bluuebunny/crossref_metadata_2025_split huggingface dataset:
WdCHS3
, a Gene That Encodes a Class III Chitin Synthase in
Wangiella
(
Exophiala
)
dermatitidis
, Is Expressed Differentially under Stress Conditions
Authors: Wang Zheng , Szaniszlo Paul J. 
Published: July 2002

ABSTRACT

Class III chitin synthases are important for hyphal growth in some filamentous fungi but are not found in yeasts. Using a specific PCR product that encodes a portion of the class III chitin synthase of
W. dermatitidis
as a probe, we isolated the chitin synthase gene,
WdCHS3
, from this polymorphic melanized pathogen of humans. Northern blotting showed that
WdCHS3
was highly expressed under stress conditions, such as the shift of cells to temperatures commensurate with infection, or to conditions that induce cellular morphogenesis in this fungus. Analysis of the 5′ upstream sequence of
WdCHS3
provided evidence for a negative regulatory element at between −780 and −1600 bp. Western blotting indicated that the production of the WdChs3p was temperature dependent and temporally regulated. Disruption of
WdCHS3
in a wild-type strain and in two temperature-sensitive morphological mutants resulted in significantly reduced chitin synthase activities but did not obviously affect their morphologies, growth rates, chitin contents, or virulence. This paradox suggested that the contributions of the high levels of
WdCHS3
gene expression and WdChs3p production in strains subjected to stress reside in unknown or unexamined parts of the life cycle of this ecologically poorly known member of the Fungi Imperfecti. Nonetheless, this report presents the first evidence that transcription of a chitin synthase gene is regulated by a negative regulatory element in its 5′ upstream sequence.
Natural Genetic Competence in
Bacillus subtilis
Natto OK2
Authors: Ashikaga Sayaka , Nanamiya Hideaki , Ohashi Yoshiaki , Kawamura Fujio 
Published: July 2002

ABSTRACT

We isolated a
Bacillus subtilis
natto strain, designated OK2, from a lot of commercial fermented soybean natto and studied its ability to undergo natural competence development using a
comG-lacZ
fusion at the
amyE
locus. Although transcription of the late competence genes was not detected in the
B. subtilis
natto strain OK2 during competence development, these genes were constitutively transcribed in the OK2 strain carrying either the
mecA
or the
clpC
mutation derived from
B. subtilis
168. In addition, both OK2 mutants exhibited high transformation frequencies, comparable with that observed for
B. subtilis
168. Moreover, as expected from these results, overproduction of ComK derived from strain 168 in strain OK2 resulted in a high transformation frequency as well as in induction of the late competence genes. These results clearly indicated that ComK produced in both the
mecA
and
clpC
mutants of strain OK2 (ComK
OK2
) could activate the transcription of the whole set of late competence genes and suggested that ComK
OK2
was not activated in strain OK2 during competence development. We therefore sequenced the
comS
gene of OK2 and compared it with that of 168. The
comS
OK2
had a single-base change, resulting in the replacement of Ser (strain 168) by Cys (strain OK2) at position 11.
An
n
-Alkane-Responsive Promoter Element Found in the Gene Encoding the Peroxisomal Protein of
Candida tropicalis
Does Not Contain a C
6
Zinc Cluster DNA-Binding Motif
Authors: Kanai Tamotsu , Hara Akihiro , Kanayama Naoki , Ueda Mitsuyoshi , Tanaka Atsuo 
Published: July 2002

ABSTRACT

When an asporogenic diploid yeast,
Candida tropicalis
, is cultivated on
n
-alkane, the expression of the genes encoding enzymes of the peroxisomal β-oxidation pathway is highly induced. An upstream activation sequence (UAS) which can induce transcription in response to
n
-alkane (UAS
ALK
) was identified on the promoter region of the peroxisomal 3-ketoacyl coenzyme A (CoA) thiolase gene of
C. tropicalis
(
CT-T3A
). The 29-bp region (from −289 to −261) present upstream of the TATA sequence was sufficient to induce
n
-alkane-dependent expression of a reporter gene. Besides
n
-alkane, UAS
ALK
-dependent gene expression also occurred in the cells grown on oleic acid. Several kinds of mutant UAS
ALK
were constructed and tested for their UAS activity. It was clarified that the important nucleotides for UAS
ALK
activity were located within 10-bp region from −273 to −264 (5′-TCCTGCACAC-3′). This region did not contain a CGG triplet and therefore differed from the sequence of the oleate-response element (ORE), which is a UAS found on the promoter region of 3-ketoacyl-CoA thiolase gene of
Saccharomyces cerevisiae
. Similar sequences to UAS
ALK
were also found on several peroxisomal enzyme-encoding genes of
C. tropicalis.
A Long T · A Tract in the
upp
Initially Transcribed Region Is Required for Regulation of
upp
Expression by UTP-Dependent Reiterative Transcription in
Escherichia coli
Authors: Cheng Yulin , Dylla Sara M. , Turnbough Charles L. 
Published: July 2002

