bluuebunny/crossref_metadata_2025 huggingface dataset:
Yeast Rpi1 Is a Putative Transcriptional Regulator That Contributes to Preparation for Stationary Phase
Authors: Sobering Andrew K. , Jung Un Sung , Lee Kyung S. , Levin David E.
Published: December 2002

The
RPI1
gene of
Saccharomyces cerevisiae
was identified initially as a dosage suppressor of the heat shock sensitivity associated with overexpression of
RAS2
(J. Kim and S. Powers, Mol. Cell. Biol. 11:3894–3904, 1991). Based on its failure to suppress mutationally activated
RAS2
,
RPI1
was proposed to be a negative regulator of the Ras/cyclic AMP (cAMP) pathway that functions at a point upstream of Ras. We isolated
RPI1
as a high-copy-number suppressor of the cell lysis defect associated with a null mutation in the
MPK1
gene, which encodes the mitogen-activated protein kinase of the cell wall integrity-signaling pathway. Although the sequence of Rpi1 is not informative about its function, we present evidence that this protein resides in the nucleus, possesses a transcriptional activation domain, and affects the mRNA levels of several cell wall metabolism genes. In contrast to the previous report, we found that
RPI1
overexpression suppresses defects associated with mutational hyperactivation of the Ras/cAMP pathway at all points including constitutive mutations in the cAMP-dependent protein kinase. We present additional genetic and biochemical evidence that Rpi1 functions independently of and in opposition to the Ras/cAMP pathway to promote preparations for the stationary phase. Among these preparations is a fortification of the cell wall that is antagonized by Ras pathway activity. This observation reveals a novel link between the Ras/cAMP pathway and cell wall integrity. Finally, we propose that inappropriate expression of
RPI1
during log phase growth drives fortification of the cell wall and that this behavior is responsible for suppression of the
mpk1
cell lysis defect.
Adenylyl Cyclase Functions Downstream of the Gα Protein Gpa1 and Controls Mating and Pathogenicity of
Cryptococcus neoformans
Authors: Alspaugh J. Andrew , Pukkila-Worley Read , Harashima Toshiaki , Cavallo Lora M. , Funnell Deanna , Cox Gary M. , Perfect John R. , Kronstad James W. , Heitman Joseph
Published: December 2002

The signaling molecule cyclic AMP (cAMP) is a ubiquitous second messenger that enables cells to detect and respond to extracellular signals. cAMP is generated by the enzyme adenylyl cyclase, which is activated or inhibited by the Gα subunits of heterotrimeric G proteins in response to ligand-activated G-protein-coupled receptors. Here we identified the unique gene (
CAC1
) encoding adenylyl cyclase in the opportunistic fungal pathogen
Cryptococcus neoformans
. The
CAC1
gene was disrupted by transformation and homologous recombination. In stark contrast to the situation for
Saccharomyces cerevisiae
, in which adenylyl cyclase is essential,
C. neoformans cac1
mutant strains were viable and had no vegetative growth defect. Furthermore,
cac1
mutants maintained the yeast-like morphology of wild-type cells, in contrast to the constitutively filamentous phenotype found upon the loss of adenylyl cyclase in another basidiomycete pathogen,
Ustilago maydis
. Like
C. neoformans
mutants lacking the Gα protein Gpa1,
cac1
mutants were mating defective and failed to produce two inducible virulence factors: capsule and melanin. As a consequence,
cac1
mutant strains were avirulent in animal models of cryptococcal meningitis. Reintroduction of the wild-type
CAC1
gene or the addition of exogenous cAMP suppressed
cac1
mutant phenotypes. Moreover, the overexpression of adenylyl cyclase restored mating and virulence factor production in
gpa1
mutant strains. Physiological studies revealed that the Gα protein Gpa1 and adenylyl cyclase controlled cAMP production in response to glucose, and no cAMP was detectable in extracts from
cac1
or
gpa1
mutant strains. These findings provide direct evidence that Gpa1 and adenylyl cyclase function in a conserved signal transduction pathway controlling cAMP production, hyphal differentiation, and virulence of this human fungal pathogen.
Isolation and Characterization of Two Ammonium Permease Genes,
meaA
and
mepA
, from
Aspergillus nidulans
Authors: Monahan Brendon J. , Fraser James A. , Hynes Michael J. , Davis Meryl A.
Published: December 2002

