whereas there was no equivalent difference for female chicks.

5. These results suggest that sons are energetically more demanding on their parents than daughters, and the more expensive sex fledges in significantly poorer condition when the parents are young. Therefore, it is predicted that optimal offspring sex ratio will vary with parental age.
Field and laboratory evidence for acclimation without costs in an egg parasitoid
Authors: Thomson L. J. , Robinson M. , Hoffmann A. A.
Published: March 2003

1. Acclimation responses are normally assumed to increase the fitness of an organism, but supporting evidence is generally lacking, especially under field conditions. Even where an advantage arises from acclimation, this can be offset by other fitness costs. Heat hardening is a well‐known form of acclimation in many invertebrates where exposure to high but sub‐lethal temperatures protects against subsequent heat‐induced death.

2. Previous laboratory work has shown that hardening occurs in wasp egg parasitoids of the genus Trichogramma because pretreatment with a mild temperature shock can increase survival at high temperatures. This system allowed fitness benefits and costs of hardening under the more stringent conditions in nature to be tested.

3. Heat hardening in Trichogramma carverae was considered at the pupal and adult stages and it was shown that hardening had a beneficial effect on heat resistance in the laboratory. Moreover, hardening enhanced adult fitness in the field under hot conditions. No costs of acclimation were detected under mild field and laboratory conditions. Conditions leading to hardening without costs were different from another Trichogramma species.

4. Hardening can therefore have fitness benefits without costs under field and laboratory conditions, and this process can be used to enhance parasitism rates in inundative commercial releases of Trichogramma against moth pests.
Functional traits, productivity and effects on nitrogen cycling of 33 grassland species
Authors: Craine J. M. , Tilman D. , Wedin D. , Reich P. , Tjoelker M. , Knops J.
Published: March 2003

Our goal was to determine the relationships among ecophysiological, whole‐plant and ecosystem traits of a wide variety of grassland species grown under field conditions in the long term. We measured 87 traits for 33 species (32 perennial, one annual) grown in monoculture for 5 years on sandy soils, and determined the relationship among traits and their correspondence with current functional classifications.

Among non‐legumes, species that produced and maintained large amounts of biomass had tough, low‐activity leaves and roots, high root : shoot ratios, and low extractable inorganic nitrogen and N mineralization in their soils. The set of correlations among the functional traits of fine roots for non‐legumes parallels the set of correlations for leaf functional traits. Low‐N species maintained greater biomass than high‐N species, more by producing tissues with low N concentrations and greater longevity than by acquiring more N. Greater relative production below ground, and the production of long‐lived below‐ground structures, were both important in determining the high root : shoot ratio of species.

For legumes, N2 fixation not only led to greater above‐ground biomass production, but also was associated with low fine root production; greater relative production of stem biomass; and accelerated ecosystem N cycling compared to non‐legumes.

The measured traits, as condensed via principal components analysis, differentiated the 32 species into groups that corresponded with a common grassland functional classification scheme (C3 grasses, C4 grasses, forbs, legumes, woody species) as well as an alternative, continuous approach. For all traits, species can be arrayed well along two continuous axes. The first axis separates cool‐season and warm‐season legumes; the second low‐N and high‐N non‐legumes.

These continuous classifications show the generality of the two strategies for dealing with low nitrogen availability (N2 fixation and the low‐N suite of traits) and extends the strategies to span organ‐level traits to ecosystem processes including roots, whole‐plant patterns of productivity, and nutrient cycling. The correlations of traits among species will also be useful in predicting a large number of important parameters associated with plant growth from the measurement of a few, key traits.
Environmental randomness underlies morphological complexity of colonial diatoms
Authors: Passy S. I.
Published: March 2003

The morphology and distribution of six benthic colonial diatoms were investigated from the perspective of fractal geometry and stream ecology to test whether colonial complexity of benthic diatoms is associated with a tolerance to environmental variability, that is, if a random force, such as the unpredictability of current velocity, could be responsible for the development of a high morphological organization.

The fractal dimension of diatom colony perimeters ranged from 1·06 to 1·54, indicating a vast morphological variation from simple geometric shapes to very complex outlines. The niche breadth of the six colonial diatoms, defined from regression models of species abundance along a current velocity gradient, also showed a substantial variation, from 0·48 to 0·79 m s−1.

There was a strong positive relationship between diatom morphological complexity and species niche breadth, suggesting that increased morphological complexity in colonial diatoms is a possible evolutionary strategy for survival in unpredictable environments.
Leaf senescence and resorption as mechanisms of maximizing photosynthetic production during canopy development at N limitation
Authors: Franklin O. , Ågren G. I.
Published: March 2003

A canopy N distribution model optimizing photosynthesis was combined with mechanisms for senescence and N resorption to predict canopy leaf area index (LAI) development as a function of canopy nutrient content, Nc.

Shedding of leaves at the bottom of the canopy was initiated when it increased canopy photosynthesis, through resorption and redistribution of N from lost leaves. The amount of N resorbed was modelled as a fraction (Rf) of the N in the leaf prior to senescence.

For a fixed Nc, the LAI at which leaf shedding was initiated was calculated for different Rfs for canopies of Amaranthus cruentus, Glycine max, Oryza sativa and Sorghum bicolor.

Predicted LAIs exceeded optimal LAIs estimated without leaf shedding and N resorption. LAI increased with increasing Rf. For all four species, the model closely predicted the observed LAIs with Rf = 0·7.