ABSTRACT

In
Escherichia coli
, pyrimidine-mediated regulation of
upp
expression occurs by UTP-sensitive selection of alternative transcriptional start sites, which produces transcripts that differ in the ability to be elongated. The
upp
initially transcribed region contains the sequence GATTTTTTTTG (nontemplate strand). Initiation can occur at either the first or the second base in this sequence (designated G6 and A7, with numbering from the promoter −10 region). High intracellular UTP levels favor initiation at position A7; however, the resulting transcripts are subject to reiterative transcription (i.e., repetitive UMP addition) within the 8-bp T · A tract in the initially transcribed region and are aborted. In contrast, low intracellular UTP levels favor initiation at position G6, which results in transcripts that can, in part, avoid reiterative transcription and be elongated normally. In this study, we examined the regulatory requirement for the long T · A tract in the
upp
initially transcribed region. We constructed
upp
promoter mutations that shorten the T · A tract to 7, 6, 5, 4, 3, or 2 bp and examined the effects of these mutations on
upp
expression and regulation. The results indicate that pyrimidine-mediated regulation is gradually reduced as the T · A tract is shortened from 7 to 3 bp; at which point regulation ceases. This reduction in regulation is due to large-percentage increases in
upp
expression in cells grown under conditions of pyrimidine excess. Quantitation of cellular transcripts and in vitro transcription studies indicate that the observed effects of a shortened T · A tract on
upp
expression and regulation are due to increases in the fraction of both G6- and A7-initiated transcripts that avoid reiterative transcription and are elongated normally.
Evidence against an Interaction between the mRNA Downstream Box and 16S rRNA in Translation Initiation
Authors: Moll Isabella , Huber Michael , Grill Sonja , Sairafi Pooneh , Mueller Florian , Brimacombe Richard , Londei Paola , Bläsi Udo 
Published: July 2002

ABSTRACT

Based on the complementarity of the initial coding region (downstream box [db]) of several bacterial and phage mRNAs to bases 1469 to 1483 in helix 44 of 16S rRNA (anti-downstream box [adb]), it has been proposed that db-adb base pairing enhances translation in a way that is similar to that of the Shine-Dalgarno (SD)/anti-Shine-Dalgarno (aSD) interaction. Computer modeling of helix 44 on the 30S subunit shows that the topography of the 30S ribosome does not allow a simultaneous db-adb interaction and placement of the initiation codon in the ribosomal P site. Thus, the db-adb interaction cannot substitute for the SD-aSD interaction in translation initiation. We have always argued that any contribution of the db-adb interaction should be most apparent on mRNAs devoid of an SD sequence. Here, we show that 30S ribosomes do not bind to leaderless mRNA in the absence of initiator tRNA, even when the initial coding region shows a 15-nucleotide complementarity (optimal fit) with the putative adb. In addition, an optimized db did not affect the translational efficiency of a leaderless λ
c
I-
lacZ
reporter construct. Thus, the db-adb interaction can hardly serve as an initial recruitment signal for ribosomes. Moreover, we show that different leaderless mRNAs are translated in heterologous systems although the sequence of the putative adb's within helix 44 of the 30S subunits of the corresponding bacteria differ largely. Taken our data together with those of others (M. O'Connor, T. Asai, C. L. Squires, and A. E. Dahlberg, Proc. Natl. Acad. Sci. USA 96:8973–8978, 1999; A. La Teana, A. Brandi, M. O'Connor, S. Freddi, and C. L. Pon, RNA 6:1393–1402, 2000), we conclude that the db does not base pair with the adb.
Cs
+
Induces the
kdp
Operon of
Escherichia coli
by Lowering the Intracellular K
+
Concentration
Authors: Jung Kirsten , Krabusch Mechthild , Altendorf Karlheinz 
Published: July 2002

