bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mapping and Sequencing of the Canine NRAMP1 Gene and Identification of Mutations in Leishmaniasis-Susceptible Dogs
Authors: Altet Laura , Francino Olga , Solano-Gallego Laia , Renier Corinne , Sánchez Armand 
Published: July 2002

ABSTRACTThe NRAMP1 gene (Slc11a1) encodes an ion transporter protein involved in the control of intraphagosomal replication of parasites and in macrophage activation. It has been described in mice as the determinant of natural resistance or susceptibility to infection with antigenically unrelated pathogens, includingLeishmania. Our aims were to sequence and map the canineSlc11a1gene and to identify mutations that may be associated with resistance or susceptibility toLeishmaniainfection. The canineSlc11a1gene has been mapped to dog chromosome CFA37 and covers 9 kb, including a 700-bp promoter region, 15 exons, and a polymorphic microsatellite in intron 1. It encodes a 547-amino-acid protein that has over 87% identity with the Slc11a1 proteins of different mammalian species. A case-control study with 33 resistant and 84 susceptible dogs showed an association between allele 145 of the microsatellite and susceptible dogs. Sequence variant analysis was performed by direct sequencing of the cDNA and the promoter region of four unrelated beagles experimentally infected withLeishmania infantumto search for possible functional mutations. Two of the dogs were classified as susceptible and the other two were classified as resistant based on their immune responses. Two important mutations were found in susceptible dogs: a G-rich region in the promoter that was common to both animals and a complete deletion of exon 11, which encodes the consensus transport motif of the protein, in the unique susceptible dog that needed an additional and prolonged treatment to avoid continuous relapses. A study with a larger dog population would be required to prove the association of these sequence variants with disease susceptibility.
Helicobacter pyloriDoes Not Require Lewis X or Lewis Y Expression To Colonize C3H/HeJ mice
Authors: Takata Tohru , El-Omar Emad , Camorlinga Margarita , Thompson Stuart A. , Minohara Yutaka , Ernst Peter B. , Blaser Martin J. 
Published: July 2002

ABSTRACTHelicobacter pyloristrains frequently express Lewis X (Lex) and/or Leyon their cell surfaces as constituents of the O antigens of their lipopolysaccharide molecules. To assess the effect of Lexand Leyexpression on the ability ofH. pylorito colonize the mouse stomach and to adhere to epithelial cells, isogenic mutants were created in whichfucT1alone orfucT1andfucT2, which encode the fucosyl transferases necessary for Lexand Leyexpression, were deleted. C3H/HeJ mice were experimentally challenged with either wild-type 26695H. pylorior its isogenic mutants. All strains, whether passaged in the laboratory or recovered after mouse passage, colonized the mice well and without consistent differences. During colonization by the mutants, there was no reversion to wild type. Similarly, adherence to AGS and KatoIII cells was unaffected by the mutations. Together, these findings indicate that Le expression is not necessary for mouse gastric colonization or forH. pyloriadherence to epithelial cells.
Expression of the Neutral Protease Gene from a Thermophilic
Bacillus
sp. BT1 Strain in
Bacillus subtilis
and Its Natural Host: Identification of a Functional Promoter
Authors: Večerek Branislav , Venema Gerard 
Published: July 2002

ABSTRACT

The expression of the neutral protease gene (
npr
) from the thermophilic
Bacillus
sp. BT1 strain was studied in its natural host and in mesophilic
Bacillus subtilis
. In the thermophilic BT1 strain, the transcription of the protease gene is initiated from its own promoter, just 5′ to the gene. In contrast, in heterologous
B. subtilis
this thermophilic
npr
promoter does not function, and expression of the
npr
gene results from transcription originating upstream of an adjacent gene, open reading frame X (ORF X). A functional promoter was identified 5′ to ORF X that is required for efficient expression of the
npr
gene in
Bacillus subtilis
as verified by primer extension, reverse transcription-PCR, and 5′ rapid amplification of cDNA ends experiments. These data suggest that transcriptional signals used in thermophilic
Bacillus
sp. BT1 strain are different from those used in
B. subtilis
.
Self-Assembly of the
Agrobacterium tumefaciens
VirB11 Traffic ATPase
Authors: Rashkova Svetlana , Zhou Xue-Rong , Chen Jun , Christie Peter J. 
Published: July 2002