Ammonium and the analogue methylammonium are taken into the cell by active transport systems which constitute a family of transmembrane proteins that have been identified in fungi, bacteria, plants, and animals. Two genes from
Aspergillus
nidulans
,
mepA
and
meaA
, which encode ammonium transporters with different affinities have been characterized. The MepA transporter exhibits the highest affinity for methylammonium (
K

m
0

, 44.3 μM); in comparison, the
K

m

for MeaA is 3.04 mM. By use of targeted gene replacement strategies,
meaA
and
mepA
deletion mutants were created. Deletion of both
meaA
and
mepA
resulted in the inability of the strain to grow on ammonium concentrations of less than 10 mM. The single
meaA
deletion mutant exhibited reduced growth at the same concentrations, whereas the
mepA
deletion mutant displayed wild-type growth. Interestingly, multiple copies of
mepA
were found to complement the methylammonium resistance phenotype conferred by the deletion of
meaA
. The expression profiles for
mepA
and
meaA
differed; the
mepA
transcript was detected only in nitrogen-starved cultures, whereas
meaA
was expressed under both ammonium-sufficient and nitrogen starvation conditions. Together, these results indicate that MeaA constitutes the major ammonium transport activity and is required for the optimal growth of
A
.
nidulans
on ammonium as the sole nitrogen source and that MepA probably functions in scavenging low concentrations of ammonium under nitrogen starvation conditions.
CDC42 Is Required for Polarized Growth in Human Pathogen
Candida albicans
Authors: Ushinsky Sophia C. , Harcus Doreen , Ash Josee , Dignard Daniel , Marcil Anne , Morchhauser Joachim , Thomas David Y. , Whiteway Malcolm , Leberer Ekkehard
Published: December 2002

Cdc42p is a member of the RAS superfamily of GTPases and plays an essential role in polarized growth in many eukaryotic cells. We cloned the
Candida albicans CaCDC42
by functional complementation in
Saccharomyces cerevisiae
and analyzed its function in
C. albicans
. A double deletion of
CaCDC42
was made in a
C. albicans
strain containing
CaCDC42
under the control of the
PCK1
promoter. When expression of the heterologous copy of
CaCDC42
was repressed in this strain, the cells ceased proliferation. These arrested cells were large, round, and unbudded and contained predominantly two nuclei. The
PCK1
-mediated overexpression of wild-type CaCdc42p had no effect on cells. However, in cells overexpressing CaCdc42p containing the dominant-negative D118A substitution, proliferation was blocked and the arrested cells were large, round, unbudded, and multinucleated, similar to the phenotype of the
cdc42
double-deletion strain. Cells overexpressing CaCdc42p containing the hyperactive G12V substitution also ceased proliferation in yeast growth medium; in this case the arrested cells were multinucleated and multibudded. An intact CAAX box is essential for the phenotypes associated with either CaCdc42p
G12V
or CaCdc42p
D118A
ectopic expression, suggesting that membrane attachment is involved in CaCdc42p function. In addition, the lethality caused by ectopic expression of CaCdc42p
G12V
was suppressed by deletion of
CST20
but not by deletion of
CaCLA4
. CaCdc42p function was also examined under hypha-inducing conditions. Cdc42p depletion prior to hyphal induction trapped cells in a round, unbudded state, while depletion triggered at the same time as hyphal induction permitted the initiation of germ tubes that failed to be extended. Ectopic expression of either the G12V or D118A substitution protein modified hyphal formation in a CAAX box-dependent manner. Thus, CaCdc42p function appears important for polarized growth of both the yeast and hyphal forms of
C. albicans
.
Heat Stress Activates the Yeast High-Osmolarity Glycerol Mitogen-Activated Protein Kinase Pathway, and Protein Tyrosine Phosphatases Are Essential under Heat Stress
Authors: Winkler Astrid , Arkind Christopher , Mattison Christopher P. , Burkholder Anne , Knoche Kathryn , Ota Irene
Published: December 2002