Area‐based resorption efficiency increased and litter N concentration decreased with increasing LAI and average canopy N concentration (mol N m−2 leaf). A reduction of Rf, e.g. in response to increased soil N availability, decreases resorption efficiency.

The model provides a mechanistic basis for interpreting plant nutrient–resorption–LAI relations.
Nutrients, arbuscular mycorrhizas and competition interact to influence seed production and germination success in Achillea millefolium
Authors: Allison V. J.
Published: March 2003

Environmental variables that positively affect one aspect of plant fitness, may have no effect, or even negatively affect some other component of fitness.

Using individuals of Achillea millefolium grown under field conditions in Michigan, USA, the hypothesis was tested that seed number was determined largely by plant biomass, while seed germination success depends on tissue nutrient concentrations. The impact was assessed of four biotic and abiotic factors on seed number and germination success: root competition, shoot competition, fertilizer, and removal of fungi by fungicide application.

Using a path analysis, it was found that total plant biomass positively affected both seed number and germination success, while inclusion of other variables did not greatly affect the amount of variation the model was able to explain. Fertilizer and fungicide increased, while root and shoot competition decreased both seed number and germination.

Fungicide applied to the maternal plant increased biomass but decreased tissue phosphorus concentrations. In species where germination responds to nutrient concentrations, the potential exists for opposing impacts of environmental treatments on different components of fitness.

This study suggests that environmental impacts on seed number will outweigh impacts on germination success under field conditions, and that biomass is an adequate surrogate for fitness in herbaceous plants.
The physiology of host feeding in parasitic wasps: implications for survival
Authors: Giron D. , Rivero A. , Mandon N. , Darrouzet E. , Casas J.
Published: March 2003

Models developed to predict behavioural and life‐history decisions of parasitoids rely heavily on assumptions concerning the nature and function of the nutrients that the female obtains from the host.

Using a combination of colorimetric and thin‐layer chromatography techniques, we analysed the composition of the host‐feeding fluid consumed by the parasitoidEupelmus vuillettiand found it to be consistent with the composition of the host's haemolymph.

The analysis of the haemolymph revealed that it was rich in proteins but also rich in sugars, trehalose and sucrose in particular.

Injections of these sugars at haemolymph concentrations directly into the parasitoid's haemolymph showed that these sugars are solely responsible for the increased longevity observed in host‐fed females.

This study is the first to identify the constituents of host‐feeding meals and their impact on parasitoid longevity and provide further evidence of the extent to which parasitoid behavioural decisions are determined by physiological variables. Our study demonstrates that the integration of nutritional physiology, behaviour, life history and population dynamics is essential in order to understand parasitoid foraging ecology.
Measures of reproductive allometry are sensitive to sampling bias
Authors: Bonnet X. , Shine R. , Lourdais O. , Naulleau G.
Published: March 2003

In both interspecific and intraspecific comparisons, maternal body size is the strongest predictor of fecundity for many kinds of animals. However, it has not been widely appreciated that the usual empirical descriptors of this relationship (correlation coefficient, slope andP‐value of the linear regression between maternal body size and offspring number) are sensitive to a factor that is very labile and subject to methodological bias: the degree of maternal investment, specifically the ratio of litter mass to maternal body mass (relative clutch mass, RCM).

Samples of females used to assess reproductive allometry may often be biased with respect to RCM. For example, RCMs may vary through time within a single population as a consequence of prey availability, or may vary geographically among populations. Also, females with low RCMs may be more difficult to capture, or may be discarded by researchers who do not realize that they are reproductive.

Our analyses on 173 litters of aspic vipers (Vipera aspisLinné) from central western France show that estimates of reproductive allometry are very sensitive to RCM: samples composed of high‐RCM females show a positive and highly significant reproductive allometry, whereas samples composed of low RCM females do not. Conclusions also depend on the method of regression analysis used. This result has strong implications for methodology (i.e. selection of samples, choice of analytical methods and timescales of study) in this field of research.
A measure of the mitotic index: studies of the abundance and half‐life of p34cdc2 in cultured cells and normal and neoplastic tissues
Authors: Gannon Julian V. , Nebreda Angel , Goodger  , Morgan  , Hunt Tim
Published: March 2003

Background

The cdc2 gene encodes a protein kinase, p34cdc2, that is essential for mitosis, and is present at high levels in dividing cells. Classical studies of the levels of this protein in dividing and resting cells used antibodies that cross‐react with other members of the CDK family, in particular with CDK2. We have therefore re‐examined the abundance of p34cdc2 in a variety of tissues and cell lines, using a highly specific, epitope‐mapped monoclonal antibody that does not react with CDK2.

Results

We observed high levels of p34cdc2 in proliferating cells, especially those in neoplastic tissues. Cells that have withdrawn from the cell cycle have low or undetectable levels. At the end of mitosis, the level of p34cdc2 declines, with simple first‐order kinetics, with a half‐life which is never less than 6 h and is more typically about 18 h. The persistence of p34cdc2 after the last cell division is comparable to that of PCNA, a commonly used marker of proliferation.

