bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitochondrial DNA phylogeography of lake cisco (Coregonus artedi): evidence supporting extensive secondary contacts between two glacial races
Authors: Turgeon J. , Bernatchez L.
Published: March 2003

The comparative molecular phylogeography of regional fish fauna has revealed the wide distribution of young clades in freshwater fishes of formerly glaciated areas as well as interspecific incongruences in their refugial origins and recolonization routes. In this study, we employed single‐strand conformation polymorphism (SSCP) and sequence analyses to describe mitochondrial DNA (mtDNA) polymorphism among 27 populations of the lake cisco (Coregonus artedi) from its entire range of distribution in order to evaluate the hypothesis of dual glacial refuges proposed by Bernatchez & Dodson against the traditional view that this species is solely of Mississippian origin. Results indicate that this taxon is composed of two closely related groups that are widely distributed and intermixed over most of the sampled range. The estimated level of divergence (0.48%), the contrast in the geographical distribution of each group, as well as the general distribution of C. artedi in North America together support the hypothesis that one group dispersed from a Mississippian refuge via the proglacial lakes, while the other is of Atlantic origin and also took advantages of earlier dispersal routes towards eastern Hudson Bay drainages. However, the signal of past range fragmentation revealed by a nested clade analysis was weak, and did not allow to formally exclude the hypothesis of a single Mississippian origin for both lineages. Comparisons with the phylogeographic patterns of other Nearctic freshwater fishes suggest that the salinity tolerance and thermal sensitivity of lake cisco may have been determinant for its extensive postglacial dispersal. The presence or co‐occurrence of sympatric or allopatric eco/morphotypes were not found to be necessarily associated with the presence of both haplotype groups.
The ‘rare allele phenomenon’ in a ribosomal spacer
Authors: Schilthuizen Menno , Hoekstra Rolf F. , Gittenberger Edmund
Published: March 2003

We describe the increased frequency of a particular length variant of the internal transcribed spacer 1 (ITS‐1) of the ribosomal DNA in a hybrid zone of the land snail Albinaria hippolyti. The phenomenon that normally rare alleles or other markers can increase in frequency in the centre of hybrid zones is not new. Under the term ‘hybrizyme’ or ‘rare allele’ phenomenon it has been recorded in many organisms and different genetic markers. However, this is the first time that it has been found in a multicopy locus. On the one hand, the pattern fits well with the view that purifying selection in hybrid populations works on many loci across the genome and should thus have its effect on many independent molecular markers. On the other hand, the results are puzzling, given that the multiple copies of rDNA are not expected to respond in unison. We suggest two possible explanations for these conflicting observations.
Phylogeography and conservation genetics of the Columbia spotted frog (Rana luteiventris; Amphibia, Ranidae)
Authors: Bos David H. , Sites Jack W.
Published: March 2003

The Columbia spotted frog (Rana luteiventris) has a widespread distribution in western Canada and the western US, although the southern reach of its range is highly fragmented into several isolated populations. Threats from various factors have raised concerns regarding the long‐term survival of many small, isolated populations. Here, we report a study designed to determine the phylogeographic and conservation genetic parameters of R. luteiventris in the western US. Mitochondrial DNA (mtDNA) sequences were examined for phylogeographic structuring using phylogenetic reconstruction methods, coupled with networking and nested clade analyses. These methods permitted a distinction to be made between historic and demographic forces acting to generate geographical patterning of genetic variation. Phylogenetic analysis revealed four geographically correlated monophyletic clades. Three of these clades correspond to well‐defined, nonoverlapping geographical locations in the fragmented portion of the range. The other is comprised of all samples collected from the contiguous range and includes one isolate from northern Wyoming. Networking and nested clade analyses confirmed these results and revealed that historical processes, such as range expansion and vicariance, rather than recurrent gene flow are likely responsible for observed patterns of genetic variation. A measure of genetic variation (θ = 4Neµ) revealed that R. luteiventris populations in Utah have a relatively low amount of genetic variation compared with populations in the continuous portion of the range.
Assessment of genetic structure within and among Bulgarian populations of the common ash (Fraxinus excelsior L.)
Authors: Heuertz Myriam , Hausman Jean‐François , Tsvetkov Ivaylo , Frascaria‐Lacoste Nathalie , Vekemans Xavier
Published: March 2003

We analysed genetic variation within and between populations of the common ash from Bulgaria in order to extract biological information useful in the context of conservation management of eastern European genetic resources of noble hardwood species. A total of 321 trees from three regions of Bulgaria were typed at six highly polymorphic microsatellite loci. Analysis of within‐population inbreeding suggests an upper boundary value of 2.7% for the selfing rate. Significant spatial genetic structure consistent with models of isolation by distance was detected within four out of 10 populations as well as among populations. Estimates of neighbourhood size in the range 38–126 individual trees were obtained based on spatial genetic structure analyses at either the intrapopulation or interpopulation level. Differentiation among populations explained only about 8.7% of total genetic diversity. These results are discussed in comparison with data from social broad‐leaved species such as oak and beech.
Temporal change in genetic structure and effective population size in steelhead trout (Oncorhynchus mykiss)
Authors: Heath Daniel D. , Busch Christopher , Kelly Joanne , Atagi Dana Y.
Published: March 2003

