bluuebunny/crossref_metadata_2025 huggingface dataset:
Indo‐Pacific population structure and evolutionary history of the coconut crab Birgus latro
Authors: Lavery S. , Moritz C. , Fielder D. R.
Published: November 2003

Mitochondrial DNA variation was used to examine population structure in a widespread, marine‐dispersed species, Birgus latro. Crabs were collected from eight locations throughout the species' Indo‐Pacific distribution. Purified mtDNA from 160 individuals was cut with five restriction enzymes, revealing high haplotype diversity (0.96) and moderate nucleotide diversity (0.75%). Island populations from the Indian Ocean (Christmas I.) and Pacific Ocean were significantly different (GST= 0.37) and had distinct mtDNA lineages with a net sequence divergence of 1.4%. Pacific island populations had diverged in a manner consistent with isolation by distance, with only the most peripheral populations being significantly different. The results for mtDNA are largely concordant with those from allozymes, although estimates of gene flow between the Indian and Pacific Oceans were much lower when based on mtDNA. The mtDNA phylogeny also permitted a deeper examination of the evolutionary and demographic history of Birgus latro. Long‐term separation of populations is evident in the complete phylogenetic subdivision of mtDNA lineages between the Indian and Pacific Ocean populations sampled. The starlike phylogeny of alleles from the Pacific suggests a rapid population expansion in the Pacific during the Pleistocene. Including information about allele phylogeny, as well as distribution and frequency, obscured contemporary population structure, but provided unique insights into the evolutionary history of the species.
Is the black robin in genetic peril?
Authors: Ardern S. L. , Lambert D. M.
Published: November 2003

In 1980 the entire black robin species Petroica traversi comprised only five birds, and the current population of ≊ 200 individuals is known to be derived from a single breeding pair. We show here that levels of minisatellite DNA variation in the black robin are among the lowest reported for any avian species in the wild. Surprisingly, similarly bottlenecked control populations of a closely related species (P. australis australis) exhibit significantly higher levels of genetic variation. This suggests that the black robin's persistence in a single small population for the last 100 years, rather than the recent bottleneck itself, accounts for the low genetic variation observed. Despite apparent genetic impoverishment, survival and reproductive performance indicate that the black robin is viable under existing conditions. This example illustrates that significant levels of genetic variation are not a necessary prerequisite for endangered species' survival.
Paternity assessment and population subdivision in a natural population of the larger mouse‐eared bat Myotis myotis
Authors: Petri B. , Pääbo S. , Von Haeseler A. , Tautz D.
Published: November 2003

A hypervariable simple sequence locus and mitochondrial D‐loop sequences were used to analyse genetically a natural population of the larger mouse‐eared bat Myotis myotis in southern Bavaria. Tests for population subdivision and direct observations suggest that females return to their natal sites, while males disperse. The males present in female nursery colonies are not related to the females. Paternity assessment for 46 offspring from a particular nursery colony showed that there are no males that monopolize the reproduction, and that the resident males in the colony had only a small mating success. Instead, the results suggest that females actively seek matings outside their colony. Most interestingly, it appears that a group of males about 16 km away from the nursery colony had a relatively high mating success and that this group of males may be related to the females of the nursery colonies. If this finding can be confirmed in a larger study, it may have important consequences for future conservation strategies.
Genetic variation in an introduced aphid pest (Metopolophium dirhodum) in New Zealand and relation to individuals from europe
Authors: Nicol D. , Armstrong K. F. , Wratten S. D. , Cameron C. M. , Frampton C. , Fenton B.
Published: November 2003

RAPD‐PCR was used to determine the genetic variation of Metopolophium dirhodum collected in a winter wheat field and in a nearby 2.5‐m‐high suction trap at Lincoln, New Zealand. Over three collection dates, five distinct genotypes were identified, using two primers (OPK16 and OPC09) independently. There was a significant temporal effect on the ratio of genotypes in populations collected in the field. There was no significant spatial aggregation or association of these genotypes in the field. Two of the genotypes present in the field were also detected in the suction trap sample. Using a higher resolution method of RAPD‐PCR (with the Stoffel fragment of Taq polymerase), a total of 124 genotypes were distinguished from 142 individuals collected from Scotland and New Zealand. The Jaccard similarity index (S) was used to measure similarity between individual aphids within and between populations from both hemispheres. All populations were very diverse (S < 0.33). However, at similar crop growth stages, M. dirhodum was significantly more diverse in Scotland than in New Zealand. The results are discussed in relation to the value of monitoring aphid flights for pest forecasting, and in terms of the most appropriate RAPD‐PCR techniques.
AFLP analysis sheds light on distribution of two Salix species and their hybrid along a natural gradient
Authors: Beismann H. , Barker J. H. A. , Karp A. , Speck T.
Published: November 2003

Amplified fragment length polymorphism (AFLP) analysis of 26 trees of three Salix taxa: Salix alba L. (White Willow), S. fragilis L. (Crack Willow) and their hybrid S. x rubens Schrank, across an example of their habitat range in south‐west Germany, supported the distribution previously determined using morphological characterization. UPGMA and principal coordinates analysis of the AFLP data revealed three distinct clusters corresponding to the three taxa. In addition, AFLP analysis on individuals which were difficult to identify morphologically revealed that they were either the hybrid S. x rubens or S. fragilis. Four specimens of S. fragilis were indistinguishable with three primer combinations suggesting they are members of one clone.
Population genetic response to microsite ecological stress in wild barley, Hordeum spontaneum
Authors: Owuor Edward D. , Fahima Tzion , Beiles Avigdor , Korol Abraham , Nevo Eviatar
Published: November 2003

