NEUROSPORA 2006 PLENARY SESSION ABSTRACTS
Session I: From Genes to Populations
Tony Griffiths, Chair
Control of DNA Methylation in Neurospora
Eric Selker, Institute of Molecular Biology, University of Oregon,
Eugene, OR 97403-1229
Most methylated regions of Neurospora are products of RIP (repeat-induced point mutation), a premeiotic homology-based genome defense system that litters duplicated sequences with C:G to T:A mutations and typically leaves them methylated at remaining cytosines. I will present our current understanding about how A:T-rich DNA, such as that resulting from RIP, triggers methylation. Our efforts to elucidate the control of DNA methylation in vegetative cells have revealed mechanistic ties between modifications of DNA and histones. The DIM-2 DNA methyltransferase is directed by heterochromatin protein 1 (HP1), which in turn recognizes trimethyl-lysine 9 on histone H3, placed by the DIM-5 histone H3 methyltransferase. Results of in vitro and in vivo studies indicate that DIM-5 recognizes at least residues 8-12 of histone H3 and is sensitive to methylation of lysine 4 and phosphorylation of serine 10 in histone H3, supporting our suggestion that histones serve to integrate diverse signals to control DNA methylation. Additional support for this notion comes from our studies on mutants defective in other histone modification enzymes. DNA methylation and HP1 localization do not depend on RNAi machinery in Neurospora but do depend, in part, on deacetylation and dephosphorylation of histones. Conversely, DNA methylation can lead to deacetylation of histones, which may aid in propagation of DNA methylation and the associated silenced chromatin state.
Structural studies of protein (histone)
methylation
Xiaodong Cheng. Department of Biochemistry, Emory University School
of Medicine, Atlanta, Georgia 30322;
There is a rapidly growing appreciation that the study of covalent modification to proteins and transcriptional regulation will likely dominate the research headlines in the next decade. Protein (de)methylation plays a central role in both of these fields, as several different residues (Arg, Lys) are methylated in cells and methylation plays a central role in the "histone code" that regulates chromatin structure, impacts transcription, and responds to DNA damage. In some cases, a single lysine can be mono-, di-, or trimethylated, with different functional consequences for each of the three forms. I will review structural aspects of methylation of histone lysine residues by two enzyme families with entirely different structural scaffolding (the SET proteins and Dot1p) and methylation of protein arginine residues by PRMTs, and discuss, somewhat speculatively, their mechanisms.
Meiotic Recombination initiated by the cog hotspot in Neurospora.
Meiotic recombination in Neurospora crassa is initiated at hotspots regulated by transacting genes.
Our current focus is on recombination initiated by the hotspot cog, occurring within the his-3
locus and in flanking regions stretching proximally to lys-4 and distally to ad-3. We have
analysed recombination by measuring the frequency of His+ progeny from crosses heterozygous for auxotrophic his-3 alleles, and by following segregation in octads heterozygous for lys-4, his-3,
ad-3, cot-1, am, snp markers between his-3 and cog and additional snps both proximal
and distal of his-3. Octad analysis revealed additional hotspots that, like cog, appear to be
particularly active in initiating recombination, and showed that all reciprocal exchanges whose location
could be determined also experienced conversion nearby. We have constructed knockouts of spo11,
msh-2, msh4 and ku70. Each mutant shows disturbance of chromosome
behavior during
meiosis. Although chromosome pairing is severely affected in crosses homozygous spo11 and
aneuploidy rife amongst the progeny, we were surprised to find recombination events initiated by the
cog hotspot are somewhat elevated over normal amongst those infrequent spores that are
sufficiently genetically balanced to be viable. One interpretation of these data is that Neurospora is able to
initiate recombination, at least at cog, by mechanisms other than Spo11-induced double
strand breaks.
DNA repair and genomic instability in Neurospora
Mutants which show high sensitivity to mutagen(s) have been isolated and
characterized in Neurospora.
Based on spectra of mutagen sensitivity and epistatic relationship, they were classified into 5 groups;
excision repair, recombination repair, post replication repair, damage-checkpoint and mismatch repair. Some
of those mutants have mutator phenotype and/or growth defect phenotype. Majority of spontaneous
mutation is generated in replication process. Recently 2 different recQ genes encoding proteins with
3’-5’ helicase motif were identified in Neurospora. A double recQ mutant showed genomic instability.
Mutator or growth defect phenotype of a recQ double mutant was suppressed by mutation of either
of nonhomologous end joining (NHEJ) or homologous recombination (HR), respectively. Roles of HR and
NHEJ in double-strand breaks repair, gene targeting and spontaneous mutagenesis are discussed.
Neurospora comparative biology is enabled by phylogenetics and species recognition.
Comparative biology depends on accuracy in recognition of genetically isolated groups of organisms, in
analysis of relationships among these groups, and in dating of evolutionary events that create modern
groups. In Neurospora, outbreeding species form a single evolutionary lineage of 15 phylogenetic species in
two lineages, biological and phylogenetic species recognition are nearly equivalent, and genetic isolation
precedes reproductive isolation. Recognition and comparison of Neurospora species is unmatched in its
scope in fungi and represents a tool to enable careful comparative biology.
Research using this tool began with an examination of the evolution of microsatellites. Current projects
include: 1) Phylogenomics, where outbreeding Neurospora are providing a means of testing the claims made
for this approach in studies of yeast. 2) Evolution of reproductive isolation, where previous knowledge of
phylogenetic relationships and reproductive isolation among clades facilitated the choice of strains for QTL
analysis of reproductive isolation in hybrid matings. 3) Intraspecific or intraclade variation, where studies of
variation in fecundity and phenotypes associated with biological clocks or reproduction are being used to
dissect genetic control of variable phenotype.
Evolutionary genetics of reproductive isolation barriers separating Neurospora crassa and N.
intermedia.
