bluuebunny/crossref_metadata_2025 huggingface dataset:
The evolutionary history of Drosophila buzzatti. XXVI. Macrogeographic patterns of inversion polymorphism in New World populations
Authors: Hasson Esteban , Rodríguez Constantina , Fanara Juan J. , Naveira Horacio , Reig Osvaldo A. , Fontdevila Antonio
Published: December 2004

Inversion polymorphisms in the second and fourth chromosomes of the cactophilic Drosophila buzzatti in the native distribution range of the species are described. Over 5,000 flies from 26 localities were scored revealing interesting geographic structuring of arrangement frequencies. Multiple regression and partial correlation approaches showed that the frequencies of second and fourth chromosome arrangements vary clinically along latitudinal and altitudinal gradients and to a lesser extent with longitude. Although many non selective explanations can account for this pattern, its resemblance to the clinal pattern described in recently established Australian populations of Drosophila buzzatii, strongly suggests a selective explanation. Additionally, the correlated variation observed between the frequencies of arrangements 2St on the second chromosome and 4St on the fourth suggests a pattern of interchromosomal association, which, when considering the vast area surveyed, might be explained as the result of epistatic interactions. The analysis of population structure revealed a significant regional pattern, concordant with previously described phytogeographic regions. F‐statistics showed that the patterns of variation were different not only between the second and fourth chromosomes, but also between second chromosome arrangements, suggesting that selective differentiation might have contributed to population structure.

Since D. buzzatii breeds and feeds on the decaying tissues of diverse cactus species present in different phytogeographic regions, it is difficult to distinguish the underlying causes of the geographic patterns observed. However, inversion heterozygosity is not correlated with the diversity of potential cactus hosts.

The evidence presented suggests that differential selection may be the main cause for the population structure. It is also possible to conclude that the inversion polymorphism of D. buzzatti is flexible rather than rigid.
The differential genetic and environmental canalization of fitness components in Drosophila melanogaster
Authors: Stearns Stephen C. , Kaiser Marcel , Kawecki Tadeusz J.
Published: December 2004

Canalization describes the process by which phenotypic variation is reduced by developmental mechanisms. A trait can be canalized against environmental or genetic perturbations. Stabilizing selelction should favor improved canalization, and the degree of a trait's canalization should be positively correlated with its impact on fitness. Here we report, for Drosophila melanogaster, measurements of environmental canalization for five fitness components. We compare them with measurements of genetic canalization, and we discuss the impact of inbreeding on both. In three experiments we measured the variation of fitness components within lines nested within temperature, treatment, and experiment. Lines differed in the position of a P element insert or in genetic background. Within lines flies were genetically nearly identical. We designated trait variation within lines as environmental canalization. The canalization of the traits increased with their impact on fitness, and the pattern was similar to that found for the canalization of fitness components against genetic differences, measured as the variation among lines nested within temperature, treatment, and experiment. This suggests that developmental mechanisms buffer the phenotype against both genetic and environmental disturbance. The results also suggest, less strongly, that inbreeding weakens canalization.
Assessing population genetic structure and variability with RAPD data: Application to Vaccinium macrocarpon (American Cranberry)
Authors: Stewart C. N. , Excoffier L.
Published: December 2004

A method for estimating and comparing population genetic variation using random amplified polymorphic DNA (RAPD) profiling is presented. An analysis of molecular variance (AMOVA) is extended to accomodate phenotypic molecular data in diploid populations in Hardy‐Weinberg equilibrium or with an assumed degree of selfing. We present a two step strategy: 1) Estimate RAPD site frequencies without preliminary assumptions on the unknown population structure, then perform significance testing for population substructuring. 2) If population structure is evident from the first step, use this data to calculate better estimates for RAPD site frequencies and sub‐population variance components. A nonparametric test for the homogeneity of molecular variance (HOMOVA) is also presented. This test was designed to statistically test for differences in intrapopulational molecular variances (heteroscedasticity among populations). These theoretical developments are applied to a RAPD data set in Vaccinium macrocarpon (American cranberry) using small sample sizes, where a gradient of molecular diversity is found between central and marginal populations. The AMOVA and HOMOVA methods provide flexible population analysis tools when using data from RAPD or other DNA methods that provide many polymorphic markers with or without direct allelic data.
Sexual size dimorphism and selection in the wild in the waterstrider Aquarius remigis: Body size, components of body size and male mating success
Authors: Preziosi R. F. , Fairbairn D. J.
Published: December 2004

Sexual size dimorphism is assumed to be adaptive and is expected to evolve in response to a difference in the net selection pressures on the sexes. Although a demonstration of sexual selection is neither necessary nor sufficient to explain the evolution of sexual size dimorphism, sexual selection is generally assumed to be a major evolutionary force. If contemporary sexual selection is important in the evolution and maintenance of sexual size dimorphism then we expect to see concordance between patterns of sexual selection and patterns of sexual dimorphism. We examined sexual selection in the wild, acting on male body size, and components of body size, in the waterstrider Aquarius remigis, as part of a long term study examining net selection pressures on the two sexes in this species. Selection was estimated on both a daily and annual basis. Since our measure of fitness (mating success) was behavioral, we estimated reliabilities to determine if males perform consistently. Reliabilities were measured as ϰ statistics and range from fair to perfect agreement with substantial agreement overall. We found significant univariate sexual selection favoring larger total length in the first year of our study but not in the second. Multivariate analysis of components of body size revealed that sexual selection for larger males was not acting directly on total length but on genital length. Sexual selection for larger male body size was opposed by direct selection favoring smaller midfemoral lengths. While males of this species are smaller than females, they have longer genital segments and wider forefemora. Patterns of contemporary sexual selection and sexual size dimorphism agree only for genital length. For total length, and all other components of body size examined, contemporary sexual selection was either nonsignificant or opposed the pattern of size dimporhism. Thus, while the net pressures of contemporary selection for the species may still act to maintain sexual size dimorphism, sexual selection alone does not.
Evolutionary responses of Drosophila melanogaster life history to differences in larval density
Authors: Roper C. , Pignatelli P. , Partridge L.
Published: December 2004