Genetic diversity was studied in six subpopulations (a total of 60 individuals) of wild barley, Hordeum spontaneum, the progenitor of cultivated barley, sampled from six stations located along a transect of 300 m across the two opposing slopes of ‘Evolution Canyon’, a Mediterranean microsite at Lower Nahal Oren, Mt Carmel. The two opposing slopes are separated by between 100 and 400 m and designated SFS (South‐Facing Slope) and NFS (North‐Facing Slope) with each having three equidistant test stations. The SFS, which receives up to 300% more solar radiation, is drier, ecologically more heterogeneous, fluctuating, and more stressful than the NFS. Analysis of 12 RAPD primers, representing a total of 51 putative loci, revealed a significant inter‐ and intraslope variation in RAPD band polymorphism. A significantly higher proportion of polymorphic RAPD loci was found amongst the subpopulations on the SFS (mean P= 0.909) than on the NFS (mean P= 0.682), on the basis of the presence and absence of 22 strong bands. Polymorphism generally increased upwards from the bottom to the top of the SFS (0.636, 0.773, 0.955) and NFS (0.409, 0.500, 0.545), respectively. Gametic phase disequilibria estimates, D, revealed SFS and NFS unique predominant combinations which sharply differentiated the two slopes and indicated that there is differential interslope selection favouring slope‐specific multilocus combinations of alleles, or blocks of genes over tens to hundreds of meters. This suggests that selection overrides migration. RAPD polymorphism appears to parallel allozyme diversity which is climatically adaptive and driven by natural selection in the same subpopulations at the microsite.
A common export pathway for proteins binding complex redox cofactors?
Authors: Berks Ben C.
Published: November 2003

The precursor polypeptides of periplasmic proteins binding seven types of redox cofactor have unusually long signal sequences bearing a consensus (S/T)‐R‐R‐x‐F‐L‐K motif immediately before the hydrophobic region. Such ‘double‐arginine’ signal sequences are not, in general, found on the precursors of other periplasmic proteins. It is suggested that precursor proteins with double‐arginine signal sequences share a common specialization in their export pathway. The nature of this specialization, the structure of the double‐arginine signal sequences, and the possible relationship with the double‐arginine signal peptide‐dependent thylakoid import pathway are discussed.
Characterization of cpsF and its product CMP‐N‐acetylneuraminic acid synthetase, a group B streptococcal enzyme that can function in K1 capsular polysaccharide biosynthesis in Escherichia coli
Authors: Haft Rachel F. , Wessels Michael R. , Mebane Mary Fisk , Conaty Neil , Rubens Craig E.
Published: November 2003

Group B Streptococcus (GBS) is the foremost cause of neonatal sepsis and meningitis in the United States. A major virulence factor for GBS is its capsular polysaccharide, a high molecular weight polymer of branched oligosaccharide subunits. N‐acetylneuraminic acid (Neu5Ac or sialic acid), at the end of the polysaccharide side chains, is critical to the virulence function of the capsular polysaccharide. Neu5Ac must be activated by CMP‐Neu5Ac synthetase before it is incorporated into the polymer. We showed previously that a transposon mutant of a serotype III GBS strain which had no detectable capsular Neu5Ac was deficient in CMP‐Neu5Ac‐synthetase activity (Wessels et al., 1992). In this paper, we report the identification and characterization of cpsF, a gene interrupted by transposon insertion in the previously described Neu5Ac‐deficient mutant. The predicted amino acid sequence of the cpsF gene product shares 57% similarity and 37% identity with CMP‐Neu5Ac synthetase encoded by the Escherichia coli K1 gene, neuA. The enzymatic function of the protein encoded by cpsF was established by cloning the gene in E. coli under the control of the T7 polymerase/promoter. Lysates of E. coli in which the cpsF gene product was expressed, catalysed the condensation of CTP with Neu5Ac to form CMP‐Neu5Ac. In addition, when a CMP‐Neu5Ac synthetase‐deficient mutant of E. coli K1 was transformed with cpsF, K1 antigen expression was restored. We conclude that cpsF encodes CMP‐Neu5Ac synthetase in type III GBS, and that the GBS enzyme can function in the capsule‐synthesis of a heterologous bacterial species.
Theta‐type DNA replication stimulates homologous recombination in the Bacillus subtilis chromosome
Authors: Morel‐Deville Françoise , Ehrlich S. Dusko
Published: November 2003

To test the effects of theta‐type replication on homologous DNA recombination, we integrated in the chromosome of Bacillus subtilis a structure comprising a conditional replication region and direct repeats of ∼ 4 kb. The replicon was derived from a broad‐host‐range plasmid, pAMβ1, which replicates by a unidirectional theta mechanism and is thermosensitive. The direct repeats were derived from plasmid pBR322 and flanked the chloramphenicol‐resistance gene of plasmid pC194. Recombination between the repeats could therefore lead to a loss of the resistance gene or the appearance of additional repeats. The integrated replicon was active at the permissive temperature, and ∼ 25% of the integrated plasmids could be isolated as Y‐shaped molecules after restriction, having a branch at the replication origin. Replicon activity stimulated recombination four‐ to fivefold, as estimated from the proportion of chloramphenicol‐sensitive cells at the restrictive and permissive temperature, and also led to the appearance of additional direct repeats. We conclude that theta‐type replication stimulates homologous recombination and suggest that many or even most recombination events between long homologous sequences present in a bacterial genome may be the consequence of DNA replication.
The role of the lux autoinducer in regulating luminescence in Vibrio harveyi; control of luxR expression
Authors: Miyamoto Carol M. , Chatterjee Jaidip , Swartzman Elana , Szittner Rose , Meighen Edward A.
Published: November 2003