ABSTRACT

The
Agrobacterium tumefaciens
VirB11 ATPase is a component of a type IV transporter dedicated to T-DNA delivery to plant cells. In this study, we tested a prediction from genetic findings that VirB11 self-associates in vivo. A chimeric protein composed of VirB11 fused to the DNA binding domain of λ cI repressor protein formed dimers, as shown by immunity of
Escherichia coli
to λ superinfection. An allele encoding VirB11 fused at its C terminus to the green fluorescent protein (GFP) exerted strong negative dominance when synthesized in wild-type
A. tumefaciens
cells. Dominance was suppressed by overproduction of native VirB11, suggestive of titrating or competitive interactions between VirB11 and VirB11::GFP. In support of the titration model, a complex of native VirB11 and VirB11::GFP was recovered by precipitation with anti-GFP antibodies from detergent-solubilized
A. tumefaciens
cell extracts. VirB11 was shown by cI repressor fusion and immunoprecipitation assays to interact with VirB11 derivatives encoded by (i) 11 dominant negative alleles, (ii) recessive alleles bearing codon substitutions or deletions in the Walker A nucleotide binding motif, and (iii) alleles corresponding to the 5′ and 3′ halves of
virB11
. Further immunoprecipitation studies showed a hybrid protein composed of the N-terminal half of VirB11 fused to GFP interacted with mutant proteins exerting dominant effects and with a recessive Walker A deletion mutant (ΔGKT174-176). By contrast, a hybrid protein composed of the C-terminal half fused to GFP interacted with mutants exerting dominant effects but not the Walker A mutant protein. Together, these studies establish that VirB11 assembles as homomultimers in vivo via domains residing in each half of the protein. Furthermore, ATP binding appears to be critical for C-terminal interactions required for assembly of productive homomultimers.
Coactivation of the RpoS-Dependent
proP
P2 Promoter by Fis and Cyclic AMP Receptor Protein
Authors: McLeod Sarah M. , Xu Jimin , Johnson Reid C. 
Published: July 2002

ABSTRACT

The
Escherichia coli proP
P2 promoter, which directs the expression of an integral membrane transporter of proline, glycine betaine, and other osmoprotecting compounds, is induced upon entry into stationary phase to protect cells from osmotic shock. Transcription from the P2 promoter is completely dependent on RpoS (ς
38
) and Fis. Fis activates transcription by binding to a site centered at −41, which overlaps the promoter, where it makes a specific contact with the C-terminal domain of the α subunit of RNA polymerase (α-CTD). We show here that Fis and cyclic AMP (cAMP) receptor protein (CRP)-cAMP collaborate to activate transcription synergistically in vitro. Coactivation both in vivo and in vitro is dependent on CRP binding to a site centered at −121.5, but CRP without Fis provides little activation. The contribution by CRP requires the correct helical phasing of the CRP site and a functional activation region 1 on CRP. We provide evidence that coactivation is achieved by Fis and CRP independently contacting each of the two α-CTDs. Efficient transcription in vitro requires that both activators must be preincubated with the DNA prior to addition of RNA polymerase.
oriT
-Directed Cloning of Defined Large Regions from Bacterial Genomes: Identification of the
Sinorhizobium meliloti
pExo Megaplasmid Replicator Region
Authors: Chain Patrick S. G. , Hernandez-Lucas Ismael , Golding Brian , Finan Turlough M. 
Published: July 2002

