1. From an amoeba to fruiting body - path laid
by polyketide synthases in Dictyostelium Development. Divya Nair,
Mauld Lamarque and Rajesh S. Gokhale, National
Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067; Institute of genomics & Integrative
Biology, Mall Road, Delhi 110007.
Dictyostelium discoideum is a single-celled soil amoeba that feeds on bacteria
and yeast under natural conditions. When
faced with starvation, it undergoes a complex developmental pathway to form a
multicellular structure known as fruiting body.
It is intriguing as to how this lower eukaryote sets into motion a
series of well-synchronized differentiation events. Its genome, which was recently decoded, has
revealed an unprecedented number of a family of enzymes known as polyketide
synthases (PKSs). These are
multi-functional enzymes which are capable of synthesizing diverse metabolites
with varied biological functions.
However, the role of these enzymes in the biology of Dictyostelium remains largely
unexplored. Previous studies have
provided evidence that they may actually be involved in controlling
developmental processes through synthesis of development regulating factors
(DRFs). Through biochemical methods we
have demonstrated that one of the DRFs, MPBD, is a biosynthetic product of
DiPKS1. Interestingly, DiPKS1 and few
others show temporally differential expression through the morphogenetic
process. Spatial localization studies also
demonstrate the expression of many of these PKSs in specific cell-types of the
multicellular organization. To delve
further into the relevance of some of the PKSs, we have created their genetic
knockouts through homologous recombination.
Interestingly, all these mutants complete the differentiation cycle but
with varying phenotypic abnormalities. These observations raise several
interesting questions regarding the mechanisms by which PKSs regulate
development. We are presently
investigating these various possibilities through a combination of genetic and
biochemical analysis. A pre-requisite
for obtaining functional PKSs is their post-translational modification by
phosphopantetheinyl transferases (PPTases).
We have analyzed the specific activation of the DiPKSs by two different
classes of PPTases and shown their distinct functions and relevance in
Dictyostelium biology.
2. RNAi-mediated Epigenetic Control of the
Genome. Shiv Grewal, Laboratory of Molecular Biochemistry and
Molecular Biology, Center for Cancer Research, National Cancer Institute,
National Institutes of Health,
Transposons and their remnants, which constitute a significant proportion of the complex genomes, have greatly molded eukaryotic genome evolution. How genomes bear the burden of these repetitive DNA elements that are known to be major source of genomic instability has been a fundamental question in biology. Our previous work has suggested that in the fission yeast genome RNAi machinery targets a specific class of repeat elements to initiate the assembly of heterochromatin structures, implicated in the maintenance of genomic integrity and regulation of gene expression. We have found that heterochromatin is dynamically regulated during the cell cycle and that transcription of repeat elements during S-phase of the cell cycle is closely linked to loading of RNAi and chromatin-modifying factors involved in heterochromatin assembly. These findings highlight an emerging theme that transcription and non-coding RNAs provide the initial scaffold for the formation of heterochromatin that serves as a versatile recruiting platform for diverse factors involved in many cellular processes. In a surprising finding, we have discovered that heterochromatin factors are widely distributed across euchromatic loci and collaborate with RNAi machinery to regulate the expression of RNA polymerase II transcripts across large portions of the genome. Our recent progress in understanding the mechanisms of epigenetic genome control by RNAi and heterochromatin factors will be discussed.
Grewal,
S.I.S. and Jia, S. (2007). Heterochromatin revisited. Nat. Rev. Genet.,
8:35-46.
Chen,
E.S., Zhang, K., Nicolas, E.,
3. Control of DNA methylation through chromatin
in Neurospora. Eric Selker, Shinji Honda, Keyur Adhvaryu,
Zachary Lewis and Anthony Shiver.
Most methylated regions of Neurospora are relics of
transposons inactivated by RIP (repeat-induced point mutation), a premeiotic
homology-based genome defense system that litters duplicated sequences with C:G
to T:A mutations. A detailed analysis of
the distribution of DNA methylation in the Neurospora genome revealed that it
is most concentrated at centromeric regions.
Subtelomeric regions typically also include methylated relics of
RIP. Our genetic and biochemical studies
on the control of DNA methylation have revealed clear ties between DNA
methylation and chromatin modifications.
In vegetative cells, 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. DIM-5 is sensitive to modifications of
histones including methylation and phosphorylation and is found in a complex
with several other proteins that are essential for DNA methylation. DNA methylation is modulated by a variety of
additional factors. For example, mutants
in mdm-1 (modulator of DNA
methylation -1) show aberrant methylation of DNA and histone H3K9, with both
frequently spreading into genes adjacent to inactivated transposable
elements. Mutants defective in mdm-1 grow poorly but growth can be
restored by reduction or elimination of DNA methylation using the drug
5-azacytosine or by mutation of the DNA methyltransferase gene, dim-2.
