bluuebunny/crossref_metadata_2025 huggingface dataset:
Microbial Infections, Immunomodulation, and Drugs of Abuse
Authors: Friedman Herman , Newton Catherine , Klein Thomas W.
Published: April 2003

The use of recreational drugs of abuse has generated serious health concerns. There is a long-recognized relationship between addictive drugs and increased levels of infections. Studies of the mechanisms of actions of these drugs became more urgent with the advent of AIDS and its correlation with abused substances. The nature and mechanisms of immunomodulation by marijuana, opiates, cocaine, nicotine, and alcohol are described in this review. Recent studies of the effects of opiates or marijuana on the immune system have demonstrated that they are receptor mediated, occurring both directly via specific receptors on immune cells and indirectly through similar receptors on cells of the nervous system. Findings are also discussed that demonstrate that cocaine and nicotine have similar immunomodulatory effects, which are also apparently receptor mediated. Finally, the nature and mechanisms of immunomodulation by alcohol are described. Although no specific alcohol receptors have been identified, it is widely recognized that alcohol enhances susceptibility to opportunistic microbes. The review covers recent studies of the effects of these drugs on immunity and on increased susceptibility to infectious diseases, including AIDS.
Low Occurrence of Pathogenic
Yersinia enterocolitica
in Clinical, Food, and Environmental Samples: a Methodological Problem
Authors: Fredriksson-Ahomaa Maria , Korkeala Hannu
Published: April 2003

While Yersinia enterocolitica is an important pathogen, which can cause yersiniosis in humans and animals, its epidemiology remains obscure. The pig is the major reservoir of pathogenic Y. enterocolitica of bioserotype 4/O:3, the most common type found in humans. Y. enterocolitica is thought to be a significant food-borne pathogen, although pathogenic isolates have seldom been recovered from foods. The low isolation rate of this pathogenic bacterium in natural samples, including clinical, food, and environmental samples, may be due to the limited sensitivity of culture methods. During the last decade, numerous DNA-based methods, such as PCR and colony hybridization assays, have been designed to detect pathogenic Y. enterocolitica in natural samples more rapidly and with better sensitivity than can be achieved by culture methods. In addition, the occurrence of pathogenic Y. enterocolitica in natural samples is clearly higher with PCR than with culture methods. The methods available for detection of pathogenic Y. enterocolitica in natural samples are reviewed in this article.
Importance of Respiratory Viruses in Acute Otitis Media
Authors: Heikkinen Terho , Chonmaitree Tasnee
Published: April 2003

Acute otitis media is usually considered a simple bacterial infection that is treated with antibiotics. However, ample evidence derived from studies ranging from animal experiments to extensive clinical trials supports a crucial role for respiratory viruses in the etiology and pathogenesis of acute otitis media. Viral infection of the upper respiratory mucosa initiates the whole cascade of events that finally leads to the development of acute otitis media as a complication. The pathogenesis of acute otitis media involves a complex interplay between viruses, bacteria, and the host’s inflammatory response. In a substantial number of children, viruses can be found in the middle-ear fluid either alone or together with bacteria, and recent studies indicate that at least some viruses actively invade the middle ear. Viruses appear to enhance the inflammatory process in the middle ear, and they may significantly impair the resolution of otitis media. Prevention of the predisposing viral infection by vaccination against the major viruses would probably be the most effective way to prevent acute otitis media. Alternatively, early treatment of the viral infection with specific antiviral agents would also be effective in reducing the occurrence of acute otitis media.
Toxocariasis: Clinical Aspects, Epidemiology, Medical Ecology, and Molecular Aspects
Authors: Despommier Dickson
Published: April 2003

Toxocariasis is caused by a series of related nematode species (ascarids) that routinely infect dogs and cats throughout the world. The eggs from these ascarids are common environmental contaminants of human habitation, due largely to the fact that many kinds of dogs and cats serve as pets, while countless others run wild throughout the streets of most urban centers. The eggs, present in dog and cat feces, become infectious within weeks after they are deposited in the local environment (e.g., sandboxes, city parks, and public beaches, etc.). Humans, particularly children, frequently ingest these eggs by accident and become infected. Infection in humans, in contrast to their definitive hosts, remains occult, often resulting in disease caused by the migrating larval stages. Visceral larva migrans (VLM) and ocular larva migrans (OLM) are two clinical manifestations that result in definable syndromes and present as serious health problems wherever they occur. Diagnosis and treatment of VLM and OLM are difficult. These issues are summarized in this review, with emphasis on the ecology of transmission and control of spread to both humans and animals through public health initiatives employing treatment of pets and environmental intervention strategies that limit the areas that dogs and cats are allowed within the confines of urban centers.
Acanthamoebaspp. as Agents of Disease in Humans
Authors: Marciano-Cabral Francine , Cabral Guy
Published: April 2003