Conclusions

The immunochemical detection of p34cdc2 provides an accurate, reliable and meaningful measure of the proliferative activity of cells in tissues. We suggest that p34cdc2 should be considered as the most authentic molecular marker of the mitotic index.
Region‐specific expression of murine Hox genes implies the Hox code‐mediated patterning of the digestive tract
Authors: Sekimoto Tomohisa , Yoshinobu Kumiko , Yoshida Michio , Kuratani Shigeru , Fujimoto Shoji , Araki Masatake , Tajima Naoya , Araki Kimi , Yamamura Ken‐ichi
Published: March 2003

Background

Hox genes encode transcription factors which are involved in the establishment of regional identities along the anteroposterior (AP) body axis. To elucidate the AP patterning of the digestive tract, we have systematically examined the expression patterns of Hox genes belonging to paralogue groups 6, 7, 8 and 9 by whole‐mount in situ hybridization and by section in situ hybridization analyses.

Results

The expression patterns of these genes showed co‐linearity along the wall of the digestive tract, thereby yielding the Hox code of the gut. The expression boundaries of the Hox genes at later stages (12.5 d.p.c.) corresponded to the morphological boundaries of individual gut subdomains.

Conclusions

The visceral mesoderm‐restricted expression suggested that the Hox code primarily functions in the mesenchymal specification which eventually leads to the regional differentiation of gut subdomains as the result of epithelial–mesenchymal interactions. Overlapping expression patterns were found among the paralogous Hox genes, indicating that the paralogues may have redundant functions in the specification of the gut.
Tip60 acetylates six lysines of a specific class in core histones in vitro
Authors: Kimura Akatsuki , Horikoshi Masami
Published: March 2003

Background: Tip60, an HIV‐1‐Tat interactive protein, is a nuclear histone acetyltransferase (HAT) with unique histone substrate specificity. Since the acetylation of core histones at particular lysines mediates distinct effects on chromatin assembly and gene regulation, the identification of lysine site specificity of the HAT activity of Tip60 is an initial step in the analysis of its molecular function.

Results: Tip60 significantly acetylates amino‐terminal tail peptides of histones H2A, H3 and H4, but not H2B, consistent with substrate preference on intact histones. Preferred acetylation sites for Tip60 are the Lys‐5 of histone H2A, the Lys‐14 of histone H3, and the Lys‐5, ‐8, ‐12, ‐16 of histone H4, determined by a method which combined matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) measurements and Lys‐C endopeptidase digestion, or a method detecting the incorporation of radiolabelled acetate into synthetic peptides.

Conclusion: The lysine site specificity of Tip60 in histone amino‐terminal tail peptides in vitro has been characterized by an assay measuring the molecular mass of endopeptidase digested peptides, or a previously described assay. These results agree well with our proposed classification of lysines in core histones. The classification may be useful for an analysis of the relationships between HATs and the substrates of other uncharacterized HATs.
Enhancer and silencer binding proteins involved in the rat cdc2 promoter activation at the G1/S boundary
Authors: Takeuchi Arata , Shimizu Mamoru , Nishina Mitsue , Shirasuna Kenna , Miura Akihiko , Nakajima Takuma , Oda Kinichiro
Published: March 2003

Background

Expression of the rat cdc2 gene during G1‐S phase progression is negatively and positively regulated by the silencer and enhancer elements located upstream of the basal promoter. The silencer and enhancer sequences resemble each other, but the silencer contains extra internal AG residues.

Results

The cDNA clones encoding the enhancer binding proteins cdc2E1 and cdc2E2 were isolated by South‐Western blotting. cdc2E1 and cdc2E2 comprise 436 and 256 amino acids and have two RNA binding domains which contain an RNP1 octamer and an RNP 2 hexamer. Both cdc2E1 and cdc2E2 bind to the double‐stranded and single‐stranded silencer and enhancer sequences, but their binding affinity to the enhancer was stronger than that to the silencer. Transfection of quiescent 3Y1 cells with the cdc2 promoter‐luciferase constructs, followed by serum stimulation, showed that the promoter activation at the G1‐S phase boundary was reduced greatly by base substitutions within the enhancer, but not within the silencer. Gel shift assays with oligonucleotides containing both the silencer and enhancer showed that formation of the large complex was greatly reduced if base‐substitutions were introduced into the enhancer, but not within the silencer. The complex was supershifted completely by anti‐cdc2E1 antibody and partially by anti‐cdc2E2 antibody.

Conclusion

These results suggest that cdc2E1 and cdc2E2 preferentially form the multimeric complex at the enhancer site after the late G1 phase for activation of the cdc2 promoter.
Competitive interaction of Seven in absentia homolog‐1A and Ca2+/calmodulin with the cytoplasmic tail of group 1 metabotropic glutamate receptors
Authors: Ishikawa Kenji , Nash S. Russell , Nishimune Atsushi , Neki Akio , Kaneko Satoshi , Nakanishi Shigetada
Published: March 2003

Background

Group 1 metabotropic glutamate receptors (mGluR1 and mGluR5) are coupled to inositol trisphosphate/Ca2+ signaling via G proteins and play an important role in excitatory synaptic transmission. To explore the regulation of group 1 mGluR function, we applied the yeast two‐hybrid system using the intracellular carboxy‐terminal domain of group 1 mGluRs (group 1 ct‐mGluRs) and attempted to identify novel protein–protein interactions of group 1 mGluRs.