The study examined the effects of evolution at two different larval densities on pre‐adult and adult fitness traits. Five replicate selection lines each were cultured at either 50 or 150 larvae per vial, avoiding selection on development time, age at breeding or for adaptation to adult density, one or more of which factors has been a confounding variable in previous studies. Low density selection lines evolved extended development times at both growth densities. The extended development times were associated with greater adult body size at the lower growth density only, and particularly in females. The lines did not differ significantly in larval competitive ability at either growth density. At neither growth density did the early adult fertility of females or the lifespan of either sex differ between the lines from the two selection regimes, but at the lower growth density the late fertility of low density line females was significantly enhanced. The results suggest that larval density does have important effects on the expression and resolution of life history trade‐offs in Drosophila melanogaster, but that these may be somewhat different from those reported in previous studies.
A meta‐analysis of the heritability of developmental stability
Authors: Møller A. P. , Thornhill R.
Published: December 2004

The existence of additive genetic variance in developmental stability has important implications for our understanding of morphological variation. The heritability of individual fluctuating asymmetry and other measures of developmental stability have frequently been estimated from parent‐offspring regressions, sib analyses, or from selection experiments. Here we review by meta‐analysis published estimates of the heritability of developmental stability, mainly the degree of individual fluctuating asymmetry in morphological characters. The overall mean effect size of heritabilities of individual fluctuating asymmetry was 0.19 from 34 studies of 17 species differing highly significantly from zero (P < 0.0001). The mean heritability for 14 species was 0.27. This indicates that there is a significant additive genetic component to developmental stability. Effect size was larger for selection experiments than for studies based on parent‐offspring regression or sib analyses, implying that genetic estimates were unbiased by maternal or common environment effects. Additive genetic coefficients of variation for individual fluctuating asymmetry were considerably higher than those for character size per se. Developmental stability may be significantly heritable either because of strong directional selection, or fluctuating selection regimes which prevent populations from achieving a high degree of developmental stability to current environmental and genetic conditions.
On the evolution of heavy‐metal tolerant populations in Armeria maritima: evidence from allozyme variation and reproductive barriers
Authors: Vekemans X. , Lefèbvre C.
Published: December 2004

The process of ecological differentiation leading to the evolution of heavy‐metal tolerant populations in Armeria maritima was studied by comparing population genetic structure and pattern of gene flow between populations growing on heavy‐metal contaminated against non‐contaminated sites using allozyme markers. In addition the evolution of reproductive isolation among populations was studied by measuring pollen fertility in interpopulational hybrids. The allozyme data suggested that in A. maritima multiple independent evolutionary origins of heavy‐metal tolerant populations have occurred in the absence of strong genetic bottlenecks. The pattern of gene flow among populations was consistent with the model of isolation by distance with considerable gene flow between neighbor populations, and no reduction of gene flow between tolerant and non‐tolerant populations. Hence it appears that substantial gene flow has not hampered genetic differentiation, probably because of the high selection pressure for heavy‐metal tolerance. The pattern of reproductive isolation among populations suggests that evolution of heavy‐metal tolerant populations has not triggered the development of reproductive barriers against non‐tolerant populations. However, partial reproductive isolation has occurred under geographic separation.
Performance of two monophagous leaf feeding beetles (Coleoptera: Chrysomelidae) on each other's host plant: Do intrinsic factors determine host plant specialization?
Authors: Keese M. C.
Published: December 2004

There has been much recent debate on whether physiological tradeoffs in performance across hosts or ecological factors such as predation are the primary determinants of host plant specialization in plant‐herbivore interactions. This paper examines the relative role of intrinsic behavioral and physiological factors in host specialization of two species of leaf‐feeding beetles (Coleoptera: Chrysomelidae). Ophraella notulata and Ophraella slobodkini are sister taxa that feed exclusively on the asteraceous plants Iva frutescens and Ambrosia artemisiifolia, respectively. Ambrosia is the ancestral host plant for this pair of beetles. I performed full‐sib breeding experiments in both the laboratory and the field to assess mean responses of each species to both its native host and the host of its congener, genetic variation within each species for traits associated with using each host, and tradeoffs in performance across hosts. I reared each beetle species on each host plant and measured larval consumption, survival, development time and growth. I measured only survival and growth in the field. Genetic correlations were calculated to assess tradeoffs in performance across hosts. In the laboratory experiment, larval survival of O. slobodkini on I. frutescens was lower and development time longer than on A. artemisiifolia. Survival of O. notulata on A. artemisiifolia was marginally lower than on I. frutescens while development time did not differ. There was little genetic variation among families in host use traits for either species. None of the estimates of genetic correlations were negative. The results of the field experiment support the results of the laboratory experiment. I conclude that O. notulata, the species with the derived host association, retains considerable ability to utilize the ancestral host plant, while O. slobodkini, the species witht he ancestral host association, does not show a similar ability to ut ilize the derived host. Tradeoffs in performance across hosts were not documented for either species of Ophraella. That notulata performs so well on A. artemissifolia suggests that intrinsic factors may not provide a sufficient explanation for the host specialization of this species on I. frutescens.
Costs and limits of phenotypic plasticity: Tests with predator‐induced morphology and life history in a freshwater snail
Authors: DeWitt T. J.
Published: December 2004

