bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of Essential Genes in the Human Fungal Pathogen
Aspergillus fumigatus
by Transposon Mutagenesis
Authors: Firon Arnaud , Villalba François , Beffa Roland , d'Enfert Christophe 
Published: April 2003

ABSTRACT

The opportunistic pathogen
Aspergillus fumigatus
is the most frequent cause of deadly airborne fungal infections in developed countries. In order to identify novel antifungal-drug targets, we investigated the genome of
A. fumigatus
for genes that are necessary for efficient fungal growth. An artificial
A. fumigatus
diploid strain with one copy of an engineered
impala160
transposon from
Fusarium oxysporum
integrated into its genome was used to generate a library of diploid strains by random in vivo transposon mutagenesis. Among 2,386 heterozygous diploid strains screened by parasexual genetics, 1.2% had a copy of the transposable element integrated into a locus essential for
A. fumigatus
growth. Comparison of genomic sequences flanking
impala160
in these mutants with that of the genome of
A. fumigatus
allowed the characterization of 20 previously uncharacterized
A. fumigatus
genes. Among these, homologues of genes essential for
Saccharomyces cerevisiae
growth have been identified, as well as genes that do not have homologues in other fungal species. These results confirm that heterologous transposition using the transposable element
impala
is a powerful tool for functional genomics in ascomycota, and they pave the way for defining the complete set of essential genes in
A. fumigatus
, the first step toward target-based development of new antifungal drugs.
Novel Small-Molecule Inhibitors of RNA Polymerase III
Authors: Wu Liping , Pan Jing , Thoroddsen Vala , Wysong Deborah R. , Blackman Ronald K. , Bulawa Christine E. , Gould Alexandra E. , Ocain Timothy D. , Dick Lawrence R. , Errada Patrick , Dorr Patrick K. , Parkinson Tanya , Wood Tony , Kornitzer Daniel , Weissman Ziva , Willis Ian M. , McGovern Karen 
Published: April 2003

ABSTRACTA genetic approach utilizing the yeastSaccharomyces cerevisiaewas used to identify the target of antifungal compounds. This analysis led to the identification of small molecule inhibitors of RNA polymerase (Pol) III fromSaccharomyces cerevisiae. Three lines of evidence show that UK-118005 inhibits cell growth by targeting RNA Pol III in yeast. First, a dominant mutation in the g domain of Rpo31p, the largest subunit of RNA Pol III, confers resistance to the compound. Second, UK-118005 rapidly inhibits tRNA synthesis in wild-type cells but not in UK-118005 resistant mutants. Third, in biochemical assays, UK-118005 inhibits tRNA gene transcription in vitro by the wild-type but not the mutant Pol III enzyme. By testing analogs of UK-118005 in a template-specific RNA Pol III transcription assay, an inhibitor with significantly higher potency, ML-60218, was identified. Further examination showed that both compounds are broad-spectrum inhibitors, displaying activity against RNA Pol III transcription systems derived fromCandida albicansand human cells. The identification of these inhibitors demonstrates that RNA Pol III can be targeted by small synthetic molecules.
Isolation of Mutations in the Catalytic Domain of the Snf1 Kinase That Render Its Activity Independent of the Snf4 Subunit
Authors: Leech Anna , Nath Nandita , McCartney Rhonda R. , Schmidt Martin C. 
Published: April 2003

