Docstoc

hons_projects2011

Document Sample
hons_projects2011 Powered By Docstoc
					 Department of
    Genetics,
The University of
  Melbourne.

 Honours Research
     Projects
      2011
APPLETON GROUP

Alpaca Genetics
Alpacas originate from areas of poor pasture quality in Peru, Chile and Bolivia and
therefore thrive in the Australian environment. They most efficiently convert our poor
quality pasture into nutrients and their padded feet cause minimal damage. They do not
require special fencing or frequent drenching and do not suffer flystrike.
The Australian industry was established in the late 1980‟s and outside Peru, Australia‟s
alpaca industry leads the world in both quality and quantity. The alpaca grows a soft
handling, hard wearing, warm lightweight fibre. The Australian industry has consistently
seen increases in herd numbers, fleece value and fibre production with more than 100
tonne now produced locally per annum.

Our lab investigates the genetic basis of commercially important traits in alpaca.
Details of a possible project are listed below. This project and its availability may alter as
data is accumulated in the laboratory and there may be scope for input from interested
students on the direction of the project. It is important to keep in contact with Belinda
Appleton regarding your interest in the project closer to the Honours application
deadline.

Project BA1 - Alpaca candidate gene screening
Supervisor: Belinda Appleton
Will investigate a wide range of associations between phenotype and genotype.
This project will involve microsatellite genotyping. This work will identify (and exclude)
candidate loci for particular traits of interest. Subsequent work will be dependent on
findings, but may provide opportunity for mapping to refine the genomic location and
attempting to identify the gene responsible.


Bats, Biodiversity and Biogeography.
The current rate of deforestation occurring in south east Asia is of great concern to
many natural scientists. Between 13 and 40% of the region‟s biodiversity (flora and
fauna) is likely to be lost in the next century. Baseline data of the biodiversity that has
existed in the past is essential for the continuing work of recording current biodiversity,
the loss of diversity and to provide information to most effectively direct conservation
effort.
 
 Natural history collections in museums contain data critical to decisions in
biodiversity conservation. Collectively, these specimen-based data describe the true
distributions of taxa in time and space. The information contained within Museum
collections can be placed into a modern conservation context. It has been shown that the
recognised level of biodiversity, as described by traditional morphological taxonomy,
frequently underestimates the actual level that exists. This is important for conservation
efforts as we cannot hope to conserve biodiversity until we understand what it is we are
attempting to conserve. 
 
 Bats are often the only mammals found on islands,
especially very small islands. It is frequently believed that because bats can fly, they are
able to disperse out of threatened areas into new or residual habitats, and therefore do
not suffer the same conservation threats as other species. However, this belief is
generally incorrect. Many bat species are physically unable to disperse long distances,
and/or are adapted to a very limited range of environmental conditions.
Project BA2 - Asian Bat Biogeography
 Supervisor: Belinda Appleton
Tissue samples from a group of bats will be used to generate DNA sequence data to
define species boundaries, which will inform the level of biodiversity within the genus
in south east Asia, particularly the Philippine Islands. These results will potentially
describe species new to science and will substantially increase the knowledge of the
geographic distributions of each of these species. The information obtained will allow
the study of biogeographical processes in the region of interest and combined with
comparative information from other regions, may shed light on the evolution of the
genus across the globe.



Contact details:
Dr Belinda Appleton
Department of Genetics, The University of Melbourne
Room R.10
Phone: 8344 5137
b.appleton@unimelb.edu.au
MARTIN GROUP

Preamble
The major emphasis in my laboratory is the evolutionary genetics of Chironomus, with
some emphasis on molecular and cytological approaches.

General Project Area
Project JM - A molecular approach to determining the specific status of
the members of the Chironomus oppositus-group.
Supervisor: Jon Martin

The Chironomus oppositus-group comprises a number of morphologically very similar
taxa. Based on cytological criteria, some of these are considered separate species, while
the taxonomic status of others is uncertain. The main emphasis on this project will be on
clarifying those taxa currently included as C. oppositus itself, but in order to do this
comparisons within the broader group will be necessary.
At least five forms of C. oppositus have been recognized on the basis of the combinations
of inversion sequences that they possess, but only four will be considered in this study.
Some of these forms also differ in the chromosomal location of the dominant male
determining (MD) gene (LHS fig. below), or there may be polymorphism for MD
location within a single form. It has been postulated that the different MDs could act as
isolating mechanisms, and therefore that populations with different MD locations should
be different species. Chromosome banding pattern alone do not clarify this situation, as
the 4 forms do not form discrete clusters (RHS fig. below).