Reproductive isolation (RI) barriers between different pairs of N. crassa and N. intermedia
strains range from mild (reduced numbers of viable progeny) to severe (failure to develop perithecia). For the
N. crassa NcC clade, which is endemic in southern India, the severity of RI from N.
intermedia is biogeographically structured: crosses to sympatric N. intermedia strains show more
severe barriers. This pattern is consistent with reinforcement, the evolution of stronger RI barriers by
natural selection against hybridization. The potential fitness advantage of the putative reinforcement barrier,
the early abortion of hybrid perithecia, was demonstrated in experiments showing that early abortion can
dramatically increase the overall fecundity of NcC females when they have additional opportunities to mate
with conspecific males. We are undertaking quantitative trait locus (QTL) mapping to study the
evolutionary genetics of RI barriers separating N. crassa and N. intermedia using a mapping
population derived from a cross between strains of the N. crassa NcA and NcC clades. We have
identified loci that are significantly associated with the strength of RI in mating assays between these
f1 hybrids and N. intermedia tester strains from different geographic regions,
including a QTL on linkage group VI responsible for about 30% of the variance in development of hybrid
perithecia fertilized by N. intermedia sympatric to NcC.
Artificial selection for ascospore size in Neurospora crassa
Workshop: How to utilize new
tools and resources for Neurospora developed in the Program Project
Jay Dunlap, Organizer Knockouts
Workshop This workshop will present protocols and guidelines for
making your own knockout strains in Neurospora. In order to achieve
high-throughput production of knockout strains as part of the Program Project,
we have created novel procedures and software tools, as well as adapting,
simplifying and streamlining existing techniques.
http://www.dartmouth.edu/~neurosporagenome/primers.html Introduction to
Neurospora Microarray Methods The detection of large and small yet statistically significant differences in
gene expression in spotted DNA microarray studies is an ongoing challenge. We
will begin with discussion of experimental protocols that are designed for
investigations of differential gene expression using resources available in the
Neurospora community. We have recently developed Neurospora microarrays
comprising predicted 10,526 Neurospora genes and made them publicly available
through FGSC. Since the release of the arrays and accompanying protocols at
http://web.uconn.edu/townsend/Links/ffdatabase/downloads.html
Session II: Genomics
and Program Project Report
Kathy Borkovich, Chair
Enabling a
community to dissect an organism: Functional analysis of Neurospora as a
model filamentous fungus
The overall goal of the four interdependent projects in this Program Project is to carry out functional
genomics, annotation, and expression analyses of Neurospora crassa, the filamentous fungus that has
become a model for the assemblage of over 250,000 species of non-yeast fungi. The timeline for this effort
envisioned periodic repots to the community and public assessment of progress towards our goals and
benchmarks. Building from the completely sequenced 43 Mb Neurospora genome the first Project is
pursuing the systematic disruption of genes through targeted gene replacements, preliminary phenotyping
of these strains, and their distribution to the scientific community at large. Project #2, through a primary
focus in Annotation and Genomics, has developed a platform for electronically capturing community
feedback and data about the existing annotation, while building and maintaining a database to capture and
display information about phenotypes. Oligonucleotide-based microarrays created in Project #3 will be used
to collect baseline expression data the nearly 11,000 distinguishable transcripts in Neurospora under
various conditions of growth and development, and eventually to begin to analyze the global effects of loss
of novel genes in strains created by Project #1. cDNA libraries generated in Project #4 will illuminate
alternative splicing, alternative promoters, antisense transcripts and help to document the overall complexity
of expressed sequences in Neurospora, as well as driving the assembly of a SNP map.
Genome informatics and Neurospora crassa functional studies We have developed a high-throughput method for creating Neurospora
knockout mutants. In this procedure, yeast recombinational cloning is used to
create constructs that are then transformed into Neurospora strains
deficient in nonhomologous end-joining DNA repair (mus-51 and mus-52
mutants). Here we present the results of our initial application of the
procedure, with mutational analysis of 103 transcription factor-encoding genes.
The methodology is robust, with a >90% success rate for producing the desired
knockout mutant. The resulting mutants were screened for a variety of phenotypes
and 43% exhibited discernible defects. The genes producing phenotypes are
variously involved in growth of basal hyphae (25 genes), aerial hyphae height
and/or macroconidiation (27 genes), and differentiation of protoperithecia or
perithecia (15 genes). This analysis demonstrated roles for many uncharacterized
transcription factors and also revealed novel functions for genes that had been
previously studied. Several transcription factors are required for than one
aspect of growth or development, suggesting roles in integration of multiple
upstream signals. The observation that half of the genes did not produce obvious
defects when mutated may result from functional redundancy, which has been
reported for other transcription factor genes. The availability of this
collection of Neurospora transcription factor mutants will enable future
investigations aimed at elucidating the complexity of gene regulation in
filamentous fungi.
A High-Density SNP Map for Neurospora crassa.
A collection of SNPs (single nucleotide polymorphisms) in the Mauriceville strain of
Neurospora crassa, relative to the Oak Ridge standard, was established, randomly distributed over
the entire genome with an average density of one every 100 kb. To this end, an EST library was constructed
from Mauriceville germinating conidia and sequenced. By alignment with the published Oak Ridge genomic
sequence, a set of putative SNPs was formulated, out of which a subset was selected based on quality of
the sequencing information as well as amenability to CAPS (cleaved amplified polymorphic sequence), some
of which were experimentally validated by PCR amplification of genomic DNA followed by differential
restriction digest. We present a map containing confirmed SNPs, each one supplemented by a primer pair
and a restriction enzyme that has been shown to consistently distinguish between the Oak Ridge and
Mauriceville version. Given the possibility of high-throughput assignment of a large number of SNP markers
to individual progeny, we expect this information to be invaluable for the rapid mapping of conventionally
derived mutations, as well as in the further assembly and orientation of the genomic sequence and in the
analysis of complex traits such as QTLs.