Results

The two‐hybrid screening revealed a specific interaction between group 1 ct‐mGluRs and Siah‐1A, the mammalian homolog of Drosophila seven in absentia which is involved in photoreceptor cell differentiation via the ubiquitin/proteasome‐dependent mechanism. This interaction occurs within a homologous 27–28 amino acid stretch within group 1 ct‐mGluRs and requires the latter two‐thirds of Siah‐1A. Following coexpression in COS‐7 cells, myc‐tagged Siah‐1A was coimmunoprecipitated with the flag‐tagged ct‐mGluR1 by anti‐flag antibody. Furthermore, in vitro binding revealed that Siah‐1A and Ca2+/calmodulin (CaM) binding sites overlap, such that Siah‐1A binding is competitively inhibited by CaM in a Ca2+‐dependent manner.

Conclusions

The results demonstrate a direct interaction between group 1 mGluRs and Siah‐1A and suggest a novel modulatory mechanism mediated by a competitive interaction between Ca2+/CaM and Siah‐1A.
Molecular basis of tissue‐specific gene expression mediated by the Runt domain transcription factor PEBP2/CBF
Authors: Ito Yoshiaki
Published: March 2003

The Runt domain transcription factor PEBP2/CBF is a heterodimer of α and β subunits. Of the three mammalian α subunit genes, which are homologues of the Drosophila gene runt, PEBP2αA/CBFA1 and PEBP2αB/AML1 are critical regulators of osteogenesis and haematopoiesis, respectively. A unique functional interaction between PEBP2αB/AML1 and Ets‐1 was recently observed, and provides a clear example of context‐dependent transcriptional regulation. An elaborate mechanism of cooperation between the two factors may represent a basis for tissue‐specific gene expression, which is thought to be achieved by combinations of specific sets of transcription factors unique to each cell lineage or stage of differentiation. A possible molecular basis for the critical role(s) played by PEBP2/CBF in tissue determination is discussed in light of these observations.
amiB, a novel gene required for the growth/differentiation transition in Dictyostelium
Authors: Kon Takahide , Adachi Hiroyuki , Sutoh Kazuo
Published: March 2003

Background

The differentiation programme of Dictyostelium discoideum is initiated by starvation. Nutrient depletion triggers the differentiation of Dictyostelium cells through the transcriptional inactivation of some growth‐phase genes, as well as through the transcriptional activation of essential genes required for the aggregation of the cells. The adenylyl cyclase (ACA) gene, acaA, is one of the earliest genes expressed following starvation. ACA produces intracellular and extracellular cAMP that drives further differentiation by inducing chemotaxis, developmental gene expression and morphogenesis of Dictyostelium cells. Although several genes have been identified as being essential for the initiation of differentiation process, such as the transcriptional activation of ACA expression, the molecular mechanisms of the growth/differentiation transition remain to be explored.

Results

Using insertional mutagenesis, we have isolated a mutant that does not aggregate upon starvation. The disrupted gene, amiB (aggregation minus B), is predicted to encode a novel protein of 298.9 kDa. When starved, amiB− cells produced an undetectable level of cAMP. Analyses of gene expression showed that amiB− cells fail to turn off the expression of one of the growth‐phase genes, cprD, and to turn on the expression of ACA following starvation. The ectopic expression of ACA from a constitutive promoter rescued the differentiation and morphogenesis of amiB− mutants. Furthermore, the ectopic expression of a putative transcriptional factor DdMyb2 or a catalytic subunit of cAMP‐dependent protein kinase (PKA‐C), both of which are thought to be involved in ACA expression pathway(s), also rescued the starvation‐induced ACA expression and further differentiation of the amiB− mutant.

Conclusion

These results suggest that AmiB plays a role at the start of Dictyostelium differentiation through induction of the ACA expression which is essential for cAMP signalling.
A human homologue of yeast anti‐silencing factor has histone chaperone activity
Authors: Munakata Tsubasa , Adachi Nobuaki , Yokoyama Natsuko , Kuzuhara Takashi , Horikoshi Masami
Published: March 2003

Background

Structural changes in chromatin play essential roles in regulating eukaryotic gene expression. Silencing, potent repression of transcription in Saccharomyces cerevisiae, occurs near telomeres and at the silent mating‐type loci, as well as at rDNA loci. This type of repression relates to the condensation of chromatin that occurs in the heterochromatin of multicellular organisms. Anti‐silencing is a reaction by which silenced loci are de‐repressed. Genetic studies revealed that several factors participate in the anti‐silencing reaction. However, actions of factors and molecular mechanisms underlying anti‐silencing remain unknown.

Results

Here we report the functional activity of a highly evolutionarily conserved human factor termed CIA (CCG1‐interacting factor A), whose budding yeast homologue ASF1 has anti‐silencing activity. Using yeast two‐hybrid screening, we isolated histone H3 as an interacting factor of CIA. We also showed that CIA binds to histones H3/H4 in vitro, and that the interacting region of histone H3 is located in the C‐terminal helices. Considering the functional role of CIA as a histone‐interacting protein, we found that CIA forms a nucleosome‐like structure with DNA and histones.