Mutants defective in both mdm-1 and dim-2 display normal H3K9me3 patterns,
implying that the spread of H3K9me3 involves DNA methylation. In general, however, HP1 and DIM-2 are
dispensable for virtually all H3K9me3.
Moreover, H3K9me3 and DNA methylation are rapidly and fully
reestablished after these marks are stripped off genetically. I will summarize and discuss our recent
progress towards the elucidation of mechanisms controlling DNA methylation in
Neurospora.
4. Tracing the path of centromere evolution. Kaustuv Sanyal, Molecular Mycology Laboratory, Molecular
Biology & Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research,
Bangalore 560064, India sanyal@jncasr.ac.in
The centromere (CEN), that serves as the
chromosomal attachment site of spindle microtubules, plays a crucial role in
chromosome segregation during mitosis and meiosis. Understanding centromere
structure/function after its first molecular characterization almost three
decades ago is still far from complete. Several lines of evidence suggest that
centromere formation cannot be solely governed by the DNA sequence, rather many
other genetic and epigenetic factors are involved. To better understand the
mechanisms involved in centromere identity, its maintenance and propagation, we
have identified and analyzed centromeres of
several Candida species that are pathogenic budding yeasts. Our
studies suggest that centromere structures of two of these species, C.
albicans and C. dubliniensis, are different from those of other
organisms. We have recently shown that
in spite of having a very high degree of similarity in DNA sequence in these
two closely related yeasts, the centromere sequences diverged more rapidly than
any other regions in the genome. We
propose that this rapid evolution of centromeres, which work in highly
species-specific manner, may serve as a driving force for speciation. More
recently, we have identified centromeres of another closely related organism, C.
tropicalis. Preliminary results suggest that centromere structure of this
species provides a missing link to a simple “point” centromere of S.
cerevisiae and a more complex regional centromere of fission yeast S.
pombe.
5. Inositol pyrophosphates regulate eukaryotic
physiology via protein pyrophosphorylation.
Rashna
Bhandari, Laboratory of Cell Signalling, Centre for DNA Fingerprinting and
Diagnostics (CDFD), Nampally,
The budding yeast Saccharomyces cerevisiae is an excellent model organism to study
metabolic processes that are conserved across eukaryotes. One such metabolic pathway is the network of
phosphorylated inositols, of which more than thirty different species have been
identified to date, and more continue to be discovered. Our focus lies on the diphosphoinositol
polyphosphates, or inositol pyrophosphates, derivates of inositol that contain
one or more diphosphate moieties in addition to monophosphates. These include
diphosphoinositol pentakisphosphate (IP7) and bis-diphosphoinositol
tetrakisphosphate (IP8). In S. cerevisiae, inositol hexakisphosphate
(IP6) is converted to IP7 by the IP6 kinase
Kcs1, and the IP7 kinase Vip1 converts IP7 to IP8. Yeast lacking Kcs1 have negligible levels of
inositol pyrophosphates, and demonstrate defects in growth, endocytosis,
vacuolar morphology, stress tolerance, and telomere length maintenance. We have demonstrated that inositol
pyrophosphates including IP7 are able to transfer their beta
phosphate group onto pre-phosphorylated serine residues to form
pyrophosphoserine. Pyrophosphorylation
requires divalent cations such as Mg2+ and occurs on serine residues
present in acidic-serine sequence motifs.
It
is likely that the diverse functions
of inositol pyrophosphates are mediated at the molecular level by protein
pyrophosphorylation. Therefore, our
current objective is to understand the biochemical links between protein
pyrophosphorylation and cellular phenomena regulated by inositol
pyrophosphates. A search for pyrophosphorylation sites in the S. cerevisiae proteome revealed that 162
proteins are putative substrates for IP7. These include proteins involved in ribosome
biogenesis, cell cycle control and vesicle transport. We are currently examining S. cerevisiae strains with deletions in the enzymes that
synthesise or degrade inositol pyrophosphates, to determine whether changes in
IP7 and/or IP8 levels lead to alterations in the
pyrophosphorylation status of individual proteins, and can be correlated with
alterations in cell physiology. Our
initial data suggests that a decrease in IP7-mediated
pyrophosphorylation of certain proteins may be responsible for lowered levels
of ribosome synthesis and defects in vesicle trafficking observed in yeast
strains that lack inositol pyrophosphates.
6. Genetic and Molecular Dissection of the
Neurospora Clock: Proteomics and Epigenetics.
Jay.
