bluuebunny/crossref_metadata_2025 huggingface dataset:
Copper-Dependent Iron Assimilation Pathway in the Model Photosynthetic EukaryoteChlamydomonas reinhardtii
Authors: La Fontaine Sharon , Quinn Jeanette M. , Nakamoto Stacie S. , Page M. Dudley , Göhre Vera , Moseley Jeffrey L. , Kropat Janette , Merchant Sabeeha
Published: December 2002

The unicellular green algaChlamydomonas reinhardtiiis a valuable model for studying metal metabolism in a photosynthetic background. A search of theChlamydomonasexpressed sequence tag database led to the identification of several components that form a copper-dependent iron assimilation pathway related to the high-affinity iron uptake pathway defined originally forSaccharomyces cerevisiae. They include a multicopper ferroxidase (encoded byFox1), an iron permease (encoded byFtr1), a copper chaperone (encoded byAtx1), and a copper-transporting ATPase. A cDNA,Fer1, encoding ferritin for iron storage also was identified. Expression analysis demonstrated thatFox1andFtr1were coordinately induced by iron deficiency, as wereAtx1andFer1, although to lesser extents. In addition, Fox1 abundance was regulated at the posttranscriptional level by copper availability. Each component exhibited sequence relationship with its yeast, mammalian, or plant counterparts to various degrees; Atx1 ofC. reinhardtiiis also functionally related with respect to copper chaperone and antioxidant activities. Fox1 is most highly related to the mammalian homologues hephaestin and ceruloplasmin; its occurrence and pattern of expression inChlamydomonasindicate, for the first time, a role for copper in iron assimilation in a photosynthetic species. Nevertheless, growth ofC. reinhardtiiunder copper- and iron-limiting conditions showed that, unlike the situation in yeast and mammals, where copper deficiency results in a secondary iron deficiency, copper-deficientChlamydomonascells do not exhibit symptoms of iron deficiency. We propose the existence of a copper-independent iron assimilation pathway in this organism.
mcl1
+

, the
Schizosaccharomyces pombe
Homologue of
CTF4
, Is Important for Chromosome Replication, Cohesion, and Segregation
Authors: Williams Dewight R. , McIntosh J. Richard
Published: December 2002

The fission yeast minichromosome loss mutant
mcl1-1
was identified in a screen for mutants defective in chromosome segregation. Missegregation of the chromosomes in
mcl1-1
mutant cells results from decreased centromeric cohesion between sister chromatids.

mcl1
+

encodes a β-transducin-like protein with similarity to a family of eukaryotic proteins that includes Ctf4p from
Saccharomyces cerevisiae
, sepB from
Aspergillus nidulans
, and AND-1 from humans. The previously identified fungal members of this protein family also have chromosome segregation defects, but they primarily affect DNA metabolism. Chromosomes from
mcl1-1
cells were heterogeneous in size or structure on pulsed-field electrophoresis gels and had elongated heterogeneous telomeres.
mcl1-1
was lethal in combination with the DNA checkpoint mutations
rad3Δ
and
rad26Δ
, demonstrating that loss of Mcl1p function leads to DNA damage.
mcl1-1
showed an acute sensitivity to DNA damage that affects S-phase progression. It interacts genetically with replication components and causes an S-phase delay when overexpressed. We propose that Mcl1p, like Ctf4p, has a role in regulating DNA replication complexes.
Eng1p, an Endo-1,3-β-Glucanase Localized at the Daughter Side of the Septum, Is Involved in Cell Separation in
Saccharomyces cerevisiae
Authors: Baladrón Victoriano , Ufano Sandra , Dueñas Encarnación , Martín-Cuadrado Ana Belén , del Rey Francisco , Vázquez de Aldana Carlos R.
Published: December 2002

ENG1
(
YNR067c
), a gene encoding a new endo-1,3-β-glucanase, was cloned by screening a genomic library with a DNA probe obtained by PCR with synthetic oligonucleotides designed according to conserved regions found between yeast exo-1,3-β-glucanases (Exg1p, Exg2p, and Ssg1p). Eng1p shows strong sequence similarity to the product of the
Saccharomyces cerevisiae ACF2
gene, involved in actin assembly “in vitro,” and to proteins present in other yeast and fungal species. It is also related to plant glucan-binding elicitor proteins, which trigger the onset of a defense response upon fungal infection. Eng1p and Acf2p/Eng2p are glucan-hydrolyzing proteins that specifically act on 1,3-β linkages, with an endolytic mode of action. Eng1p is an extracellular, heavily glycosylated protein, while Acf2p/Eng2p is an intracellular protein with no carbohydrate linked by N-glycosidic bonds.
ENG1
transcription fluctuates periodically during the cell cycle; maximal accumulation occurs during the M/G
1
transition and is dependent on the transcription factor Ace2p. Interestingly,
eng1
deletion mutants show defects in cell separation, and Eng1p localizes asymmetrically to the daughter side of the septum, suggesting that this protein is involved, together with chitinase, in the dissolution of the mother-daughter septum.
A Forkhead Transcription Factor Is Important for True Hyphal as well as Yeast Morphogenesis in
Candida albicans
Authors: Bensen Eric S. , Filler Scott G. , Berman Judith
Published: December 2002