Conclusions

These results show that human CIA, whose yeast homologue ASF1 is an anti‐silencing factor, possesses histone chaperone activity. This leads to a better understanding of the relationship between chromatin structural changes and anti‐silencing processes.
Identification of an imprinted gene, Meg3/Gtl2 and its human homologue MEG3, first mapped on mouse distal chromosome 12 and human chromosome 14q
Authors: Miyoshi Naoki , Wagatsuma Hirotaka , Wakana Shigeharu , Shiroishi Toshihiko , Nomura Masashi , Aisaka Kohzoh , Kohda Takashi , Surani M. Azim , Kaneko‐Ishino Tomoko , Ishino Fumitoshi
Published: March 2003

Background

The paternal duplication of mouse distal chromosome 12 leads to late embryonal/neonatal lethality and growth promotion, whereas maternal duplication leads to late embryonal lethality and growth retardation. Human paternal or maternal uniparental disomies of chromosome 14q that are syntenic to mouse distal chromosome 12 have also been reported to show some imprinting effects on growth, mental activity and musculoskeletal morphology. For the isolation of imprinted genes in this region, a systematic screen of maternally expressed genes (Megs) was carried out by our subtraction‐hybridization method using androgenetic and normally fertilized embryos.

Results

We have isolated seven candidate clones of the mouse Meg gene. Among them, we identified a novel maternally expressed imprinted gene, Meg3, on mouse distal chromosome 12 and showed that it was identical to the Gtl2 gene. We also found that the human homologue MEG3 on chromosome 14q was also monoallelically expressed.

Conclusions

This is the first identification of the imprinting gene, both on mouse distal chromosome 12 and on human chromosome 14q, respectively. Because there are no obvious open reading frames in either the mouse Meg3/Gtl2 or human MEG3, the function of these genes remains unclear. However, this result will provide a good basis for the further investigation of several important imprinted genes in this chromosomal region.
Genes involved in the determination of the rate of inversions at short inverted repeats
Authors: Slupska Malgorzata M. , Chiang Ju‐Huei , Luther Wendy M. , Stewart Jean Lee , Amii Lisa , Conrad Alexis , Miller Jeffrey H.
Published: March 2003

Background

Not all of the enzymatic pathways involved in genetic rearrangements have been elucidated. While some rearrangements occur by recombination at areas of high homology, others are mediated by short, often interrupted homologies. We have previously constructed an Escherichia coli strain that allows us to examine inversions at microhomologies, and have shown that inversions can occur at short inverted repeats in a recB,C‐dependent fashion.

Results

Here, we report on the use of this strain to define genetic loci involved in limiting rearrangements on an F′ plasmid carrying the lac genes. Employing mini‐Tn10 derivatives to generate insertions near or into genes of interest, we detected three loci (rmuA,B,C) that, when mutated, increase inversions. We have mapped, cloned and sequenced these mutator loci. In one case, inactivation of the sbcC gene leads to an increase in rearrangements, and in another, insertions near the recE gene lead to an even larger increase. The third gene involved in limiting inversions, rmuC, has been mapped at 86 min on the E. coli chromosome and encodes a protein of unknown function with a limited homology to myosins, and some of the SMC (structural maintenance of chromosomes) proteins.

Conclusions

This work presents the first example of an anti‐mutator role of the sbcC,D genes, and defines a new gene (rmuC) involved in DNA recombination.
Mis3 with a conserved RNA binding motif is essential for ribosome biogenesis and implicated in the start of cell growth and S phase checkpoint
Authors: Kondoh Hiroshi , Yuasa Tatsuro , Yanagida Mitsuhiro
Published: March 2003

Background

In normal somatic cell cycle, growth and cell cycle are properly coupled. Although CDK (cyclin‐dependent kinase) activity is known to be essential for cell cycle control, the mechanism to ensure the coupling has been little understood.

Results

We here show that fission yeast Mis3, a novel evolutionarily highly conserved protein with the RNA‐interacting KH motif, is essential for ribosome RNA processing, and implicated in initiating the cell growth. Growth arrest of mis3‐224, a temperature sensitive mutant at the restrictive temperature, coincides with the early G2 block in the complete medium or the G1/S block in the release from nitrogen starvation, reflecting coupling of cell growth and division. Genetic interactions indicated that Mis3 shares functions with cell cycle regulators and RNA processing proteins, and is under the control of Dsk1 kinase and PP1 phosphatase. Mis3 is needed for the formation of 18S ribosome RNA, and may hence direct the level of proteins required for the coupling. One such candidate is Mik1 kinase. mis3‐224 is sensitive to hydroxyurea, and the level of Mik1 protein increases during replication checkpoint in a manner dependent upon the presence of Mis3 and Cds1.

Conclusions

Mis3 is essential for ribosome biogenesis, supports S phase checkpoint, and is needed for the coupling between growth and cell cycle. Whether Mis3 interacts solely with ribosomal precursor RNA remains to be determined.
Requirement of transfer‐messenger RNA for the growth of Bacillus subtilis under stresses
Authors: Muto Akira , Fujihara Ai , Ito Ken‐ichi , Matsuno Jun , Ushida Chisato , Himeno Hyouta
Published: March 2003

Background

Bacterial transfer‐messenger RNA (tmRNA, 10Sa RNA) is involved in a trans‐translation reaction which contributes to the degradation of incompletely synthesized peptides and to the recycling of stalled ribosomes. However, its physiological role in the cell remains elusive. In this study, an efficient system for controlling the expression of the gene for tmRNA (ssrA), as well as a tmRNA gene‐defective strain (ssrA::cat), were constructed in Bacillus subtilis. The effects of tmRNA on the growth of the cells were investigated under various physiological culture conditions using these strains.