C. Dunlap, Chris L. Baker, William J. Belden, and Jennifer J. Loros, Department of Genetics, Dartmouth Medical
School, Hanover, NH 03755 USA
Transcription/ translation feedback loops are
central to all eukaryotic circadian clocks (Dunlap et al, Cold Spring Harbor
Symp. 72: 57 – 68, 2007). In all circadian systems, proteins in these feedback
loops are regulated through a myriad of physically and temporally distinct
post-translational modifications. To better understand how this regulation
impacts a circadian oscillator we implemented a proteomics-based approach by
combining purification of endogenously produced FREQUENCY (FRQ) and its
interacting partners with quantitative mass spectrometry. We tracked
time-of-day specific protein-protein interactions in the clock and found that
these provide a platform for temporal and physical separation between the dual
roles of FRQ. By identifying nearly 100 phosphorylated residues, following the
quantitative change of phosphorylation at many of these sites over a day, and
examining the phenotypes of strains bearing mutants that have lost these sites,
we can begin to see how temporally regulated phosphorylation has opposing
effects directly on overt circadian rhythms and FRQ stability.
Expression of the circadian negative element, frq, in response to light and
time-of-day is driven by a PAS-heterodimer of WC-1 and WC-2. frq is complex, encoding alternatively
spliced sense transcripts as well as a long (> 4knt) antisense transcript, qrf.
The frq and qrf promoters show chromatin
rearrangement in response to light as well as time-of-day, and deletion of all
19 genes encoding ATP-dependent chromatin-remodeling enzymes revealed only 2
genes, clockswitch (csw-1 a homolog of yeast Fun30, mouse Etl1
and human SMARCAD genes) and chd2 (a homolog of the mammalian mi-2,
chd2 and yeast Chd1 genes), required for remodeling at frq and for normal clock function. ChIP
localizes them to frq. Unexpectedly
and unusually, however, ∆csw-1
and ∆chd2 knockouts are not
simply arrhythmic but rather become arrhythmic slowly, over days.
7. The budding yeast protein Sum1p is a novel
regulator of microtubule assembly. Sourav Sarkar, Subhash
Haldar, Sujata Hajra and
Pratima Sinha, Department of
Biochemistry, Bose Institute, Kolkata-700054, India
The budding yeast protein Sum1 is a transcription
factor that associates with the histone deacetylase Hst1p to form repressed
chromatin. SUM1 has been identified as an allele-specific dosage suppressor of
mutations in the major α-tubulin coding gene TUB1. When cloned in a 2 micron vector, SUM1 suppressed cold-sensitive and benomyl-hypersensitive
phenotypes associated with the tub1-1
mutation. The suppression was Hst1p-independent, suggesting that it was not
mediated by acetylation-deacetylation events of Hst1p. When overexpressed using
the GAL1 promoter, SUM1 was toxic to cell growth. Under
these conditions, cells had very short or no spindles at all. This was found to
be due to the inability of these cells to elongate their spindles. Cells
deleted of SUM1 showed moderate
hypersensitivity to benomyl and cold-sensitive growth. These observations
suggest a novel role for Sum1p as a regulator of microtubule function through
its association with α-tubulin. We propose that as a dosage suppressor,
Sum1p promotes the formation of microtubules by increasing the availability of
the αβ-heterodimer containing mutant α-tubulin subunit. This is
being tested.
8. Holliday Junction Binding and Processing by
Meiosis-Specific Saccharomyces cerevisiae
Hop1 Protein. K. Muniyappa, Department of
Biochemistry, Indian Institute of Science,
Saccharomyces cerevisiae HOP1, which encodes a component of symaptonemal complex, plays an
important role in both gene conversion and crossing over between homologs, as
well as enforces meiotic recombination checkpoint control over the progression
of recombination intermediates. In hop1
mutants, meiosis-specific double-strand breaks (DSBs) are reduced to10% of the
wild-type levels, and at aberrantly late times, these DSBs are processed into
intersister recombination intermediates. However, the underlying mechanism by
which Hop1 protein regulates these nuclear events is poorly understood. We
observed that Hop1 interacts selectively with the Holliday junction, changes
its global conformation and blocks the dissolution of the junction by a
helicase. The Holliday junction-Hop1 complexes were significantly more stable
than complexes containing other recombination intermediates. Structural
analyses of the Holliday junction using 2-aminopurine fluorescence emission,
DNase I footprinting and KMnO4 probing provide compelling evidence
that Hop1 binding induces significant distortion of the Holliday junction. We
propose that Hop1 might coordinate the physical monitoring of meiotic
recombination intermediates with the processes of branch migration and
resolution of the Holliday junction.
