bluuebunny/crossref_metadata_2025_split huggingface dataset:
American marten (Martes americana) in the Pacific Northwest: population differentiation across a landscape fragmented in time and space
Authors: Small Maureen P. , Stone Karen D. , Cook Joseph A. 
Published: March 2003

AbstractAmerican marten (Martes americana) have a close association with mature temperate forests, a habitat that expanded throughout the Pacific Northwest as glaciers receded at the end of the Pleistocene. Similar to several other forest‐associated mammals in North America (e.g. black bear), genetic analysis of the marten shows a deep phylogeographical subdivision that reflects populations with distinctive evolutionary histories. Using a suite of 14 microsatellite markers, we explored the genetic structure of marten populations in two reciprocally monophyletic clades in the Pacific Northwest identified previously as M. caurina and M. americana by mitochondrial haplotypes and morphology. Microsatellite phylogeographical patterns were congruent with mitochondrial analyses. These independent data sets shed light upon hybridization patterns, population structure and evolutionary histories. Hybridization between M. caurina and M. americana individuals was documented in two regions of sympatry (Kuiu Island in southeastern Alaska and southern Montana). Northern insular populations of M. caurina exhibited higher differentiation and lower variability relative to northern populations of M. americana. Greater divergence among M. caurina populations may reflect longer isolation and persistence in coastal forest habitat that was fragmented by rising sea level in the early Holocene. Lower differentiation among northern M. americana populations and close relationship to other continental M. americana populations may reflect more recent expansion into the Pacific Northwest and/or continued gene flow among populations. Differentiation among M. caurina populations was attributed to habitat fragmentation (i.e. rising sea level), as opposed to isolation‐by‐distance; oceanic straits pose significant barriers to gene flow among M. caurina populations and between populations of M. caurina and M. americana.
Nuclear DNA identification of migrating bull trout captured at the Puget Sound Energy diversion dam on the White River, Washington State
Authors: Baker J. D. , Moran P. , Ladley R. 
Published: March 2003

AbstractBull trout (Salvelinus confluentus) is a char listed as threatened under the United States Endangered Species Act throughout its range in the coterminous United States. Substantial morphological similarities between bull trout and Dolly Varden (S. malma) make field identification difficult. This has resulted in an incomplete understanding of their distribution and abundance in Washington State where these two species occur sympatrically. We used three diagnostic nuclear loci to determine the species of char collected at a trap on the White River in southern Puget Sound (Washington State, USA). Each of the 104 samples revealed the expected bull trout genotype at all three loci. This work presents three principle results: (i) the presence of a migratory bull trout population in southern Puget Sound; (ii) no evidence of migratory Dolly Varden over 3 years; and (iii) no evidence of hybridization was detected. These results also demonstrate how molecular markers can provide information essential to the conservation and management of these species.
HrpB2 and HrpF from Xanthomonas are type III‐secreted proteins and essential for pathogenicity and recognition by the host plant
Authors: Rossier Ombeline , Van den Ackerveken Guido , Bonas Ulla 
Published: March 2003

The interaction between the plant pathogen Xanthomonas campestris pv. vesicatoria and its host plants is controlled by hrp genes (hypersensitive reaction and pathogenicity), which encode a type III protein secretion system. Among type III‐secreted proteins are avirulence proteins, effectors involved in the induction of plant defence reactions. Using non‐polar mutants, we investigated the role of 12 hrp genes in the secretion of the avirulence protein AvrBs3 from X. c. pv. vesicatoria and a heterologous protein, YopE, from Yersinia pseudotuberculosis. Genes conserved among type III secretion systems (hrcQ, hrcR, hrcS and hrcT) as well as non‐conserved genes (hrpB1, hrpB2, hrpB4, hrpB5, hrpD5 and hrpD6) were shown to be required for secretion. Protein localization studies using specific antibodies showed that HrpB1 and HrpB4, as well as the putative ATPase HrcN, were mainly found in the soluble fraction of the bacterial cell. In contrast, HrpB2 and HrpF, which is related to NolX of Rhizobium fredii, are secreted into the culture medium in an hrp‐dependent manner. As HrpB2, but not HrpF, is essential for type III protein secretion, there might be a hierarchy in the secretion process. We propose that HrpF, which is dispensable for protein secretion but required for AvrBs3 recognition in planta, functions as a translocator of effector proteins into the host cell.
Characterization of the A2–A2rel gene cluster in Leishmania donovani: involvement of A2 in visceralization during infection
Authors: Zhang Wen‐Wei , Matlashewski Greg 
Published: March 2003