There is a wealth of published molecular population genetic studies, however, most do not include historic samples and thus implicitly assume temporal genetic stability. We tested for changes in genetic diversity and structure in three populations of steelhead trout (Oncorhynchus mykiss) from a northern British Columbia watershed using seven microsatellite loci over 40 years. We found little change in genetic diversity (mean allele numbers and observed and expected heterozygosity), despite large variation in the estimated numbers of steelhead returning to the watershed over the same time period. However, the temporal stability in genetic diversity is not reflected in population structure, which appears to be high among populations, yet significantly variable over time. The neighbour‐joining tree showed that, overall, two of the populations (Zymoetz and Kispiox) clustered separately from the third (Babine); a finding which was not consistent with their geographical separation. The clustering pattern was also not temporally consistent. We used the temporal method to estimate the effective number of breeders (Nb) for the three populations; our values (Nb = 17–102) were low for the large and presumed vigorous populations of steelhead trout sampled. The low Nb values were also not consistent with the generally high genetic diversity estimates, suggesting the possibility of intermittent gene flow among the three populations. The use of temporal analyses in population genetic samples should be a priority; first, to verify observed patterns in contemporary data, and second, to build a dataset of temporal analyses to allow generalizations to be made concerning temporal genetic stability and effective population size in natural populations.
Maternal inheritance of a chloroplast microsatellite marker in controlled hybrids between Fraxinus excelsior and Fraxinus angustifolia
Authors: Morand‐Prieur M. E. , Vedel F. , Raquin C. , Brachet S. , Sihachakr D. , Frascaria‐Lacoste N.
Published: March 2003

Restriction fragment length polymorphism, polymerase chain reaction–restriction fragment length polymorphism and simple sequence repeat (SSR or microsatellites) analyses were performed to detect chloroplast DNA polymorphisms between two ash species, Fraxinus excelsior and F.angustifolia. Only one SSR locus was found to be polymorphic, confirming the very close relatedness of these species. Inheritance of this marker was studied in hybrids obtained from controlled crosses between the two tree species. Results indicated, for the first time in Oleaceae, that chloroplasts are maternally inherited. This chloroplast SSR marker is now used concomitantly with nuclear markers to analyse ash populations in sympatric areas.
Fine‐scale population structure and dispersal inBiomphalaria glabrata, the intermediate snail host ofSchistosoma mansoni, in Venezuela
Authors: Mavárez J. , Amarista M. , Pointier J.‐P. , Jarne P.
Published: March 2003

Biomphalaria glabratais the main intermediate host ofSchistosoma mansoniin America and one of the most intensely studied species of freshwater snails, yet very little is known about its population biology. Here, we used seven highly polymorphic microsatellite loci to analyse genetic diversity in the Valencia lake basin, which represents the core of the endemic area for schistosomiasis in Venezuela. Populations were sampled at short spatial scale (a few kilometres), both inside the lake and in ponds or rivers near the lake. Our results indicate thatB. glabrataessentially cross‐fertilizes, with little variation in selfing rates among populations. Our markers detected considerable genetic variation, with an average heterozygosity of 0.60. More diversity per population was found within than outside the lake, suggesting an influence of connectivity among populations on the levels of genetic diversity. A marked population structure was detected and lake populations were less structured than other populations. Most individuals were assigned to their population of origin using an assignment test. No strong demographic signal (e.g. bottleneck) was detected, though lake populations are likely to experience bottlenecks more frequently than the other populations analysed. Differences in gene flow therefore seem to play an important role in population differentiation and in the restoring of genetic diversity in demographically unstable populations.
Variation at the major histocompatibility complex in Savannah sparrows
Authors: Freeman‐Gallant Corey R. , Johnson Elizabeth M. , Saponara Fiorella , Stanger Matthew
Published: March 2003

The class I and class II genes of the major histocompatibility complex (Mhc) encode dimeric glycoproteins responsible for eliciting the adaptive immune response of vertebrates. Recent work with birds suggests that the number, size, and arrangement of these genes can differ markedly across species, although the extent of this variation, and its causes and consequences, are poorly understood. We have used a 157‐base‐pair (bp) portion of the second exon of a class IIBgene to probe theMhcin a free‐living population of Savannah sparrows (Passerculus sandwichensis). Segregation analysis ofMhcbands suggests that class IIBgenes can be found in two independently assorting clusters, as previously described for domestic chickens (Gallus gallus) and ring‐necked pheasants (Phasianus colchicus) but unlike gene organization in mammals. TheMhcin Savannah sparrows appears large (with many class IIBgenes) and variable; we found 42 unique genotypes among 48 adults breeding on Kent Island, New Brunswick, Canada in 1995. Savannah sparrows are long‐distance migrants, and these results support recent predictions that migratory birds should show higher levels ofMhcpolymorphism and/or a greater number of genes than sedentary species. Savannah sparrows are also socially polygynous with high levels of extra‐pair paternity, suggesting that a history of sexual selection might also influence the size and/or structure of the avianMhc.
Population structure of plaice (Pleuronectes platessa L.) in northern Europe: microsatellites revealed large‐scale spatial and temporal homogeneity
Authors: Hoarau G. , Rijnsdorp A. D. , Van Der Veer H. W. , Stam W. T. , Olsen J. L.
Published: March 2003