Results

The cells were viable in the absence of ssrA expression under the usual culture conditions. However, the growth rate of cells without tmRNA expression, relative to that of the expressed cells, decreased with elevating temperature (> 45 °C), and at 52 °C, the highest temperature for growth of the wild‐type, cells grew depending on the expression level of tmRNA. Furthermore, the transcription level of the ssrA from the authentic promoter at a high temperature (51 °C) was about 10‐fold higher than that at a lower temperature (37 °C). tmRNA‐dependent growth and an increase in tmRNA amount were also observed in cells under other stresses, such as high concentrations of ethanol or cadmium chloride. It is also shown that alanylated tmRNA rather than tmRNA‐mediated proteolysis is required for growth at high temperature.

Conclusion

The expression of tmRNA gene (ssrA) is required for the efficient growth of B. subtilis under several strong stresses. The transcription of ssrA increases under several stressful conditions, suggesting that it is a stress‐response gene. Alanyl‐tmRNA, probably via its ability of recycling stalled ribosomes via trans‐translation, is involved in the stress tolerance of bacteria.
Involvement of fatty acid synthase in axonal development in mouse embryos
Authors: Ueno Kohji
Published: March 2003

Background

Dynamic protein palmitoylation of signalling proteins is thought to be an important step in the regulation of signal transduction in eukaryotic cells. The enzyme responsible for protein palmitoylation in vertebrates, however, has not been identified. I have previously reported that p260/270, which is expressed in embryos of the silkworm Bombyx mori, has protein palmitoylase activity.

Results

A homologue of Bombyx p260/270, mouse fatty‐acid synthase (FAS), was shown to be expressed specifically throughout most of the central and peripheral nervous system in mouse embryos. Mouse FAS was expressed specifically in cultured primary neurones in which growth‐associated protein (GAP)‐43 was expressed. GAP‐43, by protein palmitoylation, regulates Go signal transduction and neural axonal growth. In a cell‐free system, purified FAS from mouse embryos transferred palmitate to GAP‐43 through cysteine residues. Furthermore, cerulenin, an inhibitor of FAS, reduced axonal growth and in vivo palmitoylation of GAP‐43 in cultured neurones.

Conclusions

Mouse FAS was hypothesized to be responsible for the palmitoylation of GAP‐43 and subsequent regulation of axonal growth in mouse embryos.
Interaction of ribosome recycling factor and elongation factor EF‐G with E. coli ribosomes studied by the surface plasmon resonance technique
Authors: Ishino Tetsuya , Atarashi Kenji , Uchiyama Susumu , Yamami Tohru , Saihara Yukari , Yoshida Toshio , Hara Hiroto , Yokose Kazuteru , Kobayashi Yuji , Nakamura Yoshikazu
Published: March 2003

Ribosome recycling factor (RRF), in concert with elongation factor EF‐G, is required for disassembly of the post‐termination complex of a ribosome after the release of polypeptides. How RRF dissociates the complex has long been puzzling. Crystal structures of RRF molecules have been solved recently and shown to mimic a transfer RNA (tRNA) shape, which prompted us to examine whether RRF binds to the ribosome as tRNA does.

The formation of ribosome complexes on the surface‐coupled RRF and elongation factor EF‐G of Escherichia coli was monitored in real time with a BIACORE 2000 instrument based on the surface plasmon resonance technique. RRF interacted with 70S ribosomes as well as 50S and 30S subunits, although it interacted preferentially with 50S subunits, which was clearly seen under high but physiological ionic conditions. This 50S interaction was diminished by a single amino acid substitutions for Arg132 of RRF, which did not appreciably affect the protein folding but nullified the activity in vivo and in vitro. Moreover, a set of antibiotics that inhibited the RRF−50S interaction were also inhibitory to the polysome breakdown activity of RRF in vitro. The BIACORE technique also worked very well in demonstrating the action of the antibiotics thiostrepton and fusidic acid, which are inhibitory to the RRF function by freezing the pre‐ and post‐translocation intermediates catalysed by EF‐G.

These results suggest that the preferential interplay of RRF with the 50S subunit may be of biological significance, probably reflecting the mode of RRF action. The BIACORE technique proved useful for real‐time monitoring of the interaction between the ribosome and translation factors, as well as for screening of potential inhibitors for ribosome recycling factor.
Hes7: a bHLH‐type repressor gene regulated by Notch and expressed in the presomitic mesoderm
Authors: Bessho Yasumasa , Miyoshi Goichi , Sakata Ryoichi , Kageyama Ryoichiro
Published: March 2003

Background:

Whereas Notch signalling is essential for somitogenesis, mice deficient for the basic helix‐loop‐helix (bHLH) genes Hes1 and Hes5, downstream Notch effectors, display normal somite formation, indicating that there may be an as‐yet unidentified Hes1‐related bHLH gene.

Results:

We identified a novel bHLH gene, designated Hes7, from mouse embryos. Hes7 has a conserved bHLH domain in the amino‐terminal region and the WRPW domain at the carboxy‐terminal end, like Hes1. The mouse Hes7 gene is located next to Aloxe3, which is mapped to a position 37.0 cM from the centromere on chromosome 11. In a transfection analysis, Hes7 represses transcription from the N box‐ and E box‐containing promoters. In addition, Hes7 suppresses the E47‐induced transcriptional activation. Promoter analysis indicated that Hes7 expression is controlled by Notch signalling. Strikingly, Hes7 is specifically expressed in the presomitic mesoderm in a dynamic manner. We also identified two related bHLH genes from human: one is closely related to mouse Hes7 and therefore designated hHes7 and the other designated hHes4.