Potential constraints on the evolution of phenotypic plasticity were tested using data from a previous study on predator‐induced morphology and life history in the freshwater snail Physa heterostropha. The benefit of plasticity can be reduced if facultative development is associated with energetic costs, developmental instability, or an impaired developmental range. I examined plasticity in two traits for 29 families of P. heterostropha to see if it was associated with growth rate or fecundity, within‐family phenotypic variance, or the potential to produce extreme phenotypes. Support was found for only one of the potential constraints. There was a strong negative selection gradient for growth rate associated with plasticity in shell shape (β = −0.3, P < 0.0001). This result was attributed to a genetic correlation between morphological plasticity and an antipredator behavior that restricts feeding. Thus, reduced growth associated with morphological plasticity may have had unmeasured fitness benefits. The growth reduction, therefore, is equivocal as a cost of plasticity. Using different fitness components (e.g., survival, fecundity, growth) to seek constraints on plasticity will yield different results in selection gradient analyses. Procedural and conceptual issues related to tests for costs and limits of plasticity are discussed, such as whether constraints on plasticity will be evolutionarily ephemeral and difficult to detect in nature.
Cumulative effects of founding events during colonisation on genetic diversity and differentiation in an island and stepping‐stone model
Authors: Le Corre V. , Kremer A.
Published: December 2004

This paper investigates the cumulative effect of founding events on the genetic differentiation and the within‐population heterozygosity in a metapopulation increasing its size by colonisation. Two contrasting models are considered: first, an island model, where migrants and colonists are taken at random from the entire metapopulation, and second, a linear stepping‐stone model, where migrants and colonists are sampled from a limited neighbourhood. The genetic consequences of a range expansion depend on the relative magnitudes of the number of colonists and migrants, in a way similar to extinction and colonisation processes (Wade and McCauley, 1988). The cumulative effect of founding events, resulting most often in a transient increase in genetic differentiation and a gradual loss of within‐population heterozygosity, also depends on the age‐structure that is established during colonisation. It is the highest when colonists are sampled from recently founded populations and migrants are exchanged among populations of similar ages. The genetic consequences of a range expansion are therefore far more pronounced and lasting in the linear stepping‐stone model than in the island model. These two models, however, represent the two extremes between which real populations will fall.
Host immune function and sexual selection in birds
Authors: Møller A. P. , Dufva R. , Erritzøe J.
Published: December 2004

Parasites have been hypothesized to affect sexual selection of their hosts, if secondary sexual characters reliably signal absence of infectious parasites, superior parenting ability caused by the absence of parasites, or heritable resistance to parasites, for which there is some intraspecific and interspecific evidence. Measures of immune defence of hosts provide reliable information on the current infection status of individuals of the chosen sex, usually males, and correlations between immune defence and development of secondary sexual characters thus provide a novel critical test of parasite‐mediated sexual selection. In a comparative study of birds, sexually dichromatic species had higher immune defences, measured in terms of leukocyte concentration and the size of spleen and bursa of Fabricius, respectively, than closely related, monochromatic species. Male plumage brightness was consistently negatively related to the size of the spleen in males of sexually dichromatic species, but not in males of monochromatic species. Hence, the brightest males, which frequently are preferred as mates by choosy females, had low levels of immune defence, suggesting that such males were healthy. This provides evidence for a general role of parasites in sexual selection among their bird hosts.
Wolbachia affects oviposition and mating behaviour of its spider mite host
Authors: Vala F. , Egas M. , Breeuwer J. A. J. , Sabelis M. W.
Published: December 2004

Wolbachia bacteria are transmitted from mother to offspring via the cytoplasm of the egg. When mated to males infected with Wolbachia bacteria, uninfected females produce unviable offspring, a phenomenon called cytoplasmic incompatibility (CI). Current theory predicts that ‘sterilization’ of uninfected females by infected males confers a fitness advantage to Wolbachia in infected females. When the infection is above a threshold frequency in a panmictic population, CI reduces the fitness of uninfected females below that of infected females and, consequently, the proportion of infected hosts increases. CI is a mechanism that benefits the bacteria but, apparently, not the host. The host could benefit from avoiding incompatible mates. Parasite load and disease resistance are known to be involved in mate choice. Can Wolbachia also be implicated in reproductive behaviour? We used the two-spotted spider mite – Wolbachia symbiosis to address this question. Our results suggest that uninfected females preferably mate to uninfected males while infected females aggregate their offspring, thereby promoting sib mating. Our data agrees with other results that hosts of Wolbachia do not necessarily behave as innocent bystanders – host mechanisms that avoid CI can evolve.
Transport of taurocholate by mutants of negatively charged amino acids, cysteines, and threonines of the rat liver sodium‐dependent taurocholate cotransporting polypeptide Ntcp
Authors: Zahner Daniel , Eckhardt Uta , Petzinger Ernst
Published: December 2004