The A2 gene family is present in Leishmania donovani, which causes fatal visceral leishmaniasis in human patients, but is not present in Leishmania major, which causes cutaneous leishmaniasis infections. The A2 genes in L. donovani are stage specific and are expressed at high levels in the amastigote stage in the mammalian host, but are not expressed in the promastigote stage in the insect sandfly vector. The A2 genes are tandem repeated with a distinct gene family termed the A2rel genes. In order to characterize the structure and function of the A2–A2rel gene clusters, the 5′ and 3′ DNA sequences flanking the A2–A2rel cluster were isolated, sequenced and used to generate mutants through gene targeting. Although it was possible to generate partial A2–A2rel gene clusters knock‐out mutants, it was not possible to delete all the A2–A2rel gene clusters completely from the L. donovani genome, suggesting that, within this cluster, there are genes that are essential for survival in culture. Characterization of these mutants revealed that A2 and A2rel gene expression was compensated by amplifying the remaining intact A2 and A2rel genes, and the proliferation of these mutants in culture and their virulence in BALB/c mice were compromised. In order to explore further the biological role of A2, the L. donovani A2 gene was introduced into L. major. In comparison with the control L. major, the A2‐expressing L. major parasites demonstrated an increased ability to survive in the spleen of BALB/c mice. These data suggest that A2 plays a role in the visceralization of infection associated with L. donovani.
RNase II levels change according to the growth conditions: characterization of gmr, a new Escherichia coli gene involved in the modulation of RNase II
Authors: Cairrão Fátima , Chora Ângelo , Zilhão Rita , Carpousis Agamemnon J. , Arraiano Cecília M. 
Published: March 2003

In Escherichia coli, ribonucleases are effectors that rapidly modulate the levels of mRNAs for adaptation to a changing environment. Factors involved in the regulation of these ribonucleases can be relevant for mRNA stability. RNase II is one of the main ribonucleases responsible for exonucleolytic activity in E. coli extracts. We have identified and characterized a new E. coli gene, which was named gmr (gene modulating RNase II). The results demonstrate that a deletion of gmr can be associated with changes in RNase II levels and activity. Western analysis and exoribonuclease activity assays showed a threefold increase in RNase II in the gmr deletion strain. Gmr does not affect RNase II mRNA, but modulates RNase II at the level of protein stability. RNase II protein turnover is slower in the gmr deletion strain. We also show that RNase II levels change in different media, and that this regulation is abolished in a strain lacking gmr. The data presented here show that the regulation of ribonucleolytic activity can depend on growth conditions, and this regulation can be mediated by factors that are not RNases.
A guided tour: small RNA function in Archaea
Authors: Dennis Patrick P. , Omer Arina , Lowe Todd 
Published: March 2003

In eukaryotes, the C/D box family of small nucleolar (sno)RNAs contain complementary guide regions that are used to direct 2′‐O‐ribose methylation to specific nucleotide positions within rRNA during the early stages of ribosome biogenesis. Direct cDNA cloning and computational genome searches have revealed homologues of C/D box snoRNAs (called sRNAs) in prokaryotic Archaea that grow at high temperature. The guide sequences within the sRNAs indicate that they are used to direct methylation to nucleotides in both rRNAs and tRNAs. The number of sRNA genes that are detectable within currently sequenced genomes correlates with the optimal growth temperature. We suggest that archaeal sRNAs may have two functions: to guide the deposition of methyl groups at the 2′‐O position of ribose, which is an important determinant in RNA structural stability, and to serve as a molecular chaperones to help orchestrate the folding of rRNAs and tRNAs at high temperature.
The AmiE aliphatic amidase and AmiF formamidase of Helicobacter pylori: natural evolution of two enzyme paralogues
Authors: Skouloubris Stéphane , Labigne Agnès , De Reuse Hilde 
Published: March 2003