Analysis of Vibrio harveyi dark autoinducer mutants has demonstrated that the level of LuxR was much lower than that observed in wild‐type cells. Complementation with luxR fully restored luminescence suggesting that the lux autoinducer may control expression of the luxR regulatory gene. By primer extension, the transcriptional start site of luxR was located 78 bp from the initiation codon. The level of the primer‐extended product was enhanced upon addition of the lux autoinducer to the autoinducer mutants, which was confirmed by hybridization of lux mRNA with specific probes. By using chloramphenicol acetyltransferase as a reporter gene in a transcriptional fusion with luxR, the stimulatory effect of autoinducer on luxR expression was shown to occur at the level of the luxR promoter. The results provide evidence that the autoinducer signal in V. harveyi can be transduced through luxR, resulting in stimulation of luminescence.
A genetic approach for analysing surface‐exposed regions of the OmpC protein of Escherichia coli K‐12
Authors: Vakharia Hema , Misra Rajeev
Published: November 2003

Phage attachment sites on bacterial cell surfaces are provided by the exposed regions of outer membrane proteins and lipopolysaccharide (LPS). We have identified surface exposed residues of OmpC that are important for phage binding. This was accomplished by employing a genetic scheme in which two simultaneous selections enriched for ompC mutants defective in phage attachment, but retained functional channels. Mutational alterations were clustered in three regions of the OmpC protein. These regions also showed the greatest divergence from the analogous regions of the highly related OmpF and PhoE proteins. The majority of alterations (8 out of 11) occurred in a region of OmpC that is predicted to form a large exterior loop (loop 4). Interestingly, while the removal of this loop prevented phage binding, the deletion conferred enhanced channel activities.  Another type of phage‐resistant mutants synthesized defective LPS molecules. Biochemical analysis of mutant LPS revealed it to be of the Re‐type LPS, lacking the heptose moieties from the LPS inner core. As a result of this LPS defect, many outer membrane proteins were present in somewhat reduced levels. The phage resistance seen in these mutants could be a result of both the presence of defective LPS and reduced OmpC levels.
Isolation and characterization of insertional mutations in flagellin genes in the archaeon Methanococcus voltae
Authors: Jarrell Ken F. , Bayley Douglas P. , Florian Volker , Klein Albrecht
Published: November 2003

Methanococcus voltae is a flagellated member of the Domain Archaea that has four flagellin genes arranged in two transcriptional units. One transcriptional unit encodes only flaA while the second is a multi‐cistronic unit encoding three flagellin genes (flaB1flaB2, and flaB3 ) as well as at least seven other open reading frames downstream. The polymerase chain reaction was used to amplify an internal fragment of the flaA gene which was subsequently cloned into an insertion vector developed for M. voltae. Transformation of protoplasts with this vector led to the isolation of mutant strains that had insertions in flaA or flaB2. Mutant strains carrying insertions in flaA had flagella that were similar to wild‐type cells in both number and appearance when viewed using the electron microscope. In addition, some of these mutant strains had profiles identical to the wild type in immunoblots developed with antisera raised against the 31 kDa flagellin of M. voltae. All flaA mutant strains and the wild‐type cells showed immuno‐cross‐reactive bands at 33 and 31 kDa (corresponding to purified flagellins) as well as at 18 kDa. Some flaA mutant strains also showed an immuno‐cross‐reactive band at 27 kDa which probably represents a truncated flagellin produced by the insertion vector. However, both types of flaA mutant strains were less motile than the wild type in semi‐swarm plate experiments. The mutant strain with an insertion in flaB2 was non‐flagellated when examined by electron microscopy and it was non‐motile in semi‐swarm plate experiments. It represents the first structural mutant strain isolated in a methanogen. This mutant strain lacked the 33, 31, and 18 kDa immuno‐cross‐reactive bands observed in the wild type and flaA mutant strains, and instead had a novel band at 20 kDa. This band may represent an unmodified flagellin which still has an attached leader peptide. If so, then one of the downstream genes in the multi‐cistronic transcriptional unit may encode a leader peptidase for the flagellin system.
Structural analysis and proteolytic activation of Enterococcus faecalis cytolysin, a novel lantibiotic
Authors: Booth Mary C. , Bogie Charles P. , Sahl Hans‐Georg , Siezen Roland J. , Hatter Kenneth L. , Gilmore Michael S.
Published: November 2003

Clinical isolates of Enterococcus faecalis more commonly produce a cytolysin than do commensal isolates. Epidemiologic evidence and animal‐model studies have established a role for the cytolysin in the pathogenesis of enterococcal disease. The cytolysin consists of two structural subunits, CylLL and CylLs, that are activated by a third component, CylA. Genetic and biochemical characterization of CylA indicate that it is a serine protease, and that activation putatively results from cleavage of one or both cytolysin subunits. Genetic evidence also suggests that the cytolysin subunits are related to the rapidly growing class of bacteriocins termed lantibiotics. However, unlike lantibiotics, the cytolysin is lytic for eukaryotic as well as prokaryotic cells, and it consists of two structural subunits. This report describes the purification and characterization of the cytolysin subunits and detection of lanthionine‐type post‐translational modifications within their structures. Furthermore, the cleavage specificity of the CylA activator is reported and it is shown that proteolytic activation of both subunits is essential for activity.
The Rhizobium meliloti putA gene: its role in the establishment of the symbiotic interaction with alfalfa
Authors: Jiménez‐Zurdo José I. , García‐Rodríguez Fernando M. , Toro Nicolás
Published: November 2003