The relevance of functional amino acids for taurocholate transport by the sodium‐dependent taurocholate cotransporting polypeptide Ntcp was determined by site‐directed mutagenesis. cRNA from 28 single‐points mutants of the rat liver Ntcp clone was expressed in Xenopus laevis oocytes. Mutations were generated in five conserved negatively charged amino acids (aspartates and glutamates) which were present in nine members of the SBAT‐family, in two nonconserved negatively charged amino acids, in all eight Ntcp‐cysteines, and in two threonines from a protein kinase C consensus region of the Ntcp C‐terminus. Functional amino acids were Asp115, Glu257, and Cys266, which were found to be essential for the maintenance of taurocholic acid transport. Asp115 is located in the large intracellular loop III, whereas Glu257 and Cys266 are located in the large extracellular loop VI. Four mutations of threonines from the C‐terminus of the Ntcp by alanines or tyrosines showed no effects on sodium‐dependent taurocholate transport. Introduction of the FLAG® motif into several transport negative point mutations demonstrated that all mutated proteins besides one were present within the cell membrane of the oocytes and provided proof that an insertion defect has not caused transport deficiency by these Ntcp mutants. The latter was observed only with the transport negative mutant Asp24Asn. In conclusion, loop amino acids are required for sodium‐dependent substrate translocation by the Ntcp.
Identification of sites of phosphorylation by G‐protein‐coupled receptor kinase 2 in β‐tubulin
Authors: Yoshida Norihiro , Haga Kazuko , Haga Tatsuya
Published: December 2004

G‐protein‐coupled receptor kinase 2 (GRK2) is known to specifically phosphorylate the agonist‐bound forms of G‐protein‐coupled receptors (GPCRs). This strict specificity is due at least partly to activation of GRK2 by agonist‐bound GPCRs, in which basic residues in intracellular regions adjacent to transmembrane segments are thought to be involved. Tubulin was found to be phosphorylated by GRK2, but it remains unknown if tubulin can also serve as both a substrate and an activator for GRK2. Purified tubulin, phosphorylated by GRK2, was subjected to biochemical analysis, and the phosphorylation sites in β‐tubulin were determined to be Thr409 and Ser420. In addition, the Ser444 in βIII‐tubulin was also indicated to be phosphorylated by GRK2. The phosphorylation sites in tubulin for GRK2 reside in the C‐terminal domain of β‐tubulin, which is on the outer surface of microtubules. Pretreatment of tubulin with protein phosphatase type‐2A (PP2A) resulted in a twofold increase in the phosphorylation of tubulin by GRK2. These results suggest that tubulin is phosphorylated in situ probably by GRK2 and that the phosphorylation may affect the interaction of microtubules with microtubule‐associated proteins. A GST fusion protein of a C‐terminal region of βI‐tubulin (393–445 residues), containing 19 acidic residues but only one basic residue, was found to be a good substrate for GRK2, like full‐length β‐tubulin. These results, together with the finding that GRK2 may phosphorylate synuclein and phosducin in their acidic domains, indicate that some proteins with very acidic regions but without basic activation domains could serve as substrates for GRK2.
2‐Oxo acid dehydrogenase complexes in redox regulation
Authors: Bunik Victoria I.
Published: December 2004

A number of cellular systems cooperate in redox regulation, providing metabolic responses according to changes in the oxidation (or reduction) of the redox active components of a cell. Key systems of central metabolism, such as the 2‐oxo acid dehydrogenase complexes, are important participants in redox regulation, because their function is controlled by the NADH/NAD+ ratio and the complex‐bound dihydrolipoate/lipoate ratio. Redox state of the complex‐bound lipoate is an indicator of the availability of the reaction substrates (2‐oxo acid, CoA and NAD+) and thiol‐disulfide status of the medium. Accumulation of the dihydrolipoate intermediate causes inactivation of the first enzyme of the complexes. With the mammalian pyruvate dehydrogenase, the phosphorylation system is involved in the lipoate‐dependent regulation, whereas mammalian 2‐oxoglutarate dehydrogenase exhibits a higher sensitivity to direct regulation by the complex‐bound dihydrolipoate/lipoate and external SH/S‐S, including mitochondrial thioredoxin. Thioredoxin efficiently protects the complexes from self‐inactivation during catalysis at low NAD+. As a result, 2‐oxoglutarate dehydrogenase complex may provide succinyl‐CoA for phosphorylation of GDP and ADP under conditions of restricted NAD+ availability. This may be essential upon accumulation of NADH and exhaustion of the pyridine nucleotide pool. Concomitantly, thioredoxin stimulates the complex‐bound dihydrolipoate‐dependent production of reactive oxygen species. It is suggested that this side‐effect of the 2‐oxo acid oxidation at low NAD+in vivo would be overcome by cooperation of mitochondrial thioredoxin and the thioredoxin‐dependent peroxidase, SP‐22.
DNA‐binding and transcription characteristics of three cloned sigma factors from mustard (Sinapis alba L.) suggest overlapping and distinct roles in plastid gene expression
Authors: Homann Anke , Link Gerhard
Published: December 2004