Aliphatic amidases (EC 3.5.1.4) are enzymes catalysing the hydrolysis of short‐chain amides to produce ammonia and the corresponding organic acid. Such an amidase, AmiE, has been detected previously in Helicobacter pylori. Analysis of the complete H. pylori genome sequence revealed the existence of a duplicated amidase gene that we named amiF. The corresponding AmiF protein is 34% identical to its AmiE paralogue. Because gene duplication is widely considered to be a fundamental process in the acquisition of novel enzymatic functions, we decided to study and compare the functions of the paralogous amidases of H. pylori. AmiE and AmiF proteins were overproduced in Escherichia coli and purified by a two‐step chromatographic procedure. The two H. pylori amidases could be distinguished by different biochemical characteristics such as optimum pH or temperature. AmiE hydrolysed propionamide, acetamide and acrylamide and had no activity with formamide. AmiF presented an unexpected substrate specificity: it only hydrolysed formamide. AmiF is thus the first formamidase (EC 3.5.1.49) related to aliphatic amidases to be described. Cys‐165 in AmiE and Cys‐166 in AmiF were identified as residues essential for catalysis of the corresponding enzymes. H. pylori strains carrying single and double mutations of amiE and amiF were constructed. The substrate specificities of these enzymes were confirmed in H. pylori. Production of AmiE and AmiF proteins is dependent on the activity of other enzymes involved in the nitrogen metabolism of H. pylori (urease and arginase respectively). Our results strongly suggest that (i) the H. pylori paralogous amidases have evolved to achieve enzymatic specialization after ancestral gene duplication; and (ii) the production of these enzymes is regulated to maintain intracellular nitrogen balance in H. pylori.
Failure to farnesylate Rheb protein contributes to the enrichment of G0/G1 phase cells in the Schizosaccharomyces pombe farnesyltransferase mutant
Authors: Yang Wenli , Tabancay Angel P. , Urano Jun , Tamanoi Fuyuhiko 
Published: March 2003

Protein farnesylation is important for a number of physiological processes, including proliferation and cell morphology. The Schizosaccharomyces pombe mutant, cpp1–, defective in farnesylation, exhibits distinct phenotypes, including morphological changes and sensitivity to the arginine analogue, canavanine. In this work, we report a novel phenotype of this mutant, enrichment of G0/G1 phase cells. This phenotype results mainly from the inability to farnesylate the Rheb G‐protein, as normal cell cycle progression can be restored to the mutant by expressing a mutant form of SpRheb (SpRheb‐CVIL) that can bypass farnesylation. In contrast, a farnesylation‐defective mutant of SpRheb (SpRheb‐SVIA) is incapable of restoring the normal cell cycle profile to the cpp1– mutant. Inhibition of SpRheb expression leads to the accumulation of cells at the G0/G1 phase of the cell cycle. This growth arrest phenotype of the sprheb– disruption can be complemented by the introduction of wild‐type sprheb+. The complementation is dependent on farnesylation, as the farnesylation‐defective SpRheb‐SVIA mutant is incapable of complementing the sprheb– disruption. Other mutants of SpRheb, E40K and S20N, are also incapable of complementing the sprheb– disruption. Furthermore, efficient complementation can be obtained by the expression of human Rheb but not Saccharomyces cerevisiae Rheb. Our findings suggest that protein farnesylation is important for cell cycle progression of S. pombe cells and that farnesylated SpRheb is critical in this process.
Mutational analysis of F‐pilin reveals domains for pilus assembly, phage infection and DNA transfer
Authors: Manchak J. , Anthony . G. , Frost Laura S. 
Published: March 2003

SummaryThe F‐pilus has been implicated in recipient cell recognition during the establishment of a stable mating pair before conjugation as well as forming part of the conjugative pore for DNA transfer. The F‐pilus is the site of attachment of the filamentous phages (M13, f1 and fd), which attach to the F‐pilus tip, and the RNA phages, R17 and Qβ,
which attach to different sites exposed on the sides of the pilus. R17 has been shown
to undergo eclipse, or capsid release, outside the cell on pili attached to cells.
New and existing mutants of traA combined with natural variants of F‐pilin were assayed for pilin stability and processing, pilus elongation, transfer, phage sensitivity and R17 eclipse. Phenotypes of these mutants indicated that the F‐pilin subunit contains specific regions that can be associated with pilus assembly, phage sensitivity and DNA transport. Mutations involving lysines and phenylalanines within residues 45–60 suggest that these residues might participate in transmitting a signal down the length of the pilus that initiates DNA transfer or R17 eclipse.
Activation of Shiga toxin type 2d (Stx2d) by elastase involves cleavage of the C‐terminal two amino acids of the A2 peptide in the context of the appropriate B pentamer
Authors: Melton‐Celsa Angela R. , Kokai‐Kun John F. , O’Brien Alison D. 
Published: March 2003

