bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phylogeography and postglacial dispersal of arctic charr Salvelinus alpinus in North America
Authors: WILSON C. C. , HEBERT P. D. N. , REIST J. D. , DEMPSON J. B. 
Published: November 2003

AbstractRestriction fragment length polymorphism analysis of mitochondrial DNA (mtDNA) was used to reconstruct postglacial dispersal routes of arctic charr Salvelinus alpinus in North America. Twelve of 35 restriction enzymes detected polymorphisms among representative populations, revealing two distinct lineages with an estimated nucleotide divergence of 1.32%. Subsequent screening of 869 fish from 54 populations with four diagnostic restriction enzymes showed that these lineages have largely allopatric distributions, suggesting their dispersal from separate northern and eastern glacial refugia. In addition, geographical and genetic structure among eastern populations suggested the existence of a second eastern refuge. Among the three lineages, the most divergent (Arctic) lineage occurred from Alaska east to northern Labrador. Quebec, New Brunswick, and New England were colonized by a second (Laurentian) lineage, and Labrador by a third group. Contact between refugial groups was only detected in two Labrador populations. The Arctic lineage was highly differentiated from eastern North American and European haplotypes, and probably diverged during the early Pleistocene. By contrast, the Laurentian and Labrador groups were similar to Old World charr, suggesting a shared ancestry during the mid‐Pleistocene. In addition, the close relationship between Labrador and Laurentian charr indicates their probable divergence during the Wisconsinan glaciation.
Isolation and characterization of microsatellite loci in Swietenia humilis (Meliaceae): an endangered tropical hardwood species
Authors: WHITE G. , POWELL W. 
Published: November 2003

AbstractMicrosatellites are now firmly established as an informative marker system, with increasing popularity as a tool amongst molecular ecologists. We have developed a method of constructing an enriched microsatellite library for the tropical tree species Swietenia humilis Zucc. (Meliaceae). This method is based on a precloning enrichment of SSRs using synthetic oligonucleotide probes, bound to magnetic beads and hybridizing to complementary microsatellite core sequences in digested genomic DNA. Here we describe the isolation and characterization of 10 microsatellite loci that have been used to survey the genetic diversity within a natural population of S. humilis. A total of 97 alleles were identified with an average of 9.7 alleles over all loci. Very high levels of allelic polymorphism were detected at individual loci, with 23 alleles observed at the most variable. The mean observed heterozygosity was 0.415 (range 0.038‐0.815) exceeding levels of diversity detected in related species which used isozymes as the marker system. Subpopulation differentiation at a microgeographical scale was low (FST= 0.036) and the values of Nm, calculated from the allelic frequencies, were greater than 1 thus reflecting the extent of gene flow occurring between individual trees.
The ‘petite‐negative’ yeast Kluyveromyces lactis has a single gene expressing pyruvate decarboxylase activity
Authors: Bianchi Michele M. , Tizzani Lorenza , Destruelle Monika , Frontali Laura , Wésolowski‐Louvel Micheline 
Published: November 2003

SummaryWe cloned and sequenced the pyruvate decarboxylase (PDC; EC 4.1.1.1) structural gene KIPDCA in the yeast Kluyveromyces lactis and found it to be allelic to the previously isolated rag6 mutation. The putative amino acid sequence of the KIPdcAp appeared to be highly homologous to those of the yeast Pdc proteins identified so far. The disruption of KIPDCA indicated that it is the only PDC structural gene in K. lactis, as evidenced by the lack of PDC activity and ethanol production in the pdcAΔ strains and by the absence of growth on glucose in the presence of respiratory inhibitors. It was observed that expression of the KIPDCA gene is induced by glucose at the transcriptional level. Transcription of the gene was reduced in the rag1, rag2, rag5 and rag8 mutants, which are defective for the low‐affinity glucose permease, phosphoglucose isomerase, hexokinase, and a positive regulator of RAG1 expression, respectively.
Amino‐terminal maturation of the Bordetella pertussis filamentous haemagglutinin
Authors: Jacob‐Dubuisson Françcoise , Buisine Corinne , Mielcarek Nathalie , Clément Eve , Menozzi Franco D. , Locht Camille 
Published: November 2003