ABSTRACT

Activation of the Snf1 kinase requires at least two events, phosphorylation of the activation loop on threonine 210 and an Snf4-dependent process that is not completely defined. Snf4 directly interacts with a region of the regulatory domain of Snf1 that may otherwise act as an autoinhibitory domain. In order to gain insight into the regulation of Snf1 kinase by Snf4, deletions in the regulatory domain of the catalytic subunit were engineered and tested for their effect on Snf1 function in the absence of Snf4. Deletion of residues 381 to 488 from the Snf1 protein resulted in a kinase that was activated by glucose limitation even in the absence of the Snf4 protein. A larger deletion (amino acids 381 to 608) encompassing virtually the entire regulatory domain resulted in complete inactivation of the Snf1 kinase even in the presence of Snf4. A genetic screen for amino acid substitutions that conferred an Snf4-independent phenotype identified four point mutations in the Snf1 catalytic domain. One very conservative mutation, leucine 183 to isoleucine, conferred nearly wild-type levels of Snf1 kinase function in the absence of the Snf4 protein. Purified Snf1 kinase was inactive when isolated from
snf4
Δ cells, whereas the Snf1-L183I kinase exhibited significant activity in the absence of Snf4. Our data support the idea that Snf1 kinase activity is constrained in
cis
by an autoinhibitory domain and that the Snf4-mediated activation of Snf1 can be bypassed by subtle conformational changes in the catalytic domain of the Snf1 kinase.
Budding Yeast CTDK-I Is Required for DNA Damage-Induced Transcription
Authors: Ostapenko Denis , Solomon Mark J. 
Published: April 2003

ABSTRACT

CTDK-I phosphorylates the C-terminal domain (CTD) of the large subunit of yeast RNA polymerase II in a reaction that stimulates transcription elongation. Mutations in CTDK-I subunits—Ctk1p, Ctk2p, and Ctk3p—confer conditional phenotypes. In this study, we examined the role of CTDK-I in the DNA damage response. We found that mutation of individual CTDK-I subunits rendered yeast sensitive to hydroxyurea (HU) and UV irradiation. Treatment with DNA-damaging agents increased phosphorylation of Ser2 within the CTD repeats in wild-type but not in
ctk1
Δ mutant cells. Using microarray hybridization, we identified genes whose transcription following DNA damage is Ctk1p dependent, including several DNA repair and stress response genes. Following HU treatment, the level of Ser2-phosphorylated RNA polymerase II increased both globally and on the CTDK-I-regulated genes. The pleiotropic phenotypes of
ctk
mutants suggest that CTDK-I activity is essential during large-scale transcriptional repatterning under stress and unfavorable growth conditions.
Ceramide/Long-Chain Base Phosphate Rheostat in
Saccharomyces cerevisiae
: Regulation of Ceramide Synthesis by Elo3p and Cka2p
Authors: Kobayashi Scott D. , Nagiec Marek M. 
Published: April 2003

ABSTRACT

Sphingolipid precursors, namely, ceramide and long-chain base phosphates (LCBPs), are important growth regulators with often opposite effects on mammalian cells. A set of enzymes that regulate the levels of these precursors, referred to as a ceramide/LCBP rheostat, is conserved in all eukaryotes. In order to gain further insight into the function of the rheostat in
Saccharomyces cerevisiae
, we searched for mutants that are synthetically lethal with a deletion of the
LCB3
gene encoding LCBP phosphatase. In addition to acquiring expected mutants lacking the LCBP lyase, the screen revealed
elo3
(
sur4
) mutants that were defective in fatty acid elongation and
cka2
mutants lacking the α′ subunit of the protein kinase CK2 (casein kinase). Both mutations affected the in vivo activity of the acyl coenzyme A (acyl-CoA)-dependent and fumonisin B
1
-sensitive ceramide synthase (CS). The Elo3 protein is necessary for synthesis of C
26
-CoA, which in wild-type yeast is a source of C
26
fatty acyls found in the ceramide moieties of all sphingolipids. In the in vitro assay, CS had a strong preference for acyl-CoAs containing longer acyl chains. This finding suggests that a block in the formation of C
26
-CoA in yeast may cause a reduction in the conversion of LCBs into ceramides and lead to an overaccumulation of LCBPs that is lethal in strains lacking the Lcb3 phosphatase. In fact,
elo3
mutants were found to accumulate high levels of LCBs and LCBPs. The
cka2
mutants, on the other hand, exhibited only 25 to 30% of the in vitro CS activity found in wild-type membranes, indicating that the α′ subunit of CK2 kinase is necessary for full activation of CS. The
cka2
mutants also accumulated high levels of LCBs and had elevated levels of LCBPs. In addition, both the
elo3
and
cka2
mutants showed increased sensitivity to the CS inhibitors australifungin and fumonisin B
1
. Together, our data demonstrate that the levels of LCBPs in yeast are regulated by the rate of ceramide synthesis, which depends on CK2 kinase activity and is also strongly affected by the supply of C
26
-CoA. This is the first evidence indicating the involvement of protein kinase in the regulation of de novo sphingolipid synthesis in any organism.
Transcriptional Silencing of an Amoebapore Gene inEntamoeba histolytica: Molecular Analysis and Effect on Pathogenicity
Authors: Bracha Rivka , Nuchamowitz Yael , Mirelman David 
Published: April 2003