Little is known about the energy sources used by rhizobia during colonization, invasion and root nodule formation on leguminous plants. We have recently reported that an impaired proline metabolism in Rhizobium meliloti leads to a reduced nodulation efficiency and competitiveness on alfalfa roots. In the present study we have characterized the R. meliloti proline dehydrogenase gene (putA) and addressed the question of its role in symbiosis. This rhizobial gene encodes a 1224‐amino‐acid‐long polypeptide which is homologous to enteric bacteria, Rhodobacter capsulatus and Bradyrhizobium japonicum PutA proteins.Like the situation in these bacteria, sequence analysis identified the proline dehydrogenase (PDH) and pyrroline‐5‐carboxylate dehydrogenase (P5CDH) domains in the R. meliloti putA‐encoded protein. Beta‐galactosidase assays performed with free‐living cells carrying a putA–lacZ transcriptional fusion revealed that R. meliloti putA gene expression is induced by proline, autoregulated by its encoded product, and independent of the general nitrogen regulatory system (Ntr). In addition, analysis of putA expression during the different steps of the symbiotic interaction with alfalfa showed that expression of this gene is turned on by the root exudates (RE), during root invasion and nodule formation, but not in differentiated nitrogen‐fixing bacteroids. Furthermore, we show that the PutA− phenotype leads to a significant reduction of alfalfa root colonization by R. meliloti.
A mitochondrial‐like targeting signal on the hydrogenosomal malic enzyme from the anaerobic fungus Neocallimastix frontalis: support for the hypothesis that hydrogenosomes are modified mitochondria
Authors: Van Der Giezen Mark , Rechinger K. Bjo¨rn , Svendsen Ib , Durand Roger , Hirt Robert P. , Fèvre Michel , Embley T. Martin , Prins Rudolf A.
Published: November 2003

The hydrogenosomal malic enzyme (ME) was purified from the anaerobic fungus Neocallimastix frontalis. Using reverse genetics, the corresponding cDNA was isolated and characterized. The deduced amino acid sequence of the ME showed high similarity to ME from metazoa, plants and protists. Putative functional domains for malate and NAD+/NADP+ binding were identified. Phylogenetic analysis of the deduced amino acid sequence of the new ME suggests that it is homologous to reference bacterial and eukaryotic ME. Most interestingly, the cDNA codes for a protein which contains a 27‐amino‐acid N‐terminus which is not present on the purified mature protein. This presequence shares features with known mitochondrial targeting signals, including an enrichment in Ala, Leu, Ser, and Arg, and the presence of an Arg at position –2 relative to amino acid 1 of the mature protein. This is the first report of a mitochondrial‐like targeting signal on a hydrogenosomal enzyme from an anaerobic fungus and provides support for the hypothesis that hydrogenosomes in Neocallimastix frontalis might be modified mitochondria.
Replication of minichromosomes in a host in which chromosome replication is random
Authors: Eliasson Åsa , Nordström Kurt
Published: November 2003

Minichromosomes are plasmids with the origin of chromosome replication, oriC, as their only origin of replication. In Escherichia coli, minichromosomes are compatible with the chromosome and replicate in a cell‐cycle‐specific manner at the same time as oriC located on the chromosome initiates replication. In int strains, oriC has been inactivated and replaced by a plasmid origin. Because plasmids control their own replication, chromosome replication is uncoupled from the normal cell‐cycle control and is random with respect to the cell cycle in the int strains. We have used an intP1 strain to address the question of whether minicromosome replication is coupled to the replication of the chromosome or is governed by cell‐cycle‐specific signals. Minichromosome replication was analysed by density‐shift experiments and found not to be random in the randomly replicating intP1 host. This suggests that the cell‐cycle‐specific control functions of oriC replication are operating also in the intP1 strain.
Mutations of the coat protein gene of bacteriophage λ that overcome the necessity for the FI gene; the EFi domain
Authors: Murialdo Helios , Tzamtzis Dimitra
Published: November 2003

The functions of most of the 10 genes involved in phage λ capsid morphogenesis are well understood. The function of the FI gene is one of the exceptions. Mutants in FI fail to mature and package DNA. The gene product (gpFI) seems to act as a catalyst for the formation of an intermediate in capsid assembly called complex II, which contains a procapsid (an empty capsid precursor), terminase (the enzyme that cleaves the DNA precursor and packages it into the procapsid) and DNA. The mechanism for this stimulation remains unknown. It has also been reported that gpFI appeared to stimulate terminase‐mediated cos cleavage, in the absence of procapsids, by increasing enzyme turnover. In comparison with other head‐gene mutants, FI mutants are leaky, producing approx. 0.1 phage per infected cell. Some second‐site revertants of FI − phages, called ‘fin’, that bypass the necessity for gpFI, have been isolated and found to harbour a mutation in the genes that code for the two subunits of terminase. In the course of mapping additional fin mutants, it was discovered that some mapped outside the terminase genes. To localize the mutations, restriction fragments of fin mutant DNAs were subcloned into plasmids and their ability to contribute to fin function was determined by marker‐rescue analysis. The location of the fin mutation was further delineated by deletion analysis of a plasmid that was positive for fin. This showed that some fin mutations mapped to a region comprising genes E, D and a portion of C. The sequencing of this entire region in several fin isolates showed that the fin mutations are clustered in a small region of gene E corresponding to a portion of 26 amino acid residues of the coat protein (gpE). We have called this region of the protein the EFi domain. All the mutations result in an increase in positive charge relative to the wild‐type protein. These results suggest that DNA maturation and packaging are in part controlled by an interaction between gpFI and capsid gpE.
Relating primary structure to function in the Escherichia coli XerD site‐specific recombinase
Authors: Spiers Andrew J. , Sherratt David J.
Published: November 2003