Exploring regulatory networks in Neurospora Exploring regulatory networks is one of the main functions of post-genomics
research. As part of the Neurospora Functional Genomics Program Project grant,
we have constructed full genome oligonucleotide microarrays for the filamentous
fungus Neurospora crassa using gene annotation provided by The Broad
Institute and Munich Information for Protein Sequences (MIPS). Our goal is to
define transcriptional regulatory networks in N. crassa, as a model
organism for filamentous fungi. By the genome-wide microarrays, we can define
transcription factor targets by profiling transcription factor mutant strains.
There are at least five predicted DNA-binding transcription factors families in
N. crassa: BHLH, BZIP, C2H2, GATA and ZnII(Cys)6. Using phylogenetic
analyses of the transcription factor gene families within ascomycete fungi, we
have chosen to profile mutants in transcription factor genes that are
phylogenetically diverse, plus their closest paralogs; the vast majority of
these transcription factors and their regulatory networks are completely
uncharacterized. Thirty-five transcription factor mutant strains (generated by
the Neurospora Functional Genomics Program Project Grant) have been initially
selected for transcriptional profiling. We compare the gene expression profiles
of transcription factor knock out strains to wild type across a fungal colony
and under different stress conditions. Putative target genes of the
transcription factors are subsequently subjected to cis-element analysis. By
performing comprehensive transcriptional profiling, cis-element analysis and
chromatin immunoprecipitation, the transcription regulatory network of a model
filamentous fungus can be constructed.
Session III: Cell
Morphogenesis and Assembly
Functio Variabilis: Unravelling the diverse roles of ion transport in hyphal morphogenesis.
During tip growth (hyphal extension), Neurospora relies upon an internally generated Ca2+
gradient to maintain polar growth of hyphae. The tip-high Ca2+ gradient is created by the
action of a stretch-activated phospholipase C which produces inositol trisphosphate (IP3) that
then activates a Ca2+ channel to cause Ca2+ release at the tip.
Ca2+ sequestration behind the tip occurs into endoplasmic reticulum and into a unique
population of tip-localized mitochondria. As a walled cell, Neurospora usually relies upon hydrostatic
pressure (turgor) to cause tip extension. Upon hyperosmotic stress, an ensemble of ion transporters (the
H+ ATPase, and K+ and Cl- transporters) are activated by a MAP
kinase cascade to maintain turgor at about 500 kiloPascals. Besides turgor, there are intrahyphal osmotic
differences that cause mass flow of cytoplasm which is normally directed towards the growing tips. Thus,
ion transport at a number of organelles plays key roles in multiple functions during hyphal growth:
Ca2+ gradients mediate tip polarity, trans plasma membrane ion gradients generate the turgor
driving force, and intrahyphal ionic gradients mediate cytoplasm migration toward the growing tips.
How is calcium
sequestered and does vacuolar calcium play a role in morphogenesis?
See poster # 10
The cellular and genetic determination of Woronin body formation in apical hyphal compartments of Neurospora crassa
Glycosphingolipid structure and biosynthesis in Neurospora crassa.
Glycosphingolipids (GSLs) have been implicated in a number of studies as targets of plant defensin binding
to the fungal membrane. For example, sensitivity of the yeasts Pichia pastoris and Candida
albicans toward RsAFP2, a defensin isolated from seeds of Raphanus sativus (radish), was
found to be dependent on GCS, the gene encoding glucosylceramide synthase (UDP-Glc:ceramide beta-
glucosyltransferase). Although Neurospora crassa is not a phytopathogen, it has been used as a
system to investigate plant defensin-phytopathogen interactions. Chemically mutagenized N. crassa
strains selected for resistance to RsAFP2 were found to have dramatically altered glycolipid expression
profiles. Characterizing the true nature of these alterations has required detailed structure elucidation of GSL
components isolated from wild type and defensin-resistant mutant N. crassa strains. Key general
characteristics of fungal GSL expression at the gene and metabolic levels will be discussed and compared
with what we have learned so far from studies of GSL expression in N. crassa.
Biogenesis of mitochondria: Pathways and machineries involved in the
import of proteins The life of almost all proteins of the mitochondrion begins at ribosomes in
the cytoplasm of the cell. In order to reach their active states as constituents
of protein assemblies in one of the various mitochondrial subcompartments (the
outer membrane, the intermembrane space, the inner membrane or the matrix) they
interact with protein translocases of the mitochondria. So far six such
molecular machines have been identified: two in the outer membrane (the TOM and
TOB complexes), one in the intermembrane space (the Mia1/Erv1 machinery), and
three in the inner membrane (the Tim23, TIM22 and OXA1 complexes). In addition,
a number of chaperones and co-chaperones in the matrix are required for folding
in the matrix space as well as a series of assembly factors for the formation of
cofactor containing supramolecular protein complexes of the mitochondria.
Function and expression of Tob55/Sam50 in Neurospora crassa: An essential protein
for assembly of beta-barrels into the mitochondrial outer membrane The NAD(P)H dehydrogenases of Neurospora crassa mitochondria The proton-pumping NADH dehydrogenase or complex I is a major entry point of
electrons into the mitochondrial respiratory chain. It catalyses electron
transfer from NADH to ubiquinone through a series of protein-bound prosthetic
groups. Complex I deficiencies have been implicated in various mitochondrial
diseases. Complex I from the filamentous fungus Neurospora crassa
contains at least 39 polypeptide subunits of dual genetic origin, mostly
conserved in mammals, suggesting that the enzyme is involved in other cellular
processes beyond bioenergetics. Mutations in different subunits have been
generated in the last years, including mutations of conserved amino acid
residues of iron-sulphur proteins as found in human diseases, in order to reveal
the role of the proteins in complex I assembly and function. Complex I is likely
regulated by transitions between active (A) and de-activated (D) forms and one
of the proteins involved in this phenomenon has been identified. In addition to
complex I and depending on the organism, several non-proton-pumping alternative
NAD(P)H dehydrogenases may also be present in the inner mitochondrial membrane.