The yeast high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway has been characterized as being activated solely by osmotic stress. In this work, we show that the Hog1 MAPK is also activated by heat stress and that Sho1, previously identified as a membrane-bound osmosensor, is required for heat stress activation of Hog1. The two-component signaling protein, Sln1, the second osmosensor in the HOG pathway, was not involved in heat stress activation of Hog1, suggesting that the Sho1 and Sln1 sensors discriminate between stresses. The possible function of Hog1 activation during heat stress was examined, and it was found that the
hog1
Δ strain does not recover as rapidly from heat stress as well as the wild type. It was also found that protein tyrosine phosphatases (PTPs) Ptp2 and Ptp3, which inactivate Hog1, have two functions during heat stress. First, they are essential for survival at elevated temperatures, preventing lethality due to Hog1 hyperactivation. Second, they block inappropriate cross talk between the HOG and the cell wall integrity MAPK pathways, suggesting that PTPs are important for maintaining specificity in MAPK signaling pathways.
Altered Phosphotransfer in an Activated Mutant of the
Saccharomyces cerevisiae
Two-Component Osmosensor Sln1p
Authors: Ault A. D. , Fassler J. S. , Deschenes R. J.
Published: December 2002

The
SLN1
two-component signaling pathway of
Saccharomyces cerevisiae
utilizes a multistep phosphorelay mechanism to control osmotic stress responses via the
HOG1
mitogen-activated protein kinase pathway and the transcription factor Skn7p. Sln1p consists of a sensor kinase module that undergoes histidine autophosphorylation and a receiver module that autocatalytically transfers the phosphoryl group from histidine to aspartate. The Sln1p aspartyl phosphate is then transferred to Ypd1p, which in turn transfers the phosphoryl group to a conserved aspartate on one of two response regulators, Ssk1p and Skn7p. Activated alleles of
SLN1
(
sln1*
) were previously identified that appear to increase the level of phosphorylation of downstream targets Ssk1p and Skn7p. In principle, the phenotype of
sln1
* alleles could arise from an increase in autophosphorylation or phosphotransfer activities or a decrease in an intrinsic or extrinsic dephosphorylation activity. Genetic analysis of the activated mutants has been unable to distinguish between these possibilities. In this report, we address this issue by analyzing phosphorelay and phosphohydrolysis reactions involving the Sln1p-associated receiver. The results are consistent with a model in which the activated phenotype of the
sln1
* allele,
sln1
-
22
, arises from a shift in the phosphotransfer equilibrium from Sln1p to Ypd1p, rather than from impaired dephosphorylation of the system in response to osmotic stress.
Evidence for Lateral Transfer of Genes Encoding Ferredoxins, Nitroreductases, NADH Oxidase, and Alcohol Dehydrogenase 3 from Anaerobic Prokaryotes to
Giardia
lamblia
and
Entamoeba
histolytica
Authors: Nixon Julie E. J. , Wang Amy , Field Jessica , Morrison Hilary G. , McArthur Andrew G. , Sogin Mitchell L. , Loftus Brendan J. , Samuelson John
Published: December 2002