Candida albicans
is an important pathogen of immunocompromised patients which grows with true hyphal, pseudohyphal, and yeast morphologies. The dynamics of cell cycle progression are markedly different in true hyphal relative to pseudohyphal and yeast cells, including nuclear movement and septin ring positioning. In
Saccharomyces cerevisiae
, two forkhead transcription factors (
ScFKH1
and
ScFKH2
) regulate the expression of B-cyclin genes. In both
S. cerevisiae
and
Schizosaccharomyces pombe
, forkhead transcription factors also influence morphogenesis. To explore the molecular mechanisms that connect
C. albicans
morphogenesis with cell cycle progression, we analyzed
CaFKH2
, the single homolog of
S. cerevisiae FKH1/FKH2. C. albicans
cells lacking CaFkh2p formed constitutive pseudohyphae under all yeast and hyphal growth conditions tested. Under hyphal growth conditions levels of hyphae-specific mRNAs were reduced, and under yeast growth conditions levels of several genes encoding proteins likely to be important for cell wall separation were reduced. Together these results imply that Fkh2p is required for the morphogenesis of true hyphal as well as yeast cells. Efg1p and Cph1p, two transcription factors that contribute to
C. albicans
hyphal growth, were not required for the pseudohyphal morphology of
fkh2
mutants, implying that Fkh2p acts in pathways downstream of and/or parallel to Efg1p and Cph1p. In addition, cells lacking Fkh2p were unable to damage human epithelial or endothelial cells in vitro, suggesting that Fkh2p contributes to
C. albicans
virulence.
Dtr1p, a Multidrug Resistance Transporter of the Major Facilitator Superfamily, Plays an Essential Role in Spore Wall Maturation in
Saccharomyces cerevisiae
Authors: Felder Thomas , Bogengruber Edith , Tenreiro Sandra , Ellinger Adi , Sá-Correia Isabel , Briza Peter
Published: December 2002

The de novo formation of multilayered spore walls inside a diploid mother cell is a major landmark of sporulation in the yeast
Saccharomyces cerevisiae
. Synthesis of the dityrosine-rich outer spore wall takes place toward the end of this process. Bisformyl dityrosine, the major building block of the spore surface, is synthesized in a multistep process in the cytoplasm of the prospores, transported to the maturing wall, and polymerized into a highly cross-linked macromolecule on the spore surface. Here we present evidence that the sporulation-specific protein Dtr1p (encoded by
YBR180w
) plays an important role in spore wall synthesis by facilitating the translocation of bisformyl dityrosine through the prospore membrane.
DTR1
was identified in a genome-wide screen for spore wall mutants. The null mutant accumulates unusually large amounts of bisformyl dityrosine in the cytoplasm and fails to efficiently incorporate this precursor into the spore surface. As a result, many mutant spores have aberrant surface structures. Dtr1p, a member of the poorly characterized DHA12 (drug:H
+
antiporter with 12 predicted membrane spans) family, is localized in the prospore membrane throughout spore maturation. Transport by Dtr1p may not be restricted to its natural substrate, bisformyl dityrosine. When expressed in vegetative cells, Dtr1p renders these cells slightly more resistant against unrelated toxic compounds, such as antimalarial drugs and food-grade organic acid preservatives. Dtr1p is the first multidrug resistance protein of the major facilitator superfamily with an assigned physiological role in the yeast cell.
Maintenance of Mating Cell Integrity Requires the Adhesin Fig2p
Authors: Zhang Mingliang , Bennett Daniel , Erdman Scott E.
Published: December 2002

Fungal adhesins represent a large family of serine/threonine-rich secreted glycoproteins. Adhesins have been shown to play roles in heterotypic and homotypic cell-cell adhesion processes, morphogenetic pathways and invasive/pseudohyphal growth, frequently in response to differentiation cues. Here we address the role of the
Saccharomyces cerevisiae
mating-specific adhesin Fig2p. Cells lacking
FIG2
possess a variety of mating defects that relate to processes involving the cell wall, including morphogenetic defects, cell fusion defects, and alterations in agglutination activities. We found that mating-specific morphogenetic defects caused by the absence of
FIG2
are suppressible by increased external osmolarity and that, during mating,
fig2
Δ cells display reduced viability relative to wild-type cells. These defects result from alterations in signaling activated by the mating and cell integrity pathways. Finally, we show that
fig2
Δ zygotes also have defects in zygotic spindle positioning that are osmoremedial, whereas the requirements for
FIG2
in normal cell-cell agglutination and cell fusion during mating are insensitive to changes in the extracellular osmotic environment. We conclude that
FIG2
performs distinct functions in the mating cell wall that are separable with respect to their ability to be suppressed by changes in external osmolarity and that a fundamental role of
FIG2
in mating cells is the maintenance of cell integrity.
Conidiation in
Penicillium cyclopium
Is Induced by Conidiogenone, an Endogenous Diterpene
Authors: Roncal Tomás , Cordobés Shandra , Sterner Olov , Ugalde Unai
Published: December 2002

The filamentous fungus
Penicillium cyclopium
conidiates in the presence of calcium ions in submerged culture without nutrient limitation according to a precisely timed program. Conidiation could be prematurely induced in a nutritionally sufficient medium which had previously supported growth, suggesting that a metabolite which influenced the process was produced. A diterpenoid with conidiation-inducing activity, conidiogenone, was purified from the culture medium, along with conidiogenol, a putative derivative with delayed activity. Contrary to previous thought, the presence of calcium was demonstrated to only decrease the threshold concentration of conidiogenone required for the induction to proceed. In light of these results, a mechanism of conidiation induction is presented according to which the mycelium produces a conidiation inducer (conidiogenone) that accumulates extracellularly. When a threshold concentration is reached, induction likely takes place by interaction with a specific cellular receptor. The results indicate that conidiogenone is both sufficient and necessary to induce conidiation.
The Yeast Paf1-RNA Polymerase II Complex Is Required for Full Expression of a Subset of Cell Cycle-Regulated Genes
Authors: Porter Stephanie E. , Washburn Taylor M. , Chang Meiping , Jaehning Judith A.
Published: December 2002