The fungus N. crassa contains four alternative NAD(P)H dehydrogenases:
the main external NAD(P)H dehydrogenase, an external calcium-dependent NADPH dehydrogenase, a third external enzyme and the single internal NADH
dehydrogenase. These proteins appear to have both alternative and complementary
functions. Overall, mitochondrial respiratory chain NAD(P)H dehydrogenases have
important roles in fungal development.
Session IV: Cell Signaling and Gene Regulation
Nora Plesofsky,
Chair The arginine attenuator peptide: a regulator of translation and mRNA
levels. Group I and group II introns are commonly found inserted in fungal mtDNA
genes. These introns self-splice in vitro, but require proteins for
efficient splicing in vivo to help fold the intron RNA into the
catalytically active structure. Protein factors that function in splicing group
I introns were first identified by mutational analysis in Neurospora crassa.
The Neurospora mitochondrial tyrosyl-tRNA synthetase (CYT-18) binds
specifically to group I introns RNAs and promotes RNA splicing by stabilizing
the catalytically active RNA structure. The N. crassa CYT-19 protein
functions in conjunction with CYT-18 and is DEAD-box protein that acts as an
ATP-dependent RNA chaperone to disrupt stable inactive structures that are
kinetic traps during CYT-18-assisted RNA folding. Studies with CYT-18 and CYT-19
have suggested general paradigms for how proteins function in RNA folding and
facilitate RNA-catalyzed reactions. They also show how cellular RNA bindings
proteins can evolve to function in RNA splicing, and more generally, how
essential proteins can acquire new functions.
G proteins and histidine kinases differentially
regulate sexual development Heterotrimeric G protein pathways and two-component
regulatory systems control environmental responses in fungi. Our laboratory has
demonstrated roles for both of these pathways in regulation of sexual
development in Neurospora. The G protein coupled receptors (GPCRs) PRE-1
and PRE-2, along with their cognate pheromone ligands, MFA-1 and CCG-4, are
essential for initial recognition between opposite mating type females and males
during mating. The heterotrimeric Ga protein GNA-1
and the Gbg dimer GNB-1/GNG-1 are also necessary for
female fertility, but in a mating type independent manner. Loss of the G protein
subunits or the receptor leads to trichogyne "blindness" in females, while males
that do not produce pheromone are unable to attract trichogynes. A change in
identity can be accomplished by heterologous expression of the receptor or
pheromone in cells of opposite mating type and co-expression of a cognate
receptor-pheromone pair leads to self-stimulation. The GPCR GPR-1 is required
for proper formation of beaks and ostioles during perithecial development; the
coupled Ga is not currently known, but available
genetic data points to GNA-1 as the most likely candidate. We have begun
analysis of two-component regulatory systems with characterization of the
response regulator RRG-1. rrg-1 mutants have an early block in female
fertility, in that they do not produce protoperithecia. This defect correlates
with loss of stimulation of a downstream mitogen-activated protein kinase
pathway in Neurospora
Cytology of conidial anastomosis induction, homing and fusion in Neurospora crassa Glycogen metabolism and stress response in Neurospora crassa.
Regulation of the gsn gene. Heterokaryon incompatibility Nonself recognition in filamentous fungi is conferred by genetic differences
at het (heterokaryon incompatibility) loci. When individuals that
differ in het specificity undergo hyphal fusion, the heterokaryon
undergoes a programmed cell death reaction or is highly unstable. In
Neurospora crassa, three allelic specificities at the het-c locus are
conferred by a highly polymorphic domain. This domain shows trans-species
polymorphisms indicative of balancing selection, consistent with the role of
het loci in nonself recognition. We determined that a locus closely linked
to het-c, called pin-c (partner for incompatibility
with het-c) was required for het-c nonself
recognition and heterokaryon incompatibility (HI). The pin-c alleles in
isolates that differ in het-c specificity were extremely polymorphic.
Heterokaryon and transformation tests showed that nonself recognition was
mediated by synergistic non-allelic interactions between het-c and
pin-c, while allelic interactions at het-c increased the severity of
the HI phenotype. The pin-c locus encodes a protein containing a HET
domain. These data suggest non-allelic interactions may be important in nonself
recognition in filamentous fungi and that proteins containing a HET domain may
be a key factor in these interactions. Functional VIB-1, which is a putative
transcription factor, is required for expression of pin-c, het-6 and
tol, all of which encode HET domain proteins. These observations explain why
mutations at vib-1 suppress het-c, het-6 and mat
incompatibility.
MAK-2 MAP Kinase and cAMP signaling pathways interact to control aerial
growth and conidiophore development
Session V: Clocks, Light, and Oxygen
Deborah Bell-Pedersen, Chair
The ascomycete Neurospora crassa has a long standing as a model organism for investigating both
circadian rhythms and photoreception (J. J. Loros and J. C. Dunlap, Circadian Rhythms, Photobiology and
Functional Genomics in Neurospora. Ch.4, pp 53- 74 in Volume XIII The Mycota. "Fungal
Genomics". Editor Alistair J P Brown, 2005). The circadian system of N. crassa involves a number of
interlocked molecular feedback loops that regulate the time-of-day-specific expression of a number of output
genes, thereby generating distinct phenotypes, including the clock-dependent rhythm of macroconidiation.
Light is a major entraining signal to the clock, coordinating the organism with the diurnal environment.
Known players involved in circadian and light regulation of these processes include the products of the frq,
wc-1, wc-2, and vvd genes. Both VVD and WC-1 have been shown to be blue light photoreceptors; WC-1 is
required for circadian entrainment as well as the autoregulatory feedback loop involving the frq gene and
VVD has been shown to participate in light signaling to the clock. The recent cloning of a mutation, bd, that
allows the easily visualized rhythm in conidiation suggests an imbalance of reactive oxygen species in this
mutant strain. Recent work on the clock, light signaling and Ras will be discussed. This work was supported
by grants from the National Institutes of Health MH44651 AND NIGMS1P01 GM 068087-01 to J.C.D. and
J.J.L. and R37GM34985 to J.C.D., the National Science Foundation MCB-0084509 to J.J.L.,
and the Norris Cotton Cancer Centre core grant at Dartmouth Medical School.