Giardia
lamblia
and
Entamoeba
histolytica
are amitochondriate, microaerophilic protists which use fermentation enzymes like those of bacteria to survive anaerobic conditions within the intestinal lumen. Genes encoding fermentation enzymes and related electron transport peptides (e.g., ferredoxins) in giardia organisms and amebae are hypothesized to be derived from either an ancient anaerobic eukaryote (amitochondriate fossil hypothesis), a mitochondrial endosymbiont (hydrogen hypothesis), or anaerobic bacteria (lateral transfer hypothesis). The goals here were to complete the molecular characterization of giardial and amebic fermentation enzymes and to determine the origins of the genes encoding them, when possible. A putative giardia [2Fe-2S]ferredoxin which had a hypothetical organelle-targeting sequence at its N terminus showed similarity to mitochondrial ferredoxins and the hydrogenosomal ferredoxin of
Trichomonas
vaginalis
(another luminal protist). However, phylogenetic trees were star shaped, with weak bootstrap support, so we were unable to confirm or rule out the endosymbiotic origin of the giardia [2Fe-2S]ferredoxin gene. Putative giardial and amebic 6-kDa ferredoxins, ferredoxin-nitroreductase fusion proteins, and oxygen-insensitive nitroreductases each tentatively supported the lateral transfer hypothesis. Although there were not enough sequences to perform meaningful phylogenetic analyses, the unique common occurrence of these peptides and enzymes in giardia organisms, amebae, and the few anaerobic prokaryotes suggests the possibility of lateral transfer. In contrast, there was more robust phylogenetic evidence for the lateral transfer of
G
.
lamblia
genes encoding an NADH oxidase from a gram-positive coccus and a microbial group 3 alcohol dehydrogenase from thermoanaerobic prokaryotes. In further support of lateral transfer, the
G
.
lamblia
NADH oxidase and
adh3
genes appeared to have an evolutionary history distinct from those of
E
.
histolytica
.
The Two Nuclei ofGiardiaEach Have Complete Copies of the Genome and Are Partitioned Equationally at Cytokinesis
Authors: Yu Li Zhi , Birky C. William , Adam Rodney D.
Published: December 2002

Giardia lambliais medically important as a cause of diarrhea and malabsorption throughout the world and is thought to be one of the earliest-branching eukaryotes on a phylogenetic tree. Nevertheless, the mechanisms of inheritance are largely unknown. The trophozoites ofGiardiaand other diplomonads are interesting in their possession of two nuclei that are identical or similar in several respects. They replicate at nearly the same time, have similar quantities of DNA, and are both transcriptionally active. We used fluorescence in situ hybridization to demonstrate that genes from each of the five chromosomes are found in both nuclei, confirming that each nucleus has at least one complete copy of the genome. This raises a second question. The alleles of a gene in different nuclei are expected to accumulate different mutations, but surprisingly, the degree of heterozygosity in a clone is very low. One possible mechanism for eliminating sequence differences between nuclei is that each daughter cell receives two copies of the same nucleus at cell division. We used trophozoites with a plasmid transfected into a single nucleus to demonstrate that the two nuclei are partitioned equationally at cytokinesis. The mechanism(s) by which homozygosity is maintained will require further investigation.
Involvement of
RAD9
-Dependent Damage Checkpoint Control in Arrest of Cell Cycle, Induction of Cell Death, and Chromosome Instability Caused by Defects in Origin Recognition Complex in
Saccharomyces cerevisiae
Authors: Watanabe Keiichi , Morishita Jun , Umezu Keiko , Shirahige Katsuhiko , Maki Hisaji
Published: December 2002

Perturbation of origin firing in chromosome replication is a possible cause of spontaneous chromosome instability in multireplicon organisms. Here, we show that chromosomal abnormalities, including aneuploidy and chromosome rearrangement, were significantly increased in yeast diploid cells with defects in the origin recognition complex. The cell cycle of
orc1
-
4
/
orc1
-
4
temperature-sensitive mutant was arrested at the G
2
/M boundary, after several rounds of cell division at the restrictive temperature. However, prolonged incubation of the mutant cells at 37°C led to abrogation of G
2
arrest, and simultaneously the cells started to lose viability. A sharp increase in chromosome instability followed the abrogation of G
2
arrest. In
orc1
-
4
/
orc1
-
4 rad9
Δ/
rad9
Δ diploid cells grown at 37°C, G
2
arrest and induction of cell death were suppressed, while chromosome instability was synergistically augmented. These findings indicated that DNA lesions caused by a defect in Orc1p function trigger the
RAD9
-dependent checkpoint control, which ensures genomic integrity either by stopping the cell cycle progress until lesion repair, or by inducing cell death when the lesion is not properly repaired. At semirestrictive temperatures,
orc2
-
1
/
orc2
-
1
diploid cells demonstrated G
2
arrest and loss of cell viability, both of which require
RAD9
-dependent checkpoint control. However, chromosome instability was not induced in
orc2
-
1
/
orc2
-
1
cells, even in the absence of the checkpoint control. These data suggest that once cells lose the damage checkpoint control, perturbation of origin firing can be tolerated by the cells. Furthermore, although a reduction in origin-firing capacity does not necessarily initiate chromosome instability, the Orc1p possesses a unique function, the loss of which induces instability in the chromosome.
Myosin I Is Required for Hypha Formation in
Candida
albicans
Authors: Oberholzer U. , Marcil A. , Leberer E. , Thomas D. Y. , Whiteway M.
Published: December 2002