We have previously described an alternative form of RNA polymerase II in yeast lacking the Srb and Med proteins but including Paf1, Cdc73, Hpr1, and Ccr4. The Paf1-RNA polymerase II complex (Paf1 complex) acts in the same pathway as the Pkc1-mitogen-activated protein kinase cascade and is required for full expression of many cell wall biosynthetic genes. The expression of several of these cell integrity genes, as well as many other Paf1-requiring genes identified by differential display and microarray analyses, is regulated during the cell cycle. To determine whether the Paf1 complex is required for basal or cyclic expression of these genes, we assayed transcript abundance throughout the cell cycle. We found that transcript abundance for a subset of cell cycle-regulated genes, including
CLN1
,
HO
,
RNR1
, and
FAR1
, is reduced from 2- to 13-fold in a
paf1
Δ strain, but that this reduction is not promoter dependent. Despite the decreased expression levels, cyclic expression is still observed. We also examined the possibility that the Paf1 complex acts in the same pathway as either SBF (Swi4/Swi6) or MBF (Mbp1/Swi6), the partially redundant cell cycle transcription factors. Consistent with the possibility that they have overlapping essential functions, we found that loss of Paf1 is lethal in combination with loss of Swi4 or Swi6. In addition, overexpression of either Swi4 or Mbp1 suppresses some
paf1
Δ phenotypes. These data establish that the Paf1 complex plays an important role in the essential regulatory pathway controlled by SBF and MBF.
Role of Fig2p in Agglutination in
Saccharomyces cerevisiae
Authors: Jue Chong K. , Lipke Peter N.
Published: December 2002

In W303-derived strains, disruption of
FIG2
increased agglutinability of α cells, but not
a
cells, and did not alter expression of α-agglutinin, binding of
125
I-labeled α-agglutinin, or mating efficiency. Fig2p overexpression led to α-cell-specific suppression of agglutinability. These results imply that Fig2p is an indirect masker of the active sites in α-agglutinin.
Ex Vivo and In Vitro Identification of a Consensus Promoter for
VSG
Genes Expressed by Metacyclic-Stage Trypanosomes in the Tsetse Fly
Authors: Ginger Michael L. , Blundell Patricia A. , Lewis Alyson M. , Browitt Alison , Günzl Arthur , Barry J. David
Published: December 2002

The trypanosome variant surface glycoprotein (VSG) is first expressed during differentiation to the infective, metacyclic population in tsetse fly salivary glands. Unlike the
VSG
genes expressed by bloodstream form trypanosomes, metacyclic
VSG
s (
MVSG
s) have their own promoters. The scarcity of metacyclic cells has meant that only indirect approaches have been used to study these promoters, and not even their identities have been agreed on. Here, we isolated trypanosomes by dissection from salivary glands and used an approach involving 5′ rapid amplification of cDNA ends to identify the transcription start site of three
MVSG
s. This shows that the authentic start site is that proposed for the
MVAT
series of
MVSG
s (K. S. Kim and J. E. Donelson, J. Biol. Chem.
272:
24637-24645, 1997). In the more readily accessible procyclic trypanosome stage, where
MVSG
s are normally silent, we used reporter gene assays and linker scanning analysis to confirm that the 67 bp upstream of the start site is a promoter. This is confirmed further by accurate initiation in a homologous in vitro transcription system. We show also that
MVSG
promoters become derepressed when tested outwith their endogenous, subtelomeric loci. The
MVSG
promoters are only loosely conserved with bloodstream
VSG
promoters, and our detailed analysis of the
1.63 MVSG
promoter reveals that its activity depends on the start site itself and sequences 26 to 49 bp and 56 to 60 bp upstream. These are longer than those necessary for the bloodstream promoter.
Deletion of Individual mRNA Capping Genes Is Unexpectedly Not Lethal to
Candida albicans
and Results in Modified mRNA Cap Structures
Authors: Dunyak Donna S. , Everdeen Daniel S. , Albanese Joseph G. , Quinn Cheryl L.
Published: December 2002

Eukaryotic mRNAs are modified at the 5′ end with a cap structure. In fungal cells, the formation of the mRNA cap structure is catalyzed by three enzymes: triphosphatase, guanylyltransferase, and methyltransferase. Fungal capping enzymes have been proposed to be good antifungal targets because they differ significantly from their human counterparts and the genes encoding these enzymes are essential in
Saccharomyces cerevisiae
. In the present study,
Candida albicans
null mutants were constructed for both the mRNA triphosphatase-encoding gene (
CET1
) and the mRNA methyltransferase encoding gene (
CCM1
), proving that these genes are not essential in
C. albicans.
Heterozygous deletions were generated, but no null mutants were isolated for the guanylyltransferase-encoding gene (
CGT1
), indicating that this gene probably is essential in
C. albicans.
Whereas these results indicate that Cet1p and Ccm1p are not ideal molecular targets for development of anticandidal drugs, they do raise questions about the capping of mRNA and translation initiation in this fungus. Southern blot analysis of genomic DNA indicates that there are not redundant genes for
CET1
and
CCM1
and analysis of mRNA cap structures indicate there are not alternative pathways compensating for the function of
CET1
or
CCM1
in the null mutants. Instead, it appears that
C. albicans
can survive with modified mRNA cap structures.
Rvs161p and Sphingolipids Are Required for Actin Repolarization following Salt Stress
Authors: Balguerie Axelle , Bagnat Michel , Bonneu Marc , Aigle Michel , Breton Annick M.
Published: December 2002

