bluuebunny/crossref_metadata_2025_split huggingface dataset:
Establishing Lysogenic Transcription in the Temperate Coliphage 186
Authors: Neufing Petra J. , Shearwin Keith E. , Egan J. Barry 
Published: July 2002

ABSTRACT

A single-copy chromosomal reporter system was used to measure the intrinsic strengths and interactions between the three promoters involved in the establishment of lysogeny by coliphage 186. The maintenance lysogenic promoter
p
L
for the immunity repressor gene
c
I is intrinsically ∼20-fold weaker than the lytic promoter
p
R
. These promoters are arranged face-to-face, and transcription from
p
L
is further weakened some 14-fold by the activity of
p
R
. Efficient establishment of lysogeny requires the
p
E
promoter, which lies upstream of
p
L
and is activated by the phage CII protein to a level comparable to that of
p
R
. Transcription of
p
E
is less sensitive to converging
p
R
transcription and raises
c
I transcription at least 55-fold. The
p
E
promoter does not occlude
p
L
but inhibits lytic transcription by 50%. This interference is not due to bound CII preventing elongation of the lytic transcript. The
p
E
RNA is antisense to the anti-immune repressor gene
apl
, but any role of this in the establishment of lysogeny appears to be minimal.
Dissection of the Functional and Structural Domains of Phosphorelay Histidine Kinase A of
Bacillus subtilis
Authors: Wang Ling , Fabret Céline , Kanamaru Kyoko , Stephenson Keith , Dartois Veronique , Perego Marta , Hoch James A. 
Published: July 2002

ABSTRACT

The initiation of sporulation in
Bacillus subtilis
results primarily from phosphoryl group input into the phosphorelay by histidine kinases, the major kinase being kinase A. Kinase A is active as a homodimer, the protomer of which consists of an approximately 400-amino-acid N-terminal putative signal-sensing region and a 200-amino-acid C-terminal autokinase. On the basis of sequence similarity, the N-terminal region may be subdivided into three PAS domains: A, B, and C, located from the N- to the C-terminal end. Proteolysis experiments and two-hybrid analyses indicated that dimerization of the N-terminal region is accomplished through the PAS-B/PAS-C region of the molecule, whereas the most amino-proximal PAS-A domain is not dimerized. N-terminal deletions generated with maltose binding fusion proteins showed that an intact PAS-A domain is very important for enzymatic activity. Amino acid substitution mutations in PAS-A as well as PAS-C affected the in vivo activity of kinase A, suggesting that both PAS domains are required for signal sensing. The C-terminal autokinase, when produced without the N-terminal region, was a dimer, probably because of the dimerization required for formation of the four-helix-bundle phosphotransferase domain. The truncated autokinase was virtually inactive in autophosphorylation with ATP, whereas phosphorylation of the histidine of the phosphotransfer domain by back reactions from Spo0F∼P appeared normal. The phosphorylated autokinase lost the ability to transfer its phosphoryl group to ADP, however. The N-terminal region appears to be essential both for signal sensing and for maintaining the correct conformation of the autokinase component domains.
Bordetella
Interspecies Allelic Variation in AlcR Inducer Requirements: Identification of a Critical Determinant of AlcR Inducer Responsiveness and Construction of an
alcR
(Con) Mutant Allele
Authors: Brickman Timothy J. , Armstrong Sandra K. 
Published: July 2002

ABSTRACT

Previous studies established the critical roles of AlcR and alcaligin inducer in positive regulation of alcaligin siderophore biosynthesis and transport genes in
Bordetella pertussis
and
Bordetella bronchiseptica
. Transcriptional analyses using plasmid-borne
alcR
genes of
B. pertussis
UT25 and
B. bronchiseptica
B013N to complement the
alcR
defect of
B. bronchiseptica
strain BRM13 (Δ
alcR1 alcA
::mini-Tn
5 lacZ1
) revealed interspecies differences in AlcR inducer requirements for activation of
alcABCDER
operon transcription. Whereas the
B. pertussis
UT25 AlcR protein retained strong inducer dependence when produced from multicopy plasmids,
B. bronchiseptica
B013N
alcR
partially suppressed the alcaligin requirement for transcriptional activation. Functional analysis of AlcR chimeras produced by interspecies domain swapping and interspecies reciprocal site-specific mutagenesis determined that the phenotypic difference in AlcR inducer dependence was due to a single amino acid difference within the proposed inducer-binding and multimerization domain of AlcR. Structural predictions guided the design of a mutant AlcR protein with a single amino acid substitution at this critical position, AlcR(S103T), that was fully constitutive not only when produced from multicopy plasmids but also at a single-copy gene dosage. These results indicate that AlcR residue 103 affects a critical determinant of alcaligin inducer dependence of AlcR-mediated transcriptional activation. The
alcR
(S103T) mutant allele is the first
alcR
(Con) mutant allele identified.
Rhodospirillum rubrum
Possesses a Variant of the
bchP
Gene, Encoding Geranylgeranyl-Bacteriopheophytin Reductase
Authors: Addlesee Hugh A. , Hunter C. Neil 
Published: July 2002