SummaryShiga toxins (Stx) are potent ribosome‐inactivating toxins that are produced by Shigella dysenteriae type 1 or certain strains of Escherichia coli. These toxins are composed of one A subunit that can be nicked and reduced to an enzymatically active A1(≈27 kDa) and an A2 peptide (≈4 kDa) as well as a pentamer of B subunits (≈7 kDa/monomer) that binds the eukaryotic cell. Purified Shiga toxin type 2d is activated 10‐ to 1000‐fold for Vero cell toxicity by preincubation with mouse or human intestinal mucus or purified mouse elastase, whereas Stx2, Stx2c, Stx2e and Stx1 are not activatable. E. coli strains that produce the activatable Stx2d are more virulent in a streptomycin (str)‐treated mouse model of infection [lethal dose 50% (LD50) = 101] than are E. coli strains that produce any other type of Stx (LD50= 1010). To identify the element(s) of Stx2d that are required for mucus‐mediated activation, toxin genes were constructed such that the expressed mutant toxins consisted of hybrids of Stx2d and Stx1, Stx2 or Stx2e, contained deletions of up to six amino acids from the C‐terminus of the A2 of Stx2d or were altered in one or both of the two amino acids of the A2 of Stx2d that represent the only amino acid differences between the activatable Stx2d and the non‐activatable Stx2c. Analysis of these mutant toxins revealed that the A2 portion of Stx2d is required for toxin activation and that activation is abrogated if the Stx1 or Stx2e B subunit is substituted for the Stx2d B polypeptide. Furthermore, mass spectrometry performed on buffer‐ or elastase‐treated Stx2d indicated that the A2 peptide of the activated Stx2d was two amino acids smaller than the A2 peptide from buffer‐treated Stx2d. This finding, together with the toxin hybrid results, suggests that activation involves B pentamer‐dependent cleavage by elastase of the C‐terminal two amino acids from the Stx2d A2 peptide.
Complex formation between activator and RNA polymerase as the basis for transcriptional activation by MarA and SoxS in Escherichia coli
Authors: Martin Robert G. , Gillette William K. , Martin Nicholas I. , Rosner Judah L. 
Published: March 2003

SummaryTranscriptional activation in Escherichia coli is generally considered to proceed via the formation of an activator–DNA–RNA polymerase (RNP) ternary complex. Although the order of assembly of the three elements is thermodynamically irrelevant, a prevalent idea is that the activator–DNA complex is formed first, and recruitment of RNP to the binary complex occurs subsequently. We show here that the closely related activators, MarA, SoxS and Rob, which activate the same family of genes, are capable of forming complexes with RNP core or holoenzyme in the absence of DNA. In addition, we find that the ternary MarA–DNA–RNP and SoxS–DNA–RNP complexes are more stable than the corresponding Rob–DNA–RNP complex, although the binary Rob–DNA complex is often more stable than the corresponding MarA– or SoxS–DNA complexes. These results may help to explain certain puzzling aspects of the MarA/SoxS/ Rob system. We suggest that activator–RNP complexes scan the chromosome and bind promoters of the regulon more efficiently than either RNP or the activators alone.
Novel gene products associated with NdhD3/D4‐containing NDH‐1 complexes are involved in photosynthetic CO2 hydration in the cyanobacterium, Synechococcus sp. PCC7942
Authors: Maeda Shin‐ichi , Badger Murray R. , Price G. Dean 
Published: March 2003

SummaryCyanobacteria possess light‐dependent CO2 uptake activity that results in the net hydration of CO2 to HCO3– and may involve a protein‐mediated carbonic anhydrase (CA)‐like activity. This process is vital for the survival of cyanobacteria and may be a contributing factor in the ecological success of this group of organisms. Here, via isolation of mutants of Synechococcus sp. PCC7942 that cannot grow under low‐CO2 conditions, we have identified two novel genes, chpX and chpY, that are involved in light‐dependent CO2 hydration and CO2 uptake reactions; co‐inactivation of both these genes abolished both activities. The function and mechanism of the CO2 uptake systems supported by each chp gene product differs, with each associated with functionally distinct NAD(P)H dehydrogenase (NDH‐1) complexes. The ChpX system has a low affinity for CO2 and is de‐pendent on photosystem I cyclic electron transport, whereas the inducible ChpY system has a high affinity for CO2 and is dependent on linear electron transport. We believe that ChpX and ChpY are involved in a unique, net hydration of CO2 to HCO3–, that is coupled electron flow within the NDH‐1 complex on the thylakoid membrane.
Co‐ordinated expression of phycobiliprotein operons in the chromatically adapting cyanobacterium Calothrix PCC 7601: a role for RcaD and RcaG
Authors: Noubir Sanaâ , Luque Ignacio , Alda Jesús A. G. Ochoa de , Perewoska Irène , Marsac Nicole Tandeau de , Cobley John G. , Houmard Jean 
Published: March 2003