SummaryThe 220 kDa filamentous haemagglutinin (FHA) is a major adhesin of Bordetella pertussis and is produced from a large precursor designated FhaB. Although partly surface associated, it is also very efficiently secreted into the extracellular milieu. Its secretion depends on the outer membrane accessory protein FhaC. An 80 kDa N‐terminal derivative of FHA, named Fha44, can also be very efficiently secreted in a FhaC‐dependent manner, indicating that all necessary secre tion signals are localized in the N‐terminal region of FhaB. A comparison of predicted and apparent sizes of FHA derivatives, in addition to immunoblot analyses of cell‐associated and secreted FHA polypeptides, indicated that FhaB undergoes N‐terminal maturation by the cleavage of an 8–9 kDa segment. However, phenotypic analyses of translational lacZ and phoA fusions showed that this segment does not function as a typical signal peptide. Co‐expression of the Fha44‐encoding gene with fhaC also did not allow for secretion of Fha44 in Escherichia coli. High levels of secretion could, however, be observed when the OmpA signal peptide was fused to the N‐terminal end of Fha44. Regardless of the OmpA signal peptide‐Fha44 fusion point, the E. coli‐secreted Fha44 had the same Mr as that secreted by B. pertussis, indicating that the N‐terminal proteolytic maturation does not require a B. perfussis‐specific factor. Similar to FHA, the B. pertussis‐secreted Fha44 contains an as yet uncharacterized modification at its N‐terminus. This modification did not occur in E. coli and is therefore not required for secretion. The N‐terminus of Fha44 secreted by E. coli was determined and found to correspond to the 72nd residue after the first in‐frame methionine of FhaB. The N‐terminal modification was also found not to be required for haemagglutination or interaction with sulphated glycoconjugates.
The identification, cloning and mutagenesis of a genetic locus required for lipopolysaccharide biosynthesis in Bordetella pertussis
Authors: Allen Andrew , Maskell Duncan 
Published: November 2003

Summary Bordetella pertussis lipopolysaccharide (LPS) is biologically active, being both toxic and immunogenic. Using transposon mutagenesis we have identified a genetic locus required for the biosynthesis of LPS in B. pertussis, which has been cloned and sequenced. We have also identified equivalent loci in Bordetella bronchiseptica and Bordetella parapertussis and cloned part of it from B. parapertussis. The amino acid sequences derived from most of the genes present in the sequenced B. pertussis locus are similar to proteins required for the biosynthesis of LPS and other complex polysaccharides from a variety of bacteria. The genes are in a unique arrangement in the locus. Several of the genes identified are similar to genes previously shown to play specific roles in LPS O‐antigen biosynthesis. In particular, the amino acid sequence derived from one of the genes is similar to the enzyme encoded by rfbP from Salmonella enterica, which catalyses the transfer of galactose to the undecaprenol phosphate antigen carrier lipid as the first step in building oligosaccharide O‐antigen units, which are subsequently assembled to form polymerized O‐antigen structures. Defined mutation of this gene in the B. pertussis chromosome results in the inability to express band A LPS, possibly suggesting that the trisaccharide comprising band A is a single O‐antigen‐like structure and that B. pertussis LPS is similar to semi‐rough LPS seen in some mutants of enteric bacteria.
Physical mapping and expression of gene families encoding the N‐acetyl d‐galactosamine adherence lectin of Entamoeba histolytica
Authors: Ramakrishnan Girija , Ragland Brian D. , Purdy Jay E. , Mann Barbara J. 
Published: November 2003

SummaryAdherence of the enteric protozoan parasite Entamoeba histolytica is mediated by an N‐acetyl d‐galactosamine (GaINAc)‐specific lectin, a heterodimer of heavy (170 kDa) and light (35/31 kDa) subunits. The gene families encoding the lectin subunits were characterized using clamped homogeneous electric field (CHEF) gel electrophoresis in the strain HM1:IMSS. The heavy sub‐unit was shown to be encoded by a family of five hgl genes, which were physically mapped to five distinct Hindlll restriction fragments. The light subunit was shown to be encoded by a family of lgl genes located at six loci in the genome. Heavy and light subunit genes did not appear to be linked. Partial sequences of new members of the hgl and lgl gene families were obtained. Several different strains of E histolytica were found to contain multiple hgl loci in their genomes. Expression of hgl and lgl genes in HM1: IMSS trophozoites was examined under different growth conditions using the reverse transcription‐polymerase chain reaction (RT‐PCR). mRNA transcripts were detected from three hgl genes and three lgl genes, with no significant differences between cultured amoebae and amoebae from liver abscesses. The complexity of GaINAc lectin gene expression observed suggests distinct biological functions for the products of the individual genes during pathogenesis.
The specificity of fumarate as a switching factor of the bacterial flagellar motor
Authors: Barak Rina , Giebel Igal , Eisenbach Michael 
Published: November 2003