ABSTRACT

We have developed a procedure to directly clone large fragments from the genome of the soil bacterium
Sinorhizobium meliloti
. Specific regions to be cloned are first flanked by parallel copies of an origin of transfer (
oriT
) together with a plasmid replication origin capable of replicating large clones in
Escherichia coli
but not in the target organism. Supplying transfer genes in trans specifically transfers the
oriT
-flanked region, and in this process, site-specific recombination at the
oriT
sites results in a plasmid carrying the flanked region of interest that can replicate in
E. coli
from the inserted origin of replication (in this case, the F origin carried on a BAC cloning vector). We have used this procedure with the
oriT
of the plasmid RK2 to clone contiguous fragments of 50, 60, 115, 140, 240, and 200 kb from the
S. meliloti
pExo megaplasmid. Analysis of the 60-kb fragment allowed us to identify a 9-kb region capable of autonomous replication in the bacterium
Agrobacterium tumefaciens
. The nucleotide sequence of this fragment revealed a replicator region including homologs of the
repA
,
repB
, and
repC
genes from other
Rhizobiaceae
, which encode proteins involved in replication and segregation of plasmids in many organisms.
The Amino Terminus of
Salmonella enterica
Serovar Typhimurium ς
54
Is Required for Interactions with an Enhancer-Binding Protein and Binding to Fork Junction DNA
Authors: Kelly Mary T. , Hoover Timothy R. 
Published: July 2002

ABSTRACT

Transcription initiation by the ς
54
-RNA polymerase holoenzyme requires an enhancer-binding protein that is thought to contact ς
54
to activate transcription. To identify potential enhancer-binding protein contact sites in ς
54
, we compared the abilities of wild-type and truncated forms of
Salmonella enterica
serovar Typhimurium ς
54
to interact with the enhancer-binding protein DctD in a chemical cross-linking assay. Removal of two regions in the amino-terminal portion of ς
54
, residues 57 to 105 and residues 144 to 179, prevented cross-linking, but removal of either region alone did not. In addition, deletion of 56 amino-terminal residues of ς
54
(region I) reduced the affinity of the protein for a fork junction DNA probe.
Functional Expression in
Escherichia coli
and Membrane Topology of Porin HopE, a Member of a Large Family of Conserved Proteins in
Helicobacter pylori
Authors: Bina Jim , Bains Manjeet , Hancock Robert E. W. 
Published: July 2002

ABSTRACT

HopE is one of the smallest members of a family of 31 outer membrane proteins in
Helicobacter pylori
and has been shown to function as a porin. In this study it was cloned into
Escherichia coli
where it was expressed in the outer membrane, as confirmed by indirect immunofluorescence using HopE-specific antibodies. HopE purified from
E. coli
reconstituted channels in planar bilayer membranes that were the same size as those formed by HopE purified from
H. pylori
. A model of the membrane topology of HopE was constructed and indicated that this protein formed a β-barrel with 16 transmembrane amphipathic β-strands. The accuracy of this model was tested by linker insertion mutagenesis, assuming that, like other porins, amino acid insertions were not tolerated in the transmembrane β-strands but were tolerated in the adjoining loop regions. Generally, the results obtained with a series of 12 insertions of the sequence RSKDV and two substitutions were consistent with the topological model. The preponderance of amino acids that were conserved in the extended family of HopE paralogs were predicted to be within the membrane and comprised 45% of all residues in the membrane.
Spontaneous Erythromycin Resistance Mutation in a 23S rRNA Gene,
rrlA
, of the Extreme Thermophile
Thermus thermophilus
IB-21
Authors: Gregory Steven T. , Cate Jamie H. D. , Dahlberg Albert E. 
Published: July 2002

ABSTRACT

Spontaneous, erythromycin-resistant mutants of
Thermus thermophilus
IB-21 were isolated and found to carry the mutation A2058G in one of two 23S rRNA operons. The heterozygosity of these mutants indicates that A2058G confers a dominant or codominant phenotype in this organism. This mutation provides a valuable tool for the genetic manipulation of the 23S rRNA genes of
Thermus
.
Disulfide Bond Formation in Secreton Component PulK Provides a Possible Explanation for the Role of DsbA in Pullulanase Secretion
Authors: Pugsley Anthony P. , Bayan Nicolas , Sauvonnet Nathalie 
Published: July 2002