We have isolated and studied the cloned sigma factors SASIG1‐3 from mustard (Sinapis alba). In functional analyses using both promoter and factor mutants, the three recombinant proteins all had similar basic properties but also revealed differences in promoter preference and requirements for single nucleotide positions. Directed muta‐ genesis of SASIG1 identified critical residues within the conserved regions 2.4 and 4.2 necessary for binding of the −10 and −35 promoter elements, respectively. SASIG1 and 2, but not SASIG3, each have a typical region 2.5 for binding of the extended −10 promoter element. SASIG3 has a pro‐sequence reminiscent of σK from Bacillus subtilis, suggesting that proteolytic cleavage from an inactive precursor is involved in the regulation of plastid transcription. In addition, SASIG2 was found to be more abundant in light‐grown as compared to dark‐grown mustard seedlings, while the converse was true for SASIG3.
Improved ecdysone receptor‐based inducible gene regulation system
Authors: Palli Subba R. , Kapitskaya Mariana Z. , Kumar Mohan B. , Cress Dean E.
Published: December 2004

To develop an ecdysone receptor (EcR)‐based inducible gene regulation system, several constructs were prepared by fusing DEF domains of Choristoneura fumiferana EcR (CfEcR), C. fumiferana ultraspiracle (CfUSP), Mus musculus retinoid X receptor (MmRXR) to either GAL4 DNA binding domain (DBD) or VP16 activation domain. These constructs were tested in mammalian cells to evaluate their ability to transactivate luciferase gene placed under the control of GAL4 response elements and synthetic TATAA promoter. A two‐hybrid format switch, where GAL4 DBD was fused to CfEcR (DEF) and VP16 AD was fused to MmRXR (EF) was found to be the best combination. It had the lowest background levels of reporter gene activity in the absence of a ligand and the highest level of reporter gene activity in the presence of a ligand. Both induction and turn‐off responses were fast. A 16‐fold induction was observed within 3 h of ligand addition and increased to 8942‐fold by 48 h after the addition of ligand. Withdrawal of the ligand resulted in 50% and 80% reduction in reporter gene activity by 12 h and 24 h, respectively.
Distribution of class I, III and IV alcohol dehydrogenase mRNAs in the adult rat, mouse and human brain
Authors: Galter Dagmar , Carmine Andrea , Buervenich Silvia , Duester Gregg , Olson Lars
Published: December 2004

The localization of different classes of alcohol dehydrogenases (ADH) in the brain is of great interest because of their role in both ethanol and retinoic acid metabolism. Conflicting data have been reported in the literature. By Northern blot and enzyme activity analyses only class III ADH has been detected in adult brain specimens, while results from riboprobe in situ hybridization indicate class I as well as class IV ADH expression in different regions of the rat brain. Here we have studied the expression patterns of three ADH classes in adult rat, mouse and human tissues using radioactive oligonucleotide in situ hybridization. Specificity of probes was tested on liver and stomach control tissue, as well as tissue from class IV ADH knock‐out mice. Only class III ADH mRNA was found to be expressed in brain tissue of all three investigated species. Particularly high expression levels were found in neurons of the red nucleus in human tissue, while cortical neurons, pyramidal and granule cells of the hippocampus and dopamine neurons of substantia nigra showed moderate expression levels. Purkinje cells of cerebellum were positive for class III ADH mRNA in all species investigated, whereas granular layer neurons were positive only in rodents. The choroid plexus was highly positive for class III ADH, while no specific signal for class I or class IV ADH was detected. Our results thus support the notion that the only ADH expressed in adult mouse, rat and human brain is class III ADH.
Transactivation properties of c‐Myb are critically dependent on two SUMO‐1 acceptor sites that are conjugated in a PIASy enhanced manner
Authors: Dahle Øyvind , Andersen Tor Ø. , Nordgård Oddmund , Matre Vilborg , Del Sal Giannino , Gabrielsen Odd S.
Published: December 2004

The transcription factor v‐Myb is a potent inducer of myeloid leukemias, and its cellular homologue c‐Myb plays a crucial role in the regulation of hematopoiesis. Recently, Bies and coworkers (Bies, J., Markus, J. & Wolff, L. (2002) J. Biol. Chem, 277, 8999–9009) presented evidence that murine c‐Myb can be sumoylated under overexpression conditions in COS7 cells when cotransfected with FLAG‐tagged SUMO‐1. Here we provide independent evidence that human c‐Myb is also subject to SUMO‐1 conjugation under more physiological conditions as revealed by coimmunoprecipitation analysis of Jurkat cells and transfected CV‐1 cells. Analysis in an in vitro conjugation system showed that modification of the two sites K503 and K527 is interdependent. A two‐hybrid screening revealed that the SUMO‐1 conjugase Ubc9 is one of a few major Myb‐interacting proteins. The moderate basal level of sumoylation was greatly enhanced by cotransfection of PIASy, an E3 ligase for SUMO‐1. The functional consequence of abolishing sumoylation was enhanced activation both of a transiently transfected reporter gene and of a resident Myb‐target gene. When single and double mutants were compared, we found a clear correlation between reduction in sumoylation and increase in transcriptional activation. Enhancing sumoylation by contransfection of PIASy had a negative effect on both Myb‐induced and basal level reporter activation. Furthermore, PIASy caused a shift in nuclear distribution of c‐Myb towards the insoluble matrix fraction. We propose that the negative influence on transactivation properties by the negative regulatory domain region of c‐Myb depends on the sumoylation sites located here.
The N‐terminal cysteine pair of yeast sulfhydryl oxidase Erv1p is essential for in vivo activity and interacts with the primary redox centre
Authors: Hofhaus Götz , Lee Jeung‐Eun , Tews Ivo , Rosenberg Beate , Lisowsky Thomas
Published: December 2004