Philopatry to spawning grounds combined with well‐known migratory patterns in the flatfish Pleuronectes platessa (plaice) has led to the hypothesis that regional populations may reflect relatively discrete, genetic stocks. Using six microsatellite loci we genotyped 240 adult individuals collected from locations in Norway, the Faeroe plateau, the Irish Sea, the Femer Baelt, Denmark, and the southern North Sea, and 240 0‐class juveniles collected from five nursery‐ground locations in Iceland, northwest Scotland, two sites in the Wadden Sea, and the Bay of Vilaine in Southern Brittany. The mean number of alleles/locus ranged from 5.3 to 20.4, with a mean of 13.9. Expected heterozygosity was uniformly high across all locations (multilocus Hexp = 0.744 ± 0.02). Pairwise comparisons of θ among all 11 locations revealed significant differentiation between Iceland and all other locations (θ = 0.0290*** to 0.0456***), which is consistent with the deep–water barrier to dispersal in plaice. In contrast, no significant differentiation was found among any of the remaining continental‐shelf sampling locations. This suggests that regional stocks are themselves composed of several genetic stocks under a model of panmixia which persists even to the spawning grounds. The presence of significant heterozygote deficiencies at all locations (not due to null alleles) suggests a temporal Wahlund effect yet the absence of significant population differentiation among continental shelf localities makes this explanation alone, difficult to reconcile. Sampling of eggs at the spawning grounds will be required to resolve this issue. Causes of the mismatch between genetic and geographical stocks is discussed in the context of high gene flow.
A genetic assessment of parentage in a natural population of dollar sunfish (Lepomis marginatus) based on microsatellite markers
Authors: MacKiewicz M. , Fletcher D. E. , Wilkins S. D. , Dewoody J. A. , Avise J. C.
Published: March 2003

We employ microsatellite markers to assess mating tactics in Lepomis marginatus. Genetic assignments for 1015 progeny in 23 nests indicate that about 95% of the offspring were sired by their respective nest‐guardians, a finding consistent with the apparent absence of a brood parasitic morphotype in this species. Allopaternal care was documented in two nests, one resulting from a nest takeover, the other from cuckoldry by an adjoining nest‐tender. Clustered de novo mutations also were identified. About 2.5 females (range 1–7) contributed to the offspring pool within a typical nest. Results are compared to those for other Lepomis species.
Phylogeographic analysis of a recent radiation ofEnallagmadamselflies (Odonata: Coenagrionidae)
Authors: Turgeon J. , McPeek M. A.
Published: March 2003

A phylogenetic hypothesis revealed two recent radiations among species ofEnallagmadamselflies, and extensive ecological work suggests that both adaptive and nonadaptive processes are involved in these radiations. We analysed the geographical pattern of genetic variability at 868 bp of mitochondrial DNA (mtDNA) among 283 individuals of 5 species displaying little ecological differentiation to identify the ancestral lineage, support their independent evolutionary trajectories and identify historical events and the underlying mechanism for one of these radiations. Nested clade analysis results clearly support a past event of range fragmentation inE. hageni. These Atlantic and Continentalhageniraces experienced distinct dispersal histories and still maintain nearly nonoverlapping ranges All four other species derive from the Continentalhageni. Whereas three species endemic to the Atlantic coastal plain show little genetic variation,E. ebriumshared several haplotypes with the Continentalhageni. Contrasting levels of genetic differentiation betweenE. hageniandE. ebriumin geographical areas associated with distinct events ofE. hageni's recent history support the recent origin of this species. Altogether, our results are compatible with a process of radiation via divergence in mate recognition systems within the Continentalhagenirace following secondary contacts between putative refugial races.
Polyploidy, phylogeography and Pleistocene refugia of the rockfern Asplenium ceterach: evidence from chloroplast DNA
Authors: Trewick S. A. , Morgan‐Richards M. , Russell S. J. , Henderson S. , Rumsey F. J. , Pintér I. , Barrett J. A. , Gibby M. , Vogel J. C.
Published: March 2003

Chloroplast DNA sequences were obtained from 331 Asplenium ceterach plants representing 143 populations from throughout the range of the complex in Europe, plus outlying sites in North Africa and the near East. We identified nine distinct haplotypes from a 900 bp fragment of trnL‐trnF gene. Tetraploid populations were encountered throughout Europe and further afield, whereas diploid populations were scarcer and predominated in the Pannonian‐Balkan region. Hexaploids were encountered only in southern Mediterranean populations. Four haplotypes were found among diploid populations of the Pannonian‐Balkans indicating that this region formed a northern Pleistocene refugium. A separate polyploid complex centred on Greece, comprises diploid, tetraploid and hexaploid populations with two endemic haplotypes and suggests long‐term persistence of populations in the southern Mediterranean. Three chloroplast DNA (cpDNA) haplotypes were common among tetraploids in Spain and Italy, with diversity reducing northwards suggesting expansion from the south after the Pleistocene. Our cpDNA and ploidy data indicate at least six independent origins of polyploids.
Loss of genetic diversity in sea otters (Enhydra lutris) associated with the fur trade of the 18th and 19th centuries
Authors: Larson Shawn , Jameson Ronald , Etnier Michael , Fleming Melissa , Bentzen Paul
Published: March 2003