ABSTRACTTranscriptional silencing of the gene coding for amoebapore A (AP-A) was observed when trophozoites ofEntamoeba histolyticawere transfected with a hybrid plasmid construct containing theap-agene flanked by the upstream and downstream segments of the originalEhap-agene. Transfectants were totally devoid ofap-atranscript and AP-A protein. An identical silencing effect was observed upon transfection with a plasmid that contained only the 5′ upstream region ofap-a. Removal of the selecting antibiotic enabled the isolation of plasmidless clones, which retained in their progeny the silenced phenotype.E. histolyticacells were able to overexpressap-awhen transfected with a plasmid containing the gene flanked by the 5′ and 3′ regions of theEhRP-L21gene. This plasmid, however, could not expressap-ain the retransfected, cloned trophozoites lacking AP-A. This is the first report of gene silencing inE. histolytica, and the mechanism appears to belong to transcriptional gene silencing and not to posttranscriptional gene silencing. This conclusion is based on the following results: (i) silencing was achieved by transfection of homologous 5′ flanking sequences (470 bp of theEhap-agene), (ii) transcription initiation ofEhap-awas found to be blocked, and (iii) short double-stranded RNA fragments of theap-acoding and noncoding sequences were not detected. Trophozoites lacking AP-A are nonpathogenic and impaired in their bacteriolytic capability.
Saccharomyces cerevisiaeDap1p, a Novel DNA Damage Response Protein Related to the Mammalian Membrane-Associated Progesterone Receptor
Authors: Hand Randal A. , Jia Nan , Bard Martin , Craven Rolf J. 
Published: April 2003

ABSTRACTThe response to damage is crucial for cellular survival, and eukaryotic cells require a broad array of proteins for an intact damage response. We have found that the YPL170W (DAP1[for damage response protein related to membrane-associated progesterone receptors]) gene is required for growth in the presence of the methylating agent methyl methanesulfonate (MMS). TheDAP1open reading frame shares homology with a broadly conserved family of membrane-associated progesterone receptors (MAPRs). Deletion ofDAP1leads to sensitivity to MMS, elongated telomeres, loss of mitochondrial function, and partial arrest in sterol synthesis. Sensitivity ofdap1strains to MMS is not due to loss of damage checkpoints. Instead,dap1cells are arrested as unbudded cells after MMS treatment, suggesting that Dap1p is required for cell cycle progression following damage. Dap1p also directs resistance to itraconazole and fluconazole, inhibitors of sterol synthesis. We have found thatdap1cells have slightly decreased levels of ergosterol but increased levels of the ergosterol intermediates squalene and lanosterol, indicating thatdap1cells have a partial defect in sterol synthesis. This is the first evidence linking a MAPR family member to sterol regulation or the response to damage, and these functions are probably conserved in a variety of eukaryotes.
Yeast and Human RNA Polymerase II Elongation Complexes: Evidence for Functional Differences and Postinitiation Recruitment of Factors
Authors: Pardee Timothy S. , Ghazy Mohamed A. , Ponticelli Alfred S. 
Published: April 2003