Yeast Erv1p is a ubiquitous FAD‐dependent sulfhydryl oxidase, located in the intermembrane space of mitochondria. The dimeric enzyme is essential for survival of the cell. Besides the redox‐active CXXC motif close to the FAD, Erv1p harbours two additional cysteine pairs. Site‐directed mutagenesis has identified all three cysteine pairs as essential for normal function. The C‐terminal cysteine pair is of structural importance as it contributes to the correct arrangement of the FAD‐binding fold. Variations in dimer formation and unique colour changes of mutant proteins argue in favour of an interaction between the N‐terminal cysteine pair with the redox centre of the partner monomer.
Localization of fluorescence‐labeled poly(malic acid) to the nuclei of the plasmodium of Physarum polycephalum
Authors: Karl Miachael , Gasselmaier Bernd , Krieg René C. , Holler Eggehard
Published: December 2004

The nuclei in the plasmodium of Physarum polycephalum, as of other myxomycetes, contain high amounts of polymalate, which has been proposed to function as a scaffold for the carriage and storage of several DNA‐binding proteins [Angerer, B. and Holler, E. (1995) Biochemistry34, 14741–14751]. By delivering fluorescence‐labeled polymalate into a growing plasmodium by injection, we observed microscopic staining of nuclei in agreement with the proposed function. The fluorescence intensity was highest during the reconstruction phase of the nuclei. To examine whether the delivery was under the control of polymalatase or related proteins [Karl, M. & Holler, E. (1998) Eur. J. Biochem.251, 405–412], the cellular distribution of these proteins was also examined by staining with antibodies against polymalatase. Double‐stained plasmodia revealed a fluorescent halo around each fluorescent nucleus during the reconsititution. Fluorescent nuclei were not observed when the hydroxyl terminus of polymalate, known to be essential for the binding of polymalatase, was blocked by labeling with fluorescein‐5‐isothiocyanate. By immune precipitation, it was shown that polymalate and polymalatase or related proteins were in the precipitate. It is concluded that polymalate is delivered to the surface of nuclei in the complex with polymalatase or related proteins. The complex dissociates, and polymalate translocates into the nucleus, while polymalatase or related proteins remain at the surface.
A novel nuclear DNA helicase with high specific activity from Pisum sativum catalytically translocates in the 3′→5′ direction
Authors: Phan Tuan‐Nghia , Ehtesham Nasreen Z. , Tuteja Renu , Tuteja Narendra
Published: December 2004

A novel ATP‐dependent nuclear DNA unwinding enzyme from pea has been purified to apparent homogeneity and characterized. This enzyme is present at extremely low abundance and has the highest specific activity among plant helicases. It is a heterodimer of 54 and 66 kDa polypeptides as determined by SDS/PAGE. On gel filtration chromatography and glycerol gradient centrifugation it gives a native molecular mass of 120 kDa and is named as pea DNA helicase 120 (PDH120). The enzyme can unwind 17‐bp partial duplex substrates with equal efficiency whether or not they contain a fork. It translocates unidirectionally along the bound strand in the 3′→5′ direction. The enzyme also exhibits intrinsic single‐stranded DNA‐ and Mg2+‐dependent ATPase activity. ATP is the most favoured cofactor but other NTPs and dNTPs can also support the helicase activity with lower efficiency (ATP > GTP = dCTP > UTP > dTTP > CTP > dATP > dGTP) for which divalent cation (Mg2+ > Mn2+) is required. The DNA intercalating agents actinomycin C1, ethidium bromide, daunorubicin and nogalamycin inhibit the DNA unwinding activity of PDH120 with Ki values of 5.6, 5.2, 4.0 and 0.71 µMs, respectively. This inhibition might be due to the intercalation of the inhibitors into duplex DNA, which results in the formation of DNA–inhibitor complexes that impede the translocation of PDH120. Isolation of this new DNA helicase should make an important contribution to our better understanding of DNA transaction in plants.
Sequences downstream of the transcription initiation site are important for proper initiation and regulation of mouse ribonucleotide reductase R2 gene transcription
Authors: Kotova Irina , Chabes Anna L. , Lobov Sergei , Thelander Lars , Björklund Stefan
Published: December 2004

Ribonucleotide reductase is essential for the synthesis of all four dNTPs required for DNA replication. The enzyme is composed of two proteins, R1 and R2, which are both needed for activity. Expression of the R1 and R2 mRNAs is restricted to the S‐phase of the cell cycle, but the R1 and R2 promoters show no obvious sequence homologies that could indicate coordination of transcription. Here we study initiation of transcription at the natural mouse R2 promoter, which contains an atypical TATA‐box with the sequence TTTAAA, using a combination of in vivo reporter gene assays and in vitro transcription. Our results indicate that in constructs where sequences from the R2 5′‐UTR are present, the mouse R2 TATA‐box is dispensable both for unregulated, basal transcription from the R2 promoter and for S‐phase specific activity. Instead, initiation of R2 transcription is directed by sequences downstream from the transcription start. We report that this region contains a conserved palindrome sequence that interacts with TAFIIs. This interaction down‐regulates basal transcription from the R2 promoter, both in the absence and in the presence of the TATA‐box.
Determination of native oligomeric state and substrate specificity of rat NTPDase1 and NTPDase2 after heterologous expression in Xenopus oocytes
Authors: Failer Bernd U. , Aschrafi Armaz , Schmalzing Günther , Zimmermann Herbert
Published: December 2004