9. Lessons from
quantitative trait analysis in yeast. Himanshu
Sinha, Department of Biological Sciences, Tata Institute of Fundamental
Research, Homi Bhabha Road, Mumbai 400 005, hsinha@tifr.res.in
Quantitative traits are
ubiquitous in nature and yeast is an excellent model for studying them. In
clinical isolates of yeast, high temperature growth (Htg) is a quantitative
trait and by linkage analysis, two quantitative trait loci (QTL) contributing
to the phenotype in two clinical strains were mapped. These QTLs were dissected
by reciprocal hemizygosity analysis and four genes: MKT1, RHO2, END3 and NCS2, contributing to Htg phenotype were identified. One of the QTL
had a complex architecture with two alleles coming from the clinical strain and
one allele coming from the non-Htg laboratory strain. The second QTL had only
one allele from the clinical strain contributing to the phenotype. For all of
these alleles, coding and non-coding single nucleotide polymorphisms were
identified. Alleles of these genes showed complex genetic interactions, which
included additive and non-additive interactions. Genetic background in which
these alleles exist profoundly influenced their contribution to the phenotype.
None of these genes contributing to the phenotype have functions known to
influence growth at high temperature and they act in different cellular
pathways. Yeast is helping to uncover complexities and develop techniques for
quantitative trait analysis, which will aid to understand and dissect
quantitative traits in higher eukaryotes, including humans.
10. Asymmetric cell division through epigenetic
differentiation of sister chromatids and their selective segregation in
mitosis. Amar J.S. Klar. NCI at
Our studies with the model system of fission yeast
have discovered two new principles of biology. First, developmental asymmetry
of sister cells simply results from the inheritance of older “Watson” versus
older “Crick” chain-containing chromatids at the mat1 locus where through epigenetic means nonequivalent sister
chromatids are generated by chromosome replication. Second, epigenetic states
controlling gene repression are inherited in mitosis and meiosis as remarkably
stable conventional Mendelian markers (1). We propose that likewise asymmetric
cell divisions in higher eukaryotes might result by further postulating biased
segregation of differentiated sister chromatids of both copies of a specific
chromosome to daughter cells (2,3).
Can we explain hitherto unexplained developmental
traits/disorders in humans and vertebrates by invoking such principles? The
causes of schizophrenia and bipolar human psychiatric disorders are unknown. A
novel somatic cell genetics, SSIS (Somatic Stand-specific Imprinting
and Selective strand segregation)
model, postulated biased segregation of differentiated older “Watson” vs.
“Crick” DNA chains of a chromosome to specific daughter cells. Such an oriented
asymmetric cell division in embryogenesis may constitute the mechanism for
development of healthy, functionally nonequivalent brain hemispheres in humans.
For evidence, genetic translocations of the relevant chromosome might therefore
cause disease by disrupting the chromosome-specific biased chromatid
segregation process. This way the epialleles of a hypothetical gene controlling
brain laterality development in the translocation-containing chromosome will be
randomly distributed to sister cells. Accordingly, the model predicts that
symmetrical brain hemispheres might develop in 50% of translocation carriers.
Thus, the observation of only 50% of chromosome 1/6/9;11 translocation carriers
that do develop disease is in accord with the model (4). Likewise, the SSIS
model is also advanced for visceral laterality development in mice.
1.
Klar, A.J.S. 2007. Lessons learned from studies of fission yeast mating-type
switching and silencing. Annual Review of Genetics 41: 213-36.
2.
Armakolas, A. and A.J.S. Klar 2006. Cell type regulates selective segregation
of mouse chromosome 7 DNA strands in mitosis. Science 311: 1146-1149.
3.
Armakolas, A. and A.J.S. Klar, 2007. Left-right dynein motor implicated in
selective chromatid segregation in mouse cells. Science 315:100-1.
4.
Klar, A.J.S. 2004. A genetic mechanism implicates chromosome 11 in
schizophrenia and bipolar diseases. Genetics 167: 1833-1840.
11. Environmental Sensing: Neurospora
Photobiology. Jennifer Loros,
Light is a major environmental signal for most life
on earth. Fungal genomes encode several
proteins capable of binding chromophores with the ability to harvest light
energy as well as proteins that can interact with primary photoreceptors or
further propagate the light signal. The best understood fungal photoreceptors
are the evolutionarily conserved white collar-1 (WC-1) and white collar-2 (WC-2)
proteins, and the related Vivid (VVD) protein in Neurospora crassa. Close to 6% of Neurospora genes are expressed in
response to a light stimulus in a temporally regulated cascade that includes
several transcription factors. The White
collar proteins are essential for the vast majority of this light-mediated
expression and can bind the promoter of light-responsive submerged
protoperithecia-1 (sub-1), a GATA
family transcription factor. SUB-1 is
essential for most late light gene expression.
12. Nucleosome dynamics on
the genes transcribed by RNA polymerase III of yeast. Yatendra Kumar
and Purnima Bhargava, Centre for Cellular & Molecular Biology,
Organization of eukaryotic genome into chromatin
results in repression of transcription. Apart from the two anti-repression
mechanisms; histone modifications and chromatin remodeling, nucleosome
positioning is a mechanism used by cells to display or block the binding sites
of various transcription factors. Most
of the time it is mobilization and change in relative locations of the
nucleosmes on the genome, which is the target of the epigenetic modifications
in enforcing the regulation of gene expression.
