bluuebunny/crossref_metadata_2025_split huggingface dataset:
Toxoplasma gondiiAsexual Development: Identification of Developmentally Regulated Genes and Distinct Patterns of Gene Expression
Authors: Cleary Michael D. , Singh Upinder , Blader Ira J. , Brewer Jeremy L. , Boothroyd John C. 
Published: December 2002

ABSTRACTAsexual development inToxoplasma gondiiis a vital aspect of the parasite's life cycle, allowing transmission and avoidance of the host immune response. Differentiation of rapidly dividing tachyzoites into slowly growing, encysted bradyzoites involves significant changes in both physiology and morphology. We generated microarrays of ∼4,400ToxoplasmacDNAs, representing a minimum of ∼600 genes (based on partial sequencing), and used these microarrays to study changes in transcript levels during tachyzoite-to-bradyzoite differentiation. This approach has allowed us to (i) determine expression profiles of previously described developmentally regulated genes, (ii) identify novel developmentally regulated genes, and (iii) identify distinct classes of genes based on the timing and magnitude of changes in transcript levels. Whereas microarray analysis typically involves comparisons of mRNA levels at different time points, we have developed a method to measure relative transcript abundance between genes at a given time point. This method was used to determine transcript levels in parasites prior to differentiation and to further classify bradyzoite-induced genes, thus allowing a more comprehensive view of changes in gene expression than is provided by standard expression profiles. Newly identified developmentally regulated genes include putative surface proteins (a SAG1-related protein,SRS9, and a mucin-domain containing protein), regulatory and metabolic enzymes (methionine aminopeptidase, oligopeptidase, aminotransferase, and glucose-6-phosphate dehydrogenase homologues), and a subset of genes encoding secretory organelle proteins (MIC1, ROP1, ROP2, ROP4, GRA1, GRA5, and GRA8). This analysis permits the first in-depth look at changes in gene expression during development of this complex protozoan parasite.
Random Sequencing of
Paramecium
Somatic DNA
Authors: Sperling Linda , Dessen Philippe , Zagulski Marek , Pearlman Ron E. , Migdalski Andrzey , Gromadka Robert , Froissard Marine , Keller Anne-Marie , Cohen Jean 
Published: December 2002

ABSTRACT

We report a random survey of 1 to 2% of the somatic genome of the free-living ciliate
Paramecium tetraurelia
by single-run sequencing of the ends of plasmid inserts. As in all ciliates, the germ line genome of
Paramecium
(100 to 200 Mb) is reproducibly rearranged at each sexual cycle to produce a somatic genome of expressed or potentially expressed genes, stripped of repeated sequences, transposons, and AT-rich unique sequence elements limited to the germ line. We found the somatic genome to be compact (>68% coding, estimated from the sequence of several complete library inserts) and to feature uniformly small introns (18 to 35 nucleotides). This facilitated gene discovery: 722 open reading frames (ORFs) were identified by similarity with known proteins, and 119 novel ORFs were tentatively identified by internal comparison of the data set. We determined the phylogenetic position of
Paramecium
with respect to eukaryotes whose genomes have been sequenced by the distance matrix neighbor-joining method by using random combined protein data from the project. The unrooted tree obtained is very robust and in excellent agreement with accepted topology, providing strong support for the quality and consistency of the data set. Our study demonstrates that a random survey of the somatic genome of
Paramecium
is a good strategy for gene discovery in this organism.
Roles of
TUP1
in Switching, Phase Maintenance, and Phase-Specific Gene Expression in
Candida albicans
Authors: Zhao Rui , Lockhart Shawn R. , Daniels Karla , Soll David R. 
Published: December 2002