SummaryFumarate restores to flagella of cytoplasm‐free, CheY‐ containing envelopes of Escherichia coli and Salmonella typhimurium the ability to switch from one direction of rotation to another. To examine the specificity of this effect, we studied flagellar rotation of envelopes which contained, instead of fumarate, one of its analogues. Malate, maleate and succinate promoted switching, but to a lesser extent than fumarate. These observations were made both with wild‐type envelopes and with envelopes of a mutant which lacks the enzymes succinate dehydrogenase and fumarase, indicating that the switching‐promoting activity of the analogues was not caused by their conversion to fumarate. Aspartate and lactate did not promote switching. Using strains defective in specific enzymes of the tricarboxylic acid cycle and lacking the cytoplasmic chemotaxis proteins as well as some of the chemo‐taxis receptors, we demonstrated that, in intact bacteria, unlike the situation in envelopes, fumarate promoted clockwise rotation via its metabolites acetyl phosphate and acetyladenylate, but did not promote switching (presumably because of the presence of cytoplasmic fumarate). All of the results are consistent with the notion that fumarate acts as a switching factor, presumably by lowering the activation energy of switching. Thus fumarate and some of its metabolites may serve as a connection point between the bacterial metabolic state and chemotactic behaviour.
Cross‐talk to the genes for Bacillus anthracis capsule synthesis by atxA, the gene encoding the frans‐activator of anthrax toxin synthesis
Authors: Uchida Ikuo , Makino Sou‐ichi , Sekizaki Tsutomu , Terakado Nobuyuki 
Published: November 2003

SummaryThe two major virulence factors of Bacillus anthracis are the tripartite toxin and the polyglutamate capsule, which are encoded by genes on the large plasmids, pX01 and pX02, respectively. The genes atxA, located on pX01, and acpA, located on pX02, encode positive frans‐acting proteins that are involved in bicarbonate‐mediated regulation of toxin and capsule production, respectively. A derivative strain cured of pX01 produced less capsular substance than the parent strain harbouring both pX01 and pX02, and electroporation of the strain cured of pX01 with a plasmid containing the cloned atxA gene resulted in an increased level of capsule production. An acpA‐null mutant was complemented by not only acpA but also the atxA gene. The cap region, which is essential for encapsulation, contains three genes capB, capC, and cap A, arranged in that order. The atxA gene stimulated capsule synthesis from the cloned cap region. Transcriptional analysis of cap by RNA slot‐blot hybridization and primer‐extension analysis revealed that atxA activated expression of cap in trans at the transcriptional level. These results indicate that cross‐talk occurs, in which the pX01‐located gene, atxA, activates transcription of the cap region genes located on pX02. We identified two major apparent transcriptional start sites, designated P1 and P2, located at positions 731 bp and 625 bp, respectively, upstream of the translation‐initiation codon of capB. Transcription initiated from P1 and P2 was activated by both atxA and acpA, and activation appeared to be stimulated by bicarbonate. Deletion analysis of the upstream region of the cap
Pathogenicity islands of virulent bacteria: structure, function and impact on microbial evolution
Authors: Hacker J. , Blum‐Oehler G. , Mühldorfer I. , Tschäpe H. 
Published: November 2003

SummaryVirulence genes of pathogenic bacteria, which code for toxins, adhesins, invasins or other virulence factors, may be located on transmissible genetic elements such as transposons, plasmids or bacteriophages. In addition, such genes may be part of particular regions on the bacterial chromosome, termed‘pathogenicity islands’(Pais). Pathogenicity islands are found in Gram‐negative as well as in Gram‐positive bacteria. They are present in the genome of pathogenic strains of a given species but absent or only rarely present in those of non‐pathogenic variants of the same or related species. They comprise large DNA regions (up to 200 kb of DNA) and often carry more than one virulence gene, the G+C contents of which often differ from those of the remaining bacterial genome. In most cases, Pais are flanked by specific DNA sequences, such as direct repeats or insertion sequence (IS) elements. In addition, Pais of certain bacteria (e.g. uropathogenic Escherichia coli, Yersinia spp., Helicobacter pylori) have the tendency to delete with high frequencies or may undergo duplications and amplifications. Pais are often associated with tRNA loci, which may represent target sites for the chromosomal integration of these elements. Bacteriophage attachment sites and cryptic genes on Pais, which are homologous to phage integrase genes, plasmid origins of replication or IS elements, indicate that these particular genetic elements were previously able to spread among bacterial populations by horizontal gene transfer, a process known to contribute to microbial evolution.
Double‐glycine‐type leader peptides direct secretion of bacteriocins by ABC transporters: colicin V secretion in Lactococcus lactis
Authors: van Belkum Marco J. , Worobo Randy W. , Stiles Michael E. 
Published: November 2003