Coordination of negative and positive functions of FREQUENCY in the circadian
clock
Quelling in Neurospora: an overview
The introduction of transgenes or double-strand RNAs (dsRNAs) into a variety of
eukaryotic cells can trigger a series of post-transcriptional gene silencing
mechanisms in which dsRNA intermediate molecules, after being processed into
short interfering RNA molecules (siRNA), were identified as strong elicitors of
mRNA degradation. Two PTGS mechanisms have been identified in Neurospora:
quelling and MSUD (meiotic silencing by unpaired DNA). Quellling occurs during
the vegetative phase of the life cycle and was the first PTGS mechanism
characterized in this organism.
Chromatin-Remodeling Enzymes and Circadian Rhythms.
Neurospora crassa contains a circadian feedback loop that is controlled by daily oscillations in
transcription of the frequency gene. The transcription factors White Collar-1 (WC-1) and White
Collar-2 (WC-2) activate frq expression in a circadian and light dependent manner, and are thought to
act together as heteromeric complex. To better understand regulated events at the frq promoter, we
deleted genes homologous to the swi/snf family of ATP-dependent chromatin-remodeling enzymes.
The 19 putative chromatin-remodeling factors were knocked out by gene replacement and characterized; one
was essential for growth, another was ascospore lethal and a third had a defect in circadian regulated spore
formation, and is now designated clockswitch (csw-1). To further define events at frq, we
used ChIP and nuclease accessibility assays to examine how nucleosome modifications might regulate
circadian relevant transcription. The WC proteins do not act solely as an obligate complex because in
vivo binding of WC-2 to the frq promoter occurs in a rhythmic fashion with the peak in binding
occurring coincident with the peak in frq transcription. A basal level of WC-1 is associated with the
promoter at all circadian times and only slight increases are observed when frq is transcribed. There
is marked reduction in the level of acetylated histone H3 upon light induced transcription. Chromatin
rearrangements at frq are seen when the gene is expressed and ChIP assays indicate that CSW-1 is
localized to this region, suggesting a direct role for this chromatin-remodeling enzyme in regulating
frq expression.
Cell differentiation as a response to oxidative stress
Different stress conditions in Neurospora lead to an unstable state in which formation of reactive
oxygen species surpass the antioxidant capacity of the cell.
Cell differentiation is one possible response to this inevitably transient hyperoxidant state. The hypothesis
predicts that disruption of anti-oxidant enzymes should intensify cell differentiation processes; deletion of
pro-oxidant enzymes should inhibit them. Catalase and NADPH oxidase genes were disrupted to find that
cat-3 mutant increased asexual and sexual sporulation, cat-2 mutant increased submerged
conidiation, nox-1 decreased asexual and inhibited sexual sporulation, and nox-2 inhibited
ascospore germination (1). sod-1 deletion mutant presented circadian conidiation, enhanced aerial
and submerged conidiation, and diminished protoperithecia formation. sod-1 phenocopies
bd, which is a dominant mutation probably in ras-1. Both strains seem to produce a cyclic
oxidative stress that leads to cyclic conidiation.
1) Aguirre J; Rios-Momberg M; Hewitt D; Hansberg W (2005) Reactive oxygen species and development in
microbial eukaryotes.
Trends in Microbiology 13:111-118.
Please note, some talks were selected from posters and their abstracts appear
here. Return to the Neurospora 2006 main page
Frederick Bowring, Jane Yeadon, Hui-Yin Lee, Sue Conway and David Catcheside.
School of Biological Sciences, Flinders University, PO Box 2100, Adelaide, SA 5001. Australia.
Hirokazu Inoue (Saitama University, Saitama, Japan)
John Taylor1, Dave Jacobson1,2,
Elizabeth Turner1, Luz Beatrice
Gilbert,1 Jeremy Dettman3.1UC, Berkeley, 2Stanford U. 3U.
Toronto, Mississauga.
Elizabeth Turner & John W. Taylor, Plant & Microbial Biology, University of California, Berkeley, CA 94720
Heather H. Wilkinson, Department of Plant Pathology, Program for the Biology
of Filamentous Fungi, Texas A&M University.
We are interested in ecological and evolutionary genetic bases for life-history
trait variation in natural Neurospora crassa populations. Studies thus
far have focused on breeding well-characterized isolates from a Louisiana sugar
cane field and discerning the patterns of heritability associated with variation
in a wide variety of developmental phenotypes. As a test of concept in the
present study, crosses that yielded the smallest or the largest ascospores in
that F1 population were used to artificially select, in both directions, for
ascospore size. In total, selection on both large and small ascospores lineages
has been carried out to the F4 generation. We are exploring 1) the degree to
which evolution of the mean of one trait influences the evolution of the mean of
another (e.g. ascospore shape); and 2) the degree to which selection on the mean
of a trait influences the shape of the distribution around the mean (e.g.
standard deviation, skewness, kurtosis). The implications to the functional
ecology of a trait will be discussed.
Hildur Colot and Patrick Collopy.
We will briefly describe the overall scheme and then elaborate on certain
details, including primer design, yeast recombinational cloning for assembling
the deletion cassettes, the use of magnetic beads for isolation of yeast DNA,
the creation of mus-51 and mus-52 strains, 96-well
electroporation into Neurospora, a modified transformation medium,
mini-slants for spot-testing, the use of magnetic beads for Neurospora
DNA preps, and a custom-written application for designing Southern blots.
We have performed significant portions of the work on a pipetting robot.
However, the protocols were first developed manually and can be done without
the robot with small numbers of samples or in a 96-well format. We will provide
protocols for performing all the procedures without the need for any specialized
equipment, along with information on more expensive options suitable for high
throughput.