ABSTRACT

Cs
+
was found to induce expression of the
kdpFABC
operon, encoding a high-affinity K
+
uptake system of
Escherichia coli.
Quantitative expression analyses at the transcriptional and translational levels reveal that CsCl causes much higher induction of
kdpFABC
than does NaCl. A decrease of the intracellular K
+
concentration is found in cells exposed to CsCl. The results indicate that
kdpFABC
expression is induced when the intracellular K
+
concentration is lowered. Moreover, the results imply that the signal transduction cascade mediated by KdpD and KdpE is able to integrate multiple signals.
Control of the Arabinose Regulon in
Bacillus subtilis
by AraR In Vivo: Crucial Roles of Operators, Cooperativity, and DNA Looping
Authors: Mota Luı́s Jaime , Sarmento Leonor Morais , de Sá-Nogueira Isabel 
Published: July 2002

ABSTRACT

The proteins involved in the utilization of
l
-arabinose by
Bacillus subtilis
are encoded by the
araABDLMNPQ-abfA
metabolic operon and by the
araE
/
araR
divergent unit. Transcription from the
ara
operon,
araE
transport gene, and
araR
regulatory gene is induced by
l
-arabinose and negatively controlled by AraR. The purified AraR protein binds cooperatively to two in-phase operators within the
araABDLMNPQ-abfA
(OR
A1
and OR
A2
) and
araE
(OR
E1
and OR
E2
) promoters and noncooperatively to a single operator in the
araR
(OR
R3
) promoter region. Here, we have investigated how AraR controls transcription from the
ara
regulon in vivo. A deletion analysis of the
ara
promoters region showed that the five AraR binding sites are the key
cis
-acting regulatory elements of their corresponding genes. Furthermore, OR
E1
-OR
E2
and OR
R3
are auxiliary operators for the autoregulation of
araR
and the repression of
araE
, respectively. Analysis of mutations designed to prevent cooperative binding of AraR showed that in vivo repression of the
ara
operon requires communication between repressor molecules bound to two properly spaced operators. This communication implicates the formation of a small loop by the intervening DNA. In an in vitro transcription system, AraR alone sufficed to abolish transcription from the
araABDLMNPQ-abfA
operon and
araE
promoters, strongly suggesting that it is the major protein involved in the repression mechanism of
l
-arabinose-inducible expression in vivo. The
ara
regulon is an example of how the architecture of the promoters is adapted to respond to the particular characteristics of the system, resulting in a tight and flexible control.
Viability of
rep recA
Mutants Depends on Their Capacity To Cope with Spontaneous Oxidative Damage and on the DnaK Chaperone Protein
Authors: Bredèche Marie-Florence , Ehrlich S. Dusko , Michel Bénédicte 
Published: July 2002

ABSTRACT

Replication arrests due to the lack or the inhibition of replicative helicases are processed by recombination proteins. Consequently, cells deficient in the Rep helicase, in which replication pauses are frequent, require the RecBCD recombination complex for growth.
rep recA
mutants are viable and display no growth defect at 37 or 42°C. The putative role of chaperone proteins in
rep
and
rep recA
mutants was investigated by testing the effects of
dnaK
mutations.
dnaK756
and
dnaK306
mutations, which allow growth of otherwise wild-type
Escherichia coli
cells at 40°C, are lethal in
rep recA
mutants at this temperature. Furthermore, they affect the growth of
rep
mutants, and to a lesser extent, that of
recA
mutants. We conclude that both
rep
and
recA
mutants require DnaK for optimal growth, leading to low viability of the triple (
rep recA dnaK
) mutant.
rep recA
mutant cells form colonies at low efficiency when grown to exponential phase at 30°C. Although the plating defect is not observed at a high temperature, it is not suppressed by overexpression of heat shock proteins at 30°C. The plating defect of
rep recA
mutant cells is suppressed by the presence of catalase in the plates. The cryosensitivity of
rep recA
mutants therefore results from an increased sensitivity to oxidative damage upon propagation at low temperatures.
Structure, Expression, and Functional Analysis of the Gene Coding for Calmodulin in the Chytridiomycete
Blastocladiella emersonii
Authors: Simão Rita de Cássia Garcia , Gomes Suely Lopes 
Published: July 2002