ABSTRACT

Immobilized DNA templates, glycerol gradient centrifugation, and native gel analysis were utilized to isolate and compare functional RNA polymerase II (RNAPII) elongation complexes from
Saccharomyces cerevisiae
and human cell nuclear extracts. Yeast elongation complexes blocked by incorporation of 3′-
O
-methyl-GTP into the nascent transcript exhibited a sedimentation coefficient of 35S, were less tightly associated to the template than their human counterparts, and displayed no detectable 3′-5′ exonuclease activity on the associated transcript. In contrast, blocked human elongation complexes were more tightly bound to the template, and multiple forms were identified, with the largest exhibiting a sedimentation coefficient of 60S. Analysis of the associated transcripts revealed that a subset of the human elongation complexes exhibited strong 3′-5′ exonuclease activity. Although isolated human preinitiation complexes were competent for efficient transcription, their ability to generate 60S elongation complexes was strikingly impaired. These findings demonstrate functional and size differences between
S. cerevisiae
and human RNAPII elongation complexes and support the view that the formation of mature elongation complexes involves recruitment of nuclear factors after the initiation of transcription.
Three Mating Type-Like Loci in
Candida glabrata
Authors: Srikantha Thyagarajan , Lachke Salil A. , Soll David R. 
Published: April 2003

ABSTRACT

Candida glabrata
, the second most prevalent
Candida
species colonizing humans, possesses three mating type-like (
MTL)
loci (
MTL1
,
MTL2
, and
MTL3
). These loci contain pairs of
MTL
genes with their respective coding regions on complementary Crick and Watson DNA strands. Each pair of genes is separated by a shared intergenic promoter region, the same configuration found at the mating type loci of
Saccharomyces cerevisiae
. Two of the
MTL
loci,
MTL1
and
MTL2
, contain either the
MTL
a1/
MTL
a2 configuration or the
MTL
α1/
MTL
α2 configuration in different strains. All but one of the 38 tested
C. glabrata
strains were either aaα or aαα. One test strain was ααα. Based on the mating type genotype, the
MTL
genes at the
MTL1
or
MTL2
loci, and the size of the
Xba
I fragment harboring
MTL1
or
MTL2
, four classes of
C. glabrata
strains (I, II, III, and IV) were distinguished. Northern analysis revealed that strains were either a-expressors or α-expressors and that expression always reflected the genotype of either the
MTL1
or
MTL2
locus, depending on the class. The expression pattern in each class, therefore, is similar to that observed in
S. cerevisiae
, which harbors two silent cassette loci,
HMR
and
HML
, and the expression locus
MAT
. High-frequency phenotypic switching between core phenotypes in an α-expressing, but not in an a-expressing, strain modulated the level of
MTL
expression, suggesting a possible relationship between core phenotypic switching and mating.
Histone H1 Is Required for Proper Regulation of Pyruvate Decarboxylase Gene Expression in
Neurospora
crassa
Authors: Folco H. Diego , Freitag Michael , Ramón Ana , Temporini Esteban D. , Alvarez María E. , García Irene , Scazzocchio Claudio , Selker Eric U. , Rosa Alberto L. 
Published: April 2003

ABSTRACT

We show that
Neurospora
crassa
has a single histone H1 gene,
hH1
, which encodes a typical linker histone with highly basic N- and C-terminal tails and a central globular domain. A green fluorescent protein-tagged histone H1 chimeric protein was localized exclusively to nuclei. Mutation of
hH1
by repeat-induced point mutation (RIP) did not result in detectable defects in morphology, DNA methylation, mutagen sensitivity, DNA repair, fertility, RIP, chromosome pairing, or chromosome segregation. Nevertheless,
hH1
mutants had mycelial elongation rates that were lower than normal on all tested carbon sources. This slow linear growth phenotype, however, was less evident on medium containing ethanol. The pyruvate decarboxylase gene,
cfp
, was abnormally derepressed in
hH1
mutants on ethanol-containing medium. This derepression was also found when an ectopically integrated fusion of the
cfp
gene promoter to the reporter gene
hph
was analyzed. Thus,
Neurospora
histone H1 is required for the proper regulation of
cfp
, a gene with a key role in the respiratory-fermentative pathway.
The Hog1 Mitogen-Activated Protein Kinase Is Essential in the Oxidative Stress Response and Chlamydospore Formation inCandidaalbicans
Authors: Alonso-Monge Rebeca , Navarro-García Federico , Román Elvira , Negredo Ana I. , Eisman Blanca , Nombela César , Pla Jesús 
Published: April 2003