In
Saccharomyces cerevisiae
, the actin cytoskeleton is depolarized by NaCl stress. In this study, the response was maximal after 30 min, and then actin patches repolarized. Rvs161p was required for actin repolarization because the
rvs161Δ
mutant did not repolarize actin patches after growth in a salt medium. Mutations suppressing the
rvs161Δ
-related salt sensitivity all occurred in genes required for sphingolipid biosynthesis:
FEN1
,
SUR4
,
SUR2
,
SUR1
, and
IPT1
. These suppressors also suppressed
act1-1
-related salt sensitivity and the defect in actin repolarization of the
rvs161Δ
mutant, providing a link between sphingolipids and actin polarization. Indeed, deletion of the suppressor genes suppressed the
rvs161Δ
defect in actin repolarization in two ways: either actin was not depolarized at the wild-type level in a set of suppressor mutants, or actin was repolarized in the absence of Rvs161p in the other suppressor mutants. Rvs161p was localized as cortical patches that concentrated at polarization sites, i.e., bud emergence and septa, and was found to be associated with lipid rafts. An important link between sphingolipids and actin polarization is that Rvs161p was required for actin repolarization and was found to be located in lipid rafts.
Role of Ptc2 Type 2C Ser/Thr Phosphatase in Yeast High-Osmolarity Glycerol Pathway Inactivation
Authors: Young Christian , Mapes James , Hanneman Jennifer , Al-Zarban Sheikha , Ota Irene
Published: December 2002

Three type 2C Ser/Thr phosphatases (PTCs) are negative regulators of the yeast
Saccharomyces cerevisiae
high-osmolarity glycerol mitogen-activated protein kinase (MAPK) pathway. Ptc2 and Ptc3 are 75% identical to each other and differ from Ptc1 in having a noncatalytic domain. Previously, we showed that Ptc1 inactivates the pathway by dephosphorylating the Hog1 MAPK; Ptc1 maintains low basal Hog1 activity and dephosphorylates Hog1 during adaptation. Here, we examined the function of Ptc2 and Ptc3. First, deletion of
PTC2
and/or
PTC3
together with
PTP2
, encoding the protein tyrosine phosphatase that inactivates Hog1, produced a strong growth defect at 37°C that was dependent on
HOG1
, providing further evidence that
PTC2
and
PTC3
are negative regulators. Second, overexpression of
PTC2
inhibited Hog1 activation but did not affect Hog1-Tyr phosphorylation, suggesting that Ptc2 inactivates the pathway by dephosphorylating the Hog1 activation loop phosphothreonine (pThr) residue. Indeed, in vitro studies confirmed that Ptc2 was specific for Hog1-pThr. Third, deletion of both
PTC2
and
PTC3
led to greater Hog1 activation upon osmotic stress than was observed in wild-type strains, although no obvious change in Hog1 inactivation during adaptation was seen. These results indicate that Ptc2 and Ptc3 differ from Ptc1 in that they limit maximal Hog1 activity. The function of the Ptc2 noncatalytic domain was also examined. Deletion of this domain decreased
V
max
by 1.6-fold and increased

K
m

by 2-fold. Thus Ptc2 requires an additional amino acid sequence beyond the catalytic domain defined for PTCs for full activity.
ROX1
and
ERG
Regulation in
Saccharomyces cerevisiae
: Implications for Antifungal Susceptibility
Authors: Henry Karl W. , Nickels Joseph T. , Edlind Thomas D.
Published: December 2002

Yeasts respond to treatment with azoles and other sterol biosynthesis inhibitors by upregulating the expression of the
ERG
genes responsible for ergosterol production. Previous studies on
Saccharomyces cerevisiae
implicated the
ROX1
repressor in
ERG
regulation. We report that
ROX1
deletion resulted in 2.5- to 16-fold-lower susceptibilities to azoles and terbinafine. In untreated cultures,
ERG11
was maximally expressed in mid-log phase and expression decreased in late log phase, while the inverse was observed for
ROX1
. In azole-treated cultures,
ERG11
upregulation was preceded by a decrease in
ROX1
RNA. These inverse correlations suggest that transcriptional regulation of
ROX1
is an important determinant of
ERG
expression and hence of azole and terbinafine susceptibilities.
Cellulose Synthase (
CesA
) Genes in the Green Alga
Mesotaenium caldariorum
Authors: Roberts Alison W. , Roberts Eric M. , Delmer Deborah P.
Published: December 2002

Cellulose, a microfibrillar polysaccharide consisting of bundles of β-1,4-glucan chains, is a major component of plant and most algal cell walls and is also synthesized by some prokaryotes. Seed plants and bacteria differ in the structures of their membrane terminal complexes that make cellulose and, in turn, control the dimensions of the microfibrils produced. They also differ in the domain structures of their
CesA
gene products (the catalytic subunit of cellulose synthase), which have been localized to terminal complexes and appear to help maintain terminal complex structure. Terminal complex structures in algae range from rosettes (plant-like) to linear forms (bacterium-like). Thus, algal
CesA
genes may reveal domains that control terminal complex assembly and microfibril structure. The
CesA
genes from the alga
Mesotaenium caldariorum
, a member of the order Zygnematales, which have rosette terminal complexes, are remarkably similar to seed plant
CesA
s, with deduced amino acid sequence identities of up to 59%. In addition to the putative transmembrane helices and the D-D-D-QXXRW motif shared by all known
CesA
gene products,
M. caldariorum
and seed plant CesAs share a region conserved among plants, an N-terminal zinc-binding domain, and a variable or class-specific region. This indicates that the domains that characterize seed plant CesAs arose prior to the evolution of land plants and may play a role in maintaining the structures of rosette terminal complexes. The
CesA
genes identified in
M. caldariorum
are the first reported for any eukaryotic alga and will provide a basis for analyzing the
CesA
genes of algae with different types of terminal complexes.
Hyphal Tip-Associated Localization of Cdc42 Is F-Actin Dependent in
Candida albicans
Authors: Hazan Idit , Liu Haoping
Published: December 2002