It is established now that the relative positions of the nucleosomes
with reference to the underlying DNA sequences of the genome have a profound
effect on the activity status of the genome.
Studies on global nucleosome positioning were most fruitful in yeast
owing to the simplicity of the organism as well as the fact that most of yeast
genome is transcribed euchromatin.
Several genome-wide studies have revealed certain common patterns that
are widespread throughout the yeast genome.
For a typical yeast ORF, the upstream region is characterized by a
nucleosome-free region (NFR) encompassing the transcription start site (TSS)
flanked by two well positioned nucleosomes containing histone H2A variant
H2A.Z.
Short, non-coding genes transcribed by RNA polymerase III (pol
III) are generally considered nucleosome-free.
Our previous studies have shown that sequence of one of the genes
transcribed by pol III, U6snRNA has nucleosome positioning properties. As a result of chromatin remodeling dependent
on TFIIIC, the basal factor for yeast pol III transcription, at least one nucleosome
gets translationally positioned on the U6 gene region between its promoter
elements, boxes A and B. Genome-wide
localization studies of the chromatin modifying complexes also suggest that
chromatin may be having an important regulatory role in expression of these
genes. We have studied the nucleosome
dynamics on the pol III-transcribed genes under different conditions using high
density tiling microarrays. Results,
which will be presented, have revealed interesting mechanistic details of the
regulation of transcription from these genes.
13. The multicopy plasmids of yeast segregate
equally through cohesin mediated recognition of sisters. Santanu K. Ghosh Ph.D., Assistant Professor, School
of Bioscience and Bioengineering, IIT Bombay, Powai, Mumbai 400 076 santanughosh@iitb.ac.in
The 2 micron yeast plasmid, a benign high-copy
nuclear parasite, propagates itself with nearly the same fidelity as the
chromosomes of its host. Equal plasmid segregation is absolutely dependent on
the cohesin complex assembled at the plasmid partitioning locus STB. However, the mechanism of cohesin
action in the context of multiple plasmid copies, resident within two separate
clusters following DNA replication, is unknown. We unveil two key features of
cohesin mediated plasmid segregation. First, by using single copy derivatives
of the 2 micron plasmid, we demonstrate that recruitment of cohesin at STB during S phase indeed translates
into cohesion between plasmid molecules. Second, through binary fluorescence
tagging, we reveal that segregation of replicated plasmids occurs in a
sister-to-sister fashion. Thus, cohesin serves the same fundamental purpose in
plasmid and chromosome segregation. Furthermore, one-to-one segregation of
sisters implies a highly ordered arrangement of plasmid molecules that permits
a multi-copy cluster to template its sister cluster. In addition, our results
suggest that two sister plasmid molecules, following replication, are
topologically trapped within a single cohesin ring.
14. Development of a novel methylotrophic yeast
expression system. Partha Kumar Sarkar,
Principal
Scientist (R&D)& Project Leader, Shantha Biotechnics Limited, P.O Box
4, Medchal, RR District, Andhra Pradesh-501401, sarkar@shanthabiotech.co.in
Development of recombinant DNA technology has made
possible production of an enormous variety of useful proteins using
microorganisms. Traditionally, commercial efforts employing recombinant DNA
technology for the production of proteins have focused on the use of Escherichia
coli (E. coli) as a host organism. However, E. coli has proved to be
an unsuitable host in many situations. For example, some eukaryotic proteins
that are produced in prokaryotic cells are either unstable or lack biological
activity. Accordingly, in these cases, yeasts offer advantages over their
prokaryotic counterparts, which include an intracellular environment that is
more conducive for correct folding of eukaryotic proteins. Additionally yeasts,
unlike prokaryotic hosts, have the ability to glycosylate proteins, which is
important for both the stability and biological activity of the protein. Saccharomyces
cerevisiae was the first, and one of the most commonly employed eukaryotic
expression system because its genome and physiology have been extensively
characterized. But, S. cerevisae is not always the optimal expression
system for large-scale production of heterologous proteins because of plasmid
loss during scale-up, hyperglycosylation, and low protein yields. Methylotrophic
yeast expression systems offer several advantages over S. cerevisiae. For
example, methylotrophic yeast expression systems achieve very high cell
densities in a simple defined medium and have strong inducible promoters that
enable high level of stable expression of heterologous genes integrated into
the host genome.The most highly developed methylotrophic host systems are Pichia
pastoris (Komagataella pastoris) and
Hansenula polymorpha (Pichia
angusta). Three other methylotrophic yeast species, designated Pichia
methanolica and Candida boidinii and recently Pichia minuta have
also been developed as heterologous expression systems .Since all the above
systems are either patent protected or under material transfer agreement
attempt has been made to develop a novel methylotrophic yeast expression system
using available methylotrophic yeasts
from culture collection centers (which have not been developed as an expression
system) .This presentation will cover various steps towards the above development like cloning novel constitutive and inducible
genes and their promoters and creation of
vectors for expression of
heterologous genes in the new methylotrophic yeast expression system.