ABSTRACTCandidaalbicansmutants with mutations in mitogen-activated protein (MAP) kinaseHOG1displayed an increased sensitivity to agents producing reactive oxygen species, such as oxidants (menadione, hydrogen peroxide, or potassium superoxide), and UV light. Consistent with this finding,C.albicansHog1 was activated not only in response to an increase in external osmolarity, as happens with itsSaccharomycescerevisiaehomologue, but also in response to hydrogen peroxide. The Hog1-mediated response to oxidative stress was different from that of transcription factor Cap1, the homologue ofS.cerevisiaeYap1, as shown by the different sensitivities to oxidants and the kinetics of cell death ofcap1Δ,hog1, andhog1cap1Δ mutants. Deletion ofCAP1did not influence the level of Hog1 phosphorylation, and deletion ofHOG1did not affect Cap1 nuclear localization. Moreover, we show that theHOG1gene plays a role in chlamydospore formation, another oxygen-related morphogenetic event, as demonstrated by the fact thathog1cells were unable to generate these thick-walled structures in several media through a mechanism different from that of theEFG1regulator. This is the first demonstration of the role of the Hog1-mediated MAP kinase pathway in resistance to oxidative stress in pathogenic fungi, and it allows us to propose a molecular model for the oxidative stress response inC.albicans.
Molecular Map of theChlamydomonas reinhardtiiNuclear Genome
Authors: Kathir Pushpa , LaVoie Matthew , Brazelton William J. , Haas Nancy A. , Lefebvre Paul A. , Silflow Carolyn D. 
Published: April 2003

ABSTRACTWe have prepared a molecular map of theChlamydomonas reinhardtiigenome anchored to the genetic map. The map consists of 264 markers, including sequence-tagged sites (STS), scored by use of PCR and agarose gel electrophoresis, and restriction fragment length polymorphism markers, scored by use of Southern blot hybridization. All molecular markers tested map to one of the 17 known linkage groups ofC. reinhardtii. The map covers approximately 1,000 centimorgans (cM). Any position on theC. reinhardtiigenetic map is, on average, within 2 cM of a mapped molecular marker. This molecular map, in combination with the ongoing mapping of bacterial artificial chromosome (BAC) clones and the forthcoming sequence of theC. reinhardtiinuclear genome, should greatly facilitate isolation of genes of interest by using positional cloning methods. In addition, the presence of easily assayed STS markers on each arm of each linkage group should be very useful in mapping new mutations in preparation for positional cloning.
Role of OxyR as a Peroxide-Sensing Positive Regulator in
Streptomyces coelicolor
A3(2)
Authors: Hahn Ji-Sook , Oh So-Young , Roe Jung-Hye 
Published: April 2003

ABSTRACT

Genes encoding a homolog of
Escherichia coli
OxyR (
oxyR
) and an alkyl hydroperoxide reductase system (
ahpC
and
ahpD
) have been isolated from
Streptomyces coelicolor
A3(2). The
ahpC
and
ahpD
genes constitute an operon transcribed divergently from the
oxyR
gene
.
Expression of both
ahpCD
and
oxyR
genes was maximal at early exponential phase and decreased rapidly as cells entered mid-exponential phase. Overproduction of OxyR in
Streptomyces lividans
conferred resistance against cumene hydroperoxide and H
2
O
2
. The
oxyR
mutant produced fewer
ahpCD
and
oxyR
transcripts than the wild type, suggesting that OxyR acts as a positive regulator for their expression. Both
oxyR
and
ahpCD
transcripts increased more than fivefold within 10 min of H
2
O
2
treatment and decreased to the normal level in 50 min, with kinetics similar to those of the CatR-mediated induction of the catalase A gene (
catA
) by H
2
O
2
. The
oxyR
mutant failed to induce
oxyR
and
ahpCD
genes in response to H
2
O
2
, indicating that OxyR is the modulator for the H
2
O
2
-dependent induction of these genes. Purified OxyR protein bound specifically to the intergenic region between
ahpC
and
oxyR
, suggesting its direct role in regulating these genes. These results demonstrate that in
S. coelicolor
OxyR mediates H
2
O
2
induction of its own gene and genes for alkyl hydroperoxide reductase system, but not the catalase gene (
catA
), unlike in
Escherichia coli
and
Salmonella enterica
serovar Typhimurium.
Identification of Amino Acids That Are Critical to the Processivity Function of Respiratory Syncytial Virus M2-1 Protein
Authors: Zhou Helen , Cheng Xing , Jin Hong 
Published: April 2003