The rho-type GTPase Cdc42 is important for the establishment and maintenance of eukaryotic cell polarity. To examine whether Cdc42 is regulated during the yeast-to-hypha transition in
Candida albicans
, we constructed a green fluorescence protein (GFP)-Cdc42 fusion under the
ACT1
promoter and observed its localization in live
C. albicans
cells. As in
Saccharomyces cerevisiae
, GFP-Cdc42 was observed around the entire periphery of the cell. In yeast-form cells of
C. albicans
, it clustered to the tips and sides of small buds as well as to the mother-daughter neck region of large-budded cells. Upon hyphal induction, GFP-Cdc42 clustered to the site of hyphal evagination and remained at the tips of the hyphae. This temporal and spatial localization of Cdc42 suggests that its activity is regulated during the yeast-to-hypha transition. In addition to the accumulation at the hyphal tip, GFP-Cdc42 was also seen as a band within the hyphal tube in cells that had undergone nuclear separation. With the F-actin-assembly inhibitor latrunculin A, we found that GFP-Cdc42 accumulation at the bud site in yeast-form cells is F-actin independent, whereas GFP-Cdc42 accumulation at the hyphal tip requires F-actin. Furthermore, disruption of the F-actin cytoskeleton impaired the transcriptional induction of hypha-specific genes. Therefore, hypha formation resembles mating in
Saccharomyces cerevisiae
in that both require F-actin for GFP-Cdc42 localization and efficient signaling.
Gpa2, a G-Protein α Subunit Required for Hyphal Development in
Candida albicans
Authors: Sánchez-Martínez Cristina , Pérez-Martín José
Published: December 2002

Candida albicans
is able to respond to environmental changes by inducing a distinct morphological program, which is related to the ability to infect mammalian hosts. Although some of the signal transduction pathways involved in this response are known, it is not clear how the environmental signals are sensed and transmitted to these transduction cascades. In this work, we have studied the function of
GPA2
, a new gene from
C. albicans
, which encodes a G-protein α-subunit homologue. We demonstrate that Gpa2 plays an important role in the yeast-hypha dimorphic transition in the response of
C. albicans
to some environmental inducers. Deletion of both alleles of the
GPA2
gene causes in vitro defects in morphological transitions in Spider medium and SLAD medium and in embedded conditions but not in medium containing serum. These defects cannot be reversed by exogenous addition of cyclic AMP. However, overexpression of
HST7
, which encodes a component of the filament-inducing mitogen-activated protein kinase (MAPK) cascade, bypasses the Gpa2 requirement. We have obtained different gain-of-function and loss-of-function mutant alleles of the
GPA2
gene, which we have introduced in several
C. albicans
genetic backgrounds. Our results indicate that, in response to environmental cues, Gpa2 is required for the regulation of a MAPK signaling pathway.
Cloning and Characterization ofscon-3+, a New Member of theNeurospora crassaSulfur Regulatory System
Authors: Sizemore Steven T. , Paietta John V.
Published: December 2002

The sulfur regulatory system ofNeurospora crassaconsists of a group of sulfur-regulated structural genes (e.g., arylsulfatase) that are under coordinate control of the CYS3 positive regulator andsulfur controller(SCON) negative regulators. Here we report on the cloning ofscon-3+, which encodes a polypeptide of 171 amino acids and is a Skp1 family homolog. Repeat-induced point mutation ofscon-3+resulted in a phenotype of constitutive expression of arylsulfatase, a phenotype consistent with othersulfur controllermutants. Northern analysis indicated that, unlike other members of the sulfur regulatory system, expression ofscon-3+is not under the direct control of the CYS3 transcriptional activator. In particular,scon-3+mRNA was detectable under sulfur repressing or derepressing conditions in a Δcys-3mutant. In yeast, Skp1p and an F-box protein binding partner are core constituents of a class of E3 ubiquitin ligases known as SCF complexes. TheN. crassanegative regulator SCON2 contains an F-box motif essential for the operation of the sulfur regulatory system and suggests a role for an SCF complex in theN. crassasulfur regulatory system. A crucial set of experiments, by using a yeast two-hybrid approach with confirming coimmunoprecipitation assays, demonstrated that SCON3 interacts with SCON2 in a manner dependent upon the F-box motif of SCON2. The protein-protein interaction detected between SCON2 and SCON3 represents the initial demonstration in a filamentous fungus of functional interaction between putative core components of a SCF complex.
The Glc7p-Interacting Protein Bud14p Attenuates Polarized Growth, Pheromone Response, and Filamentous Growth in
Saccharomyces cerevisiae
Authors: Cullen Paul J. , Sprague George F.
Published: December 2002

A genetic selection in
Saccharomyces cerevisiae
for mutants that stimulate the mating pathway uncovered a mutant that had a hyperactive pheromone response pathway and also had hyperpolarized growth. Cloning and segregation analysis demonstrated that
BUD14
was the affected gene. Disruption of
BUD14
in wild-type cells caused mild stimulation of pheromone response pathway reporters, an increase in sensitivity to mating factor, and a hyperelongated shmoo morphology. The
bud14
mutant also had hyperfilamentous growth. Consistent with a role in the control of cell polarity, a Bud14p-green fluorescent protein fusion was localized to sites of polarized growth in the cell. Bud14p shared morphogenetic functions with the Ste20p and Bni1p proteins as well as with the type 1 phosphatase Glc7p. The genetic interactions between
BUD14
and
GLC7
suggested a role for Glc7p in filamentous growth, and Glc7p was found to have a positive function in filamentous growth in yeast.
Agrobacterium tumefaciens
Integrates Transfer DNA into Single Chromosomal Sites of Dimorphic Fungi and Yields Homokaryotic Progeny from Multinucleate Yeast
Authors: Sullivan Thomas D. , Rooney Peggy J. , Klein Bruce S.
Published: December 2002