       This question has been approached using allozyme polymorphism, but that data
again shows some intermixture of forms (data not shown), but when the cytological and
allozyme data are combined, the forms are distinct and samples with different MD sites
occur on different branches LHS fig. below).
         Mitochondrial (mt) DNA sequences have proven ineffective in answering this
question (RHS fig above) as there is too little variation in sequence between forms, at
least in mainland populations. This may be due in large part to hybridization between the
forms.

Thus, research to date does not answer the question as to whether they are distinct
species, although f. tyleri is now considered as such, but is providing evidence of
relationships between the various forms, and the extent to which changes in MD location
may result in genetic isolation.

        While further mt sequence is desirable, the nuclear genes may be less influenced
by hybridization, provided there is sufficient variation. Two types offer this possibility:
the internal transcribed (ITS) regions of the ribosomal genes and EPIC (Exon Primed
Intron Crossing) sequence from nuclear genes. Preliminary data for the ITS-1 region
looks hopeful, but more work is required to assess suitable EPIC primers.




CONTACT DETAILS
Supervisor
  Dr. Jon Martin
  Department of Genetics,
  The University of Melbourne
  Room B.02
  Ph: 8344 6258
  j.martin@unimelb.edu.au
HYNES GROUP




CONTROL OF CARBON SOURCE UTILISATION IN
Aspergillus nidulans
Fungi can grow on many different sources of carbon. Different sets of enzymes are
required for different carbon source with a consequent rearrangement of
metabolism. The expression of whole suites of genes is modulated by transcription
factors responding to metabolic signals.

A. nidulans provides an excellent experimental system. It is easy to grow in the laboratory,
it is haploid and so mutants can be readily isolated and it has a good genetic system. The
A. nidulans genome sequence is available allowing the rapid cloning of genes and a
variety of molecular tools, including a transformation system allow us to manipulate
sequences in vitro and then re-introduce this DNA into the organism.
We have used A. nidulans for studies of gene regulation successfully for a long time.
Other filamentous fungi are important pathogens or are used for production of antibiotics
and enzymes and knowledge of how fungi respond to their nutrient status is of great
relevance to this.

Project MH - Master regulatory genes involved in reprogramming
carbon metabolism.
Supervisor: Michael Hynes

During growth on acetate, ethanol, fatty acids and amino acids as carbon sources
Aspergillus must be able to synthesize sugars by the gluconeogenic pathway (reversal of
glycolysis). We have discovered two transcription factors that turn on genes required for
the switch from growth on glucose to growth on acetate and fats and amino acids. The
transcription factors have related Cys 6 DNA binding domains and an intriguing PAS
domain at the C-terminus.
We have reporter genes to study their action.
We know that together they bind to the core consensus sequence CCGN7CCG.
We find that this sequence is conserved in the 5' region of very many carbon metabolic
genes in many fungi. It seems that they have a very wide range of targets.
We want to confirm this by transcription analysis.

We have extensively manipulated the genes to find out how they work and how they
respond to inducing signals.
The project will involve analyzing the effects of these mutations on target gene
expression.

Background is described in this fascinating and beautifully written paper:- Hynes et al.
(2007) GENETICS 176; 139 - 150.

Come and talk to me.

CONTACT DETAILS
 Prof. Michael Hynes
 Department of Genetics, The University of Melbourne
 Room 2.13/2.21
 Phone 8344 6239
 Email: mjhynes@unimelb.edu.au
DAVIS GROUP
              Gene Regulation in Filamentous Fungi




Background
Cells respond to different nutritional conditions by altering the expression of whole
suites of genes. Changes in gene expression reflect the capacity of transcription
factors to activate or repress their target genes. In filamentous fungi the availability
of carbon and nitrogen metabolites influences the ability of the organism to grow, to
undertake different developmental pathways and, in the case of pathogenic species,
to initiate and maintain infectious growth in a host.