The pathogenic yeast
Candida
albicans
can undergo a dramatic change in morphology from round yeast cells to long filamentous cells called hyphae. We have cloned the
CaMYO5
gene encoding the only myosin I in
C
.
albicans
. A strain with a deletion of both copies of
CaMYO5
is viable but cannot form hyphae under all hypha-inducing conditions tested. This mutant exhibits a higher frequency of random budding and a depolarized distribution of cortical actin patches relative to the wild-type strain. We found that polar budding, polarized localization of cortical actin patches, and hypha formation are dependent on a specific phosphorylation site on myosin I, called the “TEDS-rule” site. Mutation of this serine 366 to alanine gives rise to the null mutant phenotype, while a S366D mutation, the product of which mimics a phosphorylated serine, allows hypha formation. However, the S366D mutation still causes a depolarized distribution of cortical actin patches in budding cells, similar to that in the null mutant. The localization of CaMyo5-GFP together with cortical actin patches at the bud and hyphal tips is also dependent on serine 366. Intriguingly, the cortical actin patches in the majority of the hyphae of the mutant expressing

Camyo5
S366D

were depolarized, suggesting that although their distribution is dependent on myosin I localization, polarized cortical actin patches may not be required for hypha formation.
Nnf1p, Dsn1p, Mtw1p, and Nsl1p: a New Group of Proteins Important for Chromosome Segregation in
Saccharomyces cerevisiae
Authors: Euskirchen Ghia M.
Published: December 2002

Previously, antibodies were raised against a nuclear envelope-enriched fraction of yeast, and the essential gene
NNF1
was cloned by reverse genetics. Here it is shown that the conditional
nnf1
-
17
mutant has decreased stability of a minichromosome in addition to mitotic spindle defects. I have identified the novel essential genes
DSN1, DSN3
, and
NSL1
through genetic interactions with
nnf1
-
17
. Dsn3p was found to be equivalent to the kinetochore protein Mtw1p. By indirect immunofluorescence, all four proteins, Nnf1p, Mtw1p, Dsn1p, and Nsl1p, colocalize and are found in the region of the spindle poles. Based on the colocalization of these four proteins, the minichromosome instability and the spindle defects seen in
nnf1
mutants, I propose that Nnf1p is part of a new group of proteins necessary for chromosome segregation.
Aspergillus
nidulans
swoF
Encodes an
N
-Myristoyl Transferase
Authors: Shaw Brian D. , Momany Cory , Momany Michelle
Published: December 2002