ABSTRACT

Candida albicans
strain WO-1 switches spontaneously and reversibly between a “white” and “opaque” phenotype that affects colony morphology, cellular phenotype, and expression of a number of phase-specific genes and virulence traits. To assess the role of the transcription regulator Tup1p in this phenotypic transition, both
TUP1
alleles were deleted in the mutant Δ
tup1
. Δ
tup1
formed “fuzzy large” colonies made up of cells growing exclusively in the filamentous form. Δ
tup1
cells did not undergo the white-opaque transition, but it did switch spontaneously, at high frequency (∼10
−3
), and unidirectionally through the following sequence of colony (and cellular) phenotypes: “fuzzy large” (primarily hyphae) → “fuzzy small” (primarily pseudohyphae) → “smooth” (primarily budding yeast) → “revertant fuzzy” (primarily pseudohyphae). Northern analysis of white-phase, opaque-phase, and hypha-associated genes demonstrated that Tup1p also plays a role in the regulation of select phase-specific genes and that each variant in the Δ
tup1
switching lineage differs in the level of expression of one or more phase-specific and/or hypha-associated genes. Using a rescued Δ
tup1
strain, in which
TUP1
was placed under the regulation of the inducible
MET3
promoter, white- and opaque-phase cells were individually subjected to a regime in which
TUP1
was first downregulated and then upregulated. The results of this experiment demonstrated that (i) downregulation of
TUP1
led to exclusive filamentous growth in both originally white- and opaque-phase cells; (ii) the white-phase-specific gene
WH11
continued to be expressed in
TUP1
downregulated cultures originating from white-phase cells, while
WH11
expression remained repressed in
TUP1
-downregulated cultures originating from opaque-phase cells, suggesting that cells maintained phase identity in the absence of
TUP1
expression; and (iii) subsequent upregulation of
TUP1
resulted in mass conversion of originally white-phase cells to the opaque phase and maintenance of originally opaque-phase cells in the opaque phase and in the resumption in both cases of switching, suggesting that
TUP1
reexpression turns on the switching system in the opaque phase.
Pheromones Stimulate Mating and Differentiation via Paracrine and Autocrine Signaling in
Cryptococcus neoformans
Authors: Shen Wei-Chiang , Davidson Robert C. , Cox Gary M. , Heitman Joseph 
Published: December 2002

ABSTRACT

Cryptococcus neoformans
is a pathogenic fungus with a defined sexual cycle involving haploid
MAT
α and
MAT
a cells. Interestingly,
MAT
α strains are more common, are more virulent than congenic
MAT
a strains, and undergo haploid fruiting in response to nitrogen limitation or
MAT
a cells. Three genes encoding the MFα pheromone were identified in the
MAT
α mating-type locus and shown to be transcriptionally induced by limiting nutrients and coculture with
MAT
a cells. The
MFα1
,
MFα2
, and
MFα3
genes were mutated, individually and in combination.
MAT
α strains lacking MFα pheromone failed to induce morphological changes in
MAT
a cells. Pheromoneless
MAT
α mutants were fusion and mating impaired but not sterile and mated at ∼1% the wild-type level. The pheromoneless
MAT
α mutants were also partially defective in haploid fruiting, and overexpression of MFα pheromone enhanced haploid fruiting. Overexpression of MFa pheromone also enhanced haploid fruiting of
MAT
α cells and stimulated conjugation tube formation in
MAT
a cells. A conserved G-protein activated mitogen-activated protein kinase signaling pathway was found to be required for both induction and response to mating pheromones. The MFα pheromone was not essential for virulence of
C. neoformans
but does contribute to the overall virulence composite. These studies define paracrine and autocrine pheromone response pathways that signal mating and differentiation of this pathogenic fungus.
A G-Protein β Subunit Required for Sexual and Vegetative Development and Maintenance of Normal Gα Protein Levels in
Neurospora crassa
Authors: Yang Qi , Poole Sheven I. , Borkovich Katherine A. 
Published: December 2002

ABSTRACT

The genome of the filamentous fungus
Neurospora crassa
contains a single gene encoding a heterotrimeric G-protein β subunit,
gnb-1
. The predicted GNB-1 protein sequence is most identical to Gβ proteins from the filamentous fungi
Cryphonectria parasitica
and
Aspergillus nidulans
.
N. crassa
GNB-1 is also 65% identical to the human GNB-1 protein but only 38 and 45% identical to Gβ proteins from budding and fission yeasts. Previous studies in animal and fungal systems have elucidated phenotypes of Gβ null mutants, but little is known about the effects of Gβ loss on Gα levels. In this study, we analyzed a
gnb-1
deletion mutant for cellular phenotypes and levels of the three Gα proteins. Δ
gnb-1
strains are female-sterile, with production of aberrant fertilized reproductive structures. Δ
gnb-1
strains conidiate more profusely and have altered mass on solid medium. Loss of
gnb-1
leads to inappropriate conidiation and expression of a conidiation-specific gene during growth in submerged culture. Intracellular cyclic AMP levels are reduced by 60% in vegetative plate cultures of Δ
gnb-1
mutants. Loss of
gnb-1
leads to lower levels of the three Gα proteins under a variety of conditions. Analysis of transcript levels for the
gna-1
and
gna-2
Gα genes in submerged cultures indicates that regulation of Gα protein levels by
gnb-1
is posttranscriptional. The results suggest that GNB-1 directly regulates apical extension rate and mass accumulation. In contrast, many other Δ
gnb-1
phenotypes, including female sterility and defective conidiation, can be explained by altered levels of the three
N. crassa
Gα proteins.
Localization, Regulation, and Substrate Transport Properties of Bpt1p, a
Saccharomyces cerevisiae
MRP-Type ABC Transporter
Authors: Sharma Kailash Gulshan , Mason Deborah L. , Liu Guosheng , Rea Philip A. , Bachhawat Anand K. , Michaelis Susan 
Published: December 2002