ABSTRACT

When expressed in
Escherichia coli
, the 15
Klebsiella oxytoca pul
genes that encode the so-called Pul secreton or type II secretion machinery promote pullulanase secretion and the assembly of one of the secreton components, PulG, into pili. Besides these
pul
genes, efficient pullulanase secretion also requires the host
dsbA
gene, encoding a periplasmic disulfide oxidoreductase, independently of disulfide bond formation in pullulanase itself. Two secreton components, the secretin pilot protein PulS and the minor pseudopilin PulK, were each shown to posses an intramolecular disulfide bond whose formation was catalyzed by DsbA. PulS was apparently destabilized by the absence of its disulfide bond, whereas PulK stability was not dramatically affected either by a
dsbA
mutation or by the removal of one of its cysteines. The pullulanase secretion defect in a
dsbA
mutant was rectified by overproduction of PulK, indicating reduced disulfide bond formation in PulK as the major cause of the secretion defect under the conditions tested (in which PulS is probably present in considerable excess of requirements). PulG pilus formation was independent of DsbA, probably because PulK is not needed for piliation.
Identification and Characterization of
gsp65
, an Organic Hydroperoxide Resistance (
ohr
) Gene Encoding a General Stress Protein in
Enterococcus faecalis
Authors: Rincé Alain , Giard Jean-Christophe , Pichereau Vianney , Flahaut Sigrid , Auffray Yanick 
Published: July 2002

ABSTRACT

The
Enterococcus faecalis
general stress protein Gsp65 has been purified from two-dimensional gel electrophoresis. Determination of its N-terminal sequence and characterization of the corresponding gene revealed that the
gsp65
product is a 133-amino-acid protein sharing homologies with organic hydroperoxide resistance (Ohr) proteins. Transcriptional analysis of
gsp65
gave evidence for a monocistronic mRNA initiated 52 nucleotides upstream of the ATG start codon and for an induction in response to hydrogen peroxide, heat shock, acid pH, detergents, ethanol, sodium chloride, and
tert
-butylhydroperoxide (tBOOH). A
gsp65
mutant showed increased sensitivity to the organic hydroperoxide tBOOH and to ethanol.
Gene Structures and Regulation of the Alkane Hydroxylase Complex in
Acinetobacter
sp. Strain M-1
Authors: Tani Akio , Ishige Takeru , Sakai Yasuyoshi , Kato Nobuo 
Published: July 2002

ABSTRACT

In the long-chain
n
-alkane degrader
Acinetobacter
sp. strain M-1, two alkane hydroxylase complexes are switched by controlling the expression of two
n
-alkane hydroxylase-encoding genes in response to the chain length of
n
-alkanes, while rubredoxin and rubredoxin ruductase are encoded by a single gene and expressed constitutively.
Proteolytic Activity of YibP Protein in
Escherichia coli
Authors: Ichimura Toshiharu , Yamazoe Mitsuyoshi , Maeda Maki , Wada Chieko , Hiraga Sota 
Published: July 2002