XerC and XerD are related 298‐amino‐acid site‐specific recombinases, each of which is responsible for the exchange of one pair of strands in Xer recombination. Both recombinases encode functions necessary for sequence‐specific DNA‐binding, co‐operative XerC/D interactions, synapsis and catalysis. These functions were related to the primary amino acid sequence by constructing and analysing internal and C‐terminal XerD deletions. An XerD derivative containing residues 1–233 was proficient in specific DNA binding, but did not interact co‐operatively with XerC. Deletion of a further five C‐terminal amino acids abolished binding to DNA. Proteins deleted for residues 32–88 and for residues 145–159 were deficient in DNA binding. Deletion of residues 244–281, a region containing amino acids necessary for catalysis, gave a protein that bound to DNA. An XerD derivative containing residues 1–268 retained co‐operative interactions with XerC; nevertheless, it did not support XerC strand exchange and was defective in XerD catalysis. Residues 1–283 retain a functional catalytic active site, though a protein lacking the five C‐terminal amino acids was still unable to mediate normal in vivo recombination, indicating that these residues are needed for a function that is not directly related to DNA binding or catalysis.
DNA binding of Escherichia coli arginine repressor mutants altered in oligomeric state
Authors: Chen Sheau‐Hu , Merican Amir F. , Sherratt David J.
Published: November 2003

The Escherichia coli arginine repressor (ArgR) controls expression of the arginine biosynthetic genes and acts as an accessory protein in Xer site‐specific recombination at cer and related plasmid recombination sites. The hexameric wild‐type protein shows L‐arginine‐dependent DNA binding. In this work, ArgR mutants that are defective in trimer–trimer interactions and bind DNA as trimers in an L‐arginine‐independent manner are isolated and characterized. Whereas the wild‐type ArgR hexamer exhibits high‐affinity binding to two repeated ARG boxes separated by 3 bp (each ARG box containing two identical dyad symmetrical 9 bp half‐sites), the trimeric mutants bind to and footprint three adjacent half‐sites of this ‘idealized’ substrate. Trimeric ArgR is impaired in its ability to repress the arginine biosynthetic genes and in Xer site‐specific recombination. In the absence of L‐arginine, residual wild‐type ArgR‐binding occurs as trimers. The binding of an N‐terminal 77‐amino‐acid DNA‐binding domain to idealized ARG boxes is also characterized.
The highly thermostable arginine repressor of Bacillus stearothermophilus: gene cloning and repressor–operator interactions
Authors: Dion Michel , Charlier Daniel , Wang Haifeng , Gigot Daniel , Savchenko Alexey , Hallet Jean‐Noël , Glansdorff Nicolas , Sakanyan Vehary
Published: November 2003

We report here the cloning of the arginine repressor gene argR of Bacillus stearothermophilus and the characterization and purification to homogeneity of its product. The deduced amino acid sequence of the 16.8‐kDa ArgR subunit shares 72% identity with its mesophilic homologue AhrC of Bacilus subtilis. Sequence analysis of B. stearothermophilus ArgR and comparisons with mesophilic arginine repressors suggest that the thermostable repressor comprises an N‐terminal DNA‐binding and a C‐terminal oligomerization and arginine‐binding region. B. stearothermophilus ArgR has been overexpressed in E. coli and purified as a 48.0‐kDa trimeric protein. The repressor inhibits the expression of a B. stearothermophilus argC–lacZ fusion in E. coli cells. In the presence of arginine, the purified protein binds tightly and specifically to the argC operator, which largely overlaps the argC promoter. The purified B. stearothermophilus repressor proved to be very thermostable with a half‐life of approximately 30 min at 90°C, whereas B. subtilis AhrC was largely inactivated at 65°C. Moreover, ArgR operator complexes were found to be remarkably thermostable and could be formed efficiently at up to 85°C, well above the optimal growth temperature of the moderate thermophile B. stearothermophilus. This pronounced resistance of the repressor–operator complexes to heat treatment suggests that the same type of regulatory mechanism could operate in extreme thermophiles.
Identification of a maintenance system on rolling circle replicating plasmid pVT736‐1
Authors: Galli Dominique M. , LeBlanc Donald J.
Published: November 2003

Distribution of plasmid molecules to the two daughter cells at cell division is of major importance for their stable inheritance. Several mechanisms that control equipartitioning of low‐copy‐number plasmids have been described in molecular terms. However, no homologous or analogous systems have been identified for intermediate or high‐copy‐number plasmids, including rolling circle replicating (RCR) plasmids. It has been suggested that distribution of such plasmids at cell division relies solely on random segregation. Plasmid pVT736‐1 is a 2 kb RCR plasmid that was isolated from the Gram‐negative capnophilic coccobacillus Actinobacillus actinomycetemcomitans. The plasmid contains a DNA region of approximately 0.8 kb that is associated with its segregational stability. An operon that consists of two genes (orf3 and orf2) is followed by a putative cis‐acting site that contains an integration host factor (IHF) binding site, flanked by several repeats. Mutations in orf2 resulted in plasmid instability. In addition, this DNA region was able to stabilize partially a heterologous replicon, p15A. Homologues or analogues of the pVT736‐1 stabilization system have been detected on numerous plasmid and bacterial genomes.
The dipeptide repeat region of the fibrinogen‐binding protein (clumping factor) is required for functional expression of the fibrinogen‐binding domain on the Staphylococcus aureus cell surface
Authors: Hartford Orla , Francois Patrice , Vaudaux Pierre , Foster Timothy. J.
Published: November 2003