SummaryMany non‐lantibiotic bacteriocins of lactic acid bacteria are produced as precursors which have N‐terminal leader peptides that share similarities in amino acid sequence and contain a conserved processing site of two glycine residues in positions ‐1 and ‐2. A dedicated ATP‐binding cassette (ABC) transporter is responsible for the proteolytic cleavage of the leader peptides and subsequent translocation of the bacteriocins across the cytoplasmic membrane. To investigate the role that these leader peptides play in the recognition of the precursor by the ABC transporters, the leader peptides of leucocin A, lactococcin A or colicin V were fused to divergicin A, a bacteriocin from Carnobacterlum divergens that is secreted via the cell's general secretion pathway. Production of divergicin was monitored when these fusion constructs were introduced into Leuconostoc gelidum, Lactococcus lactis and Escherichia coli, which carry the secretion apparatus for leucocin A, lactococcins A and B, and colicin V, respectively. The different leader peptides directed the production of divergicin in the homologous hosts. In some cases production of divergicin was also observed when the leader peptides were used in heterologous hosts. For ABC‐transporter‐dependent secretion in E. coli the outer membrane protein TolC was required. Using this strategy, colicin V was produced in L. lactis by fusing this bacteriocin behind the leader peptide of leucocin A.
Mutations in the terminase genes of bacteriophage λ that bypass the necessity for FI
Authors: Murialdo Helios , Tzamtzis Dimitra , Berrú Maribel , Fife Wendy L. , Becker Andrew 
Published: November 2003

DNA maturation in bacteriophage λ is the process by which the concatemeric precursor DNA is cleaved at sites called cos to generate mature λ DNA molecules. These DNA molecules are then packaged into procapsids, the empty capsid precursors. The enzyme that catalyses these events is λ DNA terminase. It is composed of two subunits, made of 181 and 641 amino acids, the products of genes Nu1 and A, respectively. The product of the FI gene (gpFI ) stimulates the formation of an intermediate in capsid assembly called complex II, which contains a procapsid, terminase and DNA. The mechanism of stimulation remains unknown. It has been suggested that gpFI may also stimulate terminase‐mediated cos cleavage, in the absence of procapsids, by increasing enzyme turnover. Mutants in FI fail to mature and package DNA but, in comparison with other capsid gene mutants, FI mutants are leaky. Second site mutants of FI phages, called ‘fin’ (for FI independence), bypass the necessity for gpFI. These mutants were originally localized to the region of Nu1 and A and are of two classes: finA includes those that induce the synthesis of fourfold more gene A product (gpA ) than wild‐type phages, and finB includes those that produce normal amounts of gpA. Whereas all finA mutants analysed map to Nu1, finB mutants have been found both in E and in Nu1. The existence of E mutants able to bypass the necessity for gpFI in vivo shows that gpE and gpFI interact, directly or indirectly.Here we have analysed and sequenced two finA mutants and one finB mutant. All of these map in Nu1. Of the two finA mutants, one corresponds to an Ala163Ser change and the other is a silent mutation. It is likely that the finA mutations alter mRNA conformation in a manner that results in an increase in the efficiency of A mRNA translation. The fourfold increase in gpA synthesis translates into a 10‐fold increase in terminase activity. These results show that terminase overproduction is sufficient to bypass the necessity for gpFI and that such an overproduction can be achieved by changes in the efficiency of translation of A due to subtle changes in the sequence upstream of the gene.The finBcs103 mutation is a His‐87→Tyr change in Nu1. Therefore, an alternative way in which to bypass the requirement for gpFI involves an alteration in the structure of gpNu1. It is likely that the altered gpNu1 would increase cleavage and packaging efficiency directly or indirectly.We have determined that DNA cleavage in vivo does not occur in the absence of gpFI. Therefore it seems that gpFI somehow facilitates an otherwise latent capacity of terminase to autoactivate its nucleolytic activity.
5‐HT receptor types in the rat ileum longitudinal muscle: focus on 5‐HT2 receptors mediating contraction
Authors: Briejer M. R. , Mathis C. , Schuurkes J. A. J. 
Published: November 2003