ABSTRACT

The
bchP
gene product of
Rhodobacter sphaeroides
is responsible for the reduction of the isoprenoid moiety of bacteriochlorophyll (Bchl) from geranylgeraniol (GG) to phytol; here, we show that this enzyme also catalyzes the reduction of the isoprenoid moiety of bacteriopheophytin (Bphe). In contrast, we demonstrate that a newly identified homolog of this gene in
Rhodospirillum rubrum
encodes an enzyme, GG-Bphe reductase, capable of reducing the isoprenoid moiety of Bphe only. We propose that
Rhodospirillum rubrum
is a naturally occurring
bchP
mutant and that an insertion mutation may have been the initial cause of a partial loss of function. Normal BchP function can be restored to
Rhodospirillum rubrum
, creating a new transconjugant strain possessing Bchl esterified with phytol. We speculate on the requirement of
Rhodospirillum rubrum
for phytylated Bphe and on a potential link between the absence of LH2 and of phytylated Bchl from the wild-type bacterium. The identification of a second role for the fully functional BchP in catalyzing the synthesis of phytylated Bphe strongly suggests that homologs of this enzyme may be similarly responsible for the synthesis of phytylated pheophytin in organisms possessing photosystem 2. In addition to
bchP
, other members of a photosynthesis gene cluster were identified in
Rhodospirillum rubrum
, including a
bchG
gene, demonstrated to encode a functional Bchl synthetase by complementation of a
Rhodobacter sphaeroides
mutant.
A MecA Paralog, YpbH, Binds ClpC, Affecting both Competence and Sporulation
Authors: Persuh Marjan , Mandic-Mulec Ines , Dubnau David 
Published: July 2002

ABSTRACT

ComK, the master regulator of competence, is degraded by the general stress-related protease ClpCP but must be targeted to this protease by binding to the adapter protein MecA. The genome of
Bacillus subtilis
contains a paralog of
mecA
,
ypbH
. We show in the present study that YpbH, like MecA, binds ClpC and that its elimination or overproduction affects competence and sporulation.
Sensitive and Specific Method for Rapid Identification of
Streptococcus pneumoniae
Using Real-Time Fluorescence PCR
Authors: McAvin James C. , Reilly Patricia A. , Roudabush Robert M. , Barnes William J. , Salmen Ann , Jackson Glen W. , Beninga Kathleen K. , Astorga Alicia , McCleskey Ferne K. , Huff William B. , Niemeyer Debra , Lohman Kenton L. 
Published: July 2002

ABSTRACT

Molecular surveillance of pathogens has shown the need for rapid and dependable methods for the identification of organisms of clinical and epidemiological importance. As the leading cause of community-acquired pneumonia,
Streptococcus pneumoniae
was used as a model organism to develop and refine a real-time fluorescence PCR assay and enhanced DNA purification method. Seventy clinical isolates of
S. pneumoniae
, verified by latex agglutination
,
were screened against 26 negative control clinical isolates employing a TaqMan assay on a thermocycler (LightCycler). The probe, constructed from the
lytA
gene, correctly detected all
S. pneumoniae
genomes without cross-reaction to negative controls. The speed and ease of this approach will make it adaptable to identification of many bacterial pathogens and provide potential for adaptation to direct detection from patient specimens.
Improved Detection of Methicillin-Resistant
Staphylococcus aureus
Using Phenyl Mannitol Broth Containing Aztreonam and Ceftizoxime
Authors: Wertheim Heiman , Verbrugh Henri A. , van Pelt Cindy , de Man Peter , van Belkum Alex , Vos Margreet C. 
Published: July 2002

ABSTRACT

We tested a phenyl mannitol broth containing ceftizoxime and aztreonam (PHMB
+
) for detection of methicillin-resistant
Staphylococcus aureus
(MRSA) with reference MRSA strains and, subsequently, with clinical samples (
n
= 1,098). All reference MRSA strains induced color change in PHMB
+
after 24 to 72 h of incubation. In a clinical setting, 40 MRSA strains were detected with PHMB
+
, compared with only 23 detected with a routine method. Thus, this selective broth significantly (
P
< 0.001) improved the rate of MRSA detection.
Genetic Diversity of
Pneumocystis carinii
Isolated from Human Immunodeficiency Virus-Positive Patients in Turin, Italy
Authors: Volpe Gisella , Sbaiz Luca , Avanzini Claudio , Caramello Pietro , Savoia Dianella 
Published: July 2002