Sea otter (Enhydra lutris) populations experienced widespread reduction and extirpation due to the fur trade of the 18th and 19th centuries. We examined genetic variation within four microsatellite markers and the mitochondrial DNA (mtDNA) d‐loop in one prefur trade population and compared it to five modern populations to determine potential losses in genetic variation. While mtDNA sequence variability was low within both modern and extinct populations, analysis of microsatellite allelic data revealed that the prefur trade population had significantly more variation than all the extant sea otter populations. Reduced genetic variation may lead to inbreeding depression and we believe sea otter populations should be closely monitored for potential associated negative effects.
Spatial genetic structure and clonal diversity of island populations of lady's slipper (Cypripedium calceolus) from the Biebrza National Park (northeast Poland)
Authors: Brzosko E. , Wróblewska A. , Ratkiewicz M.
Published: March 2003

Three populations of the rare and endangered plant speciesCypripedium calceoluswere included in a study of genetic diversity and spatial genetic structure in the Biebrza National Park, northeast Poland. Analysis of 11 allozyme loci indicate that the populations of this species contained high genetic variability (P = 45.5%,A= 1.73). On the other hand, the genetic differentiation (FST = 0.014,P< 0.05) amongC. calceoluspopulations was very low when compared to other species with similar life history characteristics. The observed high rate of gene flow (Nm = 18) may suggest that the populations studied derived from each other in the recent past. Five polymorphic allozyme markers identified 109 multilocus genotypes in three populations and the majority of them (67%) were population‐specific. One of the populations studied, characterized by particularly extensive vegetative reproduction, showed the lowest clonal diversity (G/N = 0.15) and heterozygosity (HO = 0.111) values and the highestFIS(0.380), when compared to other two populations (G/N = 0.26–0.27,HO= 0.166–178,FIS = 0.024–0.055). This may indicate that clonal reproduction has an important influence on the genetic structure ofC. calceoluspopulations. The longevity of genets, the out‐crossing breeding system and the presence of recruitment from seeds are factors maintaining genetic diversity inC. calceolus.
Molecular diversity of arbuscular mycorrhizal fungi and patterns of host association over time and space in a tropical forest
Authors: Husband R. , Herre E. A. , Turner S. L. , Gallery R. , Young J. P. W.
Published: March 2003

We have used molecular techniques to investigate the diversity and distribution of the arbuscular mycorrhizal (AM) fungi colonizing tree seedling roots in the tropical forest on Barro Colorado Island (BCI), Republic of Panama. In the first year, we sampled newly emergent seedlings of the understory treelet Faramea occidentalis and the canopy emergent Tetragastris panamensis, from mixed seedling carpets at each of two sites. The following year we sampled surviving seedlings from these cohorts. The roots of 48 plants were analysed using AM fungal‐specific primers to amplify and clone partial small subunit (SSU) ribosomal RNA gene sequences. Over 1300 clones were screened for random fragment length polymorphism (RFLP) variation and 7% of these were sequenced. Compared with AM fungal communities sampled from temperate habitats using the same method, the overall diversity was high, with a total of 30 AM fungal types identified. Seventeen of these types have not been recorded previously, with the remainder being similar to types reported from temperate habitats. The tropical mycorrhizal population showed significant spatial heterogeneity and nonrandom associations with the different hosts. Moreover there was a strong shift in the mycorrhizal communities over time. AM fungal types that were dominant in the newly germinated seedlings were almost entirely replaced by previously rare types in the surviving seedlings the following year. The high diversity and huge variation detected across time points, sites and hosts, implies that the AM fungal types are ecologically distinct and thus may have the potential to influence recruitment and host composition in tropical forests.
Y chromosome conserved anchored tagged sequences (YCATS) for the analysis of mammalian male‐specific DNA
Authors: Hellborg Linda , Ellegren Hans
Published: March 2003

Y chromosome haplotyping based on microsatellites or single nucleotide polymorphisms has recently proven to be a powerful approach for evolutionary studies of human populations, and also holds great promise for the studies of wild species. However, the use of the approach is hampered in most natural populations by the lack of Y chromosome markers and sequence information. Here, we report the large‐scale development of Y chromosome conserved anchor tagged sequence (YCATS) markers in mammals by a polymerase chain reaction screening approach. Exonic primers flanking 48 different introns of Y‐linked genes were developed based on human and mouse sequences, and screened on a set of 20 different mammals. On average about 10 introns were amplified for each species and a total of 100 kb of Y chromosome sequence were obtained. Intron size in humans was a reasonable predictor of intron size in other mammals (r2 = 0.45) and there was a negative correlation between human fragment size and amplification success. We discuss a number of factors affecting the possibility of developing conserved Y chromosome markers, including fast evolution of Y chromosome sequences due to male‐biased mutation and adaptive evolution of male‐specific genes, dynamic evolution of the Y chromosome due to being a nonrecombining unit, and homology with X chromosome sequences.
Phylogeny and ecological radiation of New World thistles (Cirsium, Cardueae – Compositae) based on ITS and ETS rDNA sequence data
Authors: Kelch Dean G. , Baldwin Bruce G.
Published: March 2003