The dimorphic fungi
Blastomyces dermatitidis
and
Histoplasma capsulatum
cause systemic mycoses in humans and other animals. Forward genetic approaches to generating and screening mutants for biologically important phenotypes have been underutilized for these pathogens. The plant-transforming bacterium
Agrobacterium tumefaciens
was tested to determine whether it could transform these fungi and if the fate of transforming DNA was suited for use as an insertional mutagen. Yeast cells from both fungi and germinating conidia from
B. dermatitidis
were transformed via
A. tumefaciens
by using hygromycin resistance for selection. Transformation frequencies up to 1 per 100 yeast cells were obtained at high effector-to-target ratios of 3,000:1.
B. dermatitidis
and
H. capsulatum ura5
lines were complemented with transfer DNA vectors expressing
URA5
at efficiencies 5 to 10 times greater than those obtained using hygromycin selection. Southern blot analyses indicated that in 80% of transformants the transferred DNA was integrated into chromosomal DNA at single, unique sites in the genome. Progeny of
B. dermatitidis
transformants unexpectedly showed that a single round of colony growth under hygromycin selection or visible selection of transformants by
lacZ
expression generated homokaryotic progeny from multinucleate yeast. Theoretical analysis of random organelle sorting suggests that the majority of
B. dermatitidis
cells would be homokaryons after the ca. 20 generations necessary for colony formation. Taken together, the results demonstrate that
A. tumefaciens
efficiently transfers DNA into
B. dermatitidis
and
H. capsulatum
and has the properties necessary for use as an insertional mutagen in these fungi.
Loss of the F-Actin Binding and Vesicle-Associated Protein Comitin Leads to a Phagocytosis Defect
Authors: Schreiner Thomas , Mohrs Martina R. , Blau-Wasser Rosemarie , von Krempelhuber Alfred , Steinert Michael , Schleicher Michael , Noegel Angelika A.
Published: December 2002

Comitin is an F-actin binding and membrane-associated protein from
Dictyostelium discoideum
, which is present on Golgi and vesicle membranes and changes its localization in response to agents affecting the cytoskeleton. To investigate its in vivo functions we have generated knockout mutants by gene replacement. Based on comitin's in vitro functions we examined properties related to vesicular transport and microfilament function. Whereas cell growth, pinocytosis, secretion, chemotaxis, motility, and development were unaltered, comitin-lacking cells were impaired in the early steps of phagocytosis of
Saccharomyces cerevisiae
particles and of
Escherichia coli
, whereas uptake of latex beads was unaffected. Furthermore, the lack of comitin positively affected survival of pathogenic bacteria. Mutant cells also showed an altered response to hyperosmotic shock in comparison to the wild type. The redistribution of comitin during hyperosmotic shock in wild-type cells and its presence on early phagosomes suggest a direct involvement of comitin in these processes.
A Multigene Family That Interacts with the Amino Terminus of Plasmodium MSP-1 Identified Using the Yeast Two-Hybrid System
Authors: Mello Kerrianne , Daly Thomas M. , Morrisey Joanne , Vaidya Akhil B. , Long Carole A. , Bergman Lawrence W.
Published: December 2002

Merozoite surface protein 1 (MSP-1) is a high-molecular-weight protein expressed on the surface of the malaria merozoite in a noncovalent complex with other protein molecules. MSP-1 undergoes a series of proteolytic processing events, but no precise biological role for the various proteolytic fragments of MSP-1 or for the additional proteins present in the complex is known. Through the use of the yeast two-hybrid system, we have isolated genes encoding proteins that interact with a region of the amino-terminal proteolytic fragment of MSP-1 from the mouse parasitePlasmodium yoelii. This analysis has led to the isolation of two sequence-related molecules, one of which is theP. yoeliihomologue of MSP-7 originally described inPlasmodium falciparum. BLAST analysis of theP. falciparumdatabase has revealed that there are six related protein molecules present in this species encoded near each other on chromosome 13. InP. falciparum,we designated these molecules MSRP-1 to -5. Analysis of theP. yoeliidatabase indicates a similar chromosomal organization for the two genes in the mouse parasite species. The threeP. falciparumsequences with the highest degree of homology to theP. yoeliisequences isolated in the two-hybrid screen have been characterized at the molecular level (MSRP-1 to -3). Expression analysis indicated that the mRNAs are expressed at various levels in the different asexual stages. Immunofluorescence studies colocalized the expression of the MSRP molecules and the amino-terminal portion of MSP-1 to the surfaces of trophozoites. In vitro binding experiments confirmed the interaction between MSRP-1, MSRP-2, and the amino-terminal region ofP. falciparumMSP-1.
Small Variant STEVOR Antigen Is Uniquely Located within Maurer's Clefts in
Plasmodium falciparum
-Infected Red Blood Cells
Authors: Kaviratne M. , Khan S. M. , Jarra W. , Preiser P. R.
Published: December 2002