Polar growth is a fundamental process in filamentous fungi and is necessary for disease initiation in many pathogenic systems. Previously,
swoF
was identified in
Aspergillus
nidulans
as a single-locus, temperature-sensitive (
ts
) mutant aberrant in both polarity establishment and polarity maintenance. The
swoF
gene was cloned by complementation of the
ts
phenotype and sequenced. The derived protein sequence had high identity with
N
-myristoyl transferases (NMTs) found in fungi, plants, and animals. In addition, wild-type growth at restrictive temperature was partially restored by the addition of myristic acid to the growth medium. Sequencing revealed that the mutation in
swoF
changes the conserved aspartic acid 369 to a tyrosine. The predicted
A
.
nidulans
SwoF protein, SwoFp, was homology modeled based on crystal structures of NMTs from
Saccharomyces
cerevisiae
and
Candida
albicans
. The D369Y
swoF
mutation is on the opposite face of the protein, distal to the myristoyl coenzyme A and peptide substrate binding sites. In wild-type NMTs, D369 appears to stabilize a structural β-strand bend through two hydrogen bonds and an ionic interaction. These stabilizing bonds are abolished in the D369Y mutant. We hypothesize that a substrate of SwoFp must be myristoylated for proper polarity establishment and maintenance. The mutation prevents the proper function of SwoFp at restrictive temperature and thus blocks polar growth.
The Mold-Specific
MS8
Gene Is Required for Normal Hypha Formation in the Dimorphic Pathogenic Fungus
Histoplasma capsulatum
Authors: Tian Xianbin , Shearer Glenmore
Published: December 2002

The dimorphic fungus
Histoplasma capsulatum
is the etiologic agent of one of the most common systemic mycoses of humans, histoplasmosis. In the environment,
H. capsulatum
grows in a differentiated mold form and shifts to an undifferentiated yeast form after mold fragments or spores are inhaled. This mold-to-yeast shift is required for disease. Little is known about the molecular biology of dimorphism in
Histoplasma
, and most studies have been directed toward yeast-specific genes. While it is important to examine the role of genes upregulated in the yeast morphotype, genes which are silenced in the yeast (i.e., mold-specific genes) may also play a critical role in dimorphism. To begin to examine this hypothesis, we report here the first misexpression and knockout analysis of a mold-specific gene in
Histoplasma
. The strongly expressed
MS8
gene encodes a predicted 21-kDa protein extremely rich in glycine and glutamine. Forced expression of
MS8
driven by the
TEF1
promoter in yeast did not alter the yeast morphology at 37°C or mold formation at 25°C. Yeast expressing
MS8
did exhibit clumping in liquid medium and formed “sticky” colonies on agar plates. Allelic replacement of
MS8
was accomplished by a positive-negative selection procedure.
ms8
knockout mutants formed apparently normal yeast at 37°C but gave rise to aberrant mycelia at 25°C. The mold colonies of the knockouts were less than half as large as normal, had a granular surface, produced a dark-red pigment, and formed short hyphae which were 40% wider with a distinctive twisted “zig-zag” shape.
Mating-Type-Specific and Nonspecific PAK Kinases Play Shared and Divergent Roles in
Cryptococcus neoformans
Authors: Wang Ping , Nichols Connie B. , Lengeler Klaus B. , Cardenas Maria E. , Cox Gary M. , Perfect John R. , Heitman Joseph
Published: December 2002

Cryptococcus neoformans
is an opportunistic fungal pathogen with a defined sexual cycle involving fusion of haploid
MAT
α and
MAT
a cells. Virulence has been linked to the mating type, and
MAT
α cells are more virulent than congenic
MAT
a cells. To study the link between the mating type and virulence, we functionally analyzed three genes encoding homologs of the p21-activated protein kinase family:
STE20
α,
STE20
a, and
PAK1
. In contrast to the
STE20
genes that were previously shown to be in the mating-type locus, the
PAK1
gene is unlinked to the mating type. The
STE20
α,
STE20
a, and
PAK1
genes were disrupted in serotype A and D strains of
C. neoformans
, revealing central but distinct roles in mating, differentiation, cytokinesis, and virulence.
ste20
α
pak1
and
ste20
a
pak1
double mutants were synthetically lethal, indicating that these related kinases share an essential function. In summary, our studies identify an association between the
STE20
α gene, the
MAT
α locus, and virulence in a serotype A clinical isolate and provide evidence that PAK kinases function in a MAP kinase signaling cascade controlling the mating, differentiation, and virulence of this fungal pathogen.
Distinct Signaling Pathways from the Circadian Clock Participate in Regulation of Rhythmic Conidiospore Development in
Neurospora crassa
Authors: Correa Alejandro , Bell-Pedersen Deborah
Published: December 2002