Sequence data from a portion of the external transcribed spacer (ETS) and internal transcribed spacers (ITS‐1 and ITS‐2) of 18S‐26S nuclear ribosomal DNA were used to resolve historical biogeography and ecology of true thistles (Cirsium, Cardueae, Compositae) in the New World. The 650 base‐pair, 3′ portion of the ETS examined here showed a level of variation across taxa similar to that of the ITS sequences included. A maximum‐likelihood tree based on combined ETS and ITS sequences leads us to suggest that the New World species of true thistles constitute a major lineage, which in turn comprises several smaller lineages. A western North American lineage shows weak quartet‐puzzling support, but includes a well‐supported lineage of species endemic to the California Floristic Province. Comparisons of this Californian lineage with other neoendemic angiosperm groups of the region show that the CalifornianCirsiumlineage exhibits unusually high ecological diversity for a group displaying such low levels of rDNA sequence divergence across taxa. Similarly low levels of sequence divergence were found throughout the New WorldCirsiumlineage. These results indicate either thatCirsiumunderwent a rapid ecological radiation in North America, or that rDNA evolution in North AmericanCirsiumhas been highly conservative.
Conservation genetics of the fisher (Martes pennanti) based on mitochondrial DNA sequencing
Authors: Drew R. E. , Hallett J. G. , Aubry K. B. , Cullings K. W. , Koepf S. M. , Zielinski W. J.
Published: March 2003

Translocation of animals to re‐establish extirpated populations or to maintain declining ones has often been carried out without genetic information on source or target populations, or adequate consideration of the potential effects of mixing genetic stocks. We consider the conservation status of the fisher (Martes pennanti) and evaluate the potential genetic consequences of past and future translocations on this medium‐sized carnivore by examining population variation in mitochondrial control‐region sequences. We sampled populations throughout the fisher's range in North America including five populations unaffected by translocations and two western populations that had received long‐distance translocations. Twelve haplotypes showed little sequence divergence. Haplotype frequencies differed significantly among subspecies and between populations within subspecies. Analysis of molecular variance (amova) and neighbour‐joining analyses of haplotype relationships revealed population subdivision similar to current subspecies designations, but which may reflect an isolation‐by‐distance pattern. Populations in Oregon and in Montana and Idaho received several translocations and each showed greater similarity to the populations where translocations originated than to adjacent populations. Additional sequences obtained from museum specimens collected prior to any translocations suggest historical gene flow among populations in British Columbia, Washington, Oregon, and California. Anthropogenic impacts in that region have greatly reduced and isolated extant populations in Oregon and California. Future translocations may be necessary to recover populations in Washington and portions of Oregon and California; our results indicate that British Columbia would be the most appropriate source population.
A role for the Pcl9‐Pho85 cyclin–cdk complex at the M/G1 boundary in Saccharomyces cerevisiae
Authors: Tennyson Christine N. , Lee Jinhwa , Andrews Brenda J.
Published: March 2003

PHO85 is a cyclin‐dependent kinase (CDK) with roles in phosphate and glycogen metabolism and cell cycle progression. As a CDK, Pho85 is activated by association with Pho85 cyclins (Pcls), of which 10 are known. PCL1, PCL2 and PCL9 are the only members of the Pho85 cyclin family that are expressed in a cell cycle‐regulated pattern. We found that PCL9 is expressed in late M/early G1 phase of the cell cycle and is activated by the transcription factor, Swi5. This pattern of regulation is different from PCL1 and PCL2, which are expressed later in G1 phase and are regulated primarily by the transcription factor SBF. Co‐immunoprecipitation experiments using in vitro translated proteins showed that Pcl9 and Pho85 form a complex. Furthermore, immunoprecipitated Pcl9 complexes from yeast lysates were capable of phosphorylating the exogenous substrate Pho4. The Pcl9‐associated kinase activity was dependent on PHO85, showing that Pcl9 and Pho85 form a functionally active kinase complex in vivo. Deletion of PCL9 in diploid cells caused random, rather than bipolar, budding in 18% of cells. In contrast, deletion of PCL2, the closest relative of PCL9, had no effect on the budding pattern. Deleting more members of the PCL1,2 subfamily (which includes PCL9 ) increased the percentage of random budding in the cell population. When all members of the PCL1,2 subfamily were deleted, 73% of cells budded randomly, a value similar to that obtained when the CDK partner PHO85 was deleted. Our results show that PCL9 and PHO85 form a functional kinase complex and suggest a role for Pho85 CDKs at the M/G1 boundary.
Distal genes of the nuo operon of Rhodobacter capsulatus equivalent to the mitochondrial ND subunits are all essential for the biogenesis of the respiratory NADH–ubiquinone oxidoreductase
Authors: Dupuis Alain , Darrouzet Elisabeth , Duborjal Hervé , Pierrard Béatrice , Chevallet Mireille , Van Belzen Ronald , Albracht Simon P. J. , Lunardi Joël
Published: March 2003

Seven out of the 13 proteins encoded by the mitochondrial genome of mammals (peptides ND1 to ND6 plus ND4L) are subunits of the respiratory NADH–ubiquinone oxidoreductase (complex I). The function of these ND subunits is still poorly understood. We have used the NADH–ubiquinone oxidoreductase of Rhodobacter capsulatus as a model for the study of the function of these proteins. In this bacterium, the 14 genes encoding the NADH–ubiquinone oxidoreductase are clustered in the nuo operon. We report here on the biochemical and spectroscopic characterization of mutants individually disrupted in five nuo genes, equivalent to mitochondrial genes nd1, nd2, nd5, nd6 and nd4L. Disruption of any of these genes in R. capsulatus leads to the suppression of NADH dehydrogenase activity at the level of the bacterial membranes and to the disappearance of complex I‐associated iron–sulphur clusters. Individual NUO subunits can still be immunodetected in the membranes of these mutants, but they do not form a functional subcomplex. In contrast to these observations, disruption of two ORFs (orf6 and orf7 ), also present in the distal part of the nuo operon, does not suppress NADH dehydrogenase activity or complex I‐associated EPR signals, thus demonstrating that these ORFs are not essential for the biosynthesis of complex I.
A newly identified regulator is required for virulence and toxin production in Pseudomonas syringae
Authors: Kitten Todd , Kinscherf Thomas G. , McEvoy James L. , Willis David K.
Published: March 2003