15. The Biology of Deep-Sea Fungi. Chandralata Raghukumar, National Institute of
Oceanography, Dona Paula, Goa 403 004,
A rich diversity of micro- as well as macroorganisms
has been shown in the deep sea in recent times. A number of piezotolerant and piezophilic
bacteria and archaea have been reported. The few studies that have been carried
out on deep-sea fungi in recent years have provided evidence about their
presence and activity, either by culturing or molecular signatures.
Culture-independent diversity carried out by our group showed clones matching
those recovered in culture and some uncultured fungal clones. A majority of
cultured and culture-independent fungi recovered showed homology to the species
reported in terrestrial environment indicating their possible arrival in deep
sea either with wind or terrestrial runoffs. Several of these fungi showed
abnormal morphology during the initial culturing and also when grown at 20
MPa/5oC. However, several filamentous fungi and yeasts recovered
from deep-sea sediments of the Central Indian Basin (CIB) from a depth of ~5000
m and a few terrestrial species showed growth under hydrostatic pressure of
200-400 bar (20-40 MPa) and temperature of 5oC in our laboratory.
These results suggest that terrestrial species of fungi transported to the deep
sea are initially stressed but may gradually adapt themselves for growth under
these conditions.
Recovering culturable fungi from deep-sea sediments
and other such extreme environments is always fraught with an apprehension of
contaminants and therefore their detection by direct examination of sediments
provides proof of their existence. We demonstrated the presence of fungal
hyphae in these deep-sea sediments by direct staining of the sediments with
Calcofluor, a fluorescent optical brightener. Using this method fungal biomass
contribution to deep-sediments of CIB ranged from 15-1931 ug C g-1
wet sediment at various depths of sediment sections. Presence of hyphae of a
deep-sea isolate of Aspergillus terreus (#A4634) was confirmed by using
an immunofluorescence probe also.
Direct detection of fungal hyphae in deep-sea
sediments is a daunting task as they are present in low abundance. The apparent
reason for their poor detection is their cryptic presence in macroaggregates.
Treatment with EDTA resulted in breakdown of aggregates and revealed fungal
hyphae. Hyphae of fungi grown in sediment extract medium under elevated
hydrostatic pressure and low temperature showed various stages of accretion of
particles around them, leading to the formation of aggregates. Based on these
results, it is suggested that fungi in deep-sea sediments may be involved in
aggregate formation and carbon sequestration, similar to their role in
terrestrial sediments.
Once the wind-blown or terrestrial runoff-carried
fungal spores and mycelial fragments reach deep sea, they are affected by
elevated hydrostatic pressure, low temperature and low nutrients. We examined
the effects of these on germination of spores from a few deep-sea Aspergillus
isolates. Elevated hydrostatic pressure did not affect spore germination but
low temperature of 4-5oC inhibited their germination totally. On the
other hand mycelial fragments showed growth and biomass build up under elevated
hydrostatic pressure at 5oC. These results indicated that growth and
biomass build up from spores is not a viable option for the deep-sea fungi
whereas mycelial fragments are more likely to grow. We further confirmed
survival of fungal mycelia of two species exposed at 3500 m depth for ~18 months,
in the Equatorial Indian Ocean on a mooring buoy. The above results indicate that fungi with
their multicellular filaments and unicellular spores are a unique model to
study the effect of hydrostatic pressure.
16. Fungal pathogens of humans. Marc Orbach (U.
Abstract awaited.
17. Pea pathogenicity, pisatin tolerance, and
supernumerary chromosomes in Nectria haematococca MPVI. Jeffrey J. Coleman,
The ascomycetous fungus Nectria haematococca is a member of a group of >50 species known
as the “Fusarium solani species
complex”. Members of this complex have
diverse biological properties including the ability to cause disease on >100
genera of plants and opportunistic infections in humans. Previous studies on N. haematococca mating population VI (MPVI) have demonstrated
several genes controlling the ability to colonize specific habitats are located
on supernumerary chromosomes. Optical
mapping revealed that the recently sequenced isolate has 17 chromosomes and
that the physical size of the genome, 54.43 Mb, and the number of predicted
genes, 15,707, are among the largest reported for ascomycetes. The expanded genome size is largely due to
either extra copies of genes or genes which were absent in the previously
sequenced Fusarium graminearium
genome. Some of these additional genes
appear to have resulted from gene duplication events, while others may have
been acquired through horizontal gene transfer.
Three chromosomes, 14, 15, and 17, are supernumerary and these
chromosomes contain more repeat sequences, are enriched in unique and
duplicated genes, have a different codon preference, and have a lower G+C content
when compared to the other chromosomes.