ABSTRACT

Escherichia coli
YibP protein (47.4 kDa) has a membrane-spanning signal at the N-terminal region, two long coiled-coil regions in the middle part, and a C-terminal globular domain, which involves amino acid sequences homologous to the peptidase M23/M37 family. A
yibP
disrupted mutant grows in rich medium at 37°C but not at 42°C. In the
yibP
null mutant, cell division and FtsZ ring formation are inhibited at 42°C without SOS induction, resulting in filamentous cells with multiple nucleoids and finally in cell lysis. Five percent betaine suppresses the temperature sensitivity of the
yibP
disrupted mutation. The mutant has the same sensitivity to drugs, such as nalidixic acid, ethidium bromide, ethylmethane sulfonate, and sodium dodecyl sulfate, as the parental strain. YibP protein is recovered in the inner membrane and cytoplasmic fractions, but not in the outer membrane fraction. Results suggest that the coiled-coil regions and the C-terminal globular domain of YibP are localized in the cytoplasmic space, not in the periplasmic space. Purified YibP has a protease activity that split the substrate β-casein.
Expression System for High Levels of GAG Lyase Gene Expression and Study of the
hepA
Upstream Region in
Flavobacterium heparinum
Authors: Blain Françoise , Tkalec A. Lydia , Shao Zhongqi , Poulin Catherine , Pedneault Marc , Gu Kangfu , Eggimann Bernhard , Zimmermann Joe , Su Hongsheng 
Published: July 2002

ABSTRACT

A system for high-level expression of heparinase I, heparinase II, heparinase III, chondroitinase AC, and chondroitinase B in
Flavobacterium heparinum
is described.
hepA
, along with its regulatory region, as well as
hepB
,
hepC
,
cslA
, and
cslB
, cloned downstream of the
hepA
regulatory region, was integrated in the chromosome to yield stable transconjugant strains. The level of heparinase I and II expression from the transconjugant strains was approximately fivefold higher, while heparinase III expression was 10-fold higher than in wild-type
F. heparinum
grown in heparin-only medium. The chondroitinase AC and B transconjugant strains, grown in heparin-only medium, yielded 20- and 13-fold increases, respectively, in chondroitinase AC and B expression, compared to wild-type
F. heparinum
grown in chondroitin sulfate A-only medium. The
hepA
upstream region was also studied using
cslA
as a reporter gene, and the transcriptional start site was determined to be 26 bp upstream of the start codon in the chondroitinase AC transconjugant strain. The transcriptional start sites were determined for
hepA
in both the wild-type
F. heparinum
and heparinase I transconjugant strains and were shown to be the same as in the chondroitinase AC transconjugant strain. The five GAG lyases were purified from these transconjugant strains and shown to be identical to their wild-type counterparts.
Type 4 Pilus Biogenesis and Type II-Mediated Protein Secretion byVibrio choleraeOccur Independently of the TonB-Facilitated Proton Motive Force
Authors: Bose Niranjan , Payne Shelley M. , Taylor Ronald K. 
Published: July 2002

ABSTRACTInVibrio cholerae, elaboration of toxin-coregulated pilus and protein secretion by the extracellular protein secretion apparatus occurred in the absence of both TonB systems. In contrast, the cognate putative ATPases were required for each process and could not substitute for each other.
Comparison of Roche MONITOR and Organon Teknika NucliSens Assays To Quantify Human Immunodeficiency Virus Type 1 RNA in Cerebrospinal Fluid
Authors: Spearman Paul , Fiscus Susan A. , Smith Rita M. , Shepard Robin , Johnson Benjamin , Nicotera Janet , Harris Victoria L. , Clough Lisa A. , McKinsey Joel , Haas David W. 
Published: July 2002

ABSTRACT

We compared Roche MONITOR and Organon Teknika NucliSens assays for human immunodeficiency virus type 1 (HIV-1) RNA in cerebrospinal fluid (CSF). Results of 282 assays were highly correlated (
r
= 0.826), with MONITOR values being 0.29 ± 0.4 log
10
copies/ml (mean ± standard deviation) values. Both assays can reliably quantify HIV-1 RNA in CSF.
Replacement of the Ectodomains of the Hemagglutinin-Neuraminidase and Fusion Glycoproteins of Recombinant Parainfluenza Virus Type 3 (PIV3) with Their Counterparts from PIV2 Yields Attenuated PIV2 Vaccine Candidates
Authors: Tao Tao , Skiadopoulos Mario H. , Davoodi Fatemeh , Riggs Jeffrey M. , Collins Peter L. , Murphy Brian R. 
Published: July 2002