Finally, we will give a brief tour of the relevant web-based resources,
including lists of primers used, lists of knockout strains completed and in
progress (including access to the LIMS we use to track our work), updated
protocols, and the programs for both primer design and Southern design. The web
sites include:
http://www.dartmouth.edu/~neurosporagenome/protocols.html
http://www.dartmouth.edu/~neurosporagenome/knockouts_completed.html
http://borkovichlims.ucr.edu/php/sLIMS.php
http://borkovichlims.ucr.edu/southern/
http://borkovichlims.ucr.edu/primer/primerDesign.py
Jeffrey Townsend, Takao Kasuga, Baikang Pei
we have received feedback from the Neurospora community. We will discuss
growth conditions, RNA extraction, cDNA synthesis, hybridization and acquisition
of microarray data. We will provide protocols, alternative methods and
troubleshooting methodology. From there we will explore ways to design
experiments. Multifactorial experimental designs using DNA microarrays are
becoming increasingly common. We will highlight experimental design criteria
that will maximize inferential and statistical power. In experimental design,
opportunities for transitive inference should be exploited, while always
ensuring that comparisons of greatest interest comprise direct hybridizations.
We will briefly overview productive methods for analysis for completed datasets,
including Bayesian (BAGEL) and ANOVA methods. Understanding the difference in
gene expression that is detectable as significant is a vital component of
experimental design and evaluation. The gene expression level at which there is
an empirical 50% probability of a significant call is presented as a summary
statistic for the power to detect small differences in gene expression. Lastly,
we will provide an introduction to a filamentous fungal microarray database in
construction where data may be deposited, examined, and analyzed.
http://web.uconn.edu/townsend/Links/ffdatabase/introduction.htm
Jay Dunlap1, Hildur Colot1, Kathy Borkovich2, Gloria
Turner3, Dick Weiss3, Mike Plamann4, Bruce
Birren5, Matt Sachs6, Louise Glass7, Jeffrey
Townsend9, Mary Anne Nelson8, Jennifer Loros1
1Dept. Genetics, Dartmouth Medical School, Hanover, NH 03755
2Dept. Plant Pathology, UC Riverside, Riverside, CA
3Dept. Microbiology, UCLA, Los Angeles, CA
4Dept. Biology, Univ. Missouri, Kansas City, MO
5 MIT Center for Genome Research, Cambridge, MA
6 Oregon Health Sciences University, Portland, OR
7 Dept. Plant and Microbial Biology, UC Berkeley, Berkeley, CA
8 Dept. Biology, Univ New Mexico, Albuquerque, NM
9 Dept. Molec. Cell. Biology, Univ. of Connecticut, Storrs, CT
Matthew R. Henn1, Dave DeCaprio1, Heather M. Hood2,
Steve Rounsley1, Matthew Crawford1, Phil Montgomery1,
Gloria E. Turner3, Chad Nusbaum1, Matthew S. Sachs2,
James E. Galagan1, Bruce W. Birren1. 1Broad
Institute of MIT & Harvard, Cambridge, MA 02141, 2Oregon Health &
Science University, Beaverton, OR 97006, 3Department of Chemistry and
Biochemistry, University of California, Los Angeles California 90095
The next challenges for understanding the Neurospora crassa genome
sequence are to refine the genome annotation and to actively capture, improve,
and integrate with the sequence the wealth of information that exists within the
research community. To this end, the Broad Institute as part of the NIH Program
Project, "Functional Analysis of a Model Filamentous Fungus,” has constructed
community annotation and phenotype ontology infrastructures that for the first
time provide the research community the ability to link information about
genetic features with the Neurospora genome, to refine gene structures, and to
curate all entries in a searchable database. To increase the accuracy of the
gene model, gene calling using EST-based algorithms was also implemented. To
maximize the value of EST sequencing, the Broad’s Neighborhood Quality Score
algorithm was adapted to work with fungal EST sequences and we have begun
comparing Mauriceville strain cDNAs with the Oak Ridge strain genomic sequence
to identify single nucleotide polymorphisms (SNPs) for genetic mapping. The
Broad Institute is also improving the quality of the N. crassa genome for
subsequent release. One barrier to finishing the genome is regions recalcitrant
to cloning. Initial results from pyrosequencing using 454 technology has
improved coverage of uncaptured regions. In addition, an optical map, which
represents a restriction map of the entire genome, has helped anchor significant
portions of the genome leading to better representation of complete chromosomes.
Addition by subtraction: Novel insights into Neurospora biology
obtained from transcription factor knockouts
Gyungsoon Park. University of California, Riverside.
Randy Lambreghts1, Mi Shi1, David DeCaprio2, James E.
Galagan2, Bruce W. Birren2, Jay C. Dunlap1, and
Jennifer J.
Loros1.
1Department of Genetics, Dartmouth Medical School, Hanover, NH 03755; 2
Broad Institute, Cambridge, MA 02141
Chaoguang Tian, Takao Kasuga and N. Louise Glass
Department of Plant and Microbial Biology, University of California, Berkeley,
CA 94720
Oded Yarden,Chair
Roger R. Lew
Biology Department, York University, Toronto, Canada.
planters@yorku.ca
Barry Bowman
Gregory Jedd.
Temasek Life Sciences Laboratory and Department of Biological Sciences, National University of Singapore, Singapore 117604
1Tey, W.K., North, A.J., Reyes, J.L., Lu, Y.F., Jedd, G. (2005) Polarized gene expression determines Woronin body formation at the leading edge of the fungal colony. Mol. Biol. Cell. 16, 2651-2659.
Steven B. Levery1, Chaeho Park2, Beau Bennion1, Elizabeth
Owuor1, Isabelle E.J.A. François3, Kathelijne K.A. Ferket3,
Bruno P.A. Cammue3, Karin Thevissen3. 1Department of
Chemistry, University of New Hampshire, Durham, NH, USA; 2Department of Biochemistry
and Molecular Biology, University of Georgia, Athens, GA, USA; 3Centre of Microbial and
Plant Genetics (CMPG), Katholieke Universiteit Leuven, Heverlee-Leuven, Belgium.