Aspergillus nidulans is an excellent organism for studies of gene regulation and provides a
model for other filamentous fungi which are important pathogens or which are used in
industrial processes for production of antibiotics and enzymes. Penicillium marneffei is an
opportunistic fungal pathogen of humans related to A. nidulans. Both species are haploid,
their genomes have been sequenced and a variety of molecular tools are available
including a transformation system for the introduction of DNA sequences and
manipulation of the genome.

Nitrogen is an essential component of living cells and is required for cell growth and
viability. Previous studies in A. nidulans have shown that the regulation of nitrogen
utilization involves transcriptional regulation of a range of permeases and catabolic
enzymes in order to scavenge nitrogen from the environment. The GATA transcription
factor AreA is a key nitrogen regulatory protein and AreA is essential for the shift in the
pattern of gene expression that accompanies nitrogen limitation. Nitrogen availability is
also an important factor in the development of fungal infection in plants and animals.
When P. marneffei enters a mammalian host, the spores are engulfed by macrophages, a
nutrient-poor environment. The fungus needs to utilize those nutrient sources that are
available in order to grow in this environment.

Project MDBM1 – Role of gene coding sequence in attaining successful
heterologous expression of novel cellulases
Supervisors: Meryl Davis & Brendon Monahan

Crystalline cellulose forms a large proportion of plant biomass and it is a very desirable
source of sugar for the production of second-generation biofuels. In nature the breakdown
of cellulose to sugar is mostly conducted by an array of cellulase enzymes produced by
fungi, bacteria and protozoa with the latter group being the least studied. Few protozoan
cellulases have ever been expressed as soluble functional enzymes in heterologous
organisms like Escherichia coli or Saccharomyces cerevisiae; the reason(s) for lack of
success is not known but several factors implicate the gene coding sequence and its
mismatch with that of the expression host organism. This project will investigate the
effect of target gene codon usage and frequency, and how this varies within selected
protozoan cellulase genes, and its relationship to the host codon pool. We will select
target genes from a range of novel genes we have identified from protozoa that inhabit
the gut of Australian termites. The project will likely involve the use of computational
packages for gene analysis, gene cloning and heterologous gene expression in E. coli
strains, and site directed mutagenesis.

Project MDBM2 – Molecular characterisation of a fission yeast isolate
used in industrial bioethanol production.
Supervisors: Meryl Davis & Brendon Monahan

Increasing concern about the use of fossil fuels contributing to global warming has led to
exploration of renewable fuel sources. Industrial bioethanol production from plant
biomass hydrolysates, such as molasses (a by-product of the sugar industry), has arisen as
an important fossil fuel alternative. Indeed, the production of bioethanol from molasses is
the second-largest production route of bioethanol production in Australia. This industrial
process uses fission yeast, Schizosaccharomyces pombe, to convert the sucrose sugars
present in molasses to ethanol. To produce ethanol from sucrose the yeast must co-
ordinate and regulate a complex system of metabolic events, including the extracellular
breakdown of sucrose, uptake of the resulting hexoses, and intracellular hexose
metabolism. The metabolism of sucrose and the expression of hexose transporters is
repressed in S. pombe in the presence of glucose. Considering that glucose is a product of
sucrose metabolism, such a negative feedback regulatory system may limit ethanol
production. This project will create S. pombe mutants that by-pass or inactivate the
glucose-repression system. These mutants will then be used to directly test the hypothesis
that relieving glucose repression will increase sucrose metabolism, hexose transport and
subsequent bioethanol yields. This project will provide experience in yeast molecular
genetics, headspace gas chromatography for ethanol quantification, and many molecular
biology techniques including northern-blot analysis and qRT-PCR.

You will be located in Dr. Brendon Monahan’s laboratory at CSIRO, Parkville.
CONTACT DETAILS
Departmental Supervisor (must be contacted in the first instance)
Assoc. Prof. Meryl Davis
  Department of Genetics
  The University of Melbourne
  Room 1.01
  Phone 8344 6246 Email: m.davis@unimelb.edu.au

Dr. Brendon Monahan
CSIRO
343 Royal Parade Parkville
Phone: 9662 7310
Brendon.Monahan@csiro.au
DAVIS GROUP & ANDRIANOPOULOS GROUP
Project MDAA – The differences in regulation of the fmdS gene in A.
nidulans and P. marneffei.
Supervisors: Meryl Davis & Alex Andrianopoulos

In A. nidulans the AreA transcription factor activates the expression of nitrogen-regulated
genes when cells are nitrogen limited. Loss-of-function areA mutants are unable to use
most nitrogen sources as the genes encoding enzymes to breakdown these nitrogen
sources are not expressed. One of
these enzymes is a formamidase that
allows cells to release nitrogen from
formamide. This enzyme, encoded
by the fmdS gene, is regulated by
AreA and its expression is AreA-
dependent (see Fig. 1).