Several different environmental signals can induce asexual spore development (conidiation) and expression of developmentally regulated genes in
Neurospora crassa
. However, under constant conditions, where no environmental cues for conidiation are present, the endogenous circadian clock in
N. crassa
promotes daily rhythms in expression of known developmental genes and of conidiation. We anticipated that the same pathway of gene regulation would be followed during clock-controlled conidiation and environmental induction of conidiation and that the circadian clock would need only to control the initial developmental switch. Previous experiments showed that high-level developmental induction of the clock-controlled genes
eas
(
ccg-2
) and
ccg-1
requires the developmental regulatory proteins FL and ACON-2, respectively, and normal developmental induction of
fl
mRNA expression requires ACON-2. We demonstrate that the circadian clock regulates rhythmic
fl
gene expression and that
fl
rhythmicity requires ACON-2. However, we find that clock regulation of
eas
(
ccg-2
) is normal in an
fl
mutant strain and
ccg-1
expression is rhythmic in an
acon-2
mutant strain. Together, these data point to the endogenous clock and the environment following separate pathways to regulate conidiation-specific gene expression.
Outside-In Signaling of Cellulose Synthesis by a Spore Coat Protein in
Dictyostelium
Authors: West Christopher M. , Zhang Ping , McGlynn Aiko C. , Kaplan Lee
Published: December 2002

The spore coat of
Dictyostelium
is formed de novo from proteins secreted from vesicles and cellulose synthesized across the plasma membrane as differentiating spores rise up the stalk. The mechanism by which these events are coordinated is not understood. In the course of experiments designed to test the function of the inner layer coat protein SP85 (PsB), expression of a specific partial length fragment was found to interrupt coat assembly after protein secretion and prior to cellulose synthesis in 85% of the cells. This fragment consisted of SP85's N-terminal domain, containing prespore vesicle targeting information, and its Cys-rich C1 domain. The effect of the NC1 fusion was not cell autonomous in interstrain chimeras, suggesting that it acted at the cell surface. SP85-null spores presented an opposite phenotype in which spores differentiated prematurely before reaching the top of the stalk, and cellulose was slightly overproduced in a disorganized fashion. A similar though less severe phenotype occurred when a fusion of the N and C2 domains was expressed. In a double mutant, absence of SP85 was epistatic to NC1 expression, suggesting that NC1 inhibited SP85 function. Together, these results suggest the existence of an outside-in signaling pathway that constitutes a checkpoint to ensure that cellulose synthesis does not occur until coat proteins are properly organized at the cell surface and stalk formation is complete. Checkpoint execution is proposed to be regulated by SP85, which is in turn under the influence of other coat proteins that interact with SP85 via its C1 and C2 domains.
Role of Histone Deacetylation in Developmentally Programmed DNA Rearrangements in
Tetrahymena thermophila
Authors: Duharcourt Sandra , Yao Meng-Chao
Published: December 2002

In
Tetrahymena
, as in other ciliates, development of the somatic macronucleus during conjugation involves extensive and reproducible rearrangements of the germ line genome, including chromosome fragmentation and excision of internal eliminated sequences (IESs). The molecular mechanisms controlling these events are poorly understood. To investigate the role that histone acetylation may play in the regulation of these processes, we treated
Tetrahymena
cells during conjugation with the histone deacetylase inhibitor trichostatin A (TSA). We show that TSA treatment induces developmental arrests in the early stages of conjugation but does not significantly affect the progression of conjugation once the mitotic divisions of the zygotic nucleus have occurred. Progeny produced from TSA-treated cells were examined for effects on IES excision and chromosome breakage. We found that TSA treatment caused partial inhibition of excision of five out of the six IESs analyzed but did not affect chromosome breakage at four different sites. TSA treatment greatly delayed in some cells and inhibited in most the excision events in the developing macronucleus. It also led to loss of the specialized subnuclear localization of the chromodomain protein Pdd1p that is normally associated with DNA elimination. We propose a model in which underacetylated nucleosomes mark germ line-limited sequences for excision.
Evolutionary Analyses of the Small Subunit of Glutamate Synthase: Gene Order Conservation, Gene Fusions, and Prokaryote-to- Eukaryote Lateral Gene Transfers
Authors: Andersson Jan O. , Roger Andrew J.
Published: December 2002