Malaria parasite antigens encoded by multigene families are important factors in virulence and in disease pathology. In
Plasmodium falciparum
, the virulence factor PfEMP-1 is encoded by the
var
multigene family and is exposed at the infected erythrocyte surface. PfEMP-1 is clonally variant, allowing the parasite to evade host immunity. The recently identified
P. falciparum stevor
multigene family and its products also have the potential to be involved in similar important aspects of host-parasite interactions. Here, we show tightly regulated stage-specific transcription of
stevor
occurring over just a few hours of the asexual parasite life cycle. Only a subset of
stevor
genes are transcribed in parasite populations maintained in cultures and in single micromanipulated parasites. Antibodies against STEVOR recognize proteins of the expected size (∼37 kDa) and localize STEVOR in Maurer's clefts, unique membranous structures located in the cytoplasm of infected erythrocytes. The fact that the timing of
stevor
expression and the location of STEVOR are clearly distinct from those of other parasite variant antigens suggests that this gene family may have a novel role in
P. falciparum
biology.
Inhibition of HSP90 in
Trypanosoma cruzi
Induces a Stress Response but No Stage Differentiation
Authors: Graefe Sebastian E. B. , Wiesgigl Martina , Gaworski Iris , Macdonald Andrea , Clos Joachim
Published: December 2002

The 90-kDa heat shock proteins (HSP90) are important in the regulation of numerous intracellular processes in eukaryotic cells. In particular, HSP90 has been shown to be involved in the control of the cellular differentiation of the protozoan parasite
Leishmania donovani
. We investigated the role of HSP90 in the related parasite
Trypanosoma cruzi
by inhibiting its function using geldanamycin (GA). GA induced a dose-dependent increase in heat shock protein levels and a dose-dependent arrest of proliferation. Epimastigotes were arrested in G
1
phase of the cell cycle, but no stage differentiation occurred. Blood form trypomastigotes showed conversion towards spheromastigote-like forms when they were cultivated with GA, but differentiation into epimastigotes was permanently blocked. We conclude that, similar to leishmanial HSP90, functional HSP90 is essential for cell division in
T. cruzi
and serves as a feedback inhibitor in the cellular stress response. In contrast to
L. donovani
cells, however,
T. cruzi
cells treated with GA do not begin to differentiate into relevant life cycle stages.
Chromosome Localization Changes in the
Trypanosoma cruzi
Nucleus
Authors: Elias M. Carolina Q. B. , Faria Marcella , Mortara Renato A. , Motta Maria Cristina M. , de Souza Wanderley , Thiry Marc , Schenkman Sergio
Published: December 2002

Chromosome localization in the interphase nuclei of eukaryotes depends on gene replication and transcription. Little is known about chromosome localization in protozoan parasites such as trypanosomes, which have unique mechanisms for the control of gene expression, with most genes being posttranscriptionally regulated. In the present study, we examined where the chromosomes are replicated in
Trypanosoma cruzi
, the agent of Chagas' disease. The replication sites, identified by the incorporation of 5-bromodeoxyuridine, are located at the nuclear periphery in proliferating epimastigote forms in the early S phase of the cell cycle. When the S phase ends and cells progress through the cell cycle, 5-bromodeoxyuridine labeling is observed in the nuclear interior, suggesting that chromosomes move. We next monitored chromosome locations in different stages of the cell cycle by using a satellite DNA sequence as a probe in a fluorescence in situ hybridization assay. We found two distinct labeling patterns according to the cell cycle stage. The first one is seen in the G
1
phase, in hydroxyurea-arrested epimastigotes or in trypomastigotes, which are differentiated nondividing forms. In all of these forms the satellite DNA is found in dots randomly dispersed in the nucleus. The other pattern is found in cells from the S phase to the G
2
phase. In these cells, the satellite DNA is found preferentially at the nuclear periphery. The labeling at the nuclear periphery disappears only after mitosis. Also, DNA detected with terminal deoxynucleotidyl transferase is found distributed throughout the nuclear space in the G
1
phase but concentrated at the nuclear periphery in the S phase to the G
2
phase. These results strongly suggest that
T. cruzi
chromosomes move and, after entering the S phase, become constrained at the nuclear periphery, where replication occurs.
ras2
Controls Morphogenesis, Pheromone Response, and Pathogenicity in the Fungal Pathogen
Ustilago maydis
Authors: Lee Nancy , Kronstad James W.
Published: December 2002

Ustilago maydis
, a pathogen of maize, is a useful model for the analysis of mating, pathogenicity, and the morphological transition between budding and filamentous growth in fungi. As in other fungi, these processes are regulated by conserved signaling mechanisms, including the cyclic AMP (cAMP)/protein kinase A (PKA) pathway and at least one mitogen-activated protein kinase (MAP kinase) pathway. A current challenge is to identify additional factors that lie downstream of the cAMP pathway and that influence morphogenesis in
U. maydis
. In this study, we identified suppressor mutations that restored budding growth to a constitutively filamentous mutant with a defect in the gene encoding a catalytic subunit of PKA. Complementation of one suppressor mutation unexpectedly identified the
ras2
gene, which is predicted to encode a member of the well-conserved
ras
family of small GTP-binding proteins. Deletion of the
ras2
gene in haploid cells altered cell morphology, eliminated pathogenicity on maize seedlings, and revealed a role in the production of aerial hyphae during mating. We also used an activated
ras2
allele to demonstrate that Ras2 promotes pseudohyphal growth via a MAP kinase cascade involving the MAP kinase kinase Fuz7 and the MAP kinase Ubc3. Overall, our results reveal an additional level of crosstalk between the cAMP signaling pathway and a MAP kinase pathway influenced by Ras2.
Analysis of Telomerase in
Candida albicans
: Potential Role in Telomere End Protection
Authors: Singh Sunitha M. , Steinberg-Neifach Olga , Mian I. Saira , Lue Neal F.
Published: December 2002