Acanthamoeba spp. are free-living amebae that inhabit a variety of air, soil, and water environments. However, these amebae can also act as opportunistic as well as nonopportunistic pathogens. They are the causative agents of granulomatous amebic encephalitis and amebic keratitis and have been associated with cutaneous lesions and sinusitis. Immuno compromised individuals, including AIDS patients, are particularly susceptible to infections with Acanthamoeba. The immune defense mechanisms that operate against Acanthamoeba have not been well characterized, but it has been proposed that both innate and acquired immunity play a role. The ameba's life cycle includes an active feeding trophozoite stage and a dormant cyst stage. Trophozoites feed on bacteria, yeast, and algae. However, both trophozoites and cysts can retain viable bacteria and may serve as reservoirs for bacteria with human pathogenic potential. Diagnosis of infection includes direct microscopy of wet mounts of cerebrospinal fluid or stained smears of cerebrospinal fluid sediment, light or electron microscopy of tissues, in vitro cultivation of Acanthamoeba, and histological assessment of frozen or paraffin-embedded sections of brain or cutaneous lesion biopsy material. Immunocytochemistry, chemifluorescent dye staining, PCR, and analysis of DNA sequence variation also have been employed for laboratory diagnosis. Treatment of Acanthamoeba infections has met with mixed results. However, chlorhexidine gluconate, alone or in combination with propamidene isethionate, is effective in some patients. Furthermore, effective treatment is complicated since patients may present with underlying disease and Acanthamoeba infection may not be recognized. Since an increase in the number of cases of Acanthamoeba infections has occurred worldwide, these protozoa have become increasingly important as agents of human disease.
Nuclear Import of Zinc Binuclear Cluster Proteins Proceeds through Multiple, Overlapping Transport Pathways
Authors: Nikolaev Igor , Cochet Marie-Françoise , Felenbok Béatrice
Published: April 2003

In
Aspergillus nidulans
, the high transcriptional level of the ethanol utilization pathway genes (
alc
) is regulated by the specific activator AlcR. Here we have analyzed the mechanism of the nuclear import of AlcR, as well as that of other proteins belonging to the Zn
2
Cys
6
binuclear cluster family. The nuclear localization signal of AlcR maps within the N-terminal 75 amino acid residues and overlaps with its DNA-binding domain. It consists of five clusters rich in basic residues. Four of them are necessary and sufficient for nuclear targeting. The first two basic regions are crucial for both nuclear localization and recognition of AlcR-specific DNA targets. This nuclear localization signal (NLS) motif is recognized by the nuclear transport machinery of
Saccharomyces cerevisiae
and requires both Ran/Gsp1p activity and specific transport receptors. AlcR can be imported into nuclei via multiple transport pathways mediated by a distinct set of karyopherins composed of Kap104p, Sxm1p, and Nmd5p transport receptors. The two former karyopherins interact with the NLS of AlcR directly. Other Zn binuclear cluster proteins from
S. cerevisiae
, such as Gal4p and Pdr3p, also appear to be transported to the nuclei in a nonclassical, importin-α-independent manner and can share common importin β receptors.
Exportin 1 Mediates Nuclear Export of the Kinetoplastid Spliced Leader RNA
Authors: Zeiner Gusti M. , Sturm Nancy R. , Campbell David A.
Published: April 2003

The kinetoplastid protozoan spliced leader (SL) RNA is the common substrate pre-mRNA utilized in alltrans-splicing reactions. Here we show by fluorescence in situ hybridization that the SL RNA is present in the cytoplasm ofLeishmania tarentolaeandTrypanosoma brucei. Treatment with the karyopherin-specific inhibitor leptomycin B was toxic toT. bruceiand eliminated the cytoplasmic SL RNA, suggesting that cytoplasmic SL RNA was dependent on the nuclear exporter exportin 1 (XPO1). Ectopic expression of xpo1 with a C506S mutation inT. bruceiconferred resistance to leptomycin B. A reduction in SL RNA 3′ extension removal and 5′ methylation of nucleotide U4was observed in wild-typeT. bruceitreated with leptomycin B, suggesting that the cytoplasmic stage is necessary for SL RNA biogenesis. This study demonstrates spatial and mechanistic similarities between the posttranscriptional trafficking of the kinetoplastid protozoan SL RNA and the metazoancis-spliceosomal small nuclear RNAs.
A Circadian Oscillator in
Aspergillus
spp
.
Regulates Daily Development and Gene Expression
Authors: Greene Andrew V. , Keller Nancy , Haas Hubertus , Bell-Pedersen Deborah
Published: April 2003