Clumping factor of Staphylococcus aureus is a fibrinogen‐binding protein that is located on the bacterial cell surface. The protein has an unusual repeat domain (region R) comprising mainly the dipeptide aspartate and serine. To determine if region R has a role in the surface display of the fibrinogen‐binding region A domain, deletions lacking the region R encoding region of the clfA gene were generated. To determine the minimum length of region R required for wild‐type levels of ClfA expression, variants with truncated region R domains were constructed. S. aureus cells expressing mutated clfA genes were tested for (i) proteins released by lysostaphin treatment that reacted with antisera specific for region A, (ii) clumping in soluble fibrinogen, (iii) adherence to immobilized fibrinogen and (iv) expression of the ClfA antigen on the cell surface by fluorescent activated cell sorting analysis. Each construct expressed three major immunoreactive proteins, two of which were putative N‐terminal degradation products. Region R residues greater than 40 were required between region A and W (72 residues between region A and the LPDTG sorting signal) for wild‐type levels of clumping in fibrinogen. A stepwise decrease in clumping titre was observed as the distance between region A and LPDTG was decreased from 72 to 4 residues. Similarly, a decrease in binding of anti‐ClfA serum and in binding to fibrinogen‐coated plastic surfaces was observed with cells expressing ClfA with 40 region R residues or less. Nevertheless, low levels of adherence to fibrinogen and binding to anti‐ClfA serum occurred with ClfA derivatives that lacked region R altogether. This indicates that a small proportion of the ClfA molecules are linked to peptidoglycan very close to the cell surface but that residues greater than 72 are needed to allow sufficient ClfA molecules to span the entire cell wall and to display the biologically active A domain in a form that can participate fully in fibrinogen binding.
The XcpR protein of Pseudomonas aeruginosa dimerizes via its N‐terminus
Authors: Turner Leah R. , Olson John W. , Lory Stephen
Published: November 2003

Extracellular protein secretion by the main terminal branch of the general secretory pathway in Pseudomonas aeruginosa requires a secretion machinery comprising the products of at least 12 genes. One of the components of this machinery, the XcpR protein, belongs to a large family of related proteins distinguished by the presence of a highly conserved nucleotide binding domain (Walker box A). The XcpR protein is essential for the process of extracellular secretion and amino acid substitutions within the Walker A sequence result in inactive XcpR. The same mutations exert a dominant negative effect on protein secretion when expressed in wild‐type bacteria. Transdominance of XcpR mutants suggests that this protein is involved in interactions with other components of the secretion machinery or that it functions as a multimer. In this study, the amino‐terminal portion of the cI repressor protein of phage λ was used as a reporter of dimerization in Escherichia coli following fusion to full‐length as well as a truncated form of XcpR. The cI–XcpR hybrid proteins were able to dimerize, as demonstrated by the immunity of bacteria expressing them to killing by λ phage. The full‐length XcpR as well as several deletion mutants of XcpR were able to disrupt the dimerization of the chimeric cI–XcpR protein. The disruption of cI–XcpR dimers using the deletion mutants of XcpR, combined with the analysis of their dominant negative effects on protein secretion, was used to map the minimal dimerization domain of XcpR, which is located within an 85 amino acid region in its N‐terminal domain. Taken together, the data presented in this paper suggest that the XcpR protein dimerizes via its N‐terminus and that this dimerization is essential for extracellular protein secretion.
Elevated expression of stress response genes resulting from deletion of the PHO85 gene
Authors: Timblin Barbara K. , Bergman Lawrence W.
Published: November 2003

The cyclin‐dependent protein kinase Pho85 is a known negative regulatory factor for two stress response genes, PHO5 and GSY2, which encode the inducible form of acid phosphatase and glycogen synthase, respectively, in the yeast Saccharomyces cerevisiae. Cells carrying a disruption of the PHO85 gene inappropriately express both PHO5 and GSY2, resulting in the increase in phosphate scavenging and hyperaccumulation of glycogen in nutrient‐rich conditions. Constitutive activation of PKA in a pho85 mutant suppresses the hyperaccumulation of glycogen. This work presents data to show that, at least in part, the suppression of glycogen biosynthesis upon activation of PKA in a pho85 mutant results from the suppression of GSY2 expression. In addition to GSY2, disruption of the PHO85 gene inappropriately triggers the derepression of two other stress response genes, HSP12 and UBI4. At least in the case of GSY2, regulation of transcription by Pho85 is not through the stress‐responsive cis‐promoter elements (STRE). Furthermore, Pho85 may associate with the known cyclin Pho80 in the transcriptional regulation of these genes.
Lower postprandial gastric volume response in diabetic patients with vagal neuropathy
Authors: Undeland K. A. , Hausken T. , Aanderud S. , Berstad A.
Published: November 2003

Poor relaxation in the stomach after a meal may contribute to disturbed gastric emptying and abdominal discomfort in patients with diabetes mellitus. In this study we aimed to compare barostat‐recorded postprandial volume responses in these patients to those in healthy controls, and to study the relationship between the proximal volume responses, antral filling and vagal neuropathy.