ABSTRACT

Saccharomyces cerevisiae
Bpt1p is an ATP-binding cassette (ABC) protein that belongs to the MRP subfamily and is a close homologue of the glutathione conjugate (GS conjugate) transporter Ycf1p. The function of Bpt1p has previously been evaluated only in vitro, by using nonphysiological substrates. In the present study we examined the localization, regulation, and transport properties of Bpt1p in vivo, as well as its capacity to transport a set of prototypical MRP substrates in vitro. Our results show that Bpt1p, like Ycf1p, localizes to the yeast vacuolar membrane, plays a role in cadmium detoxification and
ade2
pigmentation in vivo, and can participate in the transport of GS conjugates and glucuronate conjugates, as well as free glutathione, in vitro. However, in all of these cases the contribution of Bpt1p is substantially less than that of Ycf1p. In addition, the expression patterns of
YCF1
and
BPT1
differ significantly. Whereas
YCF1
expression is markedly increased by cadmium, adenine limitation in an
ade2
strain, or overexpression of the stress-responsive transcription factor Yap1p,
BPT1
expression is only modestly affected under these conditions. Thus, although the functional capabilities of Bpt1p and Ycf1p overlap, their differences in regulation and substrate preference imply that they contribute to cellular detoxification processes in different ways.
Early Acquisition of
Pneumocystis carinii
in Neonatal Rats as Evidenced by PCR and Oral Swabs
Authors: Icenhour Crystal R. , Rebholz Sandra L. , Collins Margaret S. , Cushion Melanie T. 
Published: December 2002

ABSTRACT

The complete life cycle of
Pneumocystis carinii
has not been defined, but accumulating evidence suggests that the mammalian host may acquire this organism early in life. In the present study, the initial time of
P. carinii
acquisition was determined in rats by amplification of
P. carinii
DNA in oral swabs from seven sets of pups and dams and from fetal tissue obtained by cesarean section of three gravid female rats. DNA extracted from all samples was amplified by using PCR primers directed to the
P. carinii
mitochondrial large subunit rRNA. Amplicons were produced from 80% (28 of 35) of pups within 2 h after birth; from 97% (34 of 35) after 24 h, and in all of the serially sampled pups by 48 h. No
P. carinii
amplicons were produced from 48 fetuses or their placentae taken by cesarean section. Thus,
P. carinii
is acquired almost immediately after birth, and placental transmission occurs rarely, if ever, in rats.
The Golgi GDPase of the Fungal Pathogen
Candida albicans
Affects Morphogenesis, Glycosylation, and Cell Wall Properties
Authors: Herrero Ana B. , Uccelletti Daniela , Hirschberg Carlos B. , Dominguez Angel , Abeijon Claudia 
Published: December 2002