ABSTRACT

The single calmodulin (CaM) gene and the corresponding cDNA of the chytridiomycete
Blastocladiella emersonii
were isolated and characterized. The CaM gene is interrupted by three introns and transcribed in a single 0.7-kb mRNA species encoding a predicted protein 91% identical to human CaM.
B. emersonii
CaM has been expressed in
Escherichia coli
as a fusion protein with gluthatione
S
-transferase (GST) and purified by affinity chromatography and cleavage from the GST portion using a site-specific protease. In the presence of Ca
2+
,
B. emersonii
CaM exhibited a shift in apparent molecular mass similar to that observed with bovine CaM and was able to activate the autophosphorylation of CaM-dependent protein kinase II (CaMKII) from rat brain. CaM expression is developmentally regulated in
B. emersonii
, with CaM mRNA and protein concentrations increasing during sporulation to maximum levels observed just prior to the release of the zoospores into the medium. Both CaM protein and mRNA levels decrease drastically at the zoospore stage, increasing again during germination. The CaM antagonists compound 48/80, calmidazolium, and W7 were shown to completely inhibit
B. emersonii
sporulation when added to the cultures at least 120, 150, and 180 min after induction, respectively. All these drugs also inhibited growth and zoospore production in this fungus. The Ca
2+
channel blocker TMB-8 and the CaMKII inhibitor KN93 completely inhibited sporulation if added up to 60 min after induction of this stage, but only KN93 affected fungal growth. The data presented suggest that the Ca
2+
-CaM complex and CaMKII play an important role during growth and sporulation in
B. emersonii
.
The Lytic Enzyme of Bacteriophage PRD1 Is Associated with the Viral Membrane
Authors: Rydman Pia S. , Bamford Dennis H. 
Published: July 2002

ABSTRACT

Bacteriophage PRD1 encodes two proteins (P7 and P15) that are associated with a muralytic activity. Protein P15 is a soluble β-1,4-
N
-acetylmuramidase that causes phage-induced host cell lysis. We demonstrate here that P15 is also a structural component of the PRD1 virion and that it is connected to the phage membrane. Small viral membrane proteins P20 and P22 modulate incorporation of P15 into the virion and may connect it to the phage membrane. The principal muralytic protein involved in PRD1 DNA entry seems to be the putative lytic transglycosylase protein P7, as the absence of protein P15 did not delay initiation of phage DNA replication in the virus-host system used. The incorporation of two different lytic enzymes into virions may reflect the broad host range of bacteriophage PRD1.
Depletion of Free 30S Ribosomal Subunits in
Escherichia coli
by Expression of RNA Containing Shine-Dalgarno-Like Sequences
Authors: Mawn Mary V. , Fournier Maurille J. , Tirrell David A. , Mason Thomas L. 
Published: July 2002

ABSTRACT

We have constructed synthetic coding sequences for the expression of poly(α,
l
-glutamic acid) (PLGA) as fusion proteins with dihydrofolate reductase (DHFR) in
Escherichia coli
. These PLGA coding sequences use both GAA and GAG codons for glutamic acid and contain sequence elements (5′-GAGGAGG-3′) that resemble the consensus Shine-Dalgarno (SD) sequence found at translation initiation sites in bacterial mRNAs. An unusual feature of DHFR-PLGA expression is that accumulation of the protein is inversely related to the level of induction of its mRNA. Cellular protein synthesis was inhibited >95% by induction of constructs for either translatable or untranslatable PLGA RNAs. Induction of PLGA RNA resulted in the depletion of free 30S ribosomal subunits and the appearance of new complexes in the polyribosome region of the gradient. Unlike normal polyribosomes, these complexes were resistant to breakdown in the presence of puromycin. The novel complexes contained 16S rRNA, 23S rRNA, and PLGA RNA. We conclude that multiple noninitiator SD-like sequences in the PLGA RNA inhibit cellular protein synthesis by sequestering 30S small ribosomal subunits and 70S ribosomes in nonfunctional complexes on the PLGA mRNA.
Characterization of GlnK
1
from
Methanosarcina mazei
Strain Gö1: Complementation of an
Escherichia coli glnK
Mutant Strain by GlnK
1
Authors: Ehlers Claudia , Grabbe Roman , Veit Katharina , Schmitz Ruth A. 
Published: July 2002

ABSTRACT

Trimeric PII-like signal proteins are known to be involved in bacterial regulation of ammonium assimilation and nitrogen fixation. We report here the first biochemical characterization of an archaeal GlnK protein from the diazotrophic methanogenic archaeon
Methanosarcina mazei
strain Gö1 and show that
M. mazei
GlnK
1
is able to functionally complement an
Escherichia coli glnK
mutant for growth on arginine. This indicates that the archaeal GlnK protein substitutes for the regulatory function of
E. coli
GlnK.
M. mazei
GlnK
1
is encoded in the

glnK
1

-

amtB
1

operon, which is transcriptionally regulated by the availability of combined nitrogen and is only transcribed in the absence of ammonium. The deduced amino acid sequence of the archaeal

glnK
1

shows 44% identity to the
E. coli
GlnK and contains the conserved tyrosine residue (Tyr-51) in the T-loop structure.