Although the origin(s) of the extra genes and the supernumerary
chromosomes is not known, the presence of unique genes on these chromosomes
might account for individual isolates having different environmental niches.
A
gene encoding a cytochrome P450 involved in detoxification of the pea
phytoalexin pisatin is encoded on a CD chromosome (chromosome 14). This enzyme, termed PDA for pistain
demethylase, is a virulence factor on garden pea and resides in a cluster of
three other genes involved in pea pathogenicity. In addition to enzymatic detoxification, a
“nondegradative” tolerance mechanism has also been identified in N. haematococca. An ABC transporter, NhABC1, was identified as
the gene responsible for this tolerance.
NhABC1 is induced by pisatin
and NhABC1 mutants are reduced in
virulence on pea to a similar degree as PDA
mutants. However, isolates lacking both PDA and NhABC1 are essentially non-pathogenic on pea and are more sensitive
to pisatin than either single mutant, demonstrating these two proteins are the
major mechanisms responsible for pisatin tolerance.
18. Glutathione utilization pathways in
fungi. Anand K Bachhawat, Ph.D.,
Scientist,
Glutathione, g-glutamyl-cysteinyl-glycine,
a tripeptide with an unusual g-glutamyl linkage, is the
principle redox buffer of almost all eukaryotic cells, and is present at high
concentrations in living cells. Although glutathione degradation has been
earlier thought to be always initiated in all living cells by the enzyme g-glutamyl transpeptidase, we have recently
demonstrated that an alternative pathway exists in S.cerevisiae that
involves 3 previously uncharacterized proteins that we have named as Dug1p,
Dug2p and Dug3p, and which appear to form a complex for glutathione
degradation. In addition to the Dug complex is a high affinity glutathione
transporter, Hgt1p, that is also required for the utilization of exogenous
glutathione. Interestingly, homologues of the Dug complex appear to be present
in all fungi, barring the fission yeast, S.pombe, while homologues of
the glutathione transporter, Hgt1p appear to be present in all fungi except C.
glabrata. Investigations into the glutathione metabolism pathways in these
and other yeasts, as well as more detailed investigations into the Dug complex
and into the transporter protein, Hgt1p, has led to some interesting new
insights of glutathione utilization in fungi.
19.
Modulation of programmed cell death in Neurospora crassa. Arnaldo
Videira, IMBC, Porto, Portugal. AVideira@ibmc.up.pt
Programmed cell death
(PCD) is a genetically-controlled process of cellular suicide, initiated by
endogenous or extrinsic signals, that is essential for the development and
homeostasis of metazoan organisms and has been implicated in a number of human
disorders, including cancer, neurodegenerative and infectious diseases. Thus,
modulation of PCD has potential implications to the medical field. The fungus Neurospora crassa can be induced to undergo an apoptosis-like cell
death with drugs like phytosphingosine or staurosporine, which lead to reduced viability,
DNA condensation and fragmentation and production of reactive oxygen species.
The sensitivity/resistance of respiratory chain complex I mutants to these
drugs point to the central role of mitochondria as central mediators of cell death and to the tight connection between bioenergetics and
PCD.
The overall analysis by microarrays of gene
expression resulting from drug-induced PCD uncovers molecular pathways
associated with the process. For instance, phytosphingosine
treatment induces a general down-regulation of genes encoding mitochondrial
proteins and an ABC transporter involved in PCD was identified after transcriptional profiling of
the genes responding to staurosporine exposure. Modulation of PCD can be
achieved with a combination of death-inducing drugs with drugs that target
specific pathways associated with PCD.
20. Some unique biological aspects of the marine
stramenopilan fungi, the Labyrinthulomycetes. Seshagiri Raghukumar, Myko
Tech Private Ltd., 313 Vainguinnim Valley, Dona Paula,
The obligately marine, eukaryotic, osmoheterotrophs,
the Labyrinthulomycetes are a group of fungi belonging to the Kingdom
Stramenopila. Despite their relatively
recent discovery of just about 70 years ago, these unicellular protists,
comprising thraustochytrids, aplanochytrids and labyrinthulids are increasingly
gaining attention for biotechnology, ecology, biochemistry, genomics and
evolution. The genus Schizochytrium, belonging to
thraustochytrids is presently a major commercial source of the omega-3
polyunsaturated fatty acid, docosahexaenoic acid (DHA). Ecological studies on the abundance of
Labyrinthulomycetes in the marine ecosystem suggest that these protists might
serve as an important source of DHA to marine crustaceans, enabling them to
grow and reproduce. In practical terms,
thraustochytrids are used as feed in aquaculture. Recent studies have shown
that Schizochytrium synthesizes its
DHA not through the conventional aerobic pathway present in eukaryotes, but
through an anaerobic polyketide synthase pathway originally discovered in
marine bacteria. This finding has been a
major catalyst for research on whole genome sequence studies of representative
Labyrinthulomycetes. Molecular taxonomic
studies using the SSU ribosomal DNA sequences of Labyrinthulomycetes have shown
that contrary to conventional morphological taxonomy, these organisms do not
belong to the Kingdom Fungi but to the Kingdom Stramenipila that is also the
lineage of the oomycetan fungi. A major
finding of recent times is that both the Labyrinthulomycetes and the Oomycetes
have probably evolved into osmoheterotrophy through the loss of
chloroplasts. One possible area of
interest is the production of an elaborate system of plasma membrane
extensions, the ectoplasmic net elements and the paranuclear body. The production of extracellular
polysaccharides and carotenoids are other areas of interest for
biotechnology. Whole genome studies on
Labyrinthulomycetes are likely to shed more light on the exciting biology of
these organisms.