NTPDase1 and NTPDase2 are two related plasma membrane‐located enzymes involved in the extracellular degradation of nucleoside 5′‐tri‐ and ‐diphosphates. They differ regarding their hydrolysis ratios for ATP and ADP. Both enzymes have a predicted transmembrane domain close to the N‐ and C‐terminus, respectively, connected by an extensive extracellular domain that carries the active site. We expressed the rat‐derived enzymes in Xenopus laevis oocytes and analyzed their quarternary structure. As revealed by application of blue native PAGE and a comparison of glutaraldehyde cross‐linking, native NTPDase1 and NTPDase2 occur in oligomeric form. Oligomer formation of the cell surface‐located pool of the enzymes was verified by surface iodination. The two enzymes differed in oligomeric structure and in oligomer complex stability. NTPDase1 preferentially occurred as a dimer that could be dissociated into monomeric forms in the presence of Coomassie Brilliant blue G‐250 and dithiothreitol whereas NTPDase2 revealed higher oligomeric forms up to tetramers, largely resistant to dithiothreitol. Our results further suggest that the enzymes exist in varying oligomeric states. In contrast to NTPDase1, substrate specificity of NTPDase2 was altered with prolonged expression time, resulting in a decrease in the ATPase/ADPase activity ratio from 10 : 1 to 2.5 : 1. This was accompanied by a transition into a higher oligomeric state. Our results suggest that despite close sequence identity, NTPDase1 and NTPDase2 differ in oligomeric structure. Dynamic alterations in oligomeric state may induce changes in substrate preference and thus influence the pattern of extracellular nucleotide degradation in situ.
Nitric oxide‐induced epidermal growth factor‐dependent phosphorylations in A431 tumour cells
Authors: Ruano María J. , Hernández‐Hernando Silvia , Jiménez Amparo , Estrada Carmen , Villalobo Antonio
Published: December 2004

Nitric oxide (NO•) strongly inhibits the proliferation of human A431 tumour cells. It also inhibits tyrosine phosphorylation of a 170‐kDa band corresponding to the epidermal growth factor receptor (EGFR) and induces the phosphorylation at tyrosine residue(s) of a 58‐kDa protein which we have denoted NOIPP‐58 (nitric oxide‐induced 58‐kDa phosphoprotein). The NO•‐induced phosphorylation of NOIPP‐58 is strictly dependent on the presence of EGF. Phosphorylation of NOIPP‐58 and inhibition of the phosphorylation of the band corresponding to EGFR are both cGMP‐independent processes. We also demonstrate that the p38 mitogen‐activated protein kinase (p38MAPK) pathway is activated by NO• in the absence and presence of EGF, whereas the activity of the extracellular signal‐regulated protein kinase 1/2 (ERK1/2) and the c‐Jun N‐terminal kinase 1/2 (JNK1/2) pathways are not significantly affected or are slightly decreased, respectively, on addition of this agent. Moreover, we show that the p38MAPK inhibitor, SB202190, induces rapid vanadate/peroxovanadate‐sensitive dephosphorylation of prephosphorylated EGFR and NOIPP‐58. We propose that the dephosphorylation of both NOIPP‐58 and EGFR are mediated by a p38MAPK‐controlled phosphotyrosine‐protein phosphatase (PYPP). Activation of the p38MAPK pathway during nitrosative stress probably prevents the operation of this PYPP, allowing NOIPP‐58, and in part EGFR, to remain phosphorylated and therefore capable of generating signalling events.
A steady‐state competition model describes the modulating effects of thrombomodulin on thrombin inhibition by plasminogen activator inhibitor‐1 in the absence and presence of vitronectin
Authors: Dekker Rob J. , Pannekoek Hans , Horrevoets Anton J. G.
Published: December 2004

Thrombomodulin (TM) slows down the interaction rate between thrombin and plasminogen activator inhibitor 1 (PAI‐1). We now show that the 12‐fold reduced inhibition rate in the presence of TM does not result from an altered distribution between PAI‐1 cleavage and irreversible complex formation. Surface plasmon resonance (SPR) revealed an over 200‐fold reduced affinity of TM for thrombin‐VR1tPA as compared to thrombin, demonstrating the importance of the VR1 loop in the interaction of thrombin with both TM and PAI‐1. Furthermore, in contrast to ATIII, PAI‐1 was not able to bind the thrombin/TM complex demonstrating complete competitive binding between PAI‐1 and TM. Kinetic modeling on the inhibitory effect of TM confirms a mechanism that involves complete steric blocking of the thrombin/PAI‐1 interaction. Also, it accurately decribes the biphasic inhibition profile resulting from the substantial reduction of the extremely fast rate of reversible Michaelis complex formation, which is essential for efficient inhibition of thrombin by PAI‐1. Vitronectin (VN) is shown to partially relieve TM inhibitory action only by vastly increasing the initial rate of interaction between free thrombin and PAI‐1. In addition, SPR established that solution‐phase PAI‐1/VN complexes and non‐native VN (extracellular matrix form) bind TM directly via the chondroitin sulphate moiety of TM. Collectively, these results show that VR1 is a subsite of exosite 1 on thrombin's surface, which regulates exclusive binding of either PAI‐1 or TM. This competition will be physiologically significant in controlling the mitogenic activity of thrombin during vascular disease.
The isopenicillin N acyltransferases of Aspergillus nidulans and Penicillium chrysogenum differ in their ability to maintain the 40‐kDa αβ heterodimer in an undissociated form
Authors: Fernández Francisco J. , Cardoza Rosa E. , Montenegro Eduardo , Velasco Javier , Gutiérrez Santiago , Martín Juan F.
Published: December 2004