ABSTRACT

By DNA sequence analysis we identified two new strain types and five novel sporadic variations among 25 isolates of
Pneumocystis carinii
f. sp.
hominis
obtained from 19 human immunodeficiency virus-positive patients. Of these, 13 were infected with a single strain and 6 were coinfected. Fifteen different combination types were identified among the 18 strains for which complete molecular typing was accomplished.
Characterization of
Mycobacterium tuberculosis
Complex Isolates from the Cerebrospinal Fluid of Meningitis Patients at Six Fever Hospitals in Egypt
Authors: Cooksey Robert C. , Abbadi Said H. , Woodley Charles L. , Sikes David , Wasfy Momtaz , Crawford Jack T. , Mahoney Frank 
Published: July 2002

ABSTRACT

Mycobacterium tuberculosis
complex isolates from cerebrospinal fluid of 67 meningitis patients were obtained from six fever hospitals in Egypt. One
M. bovis
and 66
M. tuberculosis
isolates were identified by PCR-restriction fragment length polymorphism (RFLP) analysis of
oxyR
. Among the
M. tuberculosis
isolates, 53 unique strain types (with 3 to 16 copies of IS
6110
) were found by RFLP analyses. Nine clusters (eight with two isolates each and one with six isolates) were also found. Thirty-six spoligotypes, including at least 10 that have been previously reported from other countries, were also observed. Forty-one (62.1%) of the isolates were in spoligotype clusters, and 22 (33%) of the isolates were in RFLP clusters. Fifty-one of the isolates were susceptible in vitro to all of the antituberculosis drugs tested, 11 were monoresistant to capreomycin, rifampin, isoniazid (INH), pyrazinamide, or streptomycin (STR), 4 were resistant to STR and INH, and 1 was resistant to STR, INH, and ethambutol.
Human Immunodeficiency Virus (HIV)-Positive Sera Obtained Shortly after Seroconversion Neutralize Autologous HIV Type 1 Isolates on Primary Macrophages but Not on Lymphocytes
Authors: Ruppach Horst , Nara Peter , Raudonat Ina , Elanjikal Ziju , Rübsamen-Waigmann Helga , Dietrich Ursula 
Published: July 2002

ABSTRACTThe aim of this study was to analyze the role of humoral immunity in early human immunodeficiency virus (HIV) infection. As neutralizing activities in HIV-positive sera are rarely detectable earlier than 9 to 12 months after infection using primary lymphocytes as target cells in neutralization assays, humoral immunity is generally thought not to contribute significantly to early virus control in the patients. Besides lymphocytes, cells of the monocyte/macrophage lineage are known to be important target cells for HIV in vivo during the establishment of the infection. Therefore, we studied the neutralization of early primary HIV isolates by autologous serum samples using primary macrophages as target cells in the neutralization assays. We analyzed neutralizing activities against the autologous HIV-1 isolates in 10 patients' sera taken shortly after seroconversion, both on primary macrophages and, for comparison, on lymphocytes. Viruses were isolated and expanded in primary mixed cultures containing macrophages and lymphocytes in order to avoid selection for one particular cell type. All viruses replicated to different degrees in macrophages and lymphocytes; nine had a nonsyncytium-inducing phenotype, and one was syncytium inducing. The detection of neutralizing antibodies in acute primary HIV infection depended on the target cells used. Confirming previous studies, we did not find neutralizing activities on lymphocytes at this early time point. In contrast, neutralizing activities were detectable in the same sera if primary macrophages were used as target cells. Differences in neutralizing activities on macrophages and lymphocytes were not due to different virus variants being present in the different cell systems, as gp120 sequences derived from both cell types were homogeneous. Neutralization activities on macrophages did not correlate with the amount of β-chemokines in the sera. As affinity-purified immunoglobulin G preparations from an early patient serum also exhibited neutralization of the autologous virus isolate on primary macrophages, but not on lymphocytes, neutralization is very likely due to antibodies against viral epitopes necessary for infection of macrophages but not for infection of lymphocytes. Our data suggest that, along with cell-mediated immunity, humoral immunity may contribute to the reduction of primary viremia in the patient. This was further supported by a certain association between neutralizing antibody titers on macrophages and viral load in the patients.
Avian Retrovirus DNA Internal Attachment Site Requirements for Full-Site Integration In Vitro
Authors: Chiu Roger , Grandgenett Duane P. 
Published: July 2002