The 5‐hydroxytryptamine (5‐HT) receptor(s) that mediate(s) contraction of the rat ileum longitudinal muscle was studied.5‐HT and α‐methyl‐5‐HT equipotently induced contractions, whereas 5‐methoxytryptamine and 2‐methyl‐5‐HT (partial agonist) were less potent; this rank order of potency suggests involvement of a 5‐HT2 receptor. Neither tetrodotoxin nor atropine affected the contraction to 5‐HT, suggesting a smooth muscle localization of these 5‐HT2 receptors. The presence of either a selective 5‐HT2B (SB 204741), 5‐HT3 (granisetron) or 5‐HT4 (SB 204070) antagonist, slightly affected the contractions to 5‐HT. Thus, they were also included in the organ bath solution in all subsequent experiments in order to pharmacologically isolate the main contractile component. Using (if possible) 5‐HT2A receptor‐selective concentrations, ketanserin, ritanserin, metergoline, spiperone, mianserin, methiothepin, mesulergine, methysergide and cisapride all inhibited the contractions to 5‐HT, causing a depression of the curve to 5‐HT (i.e. surmountable antagonism was not observed with any of the above agents). Comparison of the affinities of these compounds for the various 5‐HT2 receptor subtypes revealed that the receptor involved in the contractions to 5‐HT most closely resembles the 5‐HT2A receptor. However, cinanserin at a concentration expected to inhibit 5‐HT2A receptor‐mediated effects, failed to affect the contractions to 5‐HT.It is thus concluded that on the longitudinal smooth muscle of the rat ileum, at least a part of the contraction to 5‐HT is mediated by 5‐HT receptors resembling the 5‐HT2A receptor subtype.
Phytochrome chromophore‐deficient mutants
Authors: TERRY M. J. 
Published: November 2003

ABSTRACTPhytochrome chromophore‐deficient mutants have been used as phytochrome‐deficient plants to study many aspects of plant development. However, there are still a number of important questions to be resolved concerning both the targets and the phenotypic consequences of these mutations. Recently, progress has been made in our understanding of the molecular basis of the chromophore deficiency in these mutants. Biochemical assays for the committed steps of chromophore synthesis have been developed and used to demonstrate that the pcd1 and yellow‐green‐2 mutants of pea and tomato, respectively, are unable to synthesize biliverdin IXα from heme while pcd2 and aurea are deficient in phytochromobilin synthase activity. This review focuses on how this information can be used to help understand the basis of other chromophore‐deficient mutants, such as the hy1 and hy2 mutants of Arabidopsis, and discusses how the phenotype of chromophore‐deficient mutants is related to lesions in the chromophore biosynthesis pathway.
Chromophore: apoprotein interactions in phytochrome A*
Authors: SONG P.‐S. , PARK M. H. , FURUYA M. 
Published: November 2003

ABSTRACTPhytochromes are molecular light switches by virtue of their photochromic red/far‐red reversibility. The His‐324 residue next to the chromophore‐linked Cys‐323 plays a critical role in conferring photochromism to the tetrapyrrole chromophore in native phytochrome A. The chromophore appears to be enclosed between the amphiphilic α‐helical chains in a hydrophobic pocket. The absorbance maxima of both the Pr and the Pfr forms of pea phytochrome A are blue‐shifted by 10 and 20 nm, respectively, upon C‐terminal truncation. We speculate that the quaternary structure of the phytochrome A molecule involves some interactions of the C‐terminal half with the chromophore domain. The Pfr conformation of phytochrome includes an amphiphilic α‐helix of the amino terminal chain, which occurs in 113 ms after picosecond photoisomerization of the Pr form. Compared to α‐helical folding, unfolding of the α‐helix occurs faster in about 310 μs upon phototransformation of the Pfr form of phytochrome A. The photochromic transformation of phytochrome A modulates protein kinase‐catalysed phosphorylation sites in vivo and in vitro, but only a subtle local change in conformation is detectable in the phosphorylated phytochromes. This suggests that the post‐translational modification serves as a surface label, rather than a transducer‐activating trigger, for the recognition of a putative phytochrome receptor.