The genes lemA (which we here redesignate gacS) and gacA encode members of a widely conserved two‐component regulatory system. In Pseudomonas syringae strain B728a, gacS and gacA are required for lesion formation on bean, as well as for the production of protease and the toxin syringomycin. A gene, designated salA, was discovered that restored syringomycin production to a gacS mutant when present on a multiple‐copy plasmid. Disruption of chromosomal salA resulted in loss of syringomycin production and lesion formation in laboratory assays. Sequence analysis of salA suggests that it encodes a protein with a DNA‐binding motif but without other significant similarity to proteins in current databases. Chromosomal reporter fusions revealed that gacS and gacA positively regulate salA, that salA upregulates its own expression and that salA positively regulates the expression of a syringomycin biosynthetic gene, syrB. Loss of syringomycin production does not account for the salA mutant's attenuated pathogenicity, as a syrB mutant was found to retain full virulence. The salA gene did not similarly suppress the protease deficient phenotype of gacS mutants, nor were salA mutants affected for protease production. A gacS/gacA‐dependent homoserine lactone activity as detected by bioassay was also unaffected by the disruption of salA. Thus, salA appears to encode a novel regulator that activates the expression of at least two separate genetic subsets of the gacS/gacA regulon, one pathway leading to syringomycin production and the other resulting in plant disease.
Meaning and therapeutic potential of microbial recognition of host glycoconjugates
Authors: Karlsson Karl‐Anders
Published: March 2003

The microbe–host interface is currently in focus because of attempts to develop infection therapy in humans based on either natural receptor saccharide (respiratory and gastrointestinal disease) or sophisticated sialic acid analogues designed from crystal structures (influenza). Most of the known host receptors for microbes are glycoconjugates, and the diversity and selectivity of host tissue glycosylation allow for the tropism of infections. However, among the many binding specificities detected so far, the biological role has been proven only in a few infectious model systems. The existence of multiple specificities of a single microbe is both a complicating factor and a challenge, requiring expanded research with a special demand on glycoscience.
Mmicular mechanisms of cytochrome c biogenesis: three distinct systems
Authors: Kranz Robert , Lill Roland , Goldman Barry , Bonnard Géraldine , Merchant Sabeeha
Published: March 2003

The past 10 years have heralded remarkable progress in the understanding of the biogenesis of c‐type cytochromes. The hallmark of c‐type cytochrome synthesis is the covalent ligation of haem vinyl groups to two cysteinyl residues of the apocytochrome (at a Cys–Xxx–Yyy–Cys–His signature motif). From genetic, genomic and biochemical studies, it is clear that three distinct systems have evolved in nature to assemble this ancient protein. In this review, common principles of assembly for all systems and the mmicular mechanisms predicted for each system are summarized. Prokaryotes, plant mitochondria and chloroplasts use either system I or II, which are each predicted to use dedicated mechanisms for haem delivery, apocytochrome ushering and thioreduction. Accessory proteins of systems I and II co‐ordinate the positioning of these two substrates at the membrane surface for covalent ligation. The third system has evolved specifically in mitochondria of fungi, invertebrates and vertebrates. For system III, a pivotal role is played by an enzyme called cytochrome c haem lyase (CCHL) in the mitochondrial intermembrane space.
Differential expression of secreted aspartyl proteinases in a model of human oral candidosis and in patient samples from the oral cavity
Authors: Schaller Martin , Schäfer Wilhelm , Korting Hans C. , Hube Bernhard
Published: March 2003

Candida albicans, an opportunistic pathogen in humans, secretes secretory aspartyl proteinases (Saps), which have been correlated with virulence. We examined the temporal regulation of the mRNA expression of seven known members of the SAP gene family by reverse transcription polymerase chain reaction (RT–PCR) in (i) an in vitro model of oral candidosis based on reconstituted human epithelium (RHE); and (ii) clinical samples from patients with oral candidosis. SAP1 and SAP3 transcripts were first detected 42 h after inoculation of RHE, while at the same time, slight morphological alterations in the epithelium were documented by light microscopy. SAP6 expression occurred 6 h later concomitantly with germ tube formation of some infecting Candida cells and severe lesions of the epithelial tissue. SAP2 and SAP8 RT–PCR products were first detected 60 h after infection, while SAP4 and SAP5 transcripts were never discovered. Thus, a temporal progression of SAP expression in the order SAP1 and SAP3 > SAP6 > SAP2 and SAP8 was observed at the same time as increasing RHE damage occurred. At the protein level, Sap antigen was found within the C. albicans yeast cells and the epithelial cells by immunoelectron microscopy using an anti‐Sap murine monoclonal antibody directed against the gene products Sap1–3. Expression of SAP1–3 and 6 was also detected by RT–PCR in samples from patients suffering from oral candidosis. Our results suggest that the pathogenesis of experimental and clinical oral candidosis is associated with the differential and temporal regulation of SAP gene expression.
FtsK is a bifunctional protein involved in cell division and chromosome localization in Escherichia coli
Authors: Liu Guowen , Draper G. Craig , Donachie W. D.
Published: March 2003