Walter Neupert, Institute of Physiological Chemistry, University of Muenchen,
Germany
The two most recently identified protein translocases are the TOB complex and
the Mia40/Erv1 machinery. The TOB complex is responsible for the membrane
integration of β-barrel proteins of the
mitochondrial outer membrane, such as Tom40 and porin. The TOB complex interacts
with the precursors of these proteins during or after their translocation across
the outer membrane by the TOM complex. The Mia40/Erv1 machinery is essential for
the import into the intermembrane space of members of a family of small proteins
with CX3C and CX9C motifs. Oxidative folding leading to formation of disulfide
bonds appears to be a step which provides a driving force for translocation
across the outer membrane via the TOM complex.
A further focus of our interest is the ATP driven import motor of the
mitochondria which is coupled to the TIM23 translocase. Tim44 is a central
component which is peripherally associated with the membrane integrated
components Tim23 and Tim17, and which organizes the import motor. Tim44 recruits
mtHsp70 which binds and, by a ratchet-like mechanism, takes into the matrix
unfolded precursor polypeptides that have passed the TOM complex and the channel
of the TIM23 translocase. The import motor contains two further Tim44 associated
essential proteins, the J-protein, Tim14, and J-like protein, Tim16. The way how
they control the activity of the import motor appears to be key to the
understanding of the function of the TIM23 complex which is responsible for the
import of the vast majority of nuclear encoded inner membrane and matrix
proteins of mitochondria.
Dr. Frank Nargang, Dept. of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada
Arnaldo Videira. Instituto de Biologia Molecular e Celular (IBMC) and Instituto
de Ci ncias Biomédicas de Abel Salazar (ICBAS), University of Porto, Portugal
Matt Sachs, OGI School of Science and Engineering, Oregon Health & Science
University, Beaverton, OR
The Neurospora crassa arg-2 gene and its fungal homologs encode the
arginine-specific carbamoyl-phosphate synthetase (CPS-A) small subunit. Excess
arginine decreases translation of arg-2 through the action of an
evolutionarily conserved upstream open reading frame (uORF) in the mRNA. This
uORF encodes a cis-regulatory element, the arginine attenuator peptide (AAP),
which stalls ribosomes in the presence of arginine, thereby decreasing ribosome
access to the downstream CPS-A reading frame. We performed an extensive analysis
of the sequence requirements for AAP-mediated translational control of ribosome
stalling using the N. crassa cell free translation system. The results
showed that some but not all of the evolutionarily conserved residues in the AAP
sequence were crucial for regulation. Furthermore, we showed using yeast that
AAP-mediated ribosome stalling at the uORF stop codon causes the mRNA to be
destabilized by inducing the nonsense mediated mRNA decay (NMD) pathway.
Preliminary evidence using an N. crassa mutant defective in NMD indicates
that the amount of cellular arg 2 mRNA is also controlled at the level of mRNA
stability by NMD.
Involvement of the Neursopora mitochondrial
tyrosyl-tRNA synthetase and DEAD-box proteins in splicing group I and group II
introns.
Alan Lambowitz, Institute for Cellular and Molecular Biology, University of
Texas at Austin, Austin TX 78712.
Katherine A. Borkovich, Department of Plant Pathology, University of
California, Riverside.
M. Gabriela Roca and Nick D. Read.
Institute of Cell Biology, University of Edinburgh, Rutherford Building, Edinburgh, EH9 3JH, UK.
Prof. Dr. Maria Célia Bertolini. Instituto de Química, UNESP, Departamento
de Bioquímica e Tecnologia Química R. Prof. Francisco Degni, s/n 14800-900,
Araraquara, SP Brazi
The synthesis and degradation of glycogen molecules are carried out by the
concerted action of a set of enzymes, the main control being of the activities
of glycogen synthase and glycogen phosphorylase, respectively. Glycogen synthase
catalyzes the formation of the α-1,4-glycosidic
linkages of glycogen by addition of UDP-glucose units into glycogen, and is
considered to be the limiting-rate step of the glycogen synthesis. This enzyme
is regulated both by allosteric modulation, and by reversible phosphorylation.
In addition to reversible changes in the glycogen synthase activity, glycogen
levels are also correlated with physiological conditions. In Saccharomyces
cerevisiae, glycogen accumulation is induced by conditions that stress the
cells, such as heat shock. Such condition induces transcription of the gene
encoding glycogen synthase (GSY2), which explains the glycogen accumulation. We
have isolated the gene encoding the Neurospora crassa glycogen synthase (gsn)
and demonstrated that the gene transcription is repressed when cells are exposed
to temperatures varying from 30 to 45ºC (heat shock). In addition, glycogen
levels rapidly decrease in the same growth condition. Analysis of the gsn
promoter region allowed us to identify multiple regulatory elements, including
many HSE (Heat Shock Element) and two STRE (STress Responsive Element), which
are usually found in promoters of genes responsive to stress conditions. Gel
shift assays (EMSA) using nuclear extract prepared from N. crassa heat
shocked mycelia showed the presence of protein(s) that bind specifically to the
STRE elements of the gsn promoter region. Our main purpose is to identify the
protein(s) that bind to the STRE elements in order to study how these elements
function in the regulation of gene transcription. Approaches coupling EMSA and
mass spectrometry have been used to reach the goal. Results will be presented
concerning the strategies we have used to identify the protein(s).
N. Louise Glass, Karine Dementhon, Isao Kaneko and Qijun Xiang. Department
of Plant and Microbial Biology, University of California, Berkeley, CA
94720-3102
Dan Ebbole, Texas A & M University.