The areA gene of P. marneffei
encodes a similar GATA finger                +N        +/- N      -N       +N           -N
transcription factor and has a similar          Wild-type                   areA mutant
role in activating expression of
nitrogen utilization genes in this Figure 1. Expression on fmdS-lacZ in A. nidulans
species. If P. marneffei is transformed with the A. nidulans fmdS gene the expression of
this gene (as an fmdS-lacZ fusion) is expressed in a nitrogen-regulated fashion as it is in
A. nidulans (see Figure).

However, if this fmdS-lacZ gene is introduced into a P. marneffei areA mutant, it
becomes highly expressed and the expression is not regulated normally. This surprising
result suggest that there may be a factor(s) able to recognize the fmdS promoter that is
able to drive expression in P. marneffei in the absence of AreA and that this factor(s) is
not active in A. nidulans.

The aim of this project is to investigate the differences in regulation of the fmdS gene in
A. nidulans and P. marneffei.

In considering possible projects, it is most important to think about the areas of genetics
that you are interested in and the types of techniques involved. It is essential that you
discuss the background of this research and the possible areas of future work to get a
better understanding of the nature of the projects.

CONTACT DETAILS
Assoc. Prof. Meryl Davis                             Assoc. Prof. Alex Andrianopoulos
  Department of Genetics                             Department of Genetics
  The University of Melbourne                        The University of Melbourne
  Room 1.01                                          Room 2.11
  Phone 8344 6246                                    Phone 8344 5164
  Email: m.davis@unimelb.edu.au                      Email: alex.a@unimelb.edu.au
ANDRIANOPOULOS GROUP
             Molecular Genetic Control of Development




Project AAJH1 - Cloning and characterization of a zebrafish mutant,
prospero, with abnormalities in intestinal development.
Supervisors: Joan Heath & Alex Andrianopoulos

The zebrafish model has found favour in multiple genetic and biomedical research
settings, including modelling of human disease and as a well-characterised in vivo
biological system upon which to conduct a chemical screen. The zebrafish team in the
Colon Molecular and Cell Biology laboratory came into the field working from the
premise that the process of zebrafish intestinal organogenesis would provide an excellent
model for colon tumour growth.
Using an ENU-mutagenesis genetic screen, we identified a dozen zebrafish mutants with
defects in intestinal development. Extensive analysis has shown that the intestinal
epithelium in these mutants display defects in cell proliferation, cell shape, differentiation
and/or apoptosis. Over the last few years, we have successfully cloned the underlying
faulty genes in seven of these mutants.
This project aims to continue the genetic characterization of one of the remaining
unidentified mutants (prospero) using positional cloning. This will involve techniques
such as identification of zebrafish embryos displaying the prospero phenotype using a
panel of microscopy and histological techniques, extraction of genomic DNA, PCR
amplification of microsatellite markers and agarose gel electrophoresis. The student will
also learn how to interpret their results in the context of the Zebrafish Genome Project
(http://www.sanger.ac.uk/Projects/D_rerio). As several similar projects have been
successfully undertaken in the host lab, these techniques are all well established.

The project will be undertaken in the Colon Molecular and Cellular Biology Laboratory,
Ludwig Institute for Cancer Research - Parkville Branch, Royal Parade (opposite the
Howard Florey Institute)

Project AAJH2 - Role of the transcription factor, c-Myb in cell growth
and proliferation in the vertebrate intestinal epithelium.
Supervisors: Joan Heath & Alex Andrianopoulos