We have established the presence of a circadian clock in
Aspergillus flavus
and
Aspergillus nidulans
by morphological and molecular assays, respectively. In
A. flavus
, the clock regulates an easily assayable rhythm in the development of sclerotia, which are large survival structures produced by many fungi. This developmental rhythm exhibits all of the principal clock properties. The rhythm is maintained in constant environmental conditions with a period of 33 h at 30°C, it can be entrained by environmental signals, and it is temperature compensated. This endogenous 33-h period is one of the longest natural circadian rhythms reported for any organism, and this likely contributes to some unique responses of the clock to environmental signals. In
A. nidulans
, no obvious rhythms in development are apparent. However, a free running and entrainable rhythm in the accumulation of
gpdA
mRNA (encoding glyceraldehyde-3-phosphate dehydrogenase) is observed, suggesting the presence of a circadian clock in this species. We are unable to identify an
Aspergillus
ortholog of
frequency
, a gene required for normal circadian rhythmicity in
Neurospora crassa
. Together, our data indicate the existence of an
Aspergillus
circadian clock, which has properties that differ from that of the well-described clock of
N. crassa.
Rapamycin Mimics the Incompatibility Reaction in the FungusPodospora anserina
Authors: Dementhon Karine , Paoletti Mathieu , Pinan-Lucarré Bérangère , Loubradou-Bourges Nathalie , Sabourin Martine , Saupe Sven J. , Clavé Corinne
Published: April 2003

In filamentous fungi, a programmed cell death (PCD) reaction occurs when cells of unlike genotype fuse. This reaction is caused by genetic differences at specific loci termedhetloci (for heterokaryon incompatibility). Although severalhetgenes have been characterized, the mechanism of this cell death reaction and its relation to PCD in higher eukaryotes remains largely unknown. InPodospora anserina, genes induced during the cell death reaction triggered by thehet-R het-Vinteraction have been identified and termedidigenes. Herein, we describe the functional characterization of oneidigene (idi-1) and explore the connection between incompatibility and the response to nutrient starvation. We show that IDI-1 is a cell wall protein which localizes at the septum during normal growth. We found that induction ofidi-1and of the other knownidigenes is not specific of the incompatibility reaction. Theidigenes are induced upon nitrogen and carbon starvation and by rapamycin, a specific inhibitor of the TOR kinase pathway. The cytological hallmarks ofhet-R het-Vincompatibility (increased septation, vacuolization, coalescence of lipid droplets, induction of autophagy, and cell death) are also observed during rapamycin treatment. Globally the cytological alterations and modifications in gene expression occurring during the incompatibility reaction are similar to those observed during starvation or rapamycin treatment.
Identification of Essential Genes in the Human Fungal Pathogen
Aspergillus fumigatus
by Transposon Mutagenesis
Authors: Firon Arnaud , Villalba François , Beffa Roland , d'Enfert Christophe
Published: April 2003

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Mus spicilegus
is an Eastern European wild mouse species that has previously been reported to harbor an unusual infectious ecotropic murine leukemia virus (MLV) and proviral envelope genes of a novel MLV subgroup. In the present study,
M. spicilegus
neonates were inoculated with Moloney ecotropic MLV (MoMLV). All 17 inoculated mice produced infectious ecotropic virus after 8 to 14 weeks, and two unusual phenotypes distinguished the isolates from MoMLV. First, most of the
M. spicilegus
isolates grew to equal titers on
M. dunni
and SC-1 cells, although MoMLV does not efficiently infect
M. dunni
cells. The deduced amino acid sequence of a representative clone differed from MoMLV by insertion of two serine residues within the VRA of SU
env
. Modification of a molecular clone of MoMLV by the addition of these serines produced a virus that grows to high titer in
M. dunni
cells, establishing a role for these two serine residues in host range. A second unusual phenotype was found in only one of the
M. spicilegus
isolates, Spl574. Spl574 produces large syncytia of multinucleated giant cells in
M. dunni
cells, but its replication is restricted in other mouse cell lines. Sequencing and mutagenesis demonstrated that syncytium formation could be attributed to a single amino acid substitution within VRA, S82F. Thus, viruses with altered growth properties are selected during growth in
M. spicilegus
. The mutations associated with the host range and syncytium-inducing variants map to a key region of VRA known to govern interactions with the cell surface receptor, suggesting that the associated phenotypes may result from altered interactions with the unusual ecotropic virus mCAT1 receptor carried by
M. dunni
.
Cloning of the Full-Length Rhesus Cytomegalovirus Genome as an Infectious and Self-Excisable Bacterial Artificial Chromosome for Analysis of Viral Pathogenesis
Authors: Chang W. L. William , Barry Peter A.
Published: April 2003