The behaviour of Escherichia coli cells in which all or part of the ftsK gene is under inducible control shows that FtsK protein has two functional domains: an N‐terminal part that is required for cell division, and a C‐terminal part that is involved in chromosome localization within the cell.
YopT, a new Yersinia Yop effector protein, affects the cytoskeleton of host cells
Authors: Iriarte Maite , Cornelis Guy R.
Published: March 2003

Extracellular Yersinia disarm the immune system of their host by injecting effector Yop proteins into the cytosol of target cells. Five effectors have been described: YopE, YopH, YpkA/YopO, YopP and YopM. Delivery of these effectors by Yersinia adhering at the cell surface requires other Yops (translocators) including YopB. Effector and translocator Yops are secreted by the type III Ysc secretion apparatus, and some Yops also need a specific cytosolic chaperone, called Syc. In this paper, we describe a new Yop, which we have called YopT (35.5 kDa). Its secretion required an intact Ysc apparatus and SycT (15.0 kDa, pI 4.4), a new chaperone resembling SycE. Infection of macrophages with a Yersinia, producing a hybrid YopT–adenylate cyclase, led to the accumulation of intracellular cAMP, indicating that YopT is delivered into the cytosol of eukaryotic cells. Infection of HeLa cells with a mutant strain devoid of the five known Yop effectors (ΔHOPEM strain) but producing YopT resulted in the alteration of the cell cytoskeleton and the disruption of the actin filament structure. This cytotoxic effect was caused by YopT and dependent on YopB. YopT is thus a new effector Yop and a new bacterial toxin affecting the cytoskeleton of eukaryotic cells.
The Escherichia coli relBE genes belong to a new toxin–antitoxin gene family
Authors: Gotfredsen Marie , Gerdes Kenn
Published: March 2003

Toxin–antitoxin systems are defined as a group of plasmid‐ and chromosome‐encoded loci that specify a cell toxin and a protein antitoxin. Plasmid‐encoded toxin–antitoxin systems stabilize their replicons by killing plasmid‐free cells. Here, we show that the relBE genes of Escherichia coli K‐12 have all the basic features previously connected with toxin–antitoxin systems: (i) relE encodes a cytotoxin lethal or inhibitory to host cells; (ii) relB encodes an antitoxin that prevents the lethal action of the relE‐encoded toxin; (iii) the relBE genes stabilize a mini‐R1 test plasmid; and (iv) the RelB antitoxin autoregulates the relBEF operon at the level of transcription. Using database searching, we found relBE homologues on the chromosomes of E. coli K‐12, Haemophilus influenzae and Vibrio choleraeA fifth relBE homologue was identified on the enterotoxin encoding E. coli plasmid P307. Indirect evidence suggests that the toxicity of RelE may be related to the inhibition of protein synthesis. Based on these observations, we propose a model that explains the delayed relaxed phenotype associated with mutations in relB.
Type IV pili are involved in plant–microbe and fungus–microbe interactions
Authors: Dörr Juliane , Hurek Thomas , Reinhold‐Hurek Barbara
Published: March 2003

Adherence of bacteria to eukaryotic cells is essential for the initiation of infection in many animal and human pathogens, e.g. Neisseria gonorrhoeae and Pseudomonas aeruginosa. Adhesion‐mediating type IV pili, filamentous surface appendages formed by pilin subunits, are crucial virulence factors. Here, we report that type IV pilus‐dependent adhesion is also involved in plant–bacteria and fungus–bacteria interactions. Nitrogen‐fixing, endophytic bacteria, Azoarcus sp., can infect the roots of rice and spread systemically into the shoot without causing symptoms of plant disease. Formation of pili on solid media was dependent on the pilAB locus. PilA encodes an unusually short (6.4 kDa) putative pilin precursor showing 100% homology to the conserved N‐terminus of the Pseudomonas aeruginosa type IV pilin. PilB encodes for a 14.2 kDa polypeptide showing similarity to FimF, a component of type I fimbriae of Escherichia coli. It was found to be extruded beyond the cell surface by immunofluorescence studies, and it may, therefore, be part of a pilus assembly complex or the pilus itself. Both genes are involved in the establishment of bacteria on the root surface of rice seedlings, as detected by fluorescence microscopy. Moreover, both genes are necessary for bacterial adhesion to the mycelium of an ascomycete, which was isolated from the same rhizosphere as the bacteria. In co‐culture with the fungus, Azoarcus sp. forms complex intracytoplasmic membranes, diazosomes, which are related to efficient nitrogen fixation. Adhesion to the mycelium appears to be crucial for this process, as diazosomes were absent and nitrogen fixation rates were decreased in pilAB mutants in co‐culture.
Penicillin tolerance genes ofStreptococcus pneumoniae: the ABC‐type manganese permease complex Psa
Authors: Novak Rodger , Braun Johann S. , Charpentier Emmanuelle , Tuomanen Elaine
Published: March 2003