The isopenicillin N acyltransferases (IATs) of Aspergillus nidulans and Penicillium chrysogenum differed in their ability to maintain the 40‐kDa proacyltransferase αβ heterodimer in an undissociated form. The native A. nidulans IAT exhibited a molecular mass of 40 kDa by gel filtration. The P. chrysogenum IAT showed a molecular mass of 29 kDa by gel filtration (corresponding to the β subunit of the enzyme) but the undissociated 40‐kDa heterodimer was never observed even in crude extracts. Heterologous expression experiments showed that the chromatographic behaviour of IAT was determined by the source of the penDE gene used in the expression experiments and not by the host itself. When the penDE gene of A. nidulans was expressed in P. chrysogenum npe6 and npe8 or in Acremonium chrysogenum, the IAT formed had a molecular mass of 40 kDa. On the other hand, when the penDE gene originating from P. chrysogenum was expressed in A. chrysogenum, the active IAT had a molecular mass of 29 kDa. The intronless form of the penDE gene cloned from an A. nidulans cDNA library and overexpressed in Escherichia coli formed the enzymatically active 40‐kDa proIAT, which was not self‐processed as shown by immunoblotting with antibodies to IAT. This 40‐kDa protein remained unprocessed even when treated with A. nidulans crude extract. In contrast, the P. chrysogenum penDE intronless gene cloned from a cDNA library was expressed in E. coli, and the IAT was self‐processed efficiently into its α (29 kDa) and β (11 kDa) subunits. It is concluded that P. chrysogenum and A. nidulans differ in their ability to self‐process their respective proIAT protein and to maintain the α and β subunits as an undissociated heterodimer, probably because of the amino‐acid sequence differences in the proIAT which affect the autocatalytic activity.
A role for the intersubunit disulfides of seminal RNase in the mechanism of its antitumor action
Authors: Bracale Aurora , Castaldi Francesco , Nitsch Lucio , D'Alessio Giuseppe
Published: December 2004

The dimeric structure of seminal ribonuclease (BS‐RNase) is maintained by noncovalent interactions and by two intersubunit disulfide bridges. Another unusual feature of this enzyme is its antitumour action, consisting in a cytotoxic activity selective for malignant cells. This cytotoxic action is exerted when the protein reaches the cytosol of the affected cells, where it degrades ribosomal RNA, thus blocking protein synthesis and leading cells to death. The current model proposed for the mechanism of antitumour action of BS‐RNase is based on the ability of the protein to resist the neutralizing action of the cytosolic RNase inhibitor, a resistance due to the dimeric structure of the enzyme. Monomeric RNases, and monomeric derivatives of BS‐RNase, are strongly bound by the inhibitor and inactive as antitumor agents. Here we report on monomeric derivatives of BS‐RNase that, although strongly inhibited by the cytosolic RNase inhibitor, are cytotoxic towards malignant cells. These monomers are produced by reductive cleavage of the intersubunit disulfides of the native, dimeric protein followed by linking the exposed sulfhydryls to small thiols through formation of mixed disulfides. We found that sulfhydryls from cell monolayers and cell membranes can attack these mixed disulfides in the monomeric derivatives, and reconstitute, through sulfhydryl‐disulfide interchange reactions, the native dimeric protein, which is internalized as such, and displays its antitumour action.
Characterization of Trypanosoma brucei PEX14 and its role in the import of glycosomal matrix proteins
Authors: Moyersoen Juliette , Choe Jungwoo , Kumar Abhinav , Voncken Frank G. J. , Hol Wim G. J. , Michels Paul A. M.
Published: December 2004

It has been shown previously in various organisms that the peroxin PEX14 is a component of a docking complex at the peroxisomal membrane, where it is involved in the import of matrix proteins into the organelle after their synthesis in the cytosol and recognition by a receptor. Here we present a characterization of the Trypanosoma brucei homologue of PEX14. It is shown that the protein is associated with glycosomes, the peroxisome‐like organelles of trypanosomatids in which most glycolytic enzymes are compartmentalized. The N‐terminal part of the protein binds specifically to TbPEX5, the cytosolic receptor for glycosomal matrix proteins with a peroxisome‐targeting signal type 1 (PTS‐1). TbPEX14 mRNA depletion by RNA interference results, in both bloodstream‐form and procyclic, insect‐stage T. brucei, in mislocalization of glycosomal proteins to the cytosol. The mislocalization was observed for different classes of matrix proteins: proteins with a C‐terminal PTS‐1, a N‐terminal PTS‐2 and a polypeptide internal I‐PTS. The RNA interference experiments also showed that TbPEX14 is essential for the survival of bloodstream‐form and procyclic trypanosomes. These data indicate the protein's great potential as a target for selective trypanocidal drugs.