ABSTRACT

Concerted integration of retrovirus DNA termini into the host chromosome in vivo requires specific interactions between the
cis
-acting attachment (
att
) sites at the viral termini and the viral integrase (IN) in
trans
. In this study, reconstruction experiments with purified avian myeloblastosis virus (AMV) IN and retrovirus-like donor substrates containing wild-type and mutant termini were performed to map the internal
att
DNA sequence requirements for concerted integration, here termed full-site integration. The avian retrovirus mutations were modeled after internal
att
site mutations studied at the in vivo level with human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV). Systematic overlapping 4-bp deletions starting at nucleotide positions 7, 8, and 9 in the U3 terminus had a decreasing detrimental gradient effect on full-site integration, while more internal 4-bp deletions had little or no effect. This decreasing detrimental gradient effect was measured by the ability of mutant U3 ends to interact with wild-type U3 ends for full-site integration in
trans
. Modification of the highly conserved C at position 7 on the catalytic strand to either A or T resulted in the same severe decrease in full-site integration as the 4-bp deletion starting at this position. These studies suggest that nucleotide position 7 is crucial for interactions near the active site of IN for integration activity and for communication in
trans
between ends bound by IN for full-site integration. The ability of AMV IN to interact with internal
att
sequences to mediate full-site integration in vitro is similar to the internal
att
site requirements observed with MLV and HIV-1 in vivo and with their preintegration complexes in vitro.
Human Immunodeficiency Virus Type 1 Nucleocapsid Protein Can Prevent Self-Priming of Minus-Strand Strong Stop DNA by Promoting the Annealing of Short Oligonucleotides to Hairpin Sequences
Authors: Driscoll Mark D. , Hughes Stephen H. 
Published: July 2002

ABSTRACT
Understanding how viral components collaborate to convert the human immunodeficiency virus type 1 genome from single-stranded RNA into double-stranded DNA is critical to the understanding of viral replication. Not only must the correct reactions be carried out, but unwanted side reactions must be avoided. After minus-strand strong stop DNA (−sssDNA) synthesis, degradation of the RNA template by the RNase H domain of reverse transcriptase (RT) produces single-stranded DNA that has the potential to self-prime at the imperfectly base-paired TAR hairpin, making continued DNA synthesis impossible. Although nucleocapsid protein (NC) interferes with −sssDNA self-priming in reverse transcription reactions in vitro, NC alone did not prevent self-priming of a synthetic −sssDNA oligomer. NC did not influence DNA bending and therefore cannot inhibit self-priming at hairpins by directly blocking hairpin formation. Using DNA oligomers as a model for genomic RNA fragments, we found that a 17-base DNA fragment annealed to the 3′ end of the −sssDNA prevented self-priming in the presence of NC. This implies that to avoid self-priming, an RNA-DNA hybrid that is more thermodynamically stable than the hairpin must remain within the hairpin region. This suggests that NC prevents self-priming by generating or stabilizing a thermodynamically favored RNA-DNA heteroduplex instead of the kinetically favored TAR hairpin. In support of this idea, sequence changes that increased base pairing in the DNA TAR hairpin resulted in an increase in self-priming in vitro. We present a model describing the role of NC-dependent inhibition of self-priming in first-strand transfer.
Intracellular Forms of Human
NOTCH1
Functionally Activate Essential Epstein-Barr Virus Major Latent Promoters in the Burkitt's Lymphoma BJAB Cell Line but Repress These Promoters in Jurkat Cells
Authors: Cotter Murray , Callahan Jennifer , Aster Jon , Robertson Erle 
Published: July 2002