We compared 14 consecutively recruited patients with type 1 diabetes mellitus (DM) to 18 healthy controls (HC) with respect to meal‐induced gastric volume response assessed by a barostat, antral area recorded by ultrasound, and vagal tone assessed by respiratory sinus arrhythmia (RSA).

Meal‐induced volume repsponse of the proximal stomach (area under time–volume curve 0–30 min) was significantly (P = 0.04) lower in DM than in HC, 49.4 min.mL ± 60.7 vs. 114.9 min.mL ± 100.8. Antral area was significantly larger in DM than in HC, both fasting (4.3 cm2 ± 1.9 vs. 3.0 cm2 ± 0.9) and 10 min after ingestion of meat soup (11.8 cm2 ± 3.4 vs. 8.8 cm2 ± 2.9), P = 0.03 and P = 0.02, respectively. Vagal tone was significantly (P = 0.01) lower in DM than in HC, 3.7 beats min−1 ± 2.3 vs. 6.1 beats min−1 ± 2.2. No significant correlation was observed between the proximal volume responses and antral widening. Maximal gastric volume response correlated significantly with vagal tone (r = 0.77, P = 0.002). Conclusions: patients with diabetes mellitus type 1 have impaired meal‐induced volume response, possibly as a consequence of reduced vagal tone.
Pyloric motor response to central and peripheral nitric oxide in the ferret
Authors: Lingenfelser Th. , Blackshaw L. A. , Sun W. M. , Dent J.
Published: November 2003

This study has investigated the relative importance of central nervous and peripheral nitroxidergic mechanisms in the control of pyloric motility. In 10 urethane‐anaesthetized ferrets, drugs were administered directly to the CNS via a 0.5‐mm‐diameter cannula inserted into the 4th ventricle, approximately at the obex. Drugs were also given directly to the upper GI tract by close intra‐arterial (i.a.) injection at the coeliac axis. Antropyloroduodenal pressures were recorded with a five‐channel sleeve/sidehole micromanometric assembly (1.35 × 1.75 mm o.d.), which was introduced via the duodenum. Pyloric motility was stimulated throughout the main part of each study with a continuous i.v. infusion of CCK‐8 (30 pmol min−1). This infusion produced an immediate and sustained increase in tonic and phasic pyloric activity, and sustained abolition of antral pressure waves. CCK‐8 also induced a duodenal motor response, but this was short‐lived (11.4 ± 7.9 min). Coeliac axis injection of the NO donor S‐nitroso‐N‐acetyl‐penicillamine (SNAP) decreased phasic pyloric activity (from 330 ± 35 to 148 ± 21 mmHg min−1 after SNAP 5 μg, P < 0.01). By comparison, central SNAP administration over the same dose range had no effect on CCK‐stimulated pyloric motility. Inhibition of endogenous NO synthase with L‐Nitro Arginine Methyl Ester (L‐NAME, 100 mg kg−1 close i.a.) caused a marked increase of phasic pyloric motor activity from 349 ± 59 to 1044 ± 140 mmHg min−1 (P < 0.01). In addition, SNAP caused marked stimulation of pyloric tone from 2.6 ± 0.5 to 13.1 ± 2.8 mmHg (P < 0.01). Central nervous administration of L‐NAME caused modest enhancement of phasic pyloric activity (248 ± 31 to 283 ± 32 mmHg min−1 P < 0.05) and pyloric tone (2.6 ± 0.5 to 3.7 ± 0.7 mmHg, P < 0.05). Our data indicate that motor activity of the ferret pylorus is potently modulated by NO released within the upper gut. Additionally, there is potential for modulation of pyloric motility by central nervous system production of NO.
Antibody response of Echinococcus granulosus infected mice: recognition of glucidic and peptidic epitopes and lack of avidity maturation
Authors: Ferragut Gabriela , Nieto Alberto
Published: November 2003

The antibody response was followed weekly during 68 weeks in 17 Balb/c mice intraperitoneally (i.p.) infected with 2000 Echinococcus granulosus protoscoleces (PSC) and in three mice i.p. immunized with 2000 dead PSC. Antibodies against hydatid cyst fluid (HCFA) and its peptidic (periodate‐resistant) and carbohydrate (periodate‐sensitive) epitopes were titrated by ELISA. Avidity and the antigen recognition pattern of antibodies were also analysed during infection and immunization by ELISA and immunoblot, respectively.

The antibody response of infected mice showed quantitative and qualitative variations during infection, since both titre as well as recognition of peptide and carbohydrate epitopes in HCFA depended on time post infection. No avidity maturation was evident during the course of infection. Sera from infected mice recognized the 38 kDa subunit of Ag5 but did not react with the 8 kDa subunit of AgB. On the contrary, the antibody response of immunized mice showed only one peak of antibodies that recognized both peptidic and carbohydrate epitopes of HCFA. In addition, sera from these mice recognized mainly 60 and 110 kDa bands. Our results suggest that: a) avidity and antigen recognition patterns of antibodies in mice treated with live PSC are different from those treated with dead PSC; b) antibodies against HCFA glucidic or peptidic epitopes appear at different times post infection.
Differential antibody recognition of FC27‐like Plasmodium falciparum merozoite surface protein MSP2 antigens which lack 12 amino acid repeats
Authors: Ranford‐Cartwright Lisa C. , Taylor Rachel R. , Asgari‐Jirhandeh Nima , Smith Donald B , Roberts Paul E. , Robinson V.Jane , Babiker Hamza A. , Riley Eleanor M. , Walliker David , McBride Jana S.
Published: November 2003