The mak-2 and pp-1 mutants have reduced growth rate, produce short
aerial hyphae, and fail to develop protoperithecia. In addition, ascospores
carrying null mutations of either gene are inviable. Subtractive cloning was
used to isolate genes having reduced expression in the mak-2 mutant.
Expression of some of these genes is protoperithecia specific and three of them
are part of a gene cluster potentially involved in the production of a
polyketide secondary metabolite. Microarray analysis was used to extend the
analysis of gene expression in mak-2 and pp-1 mutants.
Qiyang He, and Yi Liu. University of Texas Southwestern Medical
Center, Dallas, TX.
Blue light regulates many molecular and physiological activities in a large
number of organisms. In Neurospora crassa, a eukaryotic model system
for studying blue-light responses, the transcription factor and blue-light
photoreceptor WHITE COLLAR-1 (WC-1) and its partner WC-2 are central to
blue-light sensing. Neurospora’s light responses are transient, i.e.
following an initial acute phase of induction, light-regulated processes are
down-regulated under continuous illumination, a phenomenon called
photoadaptation. The molecular mechanism(s) of photoadaptation are not well
understood. Here we show that a common mechanism controls the light-induced
transcription of immediate early genes (such as frq, al-3, and
vvd) in Neurospora, in which light induces the binding of an
identical large WC-1/WC-2 complex (L-WCC) to the light response elements (LREs)
in their promoters. Using recombinant proteins, we show that the WC
complexes are functional without the requirement of additional factors.
In vivo, WCC has a long period photocycle, indicating that it cannot be
efficiently used for repeated light activation. Contrary to previous
expectations, we demonstrate that the light-induced hyperphosphorylation of
WC proteins inhibits bindings of the L-WCC to the LREs. We show that, in
vivo, due to its rapid hyperphosphorylation, L-WCC can only bind
transiently to LREs, indicating that WCC hyperphosphorylation is a critical
process for photoadaptation. Finally, phosphorylation was also shown to
inhibit the LRE-binding activity of D-WCC (dark WC complex), suggesting that
it plays an important role in the circadian negative feedback loop.
Clocks and Light and Oxygen! Oh My!
Luis Larrondo, Bill Belden, Allan Froehlich, Jay Dunlap and Jennifer Loros.
Dept. of Biochemistry, Dartmouth Medical School, jennifer.loros@dartmouth.edu
Michael Brunner. Biochemie-Zentrum Heidelberg, Im Neuenheimer Feld 328, D 69120
Heidelberg
The Neurospora circadian clock protein FREQUENCY (FRQ) has a function in the
negative and the positive limb of interconnected feedback loops. In the negative
feedback loop FRQ inhibits its transcription factor White Collar Complex (WCC),
and in the positive loop it supports accumulation of WCC. These contradictory
functions of FRQ are confined to distinct subcellular compartments and
coordinated in temporal fashion. Negative feedback occurs early after the onset
of FRQ expression and requires nuclear FRQ. Nuclear FRQ promotes
hyperphosphorylation of WCC leading to its inactivation. Support of WCC
accumulation depends on cytosolic FRQ and occurs about 8 h after the onset of
FRQ expression when high amounts of FRQ have accumulated. The transcriptional
function of FRQ in the negative limb and its posttranslational function in the
positive limb are independent and associated with distinct regions of FRQ.
Phosphorylation of serine residues within the PEST-2 region triggers the
maturation FRQ of toward a cytoplasmic activator.
Carlo Cogoni, Dip. Biotecnologie Cellulari ed Ematologia, Sez. Genetica
Molecolare, Policlinico Umberto I Universita' degli Studi di Roma 'La Sapienza'
Roma, Italy
Several components of the quelling machinery have been identified by using
either forward or reverse genetic approaches. The identification of genes
required in the silencing process together with findings from other organisms
has led to a current model for quelling. In Neurospora, as in other organisms,
it would seem that quelling is serving to limit the expansion of transposons
since an introduced Tad element, a LINE-1-like retrotransposon, has an elevated
expansion in the absence of the quelling components QDE2 and DICER.
Natural variation and the multigenic nature of circadian behavior in
Neurospora
Kwangwon Lee. Department of Plant Pathology Cornell University
We studied two under-explored areas in Neurospora
biology, natural variation and quantitative trait loci (QTL) analysis. Natural
variation in circadian period, phase and temperature compensation optimizes the
ability of an organism to synchronize its biological processes to a local
environment. Among a world-wide collection of 144 Neurospora crassa
accessions circadian rhythms of asexual conidiation revealed significant
variation centered on a 22 hour period and morning specific phase. Consistent
with the phenotypic variation, there is significant genotypic variation among
circadian components, WHITE COLLAR-1 (WC-1), WHITE COLLAR-2, FREQUENCY, and
VIVID. Furthermore, we found significant association between circadian
parameters and molecular variation in the circadian genes. WC-1 mediates
interactions between the circadian clock and the environment, acting both as a
core clock component and a blue light photoreceptor. Our data show that a
putative activation domain in WC-1 is highly polymorphic in length, revealing a
significant association between circadian period, latitude of origin and wc-1
genotype. We suggest that environment specific natural variation at WC-1
fine-tunes circadian period.
In an attempt to characterize the polygenic nature of the
circadian clock, we performed QTL analyses in three mapping populations, which
were generated by crossing natural accessions, with 188 F1 progenies. At least
80 loci were determined by simple sequence repeat markers for their map position
in each population, covering the genome with about 1000 cM. The clock QTLs
identified from three experimental populations will be discussed.
William J. Belden, Jennifer J. Loros, and Jay C. Dunlap.
Leonardo Peraza1, Nallely Cano2, Mauricio Rios1, Jesús
Aguirre2, and Wilhelm Hansberg1
1Departamento de Bioquímica and 2Departamento de Genética Molecular,
Instituto de Fisiología Celular, Universidad Nacional Autónoma de México (UNAM) Apartado postal 70-
242, México, 04510 D. F. whansberg@ifc.unam.mx
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