21. Fifty years of fungal genetics: A
perspective from the
Founded
in 1960, the
22. Dictyostelium discoideum: A Model System For All Seasons. Eugene R. Katz, Department of Molecular Genetics
& Microbiology, Stony Brook University, Stony Brook, New York, 11794, USA.
When Dictyostelium
discoideum was first described in 1935, its life cycle seemed quite
remarkable. A free living amoeba that when starved, chemotactically aggregated
with its fellow amoebae, into a mound of about 105 cells. Over the
next 16 hours this mound transformed itself into a small plant-like fruiting
body consisting of a cellulose-containing stalk supporting a droplet of liquid
containing spore cells. So, Dictyostelium seemed to be both an animal (the
amoebae) and a plant (the fruiting body). The fruiting body contained only two
kinds of cells, both quite different from the amoeba, and the ratio of spore to
stalk cells seemed quite constant, independent of the number of amoebae in the
aggregate. Thus was born Dictyostelium as a model system for differentiation
and development. One cell type (the amoeba) making one decision, becoming two
cell types, in a highly regulated way, to produce a precise pattern in the
fruiting body. It seemed like the minimal system to study pattern formation,
and, for most of the last 75 years almost all the research effort of the
Dictyostelium community has been focused on trying to understand the molecular
mechanisms associated with that one
decision.
Over the last twenty years there has been an
explosion in our understanding some of the fundamental processes in cell
biology and the Dictyostelium amoeba, with its ease of genetic manipulation has
emerged as a powerful model system to study these processes. The Dictyostelium
system has already contributed significantly to our understanding of cell motility
and chemotaxis. Very recently there has been considerable excitement in the
area of bacterial pathogenesis as many of the fundamental problems in this area
have become tractable. Although many host-pathogen systems are available, few
permit readily genetic manipulation of the host side of the equation. Once
again the Dictyostelium amoeba is emerging as a prime model system in which to
study these interactions. This
presentation will review the Dictyostelium system with particular emphasis on
its role as a model for the processes described above.
23. Rapamycin induces autophagic cell
death in Dictyostelium discoideum.
Pynskhem Bok Swer, Rakhee Lohia and Shweta Saran,
Mechanisms leading to non-apoptotic cell
death in Dictyostelium discoideum
still remain poorly understood except for the fact that the differentiation
factor, DIF, leads the cells from starvation induced autophagy to ACD. In the
present study, we have tried to understand the process as well as the mechanism
underlying autophagy in this organism. Nutrient signaling plays an important
role in the switch over from unicellular to multicellular stages in D discoideum. We have shown that
nutrient starvation and rapamycin treatment induces ACD in D discoideum via the TOR kinase.
The drug, rapamycin, specifically binds to the intracellular FKBP12
protein to form a drug-receptor complex which then interacts with the FRB
domain present in the Tor kinase gene
resulting in its inhibition and subsequently to the induction of autophagy. We
have identified and functionally characterized the FRB domain present in the
single Tor gene in D. discoideum. Studies on the expression of the partial DdTor kinase confirm its sensitivity to
rapamycin and its involvement in the process of autophagy. Our results also
show that rapamycin suppresses proliferation by induction of cell cycle arrest
in the G1 phase. In the present study we have shown that ACD acts upstream and
induces accumulation of both ROS and (Ca2+)i. Studies
provide evidences to show that an increase in the load of either of them can
induce ACD.
24. Evolutionary basis of social behaviour in the
cellular slime moulds. Vidyanand Nanjundiah, Indian
Institute of Science,
The cellular slime moulds are free-living soil
amoebae that are found all over the world. The first part of this talk will
highlight the characteristic feature of their life cycle, namely that it
consists of both solitary and social phases. The social phase involves division
of labour and – apparently – altruistic behaviour; each poses difficulties when
examined from an evolutionary point of view. The second part of the talk will
look at how