Downregulation of the major autolysin inStreptococcus pneumoniaeleads to penicillin tolerance, a feature that is characterized by the ability to survive but not grow in the presence of antibiotic. Screening a library of mutants in pneumococcal surface proteins for the ability to survive 10 × minimum inhibitory concentration (MIC) of penicillin revealed over 10 candidate tolerance genes. One such mutant contained an insertion in the known genepsaA, which is part of thepsalocus. This locus encodes an ABC‐type Mn permease complex. Sequence analysis of adjacent DNA extended the known genetic organization of the locus to include two new open reading frames (ORFs),psaB, which encodes an ATP‐binding protein, andpsaC, which encodes a hydrophobic transmembrane protein. Mutagenesis ofpsaB,psaC,psaAand downstreampsaDresulted in penicillin tolerance. Defective adhesion and reduced transformation efficiency, as reported previously for apsaA−mutant, were phenotypes shared bypsaB −,psaC −andpsaD −knockout mutants. Western blot analysis demonstrated that the set of mutants expressed RecA, but none of them showed translation of the autolysin gene, which is located downstream ofrecA. The addition of manganese (Mn) failed to correct the abnormal physiology. These results suggest that this ABC‐type Mn permease complex has a pleiotropic effect on pneumococcal physiology including adherence and autolysis. These are the first genes suggested as being involved in triggering autolysin. The results raise the possibility that loss of function of PsaA, by vaccine‐induced antibody for instance, may promote penicillin tolerance.
The effect of oxygenated mycolic acid composition on cell wall function and macrophage growth in Mycobacterium tuberculosis
Authors: Yuan Ying , Zhu YaQi , Crane Deborah D. , Barry III Clifton E.
Published: March 2003

There are three major structural classes of mycolic acids in the cell envelope of Mycobacterium tuberculosis (MTB): alpha‐, methoxy‐ and ketomycolate. The two oxygen‐containing classes are biosynthetically related through a common α‐methyl hydroxymycolate intermediate. BCG strains that fail to produce methoxymycolate and instead produce only keto‐ and alpha‐mycolic acids show apparent defects in the O‐methyltransferase MMAS‐3. Overproduction of MMAS‐3 from MTB resulted in a complete replacement of ketomycolate by methoxymycolate in both BCG and MTB. In vitro growth of these recombinant strains lacking ketomycolate was impaired at reduced temperatures but appeared to be normal at 37°C. Glucose uptake was significantly decreased in such strains, but uptake of chenodeoxycholate and glycine was unaffected. Although sensitivity to INH remained unchanged, these cells were found to be hypersensitive to ampicillin and rifampicin. Infectivity of BCG and H37Rv wild type or MMAS‐3 overproducers in THP‐1 cells was somewhat affected, but the ability of the strains lacking ketomycolate to grow within this macrophage‐like cell line was severely compromised. In vivo labelling of mycolic acids during growth of H37Rv within THP‐1 cells revealed a substantial increase in ketomycolate and alphamycolate synthesized by intracellularly grown mycobacteria. These results establish a critical role for mycolate composition in proper cell wall function during the growth of MTB in vivo.
Yeast gene YRR1, which is required for resistance to 4‐nitroquinoline N‐oxide, mediates transcriptional activation of the multidrug resistance transporter gene SNQ2
Authors: Cui Zhifeng , Shiraki Toshiyuki , Hirata Dai , Miyakawa Tokichi
Published: March 2003

We have cloned and characterized a Saccharomyces cerevisiae gene YRR1 that is important for resistance to the mutagen 4‐nitroquinoline N‐oxide (4‐NQO). The wild‐type YRR1 gene encodes a protein that contains a Zn(II)2Cys6‐type zinc‐finger motif. Disruption of the YRR1 gene leads to hypersensitivity to 4‐NQO. A dominant mutation (YRR1‐1 ) that confers strong resistance to 4‐NQO has been identified. Epistasis analysis demonstrated that 4‐NQO resistances mediated by the YRR1 and YRR1‐1 alleles require the presence of the SNQ2 gene that encodes a multidrug resistance ATP binding cassette superfamily protein responsible for 4‐NQO export. Northern blot analysis of SNQ2 mRNA levels indicated that Yrr1p is involved in basal and drug‐induced transcriptional activation of SNQ2, whereas Pdr1p/Pdr3p transcription factors are mainly involved in basal SNQ2 expression. In the YRR1‐1 mutant, the level of SNQ2 mRNA is constitutively elevated. These results establish that Yrr1p is important for 4‐NQO resistance by mediating transcriptional activation of the SNQ2 gene in response to the stress imposed by 4‐NQO. The gain‐of‐function mutation of Yrr1‐1p was attributable to the duplication of a 12‐amino‐acid sequence generated near the carboxy terminus.
Bacillus subtilis tetA(L) gene expression: evidence for regulation by translational reinitiation
Authors: Stasinopoulos Stan J. , Farr Glen A. , Bechhofer David H.
Published: March 2003

The tetA(L) gene of Bacillus subtilis encodes a transmembrane protein that can function as a Tc‐metal/H+ antiporter, conferring low‐level resistance to tetracycline. The TetA(L) coding sequence is preceded by a leader region that contains a 20‐amino‐acid open reading frame and an appropriately spaced ribosome binding site. Expression of the gene is induced by addition of tetracycline, which is thought to act by binding to ribosomes that translate the tetA(L) leader peptide coding sequence. Here we demonstrate that induction of tetA(L) expression includes minor transcriptional and major translational components. Deletion and point mutations of the tetA(L) leader region were constructed to probe the mechanism of translational induction. To account for the observed mutant phenotypes, we propose that tetA(L) expression is regulated by a translational reinitiation mechanism.