Three alleles of the FC27‐type allelic family of the MSP2 gene of the malaria parasite Plasmodium falciparum have been sequenced from parasites from the field (The Gambia and Tanzania). These alleles lack the 12 amino acid repeat units which are usual in this family of MSP2 alleles. We have investigated the recognition by sera from an endemic area (The Gambia) of three recombinant MSP2 proteins that have 5, 1 and no copies of this repeat region. Antibody recognition of these recombinant proteins varied according to the number of repeats present. High titre antibody levels were seen with most sera using the recombinant protein with 5 × 12‐mer repeats, whereas only low responses were measured using proteins containing 1 or no 12‐mer repeats. Several sera entirely failed to recognise the protein which lacked 12‐mer repeats. The data suggest that variation in the number of tandem repeat sequences could allow the parasite to avoid high avidity antibody binding and this may allow escape from immune recognition.
Cross‐reactive idiotypes on rabbit anti‐SEA antibodies stimulate anti‐idiotype spleen and lymph node cell responses of mice infected with Schistosoma mansoni
Authors: AMANO T. , NAKAZAWA M. , OSHIMA T. , BOSSHARDT S.C. , COLLEY D.G.
Published: November 2003

Antibodies to Schistosoma mansoni soluble egg antigens (SEA) generated in outbred rabbits from two different sources were used to study cross‐reactive idiotype/anti‐idiotype (Id/anti‐Id) interactions in S. mansoni‐infected mice in two different locations. Immunoaffinity purified rabbit polyclonal anti‐SEA antibody preparations (RabId) were predominantly Ig by SDS‐PAGE and demonstrated anti‐SEA activity by ELISA and Western blot. RabId prepared from three separate rabbits and used at 40 μg/ml in vitro stimulated lymphocyte proliferative responses of spleen cells from mice with eight week infections (Mean ± SEM [E–C]) of 3869 ± 764, 18 594 ± 3046 and 8962 ± 1734  cpm. Spleen cells from uninfected mice exposed to the same RabId preparations in vitro had respective [E–C] values of 206 ± 144, 494 ± 154 and 363 ± 180. Lymph node cells from mice infected for 8 weeks demonstrated [E–C] of 123 ± 400, 5073 ± 828 and 2361 ± 656 upon exposure to these 3 RabId preparations. Lymph node cells from uninfected mice had [E–C] of 220 ± 76, 194 ± 82 and 143 ± 72 when exposed to these RabId. Maximum in vitro proliferative response to Ig from unimmunized rabbits was 761 ± 400 by spleen cells from mice with eight week infections. These data indicate the presence of cross‐reactive Id on rabbit anti‐SEA antibodies from different rabbits that can stimulate in vitro lymphoproliferative responses of spleen or lymph node cells from S. mansoni‐infected mice.
Paramyosin: a candidate vaccine antigen against Schistosoma japonicum
Authors: RAMIREZ BERNADETTE L. , KURTIS JONATHAN D. , WIEST PETER M. , ARIAS PERCIVAL , ALIGUI FE , ACOSTA LUZ , PETERS PIERRE , OLDS G.RICHARD
Published: November 2003

Paramyosin, a 97 kDa myofibrillar protein, is a candidate vaccine antigen for prevention of infection with the human parasite Schistosoma mansoni. To determine if paramyosin would also induce protection against Schistosoma japonicum, paramyosin was biochemically purified from S. japonicum adult worms. SDS‐PAGE demonstrated a single protein with a molecular weight of 97 kDa. In four separate experiments, vaccination of mice with S. japonicum paramyosin without adjuvant induced significant resistance (62%–86%, P < 0.001) against cercarial challenge as compared to controls. These data suggest that S. japonicum paramyosin may represent a candidate vaccine for immunization against schistosomiasis japonica.
Long term survival of ‘damaged’Nippostrongylus brasiliensis adult worms in the testosterone‐treated Indian soft‐furred rat, Millardia meltada
Authors: TIURIA RISA , KANEYUKI TAKAKO , HORII Y , MAKIMURA S , TSUCHIYA K , NAWA Y
Published: November 2003

When testosterone‐treated female Millardia meltada were infected with Nippostrongylus brasiliensis, adult worms persisted for over seven weeks. The kinetics of faecal egg counts showed a biphasic pattern having a transient decline at around two weeks post infection (p.i.). Thus the status of  N. brasiliensis adult worms surviving in the small intestines of testosterone‐treated M. meltada was examined. The fecundity and maturity of eggs in the uteri of female adult worms were examined at one, two, three and seven weeks p.i. Both the fecundity and maturity of eggs transiently decreased at two and three weeks p.i. and then completely recovered by seven weeks. Adoptive transfer of  N. brasiliensis adult worms into naive recipients can discriminate the status of worms. Those obtained from the stable phase of a primary infection (‘normal’ worm) can establish and survive in the recipients, whereas those obtained at the time of expulsion (‘damaged’ worm) are rapidly expelled. Therefore, 300 each of N. brasiliensis adult worms collected from the testosterone‐treated female M. meltada at one, two and seven weeks p.i. were transferred intraduodenally into normal rats to determine their status. Those collected at one week p.i. persisted for eight days, indicating that they were still ‘normal’. In contrast, worms collected at two and seven weeks p.i. were expelled within four days, indicating that they had already been ‘damaged’. Moreover, when the ‘damaged’ worms obtained from rats were intraduodenally transferred into testosterone‐treated female M. meltada, they were not expelled, suggesting that testosterone‐treatment affected the final expulsive step, but not the damaging process, of the mucosal defence of M. meltada against N. brasiliensis adult worms.