ABSTRACT

We sought to develop a live attenuated parainfluenza virus type 2 (PIV2) vaccine strain for use in infants and young children, using reverse genetic techniques that previously were used to rapidly produce a live attenuated PIV1 vaccine candidate. The PIV1 vaccine candidate, designated rPIV3-1cp45, was generated by substituting the full-length HN and F proteins of PIV1 for those of PIV3 in the attenuated
cp
45 PIV3 vaccine candidate (T. Tao et al., J. Virol. 72:2955–2961, 1998; M. H. Skiadopoulos et al., Vaccine 18:503–510, 1999). However, using the same strategy, we failed to recover recombinant chimeric PIV3-PIV2 isolate carrying the full-length PIV2 glycoproteins in a wild-type PIV3 backbone. Viable PIV3-PIV2 chimeras were recovered when chimeric HN and F open reading frames (ORFs) rather than complete PIV2 F and HN ORFs were used to construct the full-length cDNA. The recovered viruses, designated rPIV3-2CT, in which the PIV2 ectodomain and transmembrane domain were fused to the PIV3 cytoplasmic domain, and rPIV3-2TM, in which the PIV2 ectodomain was fused to the PIV3 transmembrane and cytoplasmic tail domain, possessed similar in vitro and in vivo phenotypes. Thus, it appeared that only the cytoplasmic tail of the HN or F glycoprotein of PIV3 was required for successful recovery of PIV3-PIV2 chimeras. Although rPIV3-2CT and rPIV3-2TM replicated efficiently in vitro, they were moderately to highly attenuated for replication in the respiratory tracts of hamsters, African green monkeys (AGMs), and chimpanzees. This unexpected finding indicated that chimerization of the HN and F proteins of PIV2 and PIV3 itself specified an attenuation phenotype in vivo. Despite this attenuation, these viruses were highly immunogenic and protective against challenge with wild-type PIV2 in hamsters and AGMs, and they represent promising candidates for clinical evaluation as a vaccine against PIV2. These chimeric viruses were further attenuated by the addition of 12 mutations of PIV3cp45 which lie outside of the HN and F genes. The attenuating effects of these mutations were additive with that of the chimerization, and thus inclusion of all or some of the
cp
45 mutations provides a means to further attenuate the PIV3-PIV2 chimeric vaccine candidates if necessary.
Murine Gammaherpesvirus 68 Cyclin D Homologue Is Required for Efficient Reactivation from Latency
Authors: Hoge Ann T. , Hendrickson Sara B. , Burns William H. 
Published: July 2002

ABSTRACTMurine gammaherpesvirus 68 (MHV68) is a gammaherpesvirus that was first isolated from murid rodents. MHV68 establishes a latent infection in the spleen and other lymphoid organs. Several gammaherpesviruses, including herpesvirus saimiri, human herpesvirus 8, and MHV68, encode proteins with extensive homology to the D-type cyclins. To study the function of the cyclin homologue, a recombinant MHV68 has been constructed that lacks the cyclin homologue and expresses β-galactosidase as a marker (MHV68cy−). MHV68cy−grows in vitro with kinetics and to titers similar to those of the wild type. BALB/c mice infected with mixtures of equivalent amounts of the wild type and MHV68cy−show deficient growth of the MHV68cy−in an acute infection. Infection of SCID mice with virus mixtures also showed decreased MHV68cy−virus growth, indicating that the deficiency is not mediated by T or B cells. Although mice infected with mixtures containing 100 times as much MHV68cy−had greater splenic titers of the mutant virus than wild-type virus in acute infection, at 28 days postinfection splenocytes from these mice reactivated primarily wild-type virus. Quantitative PCR data indicate that equivalent genomes were present in the latent state. Reinsertion of the cyclin homologue into the cyclin-deleted virus restored the wild-type phenotype. These results indicate that the MHV68 cyclin D homologue mediates important functions in the acute infection and is required for efficient reactivation from latency.