Conclusion:

The structure, transcriptional activity and expression pattern in the presomitic mesoderm of Hes7 are very similar to those of Hes1, suggesting that Hes7, together with Hes1, may play a role in somite formation under the control of Notch signalling.
The Drosophila UBC9 homologue lesswright mediates the disjunction of homologues in meiosis I
Authors: Apionishev Sergey , Malhotra Deepak , Raghavachari Seshasayee , Tanda Soichi , Rasooly Rebekah S.
Published: March 2003

Background

In Saccharomyces cerevisiae and other organisms, the UBC9 (ubiquitin‐conjugating 9) protein modifies the function of many different target proteins through covalent attachment of the ubiquitin‐like protein SMT‐3/SUMO.

Results

Using a second‐site suppression screen of a mutation in the nod locus with a variable meiotic phenotype, we have identified mutations in the Drosophila melanogaster UBC9 homologue, encoded by the gene lesswright (lwr). lwr mutations dominantly suppress the nondisjunction and cytological defects of female meiotic mutations that affect spindle formation. The lwr lethal phenotype is rescued by a Drosophila UBC9/lwr transgene.

Conclusions

We suggest that LWR mediates the dissociation of heterochromatic regions of homologues at the end of meiotic prophase I. Our model proposes that when there is less LWR protein, homologues remain together longer, allowing for more normal spindle formation in mutant backgrounds and therefore more accurate meiotic chromosome segregation.
Coronin forms a stable dimer through its C‐terminal coiled coil region: an implicated role in its localization to cell periphery
Authors: Asano Shiro , Mishima Masanori , Nishida Eisuke
Published: March 2003

Background

Coronin is an actin‐binding protein, which contains WD (Trp‐Asp) repeats and a coiled‐coil motif, and plays a role in regulating organization of the actin cytoskeletal network. Coronin localizes to the cell periphery, is involved in lamellipodium extension, and has an implicated role in cytokinesis, cell motility and phagocytosis.

Results

Our experiments with two different tagged forms of Xenopus coronin (Xcoronin) have shown that Xcoronin forms an oligomer. This oligomer complex is stable, resistant to 2.4 m NaCl, 0.6 m KI or 2 m urea. Physiochemical analysis of endogenous Xcoronin and the protein expressed in COS7 cells or in bacteria has revealed that the oligomer complex is an Xcoronin dimer. A C‐terminal coiled‐coil motif of Xcoronin is necessary and sufficient for the dimerization. Mutations in the coiled‐coil motif generated dimerization deficient mutants of Xcoronin. Moreover, these mutant forms of Xcoronin failed to localize to the cell periphery, suggesting that dimerization is important for the proper subcellular localization of Xcoronin.

Conclusion

Xcoronin forms a stable dimer via its C‐terminal coiled‐coil region. We propose that coronin dimerization is necessary for its proper subcellular localization and function.
p48 subunit of mouse PTF1 binds to RBP‐Jκ/CBF‐1, the intracellular mediator of Notch signalling, and is expressed in the neural tube of early stage embryos
Authors: Obata Junko , Yano Masato , Mimura Hideki , Goto Tomokazu , Nakayama Rika , Mibu Yuko , Oka Chio , Kawaichi Masashi
Published: March 2003

Background

Development of the pancreas and the nervous tissues is regulated by common transcription factors. A basic helix‐loop‐helix protein, p48 of pancreas transcription factor 1 (PTF1), is essential for differentiation of the exocrine acinar cells.

Results

We isolated PTF1 p48 from 9.5‐day mouse embryos as a binding protein of RBP‐Jκ, a mediator of Notch signalling. p48 bound to RBP‐Jκ more strongly than and in a distinct way from Notch1. In 9.5–12.5 day embryos, p48 was expressed in the dorsal part of the neural tube as well as in the pancreatic buds. Two lines of evidence suggested functions of p48 in neurogenesis: (i) expression of p48 was induced in P19 cells when they committed to neural fate upon retinoic acid treatment, and (ii) p48 over‐expressed in Xenopus embryos repressed the development of neuronal precursors. p48 inhibited the MASH1‐activated transcription from the E‐box, while p48 stimulated transcription from the PTF1 motif synergistically with E47. The p48/E47‐activated transcription from the PTF1 motif was stimulated further by RBP‐Jκ and RBP‐Jκ derivatives that mimicked the active RBP‐Jκ/Notch complex.

Conclusions

In developing embryos, p48 is expressed in both the nervous system and the pancreas. p48 inhibits neuronal differentiation. We propose possible mechanisms for this inhibition.
A single nucleotide polymorphism at the splice donor site of the human MYH base excision repair gene results in reduced translation efficiency of its transcripts
Authors: Yamaguchi Satoru , Shinmura Kazuya , Saitoh Takayuki , Takenoshita Seiichi , Kuwano Hiroyuki , Yokota Jun
Published: March 2003

Background: Adenine paired with 8‐hydroxyguanine, a major oxidatively damaged DNA lesion, is excised by mutY homologue (MYH) base excision repair protein in human cells. Since genetic polymorphisms of DNA repair genes associated with the activities and the expression levels of their products may modulate cancer susceptibility of individuals, we investigated the effect of a single nucleotide polymorphism (SNP) in the MYH gene on the difference in the expression levels of its products.