Lateral gene transfer has been identified as an important mode of genome evolution within prokaryotes. Except for the special case of gene transfer from organelle genomes to the eukaryotic nucleus, only a few cases of lateral gene transfer involving eukaryotes have been described. Here we present phylogenetic and gene order analyses on the small subunit of glutamate synthase (encoded by
gltD
) and its homologues, including the large subunit of sulfide dehydrogenase (encoded by
sudA
). The scattered distribution of the
sudA
and
sudB
gene pair and the phylogenetic analysis strongly suggest that lateral gene transfer was involved in the propagation of the genes in the three domains of life. One of these transfers most likely occurred between a prokaryote and an ancestor of diplomonad protists. Furthermore, phylogenetic analyses indicate that the gene for the small subunit of glutamate synthase was transferred from a low-GC gram-positive bacterium to a common ancestor of animals, fungi, and plants. Interestingly, in both examples, the eukaryotes encode a single gene that corresponds to a conserved operon structure in prokaryotes. Our analyses, together with several recent publications, show that lateral gene transfers from prokaryotes to unicellular eukaryotes occur with appreciable frequency. In the case of the genes for sulfide dehydrogenase, the transfer affected only a limited group of eukaryotes—the diplomonads—while the transfer of the glutamate synthase gene probably happened earlier in evolution and affected a wider range of eukaryotes.
Molecular Characterization of a Cystathionine Beta-Synthase Gene,
CBS1
, in
Magnaporthe
grisea
Authors: Lo Sze Chung Clive , Hamer Lisbeth , Hamer John E.
Published: December 2002

CBS1
from
Magnaporthe
grisea
is a structural and functional homolog of the cystathionine β-synthase (CBS) gene from
Saccharomyces
cerevisiae
. Our studies indicated that
M
.
grisea
can utilize homocysteine and methionine through a CBS-independent pathway. The results also revealed responses of
M
.
grisea
to homocysteine that are reminiscent of human homocystinuria.
Toxoplasma gondii
Cyclic GMP-Dependent Kinase: Chemotherapeutic Targeting of an Essential Parasite Protein Kinase
Authors: Donald Robert G. K. , Allocco John , Singh Suresh B. , Nare Bakela , Salowe Scott P. , Wiltsie Judyann , Liberator Paul A.
Published: December 2002

The trisubstituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl]pyridine (compound 1) has in vivo activity against the apicomplexan parasites
Toxoplasma gondii
and
Eimeria tenella
in animal models. The presumptive molecular target of this compound in
E. tenella
is cyclic GMP-dependent protein kinase (PKG). Native PKG purified from
T. gondii
has kinetic and pharmacologic properties similar to those of the
E. tenella
homologue, and both have been functionally expressed as recombinant proteins in
T. gondii
. Computer modeling of parasite PKG was used to predict catalytic site amino acid residues that interact with compound 1. The recombinant laboratory-generated mutants
T. gondii
PKG T761Q or T761M and the analogous
E. tenella
T770 alleles have reduced binding affinity for, and are not inhibited by, compound 1. By all other criteria, PKG with this class of catalytic site substitution is indistinguishable from wild-type enzyme. A genetic disruption of
T. gondii
PKG can only be achieved if a complementing copy of PKG is provided in
trans
, arguing that PKG is an essential protein. Strains of
T. gondii
, disrupted at the genomic PKG locus and dependent upon the
T. gondii
T761-substituted PKGs, are as virulent as wild type in mice. However, unlike mice infected with wild-type
T. gondii
that are cured by compound 1, mice infected with the laboratory-generated strains of
T. gondii
do not respond to treatment. We conclude that PKG represents the primary molecular target responsible for the antiparasitic efficacy of compound 1.
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

Asexual 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

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

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

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

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

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

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

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.