Telomerase is a ribonucleoprotein reverse transcriptase responsible for the maintenance of one strand of telomere terminal repeats. Analysis of the telomerase complex in the budding yeast
Saccharomyces cerevisiae
has revealed the presence of one catalytic protein subunit (Est2p/TERT) and at least two noncatalytic components (Est1p and Est3p). The genome of the pathogenic yeast
Candida albicans
contains putative orthologues of all three telomerase components. Disruption of each homologue resulted in significant but distinct telomere dysfunction in
Candida
. Similar to
S. cerevisiae
, the
Candida EST3
disruption strain exhibits progressive telomere loss over many generations, at a rate that is consistent with incomplete replication. In contrast, telomeres in both the
Candida TERT
and
EST1
disruption strains can contract rapidly, followed by partial or nearly complete recovery, suggesting a defect in telomere “capping.” We propose that these two telomerase subunits may participate in the protection of chromosomal ends in
Candida
. Analysis of telomerase-mediated primer extension in vitro indicates that only the TERT protein is absolutely essential for enzyme activity. Our results support the conservation of telomerase protein components beyond the catalytic subunit but reveal species-specific phenotypic alterations in response to loss of individual telomerase component. We also identify potential homologues of Est1p in phylogenetically diverse organisms. The Est1p sequence family possesses a conserved N-terminal domain predicted to be structurally related to tetratricopeptide repeat-containing proteins.
Loss of Compartmentalization Causes Misregulation of Lysine Biosynthesis in Peroxisome-Deficient Yeast Cells
Authors: Breitling Rainer , Sharif Orzala , Hartman Michelle L. , Krisans Skaidrite K.
Published: December 2002

To characterize the metabolic role of peroxisomes in yeast cells under physiological conditions, we performed a comprehensive meta-analysis of published microarray data. Previous studies of yeast peroxisomes have mainly been focused on the function of peroxisomes under extreme conditions, such as growth on oleate or methanol as the sole carbon source, and may therefore not be representative of the normal physiological role of yeast peroxisomes. Surprisingly, our analysis of the microarray data reveals that the only pathway responding to peroxisome deficiency in mid-log phase is lysine biosynthesis, whereas classical peroxisomal pathways such as beta-oxidation are unaffected. We show that the upregulation of lysine biosynthesis genes in peroxisome-deficient yeasts shares many characteristics with the physiological response to lysine starvation. We provide data that suggest that this is the result of a “pathological” stimulation of the Lys14p transcriptional activator by the pathway intermediate aminoadipate semialdehyde. Mistargeting of the peroxisomal lysine pathway to the cytosol increases the active concentration of aminoadipate semialdehyde, which is no longer contained in the peroxisome and can now activate Lys14p at much lower levels than in wild-type yeasts. This is the first well-documented example of pathway misregulation in response to peroxisome deficiency and will be useful in understanding the phenotypic details of human peroxisome-deficient patients (Zellweger syndrome).
Multiple Functions of
mfa-1
, a Putative Pheromone Precursor Gene of
Neurospora crassa
Authors: Kim Hyojeong , Metzenberg Robert L. , Nelson Mary Anne
Published: December 2002

A putative pheromone precursor gene of
Neurospora crassa
,
mfa-1
(which encodes mating factor
a
-1), was identified as the most abundant clone in starved mycelial and perithecial cDNA libraries. Northern analysis demonstrated high
mfa-1
expression in all mating type
a
tissues and suggested low expression levels in
mat A
tissues. The
mfa-1
gene was expressed as an approximately 1.2-kb transcript predicted to encode a 24-residue peptide, followed by a long 3′ untranslated region (3′ UTR). The predicted MFA1 sequence showed 100% sequence identity to PPG2 of
Sordaria macrospora
and structural similarity (a carboxy-terminal CAAX motif) to many hydrophobic fungal pheromone precursors. Mutants with a disrupted open reading frame (ORF) in which the critical cysteine residue had been changed to a nonprenylatable residue, tyrosine (YAAX mutants), were isolated, as were
mfa-1
mutants with intact ORFs but multiple mutations in the 3′ noncoding region (CAAX mutants). The 3′ UTR is required for the full range of
mfa-1
gene activity. Both classes of mutants showed delayed and reduced vegetative growth (which was suppressed by supplementation with a minute amount [30 μM] of ornithine, citrulline, or arginine), as well as aberrant sexual development. When crossed as female parents to wild-type males, the CAAX and YAAX mutants showed greatly reduced ascospore production. No ascospores were produced in homozygous
mfa-1
crosses. As males, YAAX
mat a
mutants were unable to attract wild-type
mat A
trichogynes (female-specific hyphae) or to initiate sexual development, while CAAX
mat a
mutants were able to mate and produce sexual progeny despite their inability to attract
mat A
trichogynes. In the
mat A
background, both CAAX and YAAX mutants showed normal male fertility but defective vegetative growth and aberrant female sexual development. Thus, the
mfa-1
gene appears to have multiple roles in
N. crassa
development: (i) it encodes a hydrophobic pheromone with a putative farnesylated and carboxymethylated C-terminal cysteine residue, required by
mat a
to attract trichogynes of
mat A
; (ii) it is involved in female sexual development and ascospore production in both mating types; and (iii) it functions in vegetative growth of both mating types.
Reproductive tourism as moral pluralism in motion
Authors: Pennings G
Published: December 2002

Reproductive tourism is the travelling by candidate service recipients from one institution, jurisdiction, or country where treatment is not available to another institution, jurisdiction, or country where they can obtain the kind of medically assisted reproduction they desire. The more widespread this phenomenon, the louder the call for international measures to stop these movements.

Three possible solutions are discussed: internal moral pluralism, coerced conformity, and international harmonisation. The position is defended that allowing reproductive tourism is a form of tolerance that prevents the frontal clash between the majority who imposes its view and the minority who claim to have a moral right to some medical service. Reproductive tourism is moral pluralism realised by moving across legal borders. As such, this pragmatic solution presupposes legal diversity.