M. mazei glnK
1

was cloned and overexpressed in
E. coli
, and GlnK
1
was purified to apparent homogeneity. A molecular mass of 42 kDa was observed under native conditions, indicating that its native form is a trimer. GlnK
1
-specific antibodies were raised and used to confirm the in vivo trimeric form by Western analysis. In vivo ammonium upshift experiments and analysis of purified GlnK
1
indicated significant differences compared to
E. coli
GlnK. First, GlnK
1
from
M. mazei
is not covalently modified by uridylylation under nitrogen limitation. Second, heterotrimers between
M. mazei
GlnK
1
and
Klebsiella pneumoniae
GlnK are not formed. Because
M. mazei
GlnK
1
was able to complement growth of an
E. coli glnK
mutant with arginine as the sole nitrogen source, it is likely that uridylylation is not required for its regulatory function.
Molecular Identification of
Campylobacter concisus
Authors: Matsheka M. I. , Lastovica A. J. , Elisha B. Gay 
Published: July 2002

ABSTRACT

A 1.6-kb DNA fragment isolated from a
Campylobacter concisus
genomic library gave
C. concisus
-specific restriction fragment length patterns when it was used as a probe in hybridization studies. All of the strains tested, including type strains and clinical isolates, contained a 0.5-kb
Hin
dIII fragment that hybridized to the probe. DNA sequencing of the 1.6-kb fragment identified three open reading frames (ORFs). One of the ORFs encodes the carboxy terminus of GyrB, and the translational products of ORF2 and ORF3 showed similarity to hypothetical proteins, previously identified in
Campylobacter jejuni
. DNA-DNA hybridization studies with a fragment internal to ORF3 showed that this sequence was responsible for the signal observed with the 0.5-kb
Hin
dIII fragment. A rapid PCR assay was developed and evaluated. Primers that annealed to the extremities of the 1.6-kb fragment were used to obtain an amplicon of the correct size from both reference and clinical strains of
C. concisus
.
Characterization of Capsid Genes, Expressed in the Baculovirus System, of Three New Genetically Distinct Strains of “Norwalk-Like Viruses”
Authors: Belliot Gaël , Noel Jacqueline S. , Li Jin-Fen , Seto Yoshiyuki , Humphrey Charles D. , Ando Tamie , Glass Roger I. , Monroe Stephan S. 
Published: July 2002

ABSTRACT

“Norwalk-like viruses” (NLVs), members of a newly defined genus of the family
Caliciviridae
, are the most common agents of outbreaks of gastroenteritis in the United States. Two features of NLVs have hindered the development of simple methods for detection and determination of serotype: their genetic diversity and their inability to grow in cell culture. To assess the immune responses of patients involved in outbreaks of gastroenteritis resulting from infection with NLVs, we previously used recombinant-expressed capsid antigens representing four different genetic clusters, but this panel proved insufficient for detection of an immune response in many patients. To extend and further refine this panel, we expressed in baculovirus the capsid genes of three additional genetically distinct viruses, Burwash Landing virus (BLV), White River virus (WRV), and Florida virus. All three expressed proteins assembled into virus-like particles (VLPs) that contained a full-length 64-kDa protein, but both the BLV and WRV VLPs also contained a 58-kDa protein that resulted from deletion of 39 amino acids at the amino terminus. The purified VLPs were used to measure the immune responses in 403 patients involved in 37 outbreaks of acute gastroenteritis. A majority of patients demonstrated a fourfold rise in the titer of immunoglobulin G to the antigen homologous to the outbreak strain, but most seroconverted in response to other genetically distinct antigens as well, suggesting no clear pattern of type-specific immune response. Further study of the antigenicity of the NLVs by use of VLPs should allow us to design new detection systems with either broader reactivity or better specificity and to define the optimum panel of antigens required for routine screening of patient sera.
Detection of Pathogenic
Yersinia enterocolitica
by a Rapid and Sensitive Duplex PCR Assay
Authors: Wannet Wim J. B. , Reessink Michiel , Brunings Henk A. , Maas Henny M. E. 
Published: July 2002

ABSTRACT

A duplex PCR assay targeting the
ail
and 16S rRNA genes of
Yersinia enterocolitica
was developed to specifically identify pathogenic
Y. enterocolitica
from pure culture. Validation of the assay was performed with 215 clinical
Yersinia
strains and 40 strains of other bacterial species. Within an assay time of 4 h, this assay offers a very specific, reliable, and inexpensive alternative to the conventional phenotypic assays used in clinical laboratories to identify pathogenic
Y. enterocolitica
.