The highly elaborate epithelial lining of the vertebrate intestine is a dynamic and self-renewing tissue
system that encompasses most aspects of cell behaviour, including cell proliferation, differentiation,
migration and apoptosis. To a large extent, the genetic mechanisms involved in establishing and
maintaining this constantly remodelling tissue system remain a mystery. Due to its many favourable
characteristics, including prolific reproduction, external development and optical transparency of
embryos, the zebrafish is an ideal model for the genetic analysis of vertebrate organogenesis.
In the zebrafish intestine, three distinct cell lineages are derived from a common multipotential stem
cell. These cells undergo a series of binary cell fate decisions to give rise to the enterocytes (nutrient
absorbing), goblet (mucous producing) and enteroendocrine (hormone producing) cells. The
mechanisms that govern these binary cell fate decisions are incompletely understood. We recently
identified a new BAC transgenic line, Tg[c-myb:YFP], which provides an exciting opportunity to
throw light on this question. In this line, the regulatory elements of the c-myb gene drive strong YFP
expression in a population of cells in the proliferative compartment of the intestinal epithelium.
The specific aim of this project is to characterize the genetic regulation of epithelial cell growth and
differentiation in the zebrafish intestine using reverse genetic approaches. Specifically, antisense
morpholino oligonucleotides, targeted to c-myb, (a transcription factor known to play a role in
intestinal development) will be injected into the yolk of 1-2 cell zebrafish embryos in order to knock-
down c-Myb function over the first few days of development. The impact of inhibiting this
transcription factor on intestinal epithelial cell development will be analysed in the first instance
using fluorescence dissecting and confocal microscopy. Other approaches will be to examine
intestinal epithelial cell development in a panel of zebrafish intestinal mutants that are currently
undergoing characterization in our laboratory using positional cloning, in situ hybridization and
immunohistochemistry. This analysis will be greatly facilitated by establishing the mutant strains
onto the transgenic Tg[c-myb:YGF] background.

The project will be undertaken in the Colon Molecular and Cellular Biology Laboratory,
Ludwig Institute for Cancer Research - Parkville Branch, Royal Parade (opposite the
Howard Florey Institute)

CONTACT DETAILS
Departmental Supervisor (must be contacted in the first instance)
Assoc. Prof. Alex Andrianopoulos
Department of Genetics
Room 2.11
Phone 8344 5164
E-mail alex.a@unimelb.edu.au

Supervisor
Assoc. Prof. Joan Heath
Colon Molecular and Cellular Biology Laboratory
Ludwig Institute for Cancer Research, Parkville Campus
Phone 9341 3150
E-mail joan.heath@ludwig.edu.au
COBBETT GROUP
Metal Homeostasis in Plants
Some heavy metals, such as zinc, iron and copper, are essential for life. In plants there are
mechanisms for the uptake of these metals from soil, their translocation from root to
shoot and their distribution to tissues and cells. In addition, there are mechanisms to deal
with toxic excess of both essential and non-essential heavy metals.

Zinc-deficient soils – particularly in Australia – have severe effects on crop production
and crop grains are an important source of dietary zinc for humans and animals. Long-
term benefits of this research may be a greater understanding of the mechanisms of zinc
uptake and distribution from the soil, particularly the delivery of zinc to developing grain.
In addition, plants must respond to toxic metals from the soil and to the oxidative stress
such metals cause. Understanding mechanisms of metal detoxification may assist in the
development of plants for the bioremediation (phytoremediation) of contaminated soils.

Plants have evolved multiple mechanisms for metal homeostasis and metal detoxification.
These mechanisms include:
    metal binding compounds – (organic acids, amino acids, peptides and proteins)
    metal transport proteins
    antioxidant compounds – (glutathione and ascorbic acid)

The main aims of our research are to identify:
    important processes in heavy metal (especially zinc) homeostasis
    important processes in excess heavy metal (especially cadmium) detoxification
    roles of antioxidant compounds (especially glutathione)

We are using the model organism Arabidopsis with genetic, physiological, biochemical
and molecular approaches to understanding these problems.

Current research in the Cobbett lab is broadly in two areas. The first is in understanding more about
the mechanisms of essential zinc homeostasis in plants while the second is in exploring the transport,
metabolism and function of the antioxidant compound, glutathione.

Molecular genetic approaches to understanding metabolism of
the essential element, Zinc, in plants
Background
The P-type ATPases are a class of transporters identified in a wide range of organisms
and are involved in the transport of essential and potentially toxic metals across cell
membranes. Some P-type ATPases can be distinguished on the basis of conserved amino
acid sequence motifs as presumptive heavy metal transporters. This family includes the
copper transporting P-type ATPases affected in Menkes and Wilsons diseases in humans.