SummaryIn the cyanobacterium Calothrix sp. PCC 7601 the cpc2 operon encoding phycocyanin 2 (PC2) is expressed if red radiations are available. RcaD was previously identified in extracts from red‐light‐grown cells as an alkaline phosphatase‐sensitive protein that binds upstream of the transcription start point (TSP) of the cpc2 operon. In this work, RcaD was purified, and the corresponding gene cloned with a PCR probe obtained using degenerated primers based on RcaD peptide sequences (accession no. AJ319541). Purified RcaD binds to the cpc2 promoter region and also to those of the constitutive cpc1 and apc1 operons that encode phycocyanin 1 and allophycocyanin. Escherichia coli‐overexpressed RcaD can bind to the cpc2 promoter region. The rcaD gene is upstream of an open reading frame (ORF) termed rcaG. Co‐transcription of both genes was demonstrated by reverse transcription (RT)‐PCR experiments, and found to be independent of the light wavelengths. A single TSP was mapped. Sequence features of RcaD and RcaG led us to propose a functional relationship between these two proteins. A rcaD mutant generated by allelic exchange exhibited altered expression of the cpc2, cpeBA, apc1 and cpc1 operons upon green to red‐light shifts. RcaD seems to be a co‐activator co‐ordinating the transcription of the phycobiliprotein operons upon changes in light spectral quality.
Salmonella enterica serotype Typhimurium ShdA is an outer membrane fibronectin‐binding protein that is expressed in the intestine
Authors: Kingsley Robert A. , Santos Renato L. , Keestra A. Marijke , Adams L. Garry , Bäumler Andreas J. 
Published: March 2003

SummaryThe shdA gene is the only determinant known to be required for persistence of Salmonella enterica serotype Typhimurium (S. Typhimurium) in the murine caecum and for efficient and prolonged shedding of the organism with the faeces. To study the biological activity of the ShdA protein, we examined its expression and binding activity. ShdA was not detected with anti‐ShdA antiserum in S. Typhimurium strain ATCC14028 grown in vitro, suggesting that this protein is not expressed under standard conditions of bacterial cultivation in the laboratory. However, in mice infected with S. Typhimurium, an immunofluorescence signal detected with anti‐ShdA antiserum co‐localized with that generated by anti‐O4 antiserum in thin sections from the caecum. Expression of the cloned shdA gene from the T7 promoter in vitro resulted in detection of ShdA in the outer membrane of S. Typhimurium and in binding of fibronectin to the bacterial surface. Binding of purified glutathione‐S‐transferase (GST)–ShdA fusion protein to fibronectin was dose dependent and could be partially inhibited by preincubation with antifibronectin antibodies. GST–ShdA bound to connective tissue and the basement membrane in thin sections from the murine caecum in situ. A similar labelling pattern was produced when thin sections of the murine caecum were stained with antifibronectin antiserum. Collectively, these data demonstrate that ShdA is a surface‐localized, fibronectin‐binding protein whose expression is induced in vivo in the murine caecum, a tissue in which a cognate receptor of this outer membrane protein is expressed.
Spc24 interacts with Mps2 and is required for chromosome segregation, but is not implicated in spindle pole body duplication
Authors: Masson Ivan Le , Saveanu Cosmin , Chevalier Anne , Namane Abdelkader , Gobin Renée , Fromont‐Racine Micheline , Jacquier Alain , Mann Carl 
Published: March 2003

SummaryMps2 (monopolar spindle protein) is a coiled‐coil protein found at the spindle pole body (SPB) and at the nuclear envelope that is required for insertion of the SPB into the nuclear envelope. We identified three proteins that interact with Mps2 in a two‐hybrid screen: Bbp1, Ynl107w and Spc24. All three proteins contain coiled‐coil motifs that appear to be required for their interaction with Mps2. In this work, we verified the Mps2–Spc24 interaction by co‐immunoprecipitation in vivo
and by the in vitro interaction of recombinant proteins. Previous two‐hybrid
screens with Spc24 as bait had identified Spc25 and Ndc80 as putative interacting
partners, and we verified these interactions in vivo by purification of TAP‐tagged
derivatives of Spc24 and Ndc80. Finally, we found that spc24 thermosensitive mutants had a chromosome segregation defect, but no apparent defect in SPB duplication. These results are consistent with recently published data showing that Spc24, Spc25 and Ndc80 are peripheral kinetochore com‐ponents required for chromosome segregation. The Mps2–Spc24 interaction may contribute to the localization of Spc24 and other kinetochore components to the inner plaque of the SPB.