ABSTRACT

The M2-1 protein of respiratory syncytial virus (RSV) is a transcription processivity factor that is essential for virus replication. The function of RSV M2-1 protein can be examined by using an RSVlacZ minigenome assay in vitro since the expression of the
lacZ
gene is dependent on M2-1. The M2-1 protein of pneumonia virus of mice (PVM), also a member of the
Pneumovirus
genus, functions poorly in the RSVlacZ minigenome assay despite conservation of the Cys
3
-His
1
motif at its N terminus and an overall 40% amino acid identity with RSV M2-1. To identify the amino acids responsible for the differences between these two proteins, two chimeric proteins were constructed. The RSV/PVM (RP) M2-1 chimera that contains the N-terminal 30 amino acids from RSV and the remaining C-terminal 148 amino acids from PVM maintained a level of activity at an ca. 36% of RSV M2-1. However, the PVM/RSV (PR) M2-1 chimera with the N-terminal 29 amino acids from PVM and 164 amino acids from RSV had an activity of <5% of RSV M2-1, indicating that the functional determinants are mainly located in the N terminus of M2-1. Mutagenesis of the N terminus of PR M2-1 and RSV M2-1 identified that Leu-16 and Asn-17 of RSV M2-1 are critical to the M2-1 function. In addition, several charged residues in the N terminus of RSV M2-1 also contributed to the functional integrity of M2-1.
Generation of Novel Syncytium-Inducing and Host Range Variants of Ecotropic Moloney Murine Leukemia Virus in
Mus spicilegus
Authors: Jung Yong Tae , Kozak Christine A. 
Published: April 2003

ABSTRACT

Mus spicilegus
is an Eastern European wild mouse species that has previously been reported to harbor an unusual infectious ecotropic murine leukemia virus (MLV) and proviral envelope genes of a novel MLV subgroup. In the present study,
M. spicilegus
neonates were inoculated with Moloney ecotropic MLV (MoMLV). All 17 inoculated mice produced infectious ecotropic virus after 8 to 14 weeks, and two unusual phenotypes distinguished the isolates from MoMLV. First, most of the
M. spicilegus
isolates grew to equal titers on
M. dunni
and SC-1 cells, although MoMLV does not efficiently infect
M. dunni
cells. The deduced amino acid sequence of a representative clone differed from MoMLV by insertion of two serine residues within the VRA of SU
env
. Modification of a molecular clone of MoMLV by the addition of these serines produced a virus that grows to high titer in
M. dunni
cells, establishing a role for these two serine residues in host range. A second unusual phenotype was found in only one of the
M. spicilegus
isolates, Spl574. Spl574 produces large syncytia of multinucleated giant cells in
M. dunni
cells, but its replication is restricted in other mouse cell lines. Sequencing and mutagenesis demonstrated that syncytium formation could be attributed to a single amino acid substitution within VRA, S82F. Thus, viruses with altered growth properties are selected during growth in
M. spicilegus
. The mutations associated with the host range and syncytium-inducing variants map to a key region of VRA known to govern interactions with the cell surface receptor, suggesting that the associated phenotypes may result from altered interactions with the unusual ecotropic virus mCAT1 receptor carried by
M. dunni
.