Rigorous investigation of many functions encoded by cytomegaloviruses (CMVs) requires analysis in the context of virus-host interactions. To facilitate the construction of rhesus CMV (RhCMV) mutants for in vivo studies, a bacterial artificial chromosome (BAC) containing an enhanced green fluorescent protein (EGFP) cassette was engineered into the intergenic region between unique short 1 (US1) and US2 of the full-length viral genome by Cre/lox-mediated recombination. Infectious virions were recovered from rhesus fibroblasts transfected with pRhCMV/BAC-EGFP. However, peak virus yields of cells infected with reconstituted progeny were 10-fold lower than wild-type RhCMV, suggesting that inclusion of the 9-kb BAC sequence impeded viral replication. Accordingly, pRhCMV/BAC-EGFP was further modified to enable efficient excision of the BAC vector from the viral genome after transfection into mammalian cells. Allelic exchange was performed in bacteria to substitute the
cre
recombinase gene for
egfp
. Transfection of rhesus fibroblasts with pRhCMV/BAC-Cre resulted in a pure progeny population lacking the vector backbone without the need of further manipulation. The genomic structure of the BAC-reconstituted virus, RhCMV-
loxP
(r), was identical to that of wild-type RhCMV except for the residual
loxP
site. The presence of the
loxP
sequence did not alter the expression profiles of neighboring open reading frames. In addition, RhCMV-
loxP
(r) replicated with wild-type kinetics both in tissue culture and seronegative immunocompetent macaques. Restriction analysis of the viral genome present within individual BAC clones and virions revealed that (i) RhCMV exhibits a simple genome structure and that (ii) there is a variable number of a 750-bp iterative sequence present at the S terminus.
Analysis of 4.3 Kilobases of Divergent Locus B of Macaque Retroperitoneal Fibromatosis-Associated Herpesvirus Reveals a Close Similarity in Gene Sequence and Genome Organization to Kaposi's Sarcoma-Associated Herpesvirus
Authors: Rose Timothy M. , Ryan Jonathan T. , Schultz Emily R. , Raden Brian W. , Tsai Che-Chung
Published: April 2003

We previously identified retroperitoneal fibromatosis-associated herpesvirus (RFHV) as a simian homolog of Kaposi's sarcoma-associated herpesvirus (KSHV) in a fibroproliferative malignancy of macaques that has similarities to Kaposi's sarcoma. In this report, we cloned 4.3 kb of divergent locus B (DL-B) flanking the DNA polymerase gene from two variants of RFHV from different species of macaque with a consensus degenerate hybrid oligonucleotide primer approach. Within the DL-B region of RFHV, viral homologs of the cellular interleukin-6, dihydrofolate reductase, and thymidylate synthase genes were identified, along with a homolog of the gammaherpesvirus open reading frame (ORF) 10. In addition, a homolog of the KSHV ORF K3, the modulator of immune recognition-1, was identified. Our data show a close similarity in sequence conservation, gene content, and genomic structure between RFHV and KSHV which strongly supports the grouping of these viral species within the same RV-1 rhadinovirus lineage and the hypothesis that RFHV is the macaque homolog of KSHV.
Mutations in the N-Terminal Region of Influenza Virus PB2 Protein Affect Virus RNA Replication but Not Transcription
Authors: Gastaminza Pablo , Perales Beatriz , Falcón Ana M. , Ortín Juan
Published: April 2003

PB2 mutants of influenza virus were prepared by altering conserved positions in the N-terminal region of the protein that aligned with the amino acids of the eIF4E protein, involved in cap recognition. These mutant genes were used to reconstitute in vivo viral ribonucleoproteins (RNPs) whose biological activity was determined by (i) assay of viral RNA, cRNA, and mRNA accumulation in vivo, (ii) cap-dependent transcription in vitro, and (iii) cap snatching with purified recombinant RNPs. The results indicated that the W49A, F130A, and R142A mutations of PB2 reduced or abolished the capacity of mutant RNPs to synthesize RNA in vivo but did not substantially alter their ability to transcribe or carry out cap snatching in vitro. Some of the mutations (F130Y, R142A, and R142K) were rescued into infectious virus. While the F130Y mutant virus replicated faster than the wild type, mutant viruses R142A and R142K showed a delayed accumulation of cRNA and viral RNA during the infection cycle but normal kinetics of primary transcription, as determined by the accumulation of viral mRNA in cells infected in the presence of cycloheximide. These results indicate that the N-terminal region of PB2 plays a role in viral RNA replication.
Susceptibility of Mink (
Mustera vision
)-Derived Cells to Replication by Human Immunodeficiency Virus Type 1
Authors: Koito Atsushi , Kameyama Yuichi , Cheng-Mayer Cecilia , Matsushita Shuzo
Published: April 2003