ABSTRACT

Cell wall mannoproteins are largely responsible for the adhesive properties and immunomodulation ability of the fungal pathogen
Candida albicans
. The outer chain extension of yeast mannoproteins occurs in the lumen of the Golgi apparatus. GDP-mannose must first be transported from the cytosol into the Golgi lumen, where mannose is transferred to mannans. GDP is hydrolyzed by a GDPase, encoded by
GDA1,
to GMP, which then exits the Golgi lumen in a coupled, equimolar exchange with cytosolic GDP-mannose. We isolated and disrupted the
C. albicans
homologue of the
Saccharomyces cerevisiae GDA1
gene in order to investigate its role in protein mannosylation and pathogenesis. CaGda1p shares four apyrase conserved regions with other nucleoside diphosphatases. Membranes prepared from the
C. albicans
disrupted
gda1
/
gda1
strain had a 90% decrease in the ability to hydrolyze GDP compared to wild type. The
gda1
/
gda1
mutants showed a severe defect in O-mannosylation and reduced cell wall phosphate content. Other cell wall-related phenotypes are present, such as elevated chitin levels and increased susceptibility to attack by β-1,3-glucanases. Our results show that the
C. albicans
organism contains β-mannose at their nonreducing end, differing from
S. cerevisiae
, which has only α-linked mannose residues in its O-glycans. Mutants lacking both alleles of
GDA1
grow at the same rate as the wild type but are partially blocked in hyphal formation in Lee solid medium and during induction in liquid by changes in temperature and pH. However, the mutants still form normal hyphae in the presence of serum and
N
-acetylglucosamine and do not change their adherence to HeLa cells. Taken together, our data are in agreement with the hypothesis that several pathways regulate the yeast-hypha transition.
Gda1
/
gda1
cells offer a model for discriminating among them.
Molecular Analysis ofCPRα, aMATα-Specific Pheromone Receptor Gene ofCryptococcus neoformans
Authors: Chung Seyung , Karos Marvin , Chang Yun C. , Lukszo Jan , Wickes Brian L. , Kwon-Chung Kyung J. 
Published: December 2002

ABSTRACTThe putativeCryptococcus neoformanspheromone receptor geneCPRαwas isolated and studied for its role in mating and filamentation.CPRαisMATαspecific and located adjacent toSTE12αat theMATαlocus. It encodes a protein which possesses high sequence similarity to the seven-transmembrane class of G-protein-coupled pheromone receptors reported for other basidiomycetous fungi. Strains containing a deletion of theCPRαgene exhibited drastic reductions in mating efficiency but were not completely sterile.Δcprα cells displayed wild-type mating efficiency when reconstituted with the wild-typeCPRαgene. Hyphal production on filament agar was not affected in theΔcprαstrain, indicating no significant role forCPRαin sensing environmental cues during haploid fruiting. The wild-typeMATα CPRαstrain produced abundant hyphae in response to syntheticMATa pheromone; however, the hyphal response to pheromone byΔcprαcells was significantly reduced. Exposure of wild-type cells to syntheticMATa pheromone for 2 h inducedMFαpheromone expression, whereas unexposed cells showed only basal levels of theMFαtranscript. TheΔcprαcells, however, exhibited only basal levels ofMFαmessage with or without pheromone exposure, suggesting thatCPRαandMFαare components of the same signaling pathway.
G-Protein Signaling Mediates Asexual Development at 25°C but Has No Effect on Yeast-Like Growth at 37°C in the Dimorphic Fungus
Penicillium marneffei
Authors: Zuber Sophie , Hynes Michael J. , Andrianopoulos Alex 
Published: December 2002

ABSTRACT

The ascomycete
Penicillium marneffei
is an opportunistic human pathogen exhibiting a temperature-dependent dimorphic switch. At 25°C,
P. marneffei
grows as filamentous multinucleate hyphae and undergoes asexual development, producing uninucleate spores. At 37°C, it forms uninucleate yeast cells which divide by fission. We have cloned a gene encoding a Gα subunit of a heterotrimeric G protein from
P. marneffei
named
gasA
with high similarity to
fadA
in
Aspergillus nidulans
. Through the characterization of a Δ
gasA
strain and mutants carrying a dominant activating or a dominant interfering
gasA
allele, we show that GasA is a key regulator of asexual development but seems to play no role in the regulation of growth. A dominant activating
gasA
mutant whose mutation results in a G42-to-R change (
gasA
G42R
) does not express
brlA
, the conidiation-specific regulatory gene, and is locked in vegetative growth, while a dominant interfering
gasA
G203R
mutant shows inappropriate
brlA
expression and conidiation. Interestingly, the
gasA
mutants have no apparent defect in dimorphic switching or yeast-like growth at 37°C. Growth tests on dibutyryl cyclic AMP (dbcAMP) and theophylline suggest that a cAMP-protein kinase A cascade may be involved in the GasA signaling pathway.
Divergent Subunit Interactions among Fungal mRNA 5′-Capping Machineries
Authors: Takagi Toshimitsu , Cho Eun-Jung , Janoo Rozmin T. K. , Polodny Vladimir , Takase Yasutaka , Keogh Michael-C. , Woo Sue-Ann , Fresco-Cohen Lucille D. , Hoffman Charles S. , Buratowski Stephen 
Published: December 2002