ABSTRACT

We have demonstrated that intracellular forms of
NOTCH1
transactivate two major Epstein-Barr virus (EBV) latent promoters, the LMP1 and Cp1 promoters in an EBV-negative B-cell line, BJAB. Truncated intracellular
NOTCH1
associated with the nuclear membrane (ΔE) transactivates the LMP1 promoter fivefold; however, the intranucleus localized form of
NOTCH1
(ICN) transactivates this promoter approximately twofold in chloroamphenicol acetyltransferase (CAT) reporter assays in BJAB cells. Additionally, ΔE activated the major Cp1 promoter 12-fold, whereas the ICN form of
NOTCH1
activates at only about half that level when compared to that of ΔE membrane-bound
NOTCH1
. This result differs from previously observed data, where intracellular
NOTCH1
bound to the nuclear membrane, ΔE, and nucleus-localized
NOTCH1
, ICN, all had similar levels of activation in 293 cells. This suggests distinct transcriptional activities in different cell types. Moreover, in Jurkat cells, a T-cell line, intranucleus localized
NOTCH1
molecules demonstrated a repressive activity against the two EBV major latent promoters. Only ΔE activated the Cp1 and LMP1 promoters at a level slightly above background, whereas intranucleus localized
NOTCH1
ICN, or the form of
NOTCH1
lacking the ankyrin repeats, ΔE
TAR
, surprisingly resulted in the repression of these promoters in Jurkat cells. Similarly, another truncated form of
NOTCH1
, referred to as ICNW, which contains the tryptophan residue W
1767
within one of the RBP-Jκ interacting domains, repressed the LMP1 promoter approximately twofold. Further analysis of the truncated
NOTCH1
molecules on the LMP1 promoter element, lacking the two RBP-Jκ binding sites, suggests that repression in Jurkat cells may be affected by the presence of the two RBP-Jκ binding sites. These studies indicate that intracellular
NOTCH1
can activate the EBV major latent promoters in BJAB cells. However, in Jurkat cells, intracellular truncated forms of
NOTCH1
lacking the RBP-Jκ binding sites repress these EBV latent promoters. Only the membrane-bound form of
NOTCH1
, ΔE, activated the EBV major latent promoters in Jurkat cells, albeit at a lower level than that seen in BJAB cells. Our data suggest that EBNA2 and truncated intracellular nuclear localized forms of
NOTCH1
may be functionally similar in their interactions with RBP-Jκ; however, these molecules may have distinctly different transcriptional partners in BJAB and Jurkat cells. Moreover, these truncated
NOTCH1
molecules may not represent the normal processed forms of
NOTCH1
in cells and may exhibit dominant negative phenotypes in the absence of the required posttranslational modifications. Further investigations are necessary to determine the similarity and differences occurring with intracellular
NOTCH1
in other B- and T-cell lines.
Disruption of the Murine Gammaherpesvirus 68 M1 Open Reading Frame Leads to Enhanced Reactivation from Latency
Authors: Clambey Eric T. , Virgin Herbert W. , Speck Samuel H. 
Published: July 2002

ABSTRACT

Murine gammaherpesvirus 68 (γHV68, or MHV-68) is a genetically tractable, small animal model for the analysis of gammaherpesvirus pathogenesis. The γHV68 genome is colinear with the genomes of other sequence gammaherpesviruses, containing large blocks of conserved genes interspersed by a number of putative genes without clear homologs in the other gammaherpesviruses. One of these putative unique genes, the M1 open reading frame (ORF), exhibits sequence homology to a poxvirus serine protease inhibitor, SPI-1, as well as to another γHV68 gene, M3, which we have recently shown encodes an abundantly secreted chemokine binding protein. To assess the contribution of the M1 ORF to γHV68 pathogenesis, we have generated a recombinant γHV68 in which the M1 ORF has been disrupted through targeted insertion of a
lacZ
expression cassette (M1.LacZ). Although M1.LacZ replicated normally in tissue culture, it exhibited decreased splenic titers at days 4 and 9 postinfection in both immunocompetent and immunodeficient mice. Despite decreased levels of acute virus replication, M1.LacZ established a latent infection comparable to wild-type (wt) γHV68, but exhibited an approximately fivefold increase in efficiency of reactivation from latency. M1.LacZ also caused severe vasculitis of the great elastic arteries in gamma interferon receptor (IFN-γR)-deficient mice with a frequency comparable to wt γHV68, but did not cause the mortality or splenic pathology observed with wt γHV68 infection of IFN-γR-deficient mice. Restoration of M1 ORF sequences into M1.LacZ (M1 marker rescue, or M1.MR) demonstrated that M1.LacZ phenotypic alterations in growth in vivo and latency were not due to the presence of additional mutations located elsewhere in the M1.LacZ genome. Generation of a second M1 mutant virus containing a deletion at the 5′ end of the M1 ORF (M1Δ511), but lacking the LacZ expression cassette, revealed the same latency phenotype observed with the M1.LacZ mutant. However, M1Δ511 was not attenuated for acute virus replication in the spleen. We conclude that (i) the induction of arteritis in γHV68-infected IFN-γR-deficient mice can occur in the absence of splenic pathology and mortality, (ii) replication during acute infection is not the primary determinant for the establishment of latent infection, and (iii) the M1 ORF, or a closely linked gene, encodes a gene product that functions to suppress virus reactivation.