In vivo studies for understanding viral transmission and replication, host immune responses, and pathogenesis of human immunodeficiency virus type 1 (HIV-1) infection would greatly benefit from the establishment of a small-animal model. In this study, we explored the potential of American mink (
Mustera vison
) as a susceptible host. We found that primary cells and cell lines derived from this species efficiently supported
trans
-activation of the HIV-1 long terminal repeat by Tat. Accordingly, the cysteine residue at position 261, which has been shown to be important for interaction of the human cyclin T1 with the HIV-1 regulatory protein Tat, is conserved in the mink homologue. No species-specific defect in Rev function could be detected in mink cells. In addition, primary splenocytes, fibroblasts, and the Mv.1.Lu cell line from American mink supported early as well as late HIV-1 gene expression following infection with vesicular stomatitis G protein-pseudotyped HIV-1 viruses, at levels comparable to those seen with permissive human cells. Furthermore, the mink Mv.1.Lu cell line stably expressing human CD4 and CCR5 receptors supported a spreading HIV-1 infection with few, if any, deficiencies compared to findings in human cell lines. This indicates the potential of HIV-1 to replicate in these cells once the blockade at the stage of virus entry has been removed. These results clearly show that cells from American mink generally pose no functional intracellular block to HIV-1 replication, and collectively they raise the possibility that this animal species could be engineered to support HIV-1 infection, providing a useful small-animal model for evaluating de novo infection by HIV-1.
Maintenance of Gammaherpesvirus Latency Requires Viral Cyclin in the Absence of B Lymphocytes
Authors: van Dyk Linda F. , Virgin Herbert W. , Speck Samuel H.
Published: April 2003

Gammaherpesviruses establish a life-long chronic infection that is tightly controlled by the host immune response. We previously demonstrated that viruses lacking the gammaherpesvirus 68 (γHV68) viral cyclin (v-cyclin) exhibited a severe defect in reactivation from latency and persistent replication. In this analysis of chronic infection, we demonstrate that the v-cyclin is required for γHV68-associated mortality in B-cell-deficient mice. Furthermore, we identify the v-cyclin as the first gene product required for maintenance of gammaherpesvirus latency in vivo in the absence of B lymphocytes. While the v-cyclin was necessary for maintenance of latency in the absence of B cells, maintenance of v-cyclin-deficient viruses was equivalent to that of wild-type γHV68 in the presence of B cells. These results support a model in which maintenance of chronic γHV68 infection requires v-cyclin-dependent reactivation and reseeding of non-B-cell latency reservoirs in the absence of B cells and raise the possibility that B cells represent a long-lived latency reservoir maintained independently of reactivation. These results highlight distinct mechanisms for the maintenance of chronic infection in immunocompetent and B-cell-deficient mice and suggest that the different latency reservoirs have distinct gene requirements for the maintenance of latency.
Minimal
cis
-Acting Elements Required for Adenovirus Genome Packaging
Authors: Ostapchuk Philomena , Hearing Patrick
Published: April 2003

The design of drugs for treatment of virus infections and the exploitation of viruses as drugs for treatment of diseases could be made more successful by understanding the molecular mechanisms of virus-specific events. The process of assembly, and more specifically packaging of the genome into a capsid, is an obligatory step leading to future infections. To enhance our understanding of the molecular mechanism of packaging, it is necessary to characterize the viral components necessary for the event. In the case of adenovirus, sequences between nucleotides 200 and 400 at the left end of the genome are essential for packaging. This region contains a series of redundant bipartite sequences, termed A repeats, that function in packaging. Synthetic packaging sequences made of multimers of a single A repeat substitute for the authentic adenovirus packaging domain. A repeats are binding sites for the CCAAT displacement protein and the viral protein IVa2. Several lines of evidence implicate these proteins in the packaging process. It was not known, however, whether other
cis
-acting elements play a role in the packaging process as well. We utilized an in vivo approach to address the role of the inverted terminal repeats and the covalently linked terminal proteins in packaging of the adenovirus genome. Our results show that these elements are not necessary for efficient packaging of the viral genome. A significant implication of these results applicable to gene therapy vector design is that the linkage of the adenovirus packaging domain to heterologous DNA sequences should suffice for targeting to the viral capsid.