ABSTRACT

The
Saccharomyces cerevisiae
mRNA capping enzyme consists of two subunits: an RNA 5′-triphosphatase (RTPase) and GTP::mRNA guanylyltransferase (GTase). The GTase subunit (Ceg1) binds to the phosphorylated carboxyl-terminal domain of the largest subunit (CTD-P) of RNA polymerase II (pol II), coupling capping with transcription. Ceg1 bound to the CTD-P is inactive unless allosterically activated by interaction with the RTPase subunit (Cet1). For purposes of comparison, we characterize here the related GTases and RTPases from the yeasts
Schizosaccharomyces pombe
and
Candida albicans
. Surprisingly, the
S. pombe
capping enzyme subunits do not interact with each other. Both can independently interact with CTD-P of pol II, and the GTase is not repressed by CTD-P binding. The
S. pombe
RTPase gene (
pct1
+
) is essential for viability. Pct1 can replace the
S. cerevisiae
RTPase when GTase activity is supplied by the
S. pombe
or mouse enzymes but not by the
S. cerevisiae
GTase. The
C. albicans
capping enzyme subunits do interact with each other. However, this interaction is not essential in vivo. Our results reveal an unexpected diversity among the fungal capping machineries.
Saccharomyces cerevisiae
Cdc42p Localizes to Cellular Membranes and Clusters at Sites of Polarized Growth
Authors: Richman Tamara J. , Sawyer Mathew M. , Johnson Douglas I. 
Published: December 2002

ABSTRACT
The Cdc42p GTPase controls polarized growth and cell cycle progression in eukaryotes from yeasts to mammals, and its precise subcellular localization is essential for its function. To examine the cell cycle-specific targeting of Cdc42p in living yeast cells, a green fluorescent protein (GFP)-Cdc42 fusion protein was used. In contrast to previous immunolocalization data, GFP-Cdc42p was found at the plasma membrane around the entire cell periphery and at internal vacuolar and nuclear membranes throughout the cell cycle, and it accumulated or clustered at polarized growth sites, including incipient bud sites and mother-bud neck regions. These studies also showed that C-terminal CAAX and polylysine domains were sufficient for membrane localization but not for clustering. Time-lapse fluorescence microscopy showed that GFP-Cdc42p clustered at the incipient bud site prior to bud emergence and at the mother-bud neck region postanaphase as a diffuse, single band and persisted as two distinct bands on mother and daughter cells following cytokinesis and cell separation. Initial clustering occurred immediately prior to actomyosin ring contraction and persisted postcontraction. These results suggest that Cdc42p targeting occurs through a novel mechanism of membrane localization followed by cell cycle-specific clustering at polarized growth sites.
GTPase-Activating Proteins for Cdc42
Authors: Smith Gregory R. , Givan Scott A. , Cullen Paul , Sprague George F. 
Published: December 2002

ABSTRACT

The Rho-type GTPase, Cdc42, has been implicated in a variety of functions in the yeast life cycle, including septin organization for cytokinesis, pheromone response, and haploid invasive growth. A group of proteins called GTPase-activating proteins (GAPs) catalyze the hydrolysis of GTP to GDP, thereby inactivating Cdc42. At the time this study began, there was one known GAP, Bem3, and one putative GAP, Rga1, for Cdc42. We identified another putative GAP for Cdc42 and named it Rga2 (Rho GTPase-activating protein 2). We confirmed by genetic and biochemical criteria that Rga1, Rga2, and Bem3 act as GAPs for Cdc42. A detailed characterization of Rga1, Rga2, and Bem3 suggested that they regulate different subsets of Cdc42 function. In particular, deletion of the individual GAPs conferred different phenotypes. For example, deletion of
RGA1
, but not
RGA2
or
BEM3
, caused hyperinvasive growth. Furthermore, overproduction or loss of Rga1 and Rga2, but not Bem3, affected the two-hybrid interaction of Cdc42 with Ste20, a p21-activated kinase (PAK) kinase required for haploid invasive growth. These results suggest Rga1, and possibly Rga2, facilitate the interaction of Cdc42 with Ste20 to mediate signaling in the haploid invasive growth pathway. Deletion of
BEM3
resulted in cells with severe morphological defects not observed in
rga1
Δ or
rga2
Δ strains. These data suggest that Bem3 and, to a lesser extent, Rga1 and Rga2 facilitate the role of Cdc42 in septin organization. Thus, it appears that the GAPs play a role in modulating specific aspects of Cdc42 function. Alternatively, the different phenotypes could reflect quantitative rather than qualitative differences in GAP activity in the mutant strains.