Results: An aberrant size of the β type nuclear form transcript was detected in a lung cancer cell line, VMRC‐LCD, by reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis. The transcript contained the intron 1 sequence, and it was due to alternative splicing resulting from IVS1+5G/C SNP. The presence of the upstream open reading frame (ORF) on the 5′‐side of the native ORF in the β type transcript from the IVS1+5C allele could reduce the translation efficiency of the transcript into the nuclear form protein. Thus, expression vectors bearing the 5′‐untranslated region sequence of either the IVS1+5G or 5C allele were constructed. In vitro translation analysis, as well as Western blot and quantitative RT‐PCR analyses of the H1299 lung cancer cell line transfected with these vectors, revealed that the translation efficiency of the IVS1+5C transcript into MYH protein was much lower (≈ 30%) than that of the IVS1+5G transcript.

Conclusions: The SNP at the splice donor site of the MYH gene resulted in reduced translation efficiency of its transcripts. This is the fourth case of single nucleotide variations that cause alterations in translation initiation sites and translation efficiencies in human cells.
The Cpx stress response system of Escherichia coli senses plasma membrane proteins and controls HtpX, a membrane protease with a cytosolic active site
Authors: Shimohata Nobuyuki , Chiba Shinobu , Saikawa Naoya , Ito Koreaki , Akiyama Yoshinori
Published: March 2003

Background: The abnormal accumulation of misfolded proteins outside the plasma (cytoplasmic or inner) membrane up‐regulates the synthesis of a class of envelope‐localized catalysts of protein folding and degradation. The pathway for this transmembrane signalling is mediated by the CpxR‐CpxA two‐component phospho‐relay mechanism.

Results: We now show that an abnormality in the plasma membrane proteins, due either to the impairment of FtsH, a protease acting against integral membrane proteins, or to the overproduction of a substrate membrane protein of FtsH, activates this stress response pathway. Under such conditions, the cpxR gene function becomes essential for cell growth. We further show that the expression of a putative protease, HtpX, in the plasma membrane, is under the control of CpxR. Synthetic growth inhibition was observed when the ftsH and htpX disruption mutations had been combined, suggesting that these gene products have some complementary or overlapping proteolytic functions. Topology analyses indicated that the metalloproteinase active site of HtpX is located on the cytosolic side of the membrane.

Conclusions: Taken together, these results suggest that the Cpx ‘extracytoplasmic’ stress response system controls the quality of the plasma membrane, even on its cytoplasmic side.
Epigenetic marks by DNA methylation specific to stem, germ and somatic cells in mice
Authors: Shiota Kunio , Kogo Yasushi , Ohgane Jun , Imamura Takuya , Urano Atsushi , Nishino Koichiro , Tanaka Satoshi , Hattori Naka
Published: March 2003

Background:  DNA methylation is involved in many gene functions such as gene‐silencing, X‐inactivation, imprinting and stability of the gene. We recently found that some CpG islands had a tissue‐dependent and differentially methylated region (T‐DMR) in normal tissues, raising the possibility that there may be more CpG islands capable of differential methylation.

Results:  We investigated the genome‐wide DNA methylation pattern of CpG islands by restriction landmark genomic scanning (RLGS) in mouse stem cells (ES, EG and trophoblast stem) before and after differentiation, and sperm as well as somatic tissues. A total of 247 spots out of 1500 (16%) showed differences in the appearance of their RLGS profiles, indicating that CpG islands having T‐DMR were numerous and widespread. The methylation pattern was specific, and varied in a precise manner according to cell lineage, tissue type and during cell differentiation.

Conclusions:  Genomic loci with altered methylation status seem to be more common than has hitherto been realized. The formation of DNA methylation patterns at CpG islands is one of the epigenetic events which underlies the production of various cell types in the body. These findings should have implications for the use of embryonic stem cells and cells derived from them therapeutically, and also for the cloning of animals by the transfer of somatic cell nuclei.
Generation of human artificial chromosomes expressing naturally controlled guanosine triphosphate cyclohydrolase I gene
Authors: Ikeno Masashi , Inagaki Hidehito , Nagata Keiko , Morita Miwa , Ichinose Hiroshi , Okazaki Tuneko
Published: March 2003

Background Human artificial chromosomes (HACs) are generated from the precursor DNA constructs containing α‐satellite DNA with CENP‐B boxes, and the process could be used for the incorporation of large genes in the HACs. Guanosine triphosphate cyclohydrolase I (GCH1) is the first and rate‐limiting enzyme for the biosynthesis of tetrahydrobiopterin, the essential co‐factor of aromatic amino acid hydroxylases and nitric oxide synthase.

Results: We constructed HACs carrying a 180 kb genome segment encoding the human GCH1 gene and its control region from the bacterial artificial chromosome (BAC) with the GCH1 segment by co‐transfection with the α‐satellite DNA‐containing BAC to a human fibroblast cell line. Two cell lines carrying a HAC with GCH1 genes were obtained. Both HACs were composed of multiple copies of precursor BACs and were maintained stably in human and mouse cell lines. The GCH1 activities of the HAC‐carrying human fibroblast cell lines were elevated but still highly sensitive to IFN‐γ induction, mimicking the response of the gene expression from the authentic chromosomal genes.

Conclusion: These HACs will provide a useful system for analysis of the complex regulatory circuit of the GCH1 gene in vivo and also function as a tool for gene delivery in animal models or in therapeutic trials.