With the completion of the Arabidopsis genome sequence it is now possible to identify
the entire suite of genes encoding P-type ATPases. There are a total of eight heavy metal
transporting P-type ATPases. Each of the predicted proteins encoded by these genes
contains all the conserved motifs of P-type ATPases.
We have recently demonstrated that two of these transporters, HMA2 and HMA4 are
essential for zinc homeostasis. An hma2,hma4 double mutant has a severely zinc-
deficient phenotype that can be rescued by the addition of extra zinc to the soil.

We are extending this work to identify and study other genes involved (or thought to be
involved) in zinc homeostasis; in particular, the ZIP family of transporters.


Project CC - The role of zinc and zinc transporters in pollen
development.
Supervisor: Chris Cobbett

A striking phenotype of the hma2,hma4 double mutant is the failure to develop viable
pollen and this is consistent with the expression of both HMA2 and HMA4 in anthers
coincident with pollen development. We have also found that members of the ZIP family
of Zn-transporters are also expressed in developing flowers. Other studies indicate that
zinc levels in pollen are high and that zinc deficiency effects fertility. We aim to explore
this phenomenon in greater detail. In particular, the project will investigate the expression
of these and other genes in anthers during pollen development under various growth
conditions, particularly conditions of zinc deficiency.




CONTACT DETAILS
Supervisor:
  Prof. Chris Cobbett
  Department of Genetics, The University of Melbourne
  Room 1.06
  Phone 8344 6266
  Email:ccobbett@unimelb.edu.au
ROBIN GROUP
Project CR1 - A genome-wide analysis of insecticide resistance and cross-
resistance.
Supervisor: Dr. Charles Robin

The genome sequences of >160 isogenic Drosophila melanogaster lines became
available this year. These lines are a subset of a „living library‟ available to those in the
Drosophila community who can characterize them for whichever phenotype they desire.
Phenotype to genotype associations can then be performed to identify the genetic variants
underling traits of interest. This wonderful and unprecedented resource allows very
accurate estimation of phenotypes because many genetically identical individuals can be
scored for a trait. Furthermore these lines are being characterized for a large number of
phenotypes and so pleiotropy (and correlations between the traits) can be systematically
studied. We are currently screening these lines for DDT resistance, the genetic basis of
which is better characterized than any other insecticide resistance in the model insect,
Drosophila melanogaster. This promises to give a genome wide perspective to the
genetic basis of DDT resistance; identifying major and minor genes segregating in the
natural population from which these lines are derived. I am looking for one or possibly
two MSc(genetics)/ honours students to further characterize insecticide resistances in the
„Drosophila Genetic Reference Panel‟. The research will not only be pertinent to
understanding the problem of insecticide resistance but is a model in which to understand
the genetic architecture of adaptive evolution. One component of a project will be to
characterize resistance to a pyrethroid insecticide which acts via the same target molecule
as DDT. Thus the genes underlying two resistances can be compared and the genetic
basis for cross-resistance characterized. If there is evidence of rare alleles of large
phenotypic effect contributing to resistance then these will be identified by linkage
mapping. Novel insecticide resistance genes will be verified using transgenic approaches
that over-express them or knock them down. The project/s may include an analysis of the
role that gene copy number variation plays in the resistances, an analyses of the fitness
cost of the resistance gene, and possibly crosses exploring epistatic interactions between
the genes. They will also include analyses of population differentiation and tests looking
for signs of recent selection acting on the resistance loci.

Project CR2 - Mapping evolutionarily significant traits in interspecies crosses in
the Drosophila ananassae species complex.
Supervisor: Dr. Charles Robin

In studying insecticide resistance across many Drosophila species we stumbled upon a
cryptic species of Drosophila that is closely related to Drosophila ananassae- a
cosmopolitan species. The species differ in their body colour, male sex comb number and
their resistance to DDT and show partial reproductive isolation. However we can force
the two species to cross in the lab and have begun the process of mapping DDT
resistance. In this project we will use a next generation sequencing approach to map the
genes underlying these traits. This will be followed by a molecular population genetic
study of these genes to examine the adaptive forces that have acted in the divergence of
these of