bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of the canopy of Retama sphaerocarpa on its understorey in a semiarid environment
Authors: MORO M. J. , PUGNAIRE F. I. , HAASE P. , PUIGDEFÁBREGAS J.
Published: November 2003

1. A dense understorey of annual and perennial herbs grow under the canopy of Retama sphaerocarpa shrubs in semiarid environments of south‐east Spain, influencing plant productivity and diversity at a regional scale. We investigated the facilitation by the shrub on its understorey in field and laboratory experiments with Barley designed to explore the mechanisms of interaction between both vegetation layers and their spatial variation.

2. There was a gradient of spatial heterogeneity in soil chemical fertility under the shrub canopy, with organic matter and soil nitrogen contents higher at the centre than at the edge of the canopy. Dry mass production of Barley was also higher in soils from intermediate positions, and lower in soils from both the centre and edge of the canopy.

3. In the field, pots sown with Barley placed near the centre, at an intermediate position and at the edge of the canopy of Retama shrubs showed significant differences in productivity, suggesting a mulching effect of the canopy that also affects seedling establishment.

4. Micro‐climatic measurements showed significant differences in total radiation reaching the soil, mean air and soil temperatures and maximum temperature among different positions in the understorey, increasing radially from the centre to the edge of the canopy.

5. These results and field observations suggest that the optimal association of climatic factors under the canopy would combine with a high soil fertility mediated by litter decomposition to increase biomass production mid‐way between the centre and the edge of the canopy. Overstorey and understorey thus interact to increase nutrient retention locally, which benefits both the shrub and the herb layer.
Allelopathic effect of Cistus ladanifer on seed germination
Authors: CHAVES N. , ESCUDERO J. C.
Published: November 2003

1. The allelopathic effect of the exudate secreted by Cistus ladanifer leaves was tested on different plant species. Cynodon dactylon and Rumex crispus, species absent from C. ladanifer scrub (jarales), were clearly inhibited by the exudate.

2. Species present in the jarales, Medicago polymorpha and Lolium rigidum, showed no direct inhibition of germination, but the process this was delayed, and cotyledon and root size was diminished. This implies inhibition of seedling growth, and may explain the low abundance of these species inside the jarales.

3. The active constituents in the inhibition of germination are low molecular weight phenolic compounds. The flavonoids appear to exert no direct effect on germination of the species tested, but do provoke a reduction in cotyledon and root size, and could thereby inhibit subsequent seedling development.
Increased drag, and thus lower speed: a cost for morphological defence in Bosmina (Eubosmina) (Crustacea: Cladocera)
Authors: LAGERGREN R. , HELLSTEN M. , STENSON J. A. E.
Published: November 2003

1. The swimming speed of two forms, an extreme and a typical, within the cladoceran subgenus Eubosmina were examined using a three‐dimensional video‐technique. The extreme form has a very high carapax and extremely long antennule, features probably involved in predator defence.

2. It was found that the extreme form swam almost 40% slower than the typical form.

3. Calculations show that the extreme form had to work at least 12% harder to swim at the same speed, or if it used the same amount of energy to swim, the extreme form would swim 6% slower. Increased drag, because of its distinguishing carapax and antennule, is thus the most likely explanation for the slower swimming speed of the extreme form, assuming it selects the same power output.

4. Swimming speed can be correlated to food intake either by the frequency of hits to edible food particles or by the time to swim from a poor food patch to a good one. So the reduced speed is probably a great cost for the extreme form.
Nestling obesity and variability of food delivery in Manx Shearwaters, Puffinus puffinus
Authors: HAMER K. C. , HILL J. K.
Published: November 2003

1. Procellariiform seabirds such as Manx Shearwaters, Puffinus puffinus, accumulate large quantities of lipid during the nestling period, the purpose of which remains unclear. This paper examines temporal and age‐specific variation in the pattern of food delivery to nestling Manx Shearwaters, to determine whether food delivery was likely to be determined primarily by the availability of food to adults at sea or by a chick’s short‐term nutritional requirements. The variability in daily food delivery to chicks in the context of nestling obesity is then discussed.

2. For all but the youngest and oldest age classes, individual meals delivered by adults averaged 49 g, which was 12% of mean adult mass over the same period. More than 80% of chicks were fed each night and the maximum interval between feeding events for any chick was four nights. Feeding conditions did exert some influence over the ability of adults to feed their offspring, but deviations from expected values for both feeding frequency and meal size were restricted to a small number of nights, included values both higher and lower than expected and did not persist for more than one night in succession. These results were not compatible with the notion that large lipid reserves accumulated by chicks during their development provide insurance against prolonged interruptions to feeding.

3. Although adults did not appear to modulate food delivery over the nestling period, there was some evidence of short‐term adjustment of feeding frequency (but not meal size) according to a chick’s immediate nutritional requirements. This suggests that variation in the provisioning rates of chicks did not result solely from stochastic variation in parental foraging success. However, the effect was slight and despite such minor adjustments, the coefficient of variation in daily provisioning rate was not substantially lower than in other species where the chick has no influence on parental food delivery. This suggests that even among those species that feed their chicks at comparatively frequent intervals, lipid accumulation among Procellariiformes may result from chronic overfeeding of chicks in order to reduce the impact of variable food provisioning by individual parents.
Nitrogen fertilization affects interactions between the components of an insect–fungus–plant tripartite system
Authors: HATCHER P. E. , PAUL N. D. , AYRES P. G. , WHITTAKER J. B.
Published: November 2003

1. The chrysomelid beetle, Gastrophysa viridula and the rust fungus, Uromyces rumicis both occur on leaves of Rumex obtusifolius growing in a wide range of soil nutrient conditions. We investigated the effect of fertilizing plants with eight nitrate and four ammonium concentrations on the components of this tripartite interaction in a controlled environment.

2. Leaf weight, area and total plant weight increased as both nitrate and ammonium concentrations increased up to 15mmol l–1. Between 15 and 50mmol l–1 added nitrate, leaf and total plant weight decreased. Total plant weight was unaffected by increasing ammonium fertilization from 15 to 25mmol l–1.

3. The density and percentage of U. rumicis pustules sporulating 8 days after infection decreased with increasing nitrate but were unaffected by increasing the concentration of ammonium fertilization.

4. Leaf area consumed and number of eggs laid by G. viridula decreased as the concentration of nitrate fertilization increased. Increasing the concentration of ammonium decreased leaf area eaten.

5. First instar mortality of G. viridula was increased and gregariousness and adult fecundity was decreased additively by the combination of U. rumicis infection and decreasing the nitrate concentration fed to plants from 10 to 1mmol l–1.
Pollen dynamics of bumble‐bee visitation on Echium vulgare
Authors: RADEMAKER M. C. J. , DE JONG T. J. , KLINKHAMER P. G. L.
Published: November 2003

1. We quantified pollen deposition on the stigma, pollen removal from the anthers and pollen losses in Echium vulgare, visited by workers of Bombus terrestris under controlled conditions. We used dye as a pollen analogue. Bumble‐bees were trained to visit a sequence of non‐emasculated flowers to estimate pollen carryover and to visit individual flowers to estimate pollen loss.

2. Carryover of pollen grains and dye particles between flowers was similar, which justifies using dye as a pollen analogue. On average 93·8% of the dye particles on the bee were carried over to the next flower. Only a small fraction of the pollen grains was deposited on the stigma (0·15%). A much larger fraction (6·1%) was lost in another way: passively during flight, through grooming or on floral parts other than the stigma. The bees removed 44% of the pollen grains from a fresh flower and 50·3% of this removed pollen adhered to the bee.

3. We predict that, using the parameters mentioned above, during a single visit to a newly opened flower, a bee collects an amount of pollen grains which will bring about 60% geitonogamous self‐pollination in the next flower visited. The expected percentage of self‐pollination is considerably less if bees visit flowers that have been visited before.
A functional model for maximum growth of Atlantic Salmon parr, Salmo salar, from two populations in northwest England
Authors: ELLIOTT J. M. , HURLEY M. A.
Published: November 2003

1. The chief objective was to develop a functional model for the growth of parr (0+, 1+) of Atlantic Salmon, Salmo salar, from two populations (Rivers Leven and Lune in northwest England), using a model similar to that developed previously for Brown Trout, Salmo trutta. Parr bred from Leven parents were acclimatized to nine fairly constant temperatures (two 0+, two 1+ parr per temperature) in the range 3·8–21·7 °C (variation ± 0·3 at 3·8 °C to ± 1·0 at 21·7 °C). Each fish was kept in a separate tank and fed to satiation on shrimps. The mass and length of each fish was recorded at the start and finish of a growth period of 30 days. Parr (0+, 1+) bred from Lune parents were separated into slow and fast growers, and acclimatized to six very constant temperatures. There were three slow and three fast growers at each of 5·0, 10·0, 15·0 °C for 0+ parr and each of 5·0, 10·0, 13·0, 15·0, 18·0, 20·0 °C for 1+ parr. Procedures were the same as for Leven parr except that the growth period lasted 30 days for the 18 0+ parr and 42 days for the 36 1+ parr.

2. The growth model was an excellent fit (P < 0·001) with no significant differences between parr from the two rivers, fast‐growing parr of different ages (0+, 1+), or fast‐ and slow‐growing 1+ parr. The optimum temperature for the combined data (81 parr) was 15·9 °C with a range for growth of 6·0–22·5 °C. The model failed to fit the data for slow‐growing 0+ Lune parr in experiments from November to February. Growth was reduced in these fish, even though the temperatures were suitable for growth (10, 15°C).

3. The model described approximately the growth of three year‐classes of Atlantic Salmon parr in the River Eden in northwest England. Some of the discrepancies between actual masses and those predicted from the model were consistent and possible reasons for this are discussed.
The optimal shape of avian eggs
Authors: BARTA Z. , SZÉKELY T.
Published: November 2003

1. The shape of avian eggs is often explained by involving the mechanical properties of eggshell such as resistance to breakage or invoking physiological forces operating during egg development.

2. Here we take a different approach and investigate the efficient use of space. According to this approach the optimal egg shape is the one that produces the most compact fit under the incubating parent.

3. We extend the model of Andersson (1978) and use a numerical technique to investigate egg shapes in clutches of 1 to 10 eggs. In our model the shape of eggs is described by four parameters in a two‐dimensional space. These parameters are free to vary – but eggs cannot be concave.

4. The optimal egg shape for each clutch size was found by a genetic algorithm.

5. The model predicts that egg shape should depend on clutch size. In particular, if the clutch consists of one egg then this egg should be spherical; whereas in clutches of two or three eggs the eggs should be biconical. In clutches of four the eggs should be pointed. The model also predicts that in clutches of over seven eggs the optimal egg shape should be approximately spherical.

6. These predictions are valuable because they point out that some of the variation in avian egg shapes may be explained solely by the efficient use of the brood patch area of the incubating parent.
The influence of the rate of temperature change on the activation of dormant seeds of Rumex obtusifolius L.
Authors: VAN NERUM J. A. VAN ASSCHE & D. M.
Published: November 2003

1. One temperature shift from 20 to 30°C in darkness induces 30–40% germination in Rumex obtusifolius seeds. The same germination percentages are found with heat treatment varying between 1 and 6h duration, indicating that the total heat sum of the temperature shift is not important.

2. Germination is greatly enhanced by three consecutive heat shifts of 1h at 30°C separated by 1h periods at 20°C.

3. The seeds are activated to a small extent after a slow warming (+2°Ch–1) from 20 to 30°C, followed by incubation for 1h at 30°C. Germination is much higher after rapid heating (+10°Ch–1) to 30°C, followed by 1h incubation at this temperature. Repeated fast heating treatments on four consecutive days enhances germination. Moderately rapid heatings (+3·3°Ch–1) give intermediate results.

4. The rate of cooling does not influence the germination percentage. Cooling alone cannot induce germination.

5. Heating alone from 15 to 25°C without cooling also activates germination. In this temperature range the seeds are more activated by rapid warming than by slow warming.

6. The ecological relevance of the response to different warming rate is discussed. The insensitivity of seeds to a slow warming might keep deeply buried seeds in a dormant stage.
Changes in haematological parameters in relation to prey switching in a wild population of harbour seals
Authors: THOMPSON P. M. , TOLLIT D. J. , CORPE H. M. , REID R. J. , ROSS H. M.
Published: November 2003

1. Previous studies have found marked inter‐annual variation in winter food availability, diet composition and body condition in a population of harbour seals (Phoca vitulina L.) in Northeast Scotland. This study aimed to determine whether there were other physiological consequences of prey switching by comparing haematological parameters in years when the clupeids herring and sprat dominated the diet and in years when seals switched to alternative prey.

2. There were significant differences in leukocyte and erythrocyte parameters in relation to diet composition. In contrast, indices of body condition did not explain the variation in haematological parameters, suggesting that the observed changes did not result from differences in the energetic content of the prey.

3. Leukocyte counts were significantly higher after ‘good’ clupeid years, although the differences in mean counts were small. Such differences could have resulted either from immuno‐suppression, for example because of differences in prey nutrient or contaminant levels, or from differences in the pathogen challenge resulting from geographical variations in water quality.

4. The differences in erythrocyte parameters were more marked, and there was evidence of widespread macrocytic anaemia when seals switched from clupeids to alternative prey. Such differences could result either from acclimation, as a result of prey‐specific foraging strategies, or from differences in the nutritional quality of prey.

5. These results indicate that generalist predators such as the harbour seal may exhibit physiological responses to changes in the composition of their diet. These data highlight the need to consider the long‐term physiological effects of variations in food availability on the population dynamics of generalist marine top predators. In particular, it is hypothesized that fish‐induced anaemia may be responsible for declines in certain pinniped populations.
Identical origin of adrenal cortex and gonad revealed by expression profiles of Ad4BP/SF‐1
Authors: Hatano Osamu , Takakusu Akira , Nomura Masatoshi , Morohashi Ken‐ichirou
Published: November 2003

Background: Ad4BP/SF‐1 was originally identified as a steroidogenic tissue‐specific transcription factor. Recent gene disruption studies with the mammalian Ftz‐F1 gene encoding Ad4BP/SF‐1 clearly revealed the essential function of the factor for adrenal and gonadal differentiation.

Results: In this study, we examined the early development of these tissues using Ad4BP/SF‐1 as the marker. In rat foetuses of 11.5 days post‐coitum (d.p.c.), a cell population designated adreno‐genital primordium was firstly observed on symmetrical lines extending from the dorsal aorta to the dorsal coelomic epithelia of the primitive urogenital ridges. From 12.5 d.p.c., the rostral half of the adreno‐genital primordium started to separate into two distinct cell populations. Judging from the distribution of primordial germ cells, the cell population on the dorsal aortal side is a primordium for the adrenal cortex whereas that on the coelomic epithelial side is for the gonads. At 13.5 d.p.c., these two primordia have separated completely.

Conclusion: These observations clearly identified a novel adreno‐genital primordium from which both the adrenal cortex and the gonads originate. An RT‐PCR study conducted to detect adrenal‐ and gonad‐specific mRNAs supported the above observations.
Sensing of DNA non‐homology lowers the initiation of meiotic recomination in yeast
Authors: Rocco Vincenzo , Nicolas Alain
Published: November 2003

Background: Meiotic recombination between homologous chromosomes in the yeast Saccharomyces cerevisiae is initiated by the formation of DNA double‐strand breaks (DSBs). The mechanism of DSB formation and the factors that determine their frequency and location have yet to be elucidated. Current studies of meiotic recombination are also concerned with the question of the functional relationship between DSB formation and the other meiotic processes of homology searching, pairing and synapsis of homologues.

Results: To test if DNA identity is required for high levels of DSBs and recombination, we have asked whether small DNA heterologies (140–547 bp) located within the well characterized ARG4 initiator of meiotic recombination, can affect DSB formation and gene conversion events in the ARG4 locus. The present physical and genetic analyses show that some heterologies reduced recombination frequencies without altering DSB formation, whereas others reduced both DSB and gene conversion frequencies.

Conclusions: These results suggest that DNA heterologies overlapping a recombination initiator impair meiotic gene conversion at two levels. First, some heterologies affect the level of DSB formation, revealing the existence of an anti‐initiation process sensing the presence of sequence non‐homology between the homologous chromosomes. Second, heterologies can impair the successful processing of the recombination intermediates once DSBs are made. We present a model for interhomologue cross‐talks involving chromosomal and DNA/DNA interactions.
D‐type cyclin expression is decreased and p21 and p27 CDK inhibitor expression is increased when tsBN462 CCG1/TAFII250 mutant cells arrest in G1 at the restrictive temperature
Authors: Sekiguchi Takeshi , Noguchi Eishi , Hayashida Toshiro , Nakashima Torahiko , Toyoshima Hideo , Nishimoto Takeharu , Hunter Tony
Published: November 2003

Background: The tsBN462 temperature‐sensitive mutant hamster cell line exhibits cell cycle arrest and apoptosis at the restrictive temperature of 39.5 °C, due to a point mutation in the CCG1/TAFII 250 gene, which encodes a component of the general transcription factor TFIID.

Results: We now report that CCG1/TAFII 250 persisted as a complex with TBP and associated proteins (TAFs) in tsBN462 cells at the restrictive temperature. FACScan analysis revealed that the tsBN462 mutation resulted in a failure to progress out of G0 into G1. Using two‐dimensional gel electrophoresis we observed a decrease in the synthesis of several proteins, starting in the middle of the G1 phase, becoming very pronounced during late G1. The expression of the immediate early genes c‐fos, c‐jun and c‐myc was normally induced by serum treatment of quiescent cells at the restrictive temperature, whereas expression of cyclins A, D1 and D3 was reduced. Expression of the cyclin‐dependent kinase (CDK) inhibitor proteins p21 and p27 was enhanced. Consistent with the decreased cyclin D and increased p21/p27 expression, we found that phosphorylation of Rb was decreased at 39.5 °C. Cyclin A‐, E‐ and Cdk2‐associated histone H1 kinase activity was reduced concomitantly with the increase in p21 protein.

Conclusion: Decreased cyclin/Cdk kinase activity and decreased Rb phosphorylation are possible causes of G1 cell cycle arrest in tsBN462 cells at the restrictive temperature.
Phosphorylation of Ser‐3 of cofilin regulates its essential function on actin
Authors: Moriyama Kenji , Iida Kazuko , Yahara Ichiro
Published: November 2003

Background:  Cofilin is a low‐molecular weight actin‐modulating protein, and is structurally and functionally conserved in eucaryotes from yeast to mammals. The functions of cofilin appear to be regulated by phosphorylation and dephosphorylation.

Results:  A proteolytic study of phosphorylated porcine cofilin and expression of a mutated cofilin in cultured cells revealed that Ser‐3 is the unique phosphorylation site. Phosphorylated cofilin was found not to bind to either F‐or G‐actin while unphosphorylated cofilin binds to both. S3D‐cofilin, in which Ser‐3 was replaced with Asp, did not bind in vitro to actin while S3A‐cofilin did. The transient overexpression of wild‐type or S3A‐cofilin in cultured cells caused disruption of pre‐existing actin structures and induced cytoplasmic actin bundles. Heat shock‐induced nuclear or NaCl buffer‐induced cytoplasmic actin/cofilin rods contained the expressed cofilin. In contrast, the overexpression of S3D‐cofilin did not alter the actin structures. Induced actin rods did not contain S3D‐cofilin. S3D‐porcine cofilin did not complement the lethality associated with Δcof1 mutations in Saccharomyces cerevisiae while wild‐type and S3A‐cofilin did. Furthermore, we found that S2A/S4D‐ and S2D/S4D‐yeast cofilin mutants were not viable.

Conclusion:  We conclude that the function of cofilin is negatively regulated in vivo by phosphorylation of Ser‐3 and that cells require the functions of unphosphorylated cofilin for viability.
HALF‐1, a bZIP‐type protein, interacting with the wheat transcription factor HBP‐1a contains a novel transcriptional activation domain
Authors: Okanami Masahiro , Meshi Tetsuo , Tamai Hiroki , Iwabuchi Masaki
Published: November 2003

Background: Nuclear factors bind to cis‐acting elements and mediate transcriptional regulation through protein–protein interactions with other factors. The bZIP‐type wheat nuclear protein HBP‐1a(17) is a putative transcriptional activator specifically binding to the Hex (ccACGTCA) and G‐box (CCACGTGG) motifs, which are often found in the cis‐acting elements critical for various responses in plants.

Results: In order to investigate the mechanisms for gene expression mediated via the Hex and G‐box motifs, we attempted to isolate proteins interacting with HBP‐1a(17) based on protein–protein interactions. A cDNA expression library from wheat seedlings was screened with 32P‐labelled HBP‐1a(17), and a bZIP‐type protein, termed HALF‐1 (HBP‐1‐associated leucine‐zipper factor‐1), was isolated. GST‐pulldown assay, yeast two‐hybrid system and EMSA showed that HALF‐1 and HBP‐1a(17) interact with each other through their leucine‐zipper regions. Dissection experiments showed that HALF‐1 has at least one potential trans‐activation domain which includes a nine amino acid motif conserved between several plant bZIP‐type proteins. This motif, named GCB (GBF‐conserved box; consensus, NLNIGMDXW), activated the expression of a reporter gene, when fused to the GAL4 DNA‐binding domain. The corresponding region of Arabidopsis GBF1 also stimulated transcription. However, the trans‐activation domain of HALF‐1 did not function in yeast.

Conclusions: We identified a novel trans‐activation domain which contains the GCB motif conserved among plant bZIP‐type factors. The trans‐activation appears to be mediated by interaction between the GCB motif and a factor conserved in plants.
The shortest path from the surface to the nucleus: RBP‐Jκ/Su(H) transcription factor
Authors: Honjo Tasuku
Published: November 2003

Communication between cell surface receptors and nuclear transcription factors is of primary importance to multicellular organisms. Since there are numerous molecules involved in this process, their mutual interaction forms complex networks of informational regulation, which is still under extensive investigation. The RBP‐Jκ transcription factor interacts directly with the Notch receptor involved in cell lineage commitment, implicating the presence of a uniquely simple communication strategy between the surface receptor and the nucleus.
A new view of V(D)J recombination
Authors: Gellert Martin
Published: November 2003

The DNA rearrangement that assembles antibody and T cell receptor genes is beginning to be understood. New work shows that the first step is a site‐specific cleavage which can be performed by purified RAG1 and RAG2 proteins. The later steps largely overlap with the repair of radiation‐induced DNA double‐strand breaks, as indicated by the identity of several newly cloned factors. These developments open the way for a thorough biochemical study of V(D)J recombination.
The sbcC and sbcD genes of Escherichia coli encode a nuclease involved in palindrome inviability and genetic recombination
Authors: Connelly John C. , Leach David R. F.
Published: November 2003

Background: Long DNA palindromes have the potential to adopt hairpin or cruciform secondary structures that inhibit DNA replication. In Escherichia coli, this palindrome‐mediated inviability results from the activity of the sbcC and sbcD genes, and genetic observations have suggested that they may encode a nuclease. Mutations in these genes also restore the defect in genetic recombination associated with recBC sbcB mutants.

Results: We have purified the E. coli SbcCD protein from an overexpressing strain and have shown that it has an ATP‐dependent DNA double‐strand exonuclease activity. Co‐purification of nuclease with protein, antibody inhibition and the absence of activity in extracts lacking the sbcCD genes confirm that the activity is intrinsic to SbcCD. The purified protein also has an ATP‐independent single‐strand DNA endonuclease activity.

Conclusions: We have shown that sbcCD encodes a nuclease. The purified protein has double‐strand DNA exonuclease and single‐strand DNA endonuclease activities. We propose that SbcCD cleaves secondary structures formed at replication forks and that the broken forks can be repaired by homologous recombination.
Mechanism responsible for glucose–lactose diauxie in Escherichia coli: challenge to the cAMP model
Authors: Inada Toshifumi , Kimata Keiko , Aiba Hiroji
Published: November 2003

Background : The inhibition of β‐galactosidase expression in glucose–lactose diauxie is a typical example of the glucose effect in Escherichia coli. It is generally believed that glucose exerts its effect at least partly by reducing the intracellular cAMP level. However, there is no direct evidence that the inhibitory effect of glucose on the expression of the lac operon is mediated by a reduction of the cAMP level in the glucose–lactose system.

Results : To examine the roles of cAMP and the cAMP receptor protein (CRP) in the glucose effect, the intracellular levels of these factors were determined during diauxic growth in a glucose–lactose medium. We found that the levels of cAMP and CRP in a lactose‐grown phase were not higher than those in a glucose‐grown phase, although the cAMP levels increased transiently during the lag phase. The addition of exogenous cAMP eliminated diauxic growth but did not eliminate glucose repression. Glucose repression and diauxie were observed in cells which lack cAMP but produce a cAMP‐independent CRP. In addition, inactivation of the lac repressor by the disruption of the lacI gene or the addition of IPTG, eliminated glucose repression.

Conclusion : We conclude that the repression of β‐galactosidase expression by glucose is not due to the reduction of the cAMP‐CRP level but due to an inducer exclusion mechanism which is mediated by the phosphoenolpyruvate‐dependent sugar phosphotransferase system.
RNA‐dependent association of the Drosophila maleless protein with the male X chromosome
Authors: Richter Liz , Bone James R. , Kuroda Mitzi I.
Published: November 2003

Background: Dosage compensation results in equivalent X‐linked gene expression in males (XY) and females (XX). In Drosophila, both X chromosomes are active in females, and the single male X must double its transcriptional activity to allow male development. Four proteins (encoded by the male‐specific lethal genes) are required for dosage compensation and associate with the X chromosome in males but not in females.

Results : In this report, we focus on the maleless (MLE) protein. The MLE protein sequence contains motifs common to members of a family of RNA‐dependent ATPases. We have found that association of MLE with the male X chromosome is RNase sensitive, and that mutations in the ATPase motifs affect MLE function. Overexpression of MLE or its carboxyl terminus, which includes glycine‐rich repeats, reveals an RNase‐sensitive affinity for all chromosome arms.

Conclusions: Our results suggest that nascent transcripts or a hypothetical RNA component of chromatin play a critical role in the biochemical mechanism of dosage compensation. The potential relationship between interaction with RNA and transcriptional control of the X chromosome suggests that the mechanism of dosage compensation is distinct from classical models for transcriptional activation.
The Schizosaccharomyces pombe mra1 gene, which is required for cell growth and mating, can suppress the mating inefficiency caused by a deficit in the Ras1 activity
Authors: Hakuno Fumihiko , Hughes David A. , Yamamoto Masayuki
Published: November 2003

Background
Schizosaccharomyces pombe Ras1 regulates two downstream pathways, namely the Byr2/Byr1/Spk1 mitogen‐activated protein kinase cascade and the Cdc42sp small G protein pathway. The former is relevant to mating and sporulation, whereas the latter is relevant to mating, cell growth and cell morphology. We addressed whether Ras1 has any additional role in the regulation of cell physiology.

Results: Using a specific mutation in the effector region of Ras1, we isolated a high‐copy‐number suppressor of the mating deficiency caused by a decrease of the Ras1 activity. The isolated gene, named mra1, encodes a novel protein of 359 amino acids, which has apparent homologues in rice and budding yeast. Disruption of mra1 indicated that it is essential for cell growth, and mutational analysis indicated that it is required for the promotion of mating. These two functions could be separated by mutations, suggesting that Mra1 is bifunctional. Overexpression of mra1 could also suppress the mating inefficiency caused by either overexpression of gap1, which is a downregulator of Ras1, or loss of function of zfs1, which is a gene relevant to the mating pheromone signalling. However, it could not suppress null mutations in genes involved in the two known pathways downstream of Ras1.

Conclusions: Mra1 is an apparent downstream factor of Ras1, which is essential for cell growth and relevant to mating but is not involved in the maintenance of cell morphology. Mra1 is unlikely to interact directly with the known pathways downstream of Ras1, implying that it may be a factor constituting a third pathway regulated by Ras1.
The HGF receptor family: unconventional signal transducers for invasive cell growth
Authors: Comoglio Paolo M. , Boccaccio Carla
Published: November 2003

The HGF receptor family includes tyrosine kinases encoded by three oncogenes: METSEA and RON. The members of this gene family share a unique functional feature: they mediate cell dissociation and motility (‘scattering‘) in physiological conditions, and invasiveness in their activated versions. The MetRon and Sea receptors display a distinctive signal transduction behaviour. Unlike conventional growth factor receptors, their cytoplasmic tails contain a multifunctional docking site. Upon autophosphorylation, this sequence simultaneously binds and activates multiple SH2‐containing transducers, including Ras and PI 3‐kinase. A deregulated activation of this ‘supersite’ triggers a dramatic pleiotropic signal which is responsible for invasive cell growth.
A single Drosophila melanogaster myosin light chain kinase gene produces multiple isoforms whose activities are differently regulated
Authors: Kojima Shin‐ichiro , Mishima Masanori , Mabuchi Issei , Hotta Yoshiki
Published: November 2003

Background: Myosin light chain kinase (MLCK) specifically phosphorylates the myosin regulatory light chain in a calcium/calmodulin (Ca2+/CaM)‐dependent manner in animal cells. The roles of MLCK are not fully understood, particularly in nonmuscle cells. Therefore, we cloned and characterized a Drosophila MLCK gene as the first step in a genetic analysis of this process.

Results: Four transcripts are produced from this gene. These transcripts encode at least three isoforms (isoform‐I, ‐II and ‐III), which share a kinase domain, a fibronectin type III motif and an immunoglobulin C2 motif. However, regulatory regions differ between isoform‐I/II and ‐III due to the alternative splicing of the exon encoding a CaM‐binding domain. As a result, isoform‐I and ‐II are Ca2+/CaM‐dependent forms, whereas isoform‐III is a Ca2+/CaM‐independent form. Northern blotting and in situ hybridization showed that the expressions of these isoforms are distinctly regulated in stage‐ and tissue‐dependent manners. Isoform‐I seems to be expressed ubiquitously, while isoform‐III is expressed predominantly in muscle tissues. In contrast to these isoforms, isoform‐II is specific to late pupa and adult.

Conclusion: In Drosophila, a single MLCK gene produces multiple isoforms whose regulatory regions and expression patterns are different. These differences suggest various cellular functions of MLCK in Drosophila.
Nik1: a Nim1‐like protein kinase of S. cerevisiae interacts with the Cdc28 complex and regulates cell cycle progression
Authors: Tanaka Seiji , Nojima Hiroshi
Published: November 2003

Background: The eukaryotic cell cycle is driven by CDK‐cyclin complexes. A number of proteins interact either with CDK or the CDK complex to regulate CDK activity. A search for novel cell cycle regulators in the budding yeast Saccharomyces cerevisiae yielded multicopy suppressors of the cdc2‐L7mutation of the fission yeast, Schizosaccharomyces pombe.

Results: One of the isolated genes was found to encode a putative protein kinase similar to Nim1 of S. pombe and was termed NIK1 (Nim1‐like kinase 1). Transcription of NIK1‐was periodic and peaked at the G1/S boundary. Although NIK1 is not essential, Δnik1 cells showed G2 delay and hydroxyurea (HU) sensitivity. Anomalously elongated buds were observed in the stationary phase or in the presence of HU. Moreover, DNA was aberrantly distributed in the Δnik1 cdc28 double mutant. Genetical and biochemical evidence suggests that Nik1 interacts with the Cdc28 complex.

Conclusions: Nik is a structural and functional homo‐logue of Nim1. Nik1 interacts with the Cdc28 complex and functions not only at the G2/M transition but also at other points of the cell cycle.
A cell‐free system of Tn3 transposition and transposition immunity
Authors: Maekawa Takafumi , Yanagihara Katsuhiko , Ohtsubo Eiichi
Published: November 2003

Background: Tn3 is a bacterial transposon, which encodes transposase required for its transposition. Tn3 has terminal inverted repeat (IR) sequences of 38 bp in length, whose inner region, called the B domain, is bound by transposase. Tn3 confers transposition immunity, a phenomenon in which Tn3 transposes to a target replicon with Tn3 much less frequently than to a target replicon with no Tn3.

Results: To understand transposition and transposition immunity at the molecular level, we constructed a cell‐free system using a plasmid as the target. Transpositional recombination occurred in a cell extract containing transposase between the target and a donor plasmid carrying mini‐Tn3 at a high frequency. The reaction required ATP, Mg2+, dNTPs and 2% polyvinyl alcohol, and was inhibited by inhibitors for DNA synthesis and DNA gyrase. In this system, when a plasmid with the IR sequence was used as the target, the frequency of transposition was significantly decreased, demonstrating that the transposition immunity conferred by Tn3 is reproduced in vitro. Preincubation of the target in the cell extract increased the level of transposition immunity. On the other hand, mutations within the B domain in the IR sequence of the target abolished transposition immunity.

Conclusions: Transposition of Tn3 and transposition immunity could be reproduced in vitro. The results demonstrate that the binding of transposase to domain B of the IR sequence in the target replicon is responsible for transposition immunity. We propose that the transposition immunity results from conversion of the normal synaptic complex formed between the donor and target molecules to another complex which is inactive for transposition, due to the interaction between transposases binding to the IR sequences in the donor and target molecules.
Specific nicking at the 3′ ends of the terminal inverted repeat sequences in transposon Tn3 by transposase and an E. coli protein ACP
Authors: Maekawa Takafumi , Yanagihara Katsuhiko , Ohtsubo Eiichi
Published: November 2003

Background: Tn3, a bacterial transposon, carries tnpA gene encoding transposase which is essential for its transposition. The transposition of Tn3 has been reproduced in vitro in a cell extract containing transposase by using a plasmid carrying mini‐Tn3 as the donor and another plasmid as the target. Transposase has the ability to bind to the 38‐bp terminal inverted repeats (IRs) of Tn3. The molecular mechanism of the initiation step of the Tn3 transposition reaction promoted by the transposase has, however, not been understood.

Results: We found that nicking occurred efficiently in the cell‐free system at each of the 3′ ends of the IRs of mini‐Tn3 in the closed circular or linear donor molecules. The nicking reaction required transposase and Mg2+, but did not require ATP, an ATP‐regenerating system, dNTPs and polyvinyl alcohol, which were the requirements for the transposition reaction. By using the nicking assay employed here, transposase was purified almost to homogeneity. Gel filtration and sedimentation analyses indicate that transposase forms a dimer in a solution containing 0.5 m NaCl. The nicking activity of the purified transposase was weak and was found to be stimulated by a host factor. The nicking stimulation factor was subsequently purified and found to be ACP, an Escherichia coli acyl carrier protein.

Conclusions: Nicking occurred efficiently at the 3′ ends of mini‐Tn3 in the reaction mixture containing transposase and ACP. ACP is known to act as a factor which modulates enzymes that are involved in several biological processes either in the acylated or unacylated form. ACP may also modulate transposase to initiate the transposition reaction with nicking at the 3′ ends of Tn3.
Purification and characterization of XRad51.1 protein, Xenopus RAD51 homologue: recombinant XRad51.1 promotes strand exchange reaction
Authors: Maeshima Kazuhiro , Morimatsu Katsumi , Horii Toshihiro
Published: November 2003

Background:
The RAD51 gene of Saccharomyces cerevisiae is homologous to the Escherichia coli recA gene and plays a key role in genetic recombination and DNA double‐strand break repair. To construct an improved experimental system of homologous recombination in higher eukaryotes, we have chosen the South African clawed frog, Xenopus laevis, whose egg extracts might be useful for the in vitro studies. We identified and characterized a Xenopus homologue of RAD51 gene, the XRAD51.1.

Results:
Recombinant XRad51.1 was expressed in E. coli. The purified XRad51.1 protein showed ssDNA‐dependent ATPase activity and promoted the DNA strand exchange reaction between two 55‐mer oligonucleotides. The binding stoichiometry of XRad51.1 to ssDNA was determined by fluorescence of poly(dεA), a chemically modified poly(dA), and was found to be about six bases/XRad51.1 monomer in a nucleoprotein filament, a similar value to E. coli RecA protein. The kinetics of the fluorescence change of poly(dεA) after XRad51.1 binding in the presence of ATP was significantly different from that observed with RecA protein. The affinity of XRad51.1 to ssDNA in the presence of ATP was higher than that of RecA protein, and the dissociation of the XRad51.1‐ssDNA complex was slower than the RecA‐ssDNA complex.

Conclusions:
Purified recombinant XRad51.1 protein promoted the strand exchange between short DNA molecules. While the binding stoichiometry of XRad51.1 protein to ssDNA was identical to that of the RecA protein, XRad51.1 has a significantly higher affinity and binding stability to ssDNA than did the RecA protein in the presence of ATP.
A hamster temperature‐sensitive G1 mutant, tsBN250 has a single point mutation in histidyl‐tRNA synthetase that inhibits an accumulation of cyclin D1
Authors: Motomura Seiichi , Fukushima Kohtaro , Nishitani Hideo , Nawata Hajime , Nishimoto Takeharu
Published: November 2003

Background:
We have previously isolated a series of temperature‐sensitive mutants for cell‐proliferation from the BHK21 cell line, derived from the golden hamster. These mutants proliferate at 33.5 °C, the permissive temperature, but not at 39.5 °C the restrictive temperature. Using DNA‐mediated gene transfer, human genes complementing these ts mutants were cloned.

Results:
At 39.5 °C the tsBN250 cell line, a temperature‐sensitive mutant of the BHK21 cell line, had a defect in the G1 phase, but not in the S phase. The human gene complementing tsBN250 cells was found to encode histidyl‐tRNA synthetase. Indeed, the tsBN250 cell line had a single base change—guanine to adenine at the second position of the 362nd codon of hamster histidyl‐tRNA‐synthetase, converting arginine to histidine. Following release from serum starvation, cyclin E, but not cyclin D1, was accumulated, while, at 39.5 °C, the mRNA of cyclin D1 was normally expressed in tsBN250 cells. A similar inhibition of cyclin D1 accumulation was observed in another ts mutant, tsBN269, which has a single point mutation in lysyl‐tRNA synthetase. Overexpression of cyclin D1 enabled tsBN250 cells to enter the S phase.

Conclusion:
tsBN250 cells have a single point mutation in histidyl tRNA synthetase that causes a loss of histidyl‐tRNA synthetase activity which in turn reduces the content of cyclin D1, but not of cyclin E, thereby resulting in G1 arrest.
Dissociation kinetics of RepA dimers: implications for mechanisms of activation of DNA binding by chaperones
Authors: Chattoraj Dhruba K. , Ghirlando Rodolfo , Park Kyusung , Dibbens Justin A. , Lewis Marc S.
Published: November 2003

Background: The replication initiator of plasmid P1, RepA, binds DNA as monomer. The binding is stimulated by the chaperones DnaJ, DnaK and GrpE of Escherichia coli. Two models of chaperone action have been proposed. (i) Chaperones dissociate RepA dimers, which are inactive in DNA binding, into active monomers. (ii) The dissociation occurs spontaneously but the monomeric products require the chaperones for refolding into the active form. The latter model was based on the observation that RepA diluted 1000‐fold below the KD for dimer dissociation, still required the chaperones for DNA binding.

Results: We have confirmed that under the condition of DNA binding experiments, the RepA dimers dissociate reversibly into monomers with a KD value of 1.1 ± 0.1 μm. In the vicinity of this concentration, the sedimentation coefficient of RepA was concentration dependent, allowing estimation of s20,w coefficients for the RepA monomer (2.95 S) and dimer (4.01 S). Dynamic light scattering experiments indicated an increase of the monomer fraction within 5 min of RepA dilution. Circular dichroism (CD) measurements were consistent with these results.

Conclusion: RepA monomerization is efficient without the mediation of chaperones. They are required to activate RepA most likely because they are needed to re‐fold RepA monomers.
Histone‐like protein HU as a specific transcriptional regulator: co‐factor role in repression of gal transcription by GAL repressor
Authors: Aki Tsunehiro , Choy Hyon E. , Adhya Sankar
Published: November 2003

Background: Transcription initiation from the two overlapping promoters of the gal operon in Escherichia coli is negatively regulated by binding of Gal repressor (GalR) to bipartite operators, which encompass the promoters. Coordinated repression of the two promoters requires GalR binding to both operators. In a purified system, GalR, nevertheless, fails to show the coordinated repression, predicting the participation of an additional factor(s) in the regulation in vivo

Results: We have purified a protein that restored the expected GalR‐mediated repression for the in vitro system and have identified this factor to be the bacterial histone‐like protein HU. In vitro transcription assays in the presence of GalR and HU show that, just as in vivo, the coordinated repression of the two gal promoters requires GalR binding to both operators and is sensitive to the inducer, d‐galactose. The GalR and HU dependent repression also requires supercoiled DNA template and prevents open complex formation.

Conclusion: We propose that HU, acting as a co‐factor, brings about the GalR‐mediated repression by forming a distinct nucleoprotein complex of higher order structure. Although how HU participates in the assembly process is unknown, there may be a cooperative effect in the formation of the repression complex.
A membrane cofactor protein transgenic mouse model for the study of discordant xenograft rejection
Authors: Yannoutsos Nikos , IJzermans Jan N. M. , Harkes Clara , Bonthuis Fred , Zhou Chun‐Yan , White David , Marquet Richard L. M. , Grosveld Frank
Published: November 2003

Background: In recent years, interest has been revived in the possibility of transplanting organs into humans from a phylogenetically disparate species such as the pig (xenotransplantation). Such discordant xenografts, however, are subject to hyperacute rejection (HAR) and activation of host complement plays a major role in this rejection. This problem may be solved through the use of transgenic technology by providing the grafted tissue with molecules that down‐regulate the action of host complement.

Results: Transgenesis with a yeast artificial chromosome (YAC) was used to produce transgenic mice with the complete genomic gene of the human complement regulator membrane cofactor protein (MCP). Transgenic mice were obtained that exhibit full regulation of MCP as normally observed in humans. Hearts from these mice were shown to be significantly protected from HAR caused by human serum in an in vivo experimental procedure.

Conclusions: We conclude that MCP can protect discordant xenografts from HAR caused by human serum and that transgenic mice can be used effectively as in vivo models for the study of the role of human complement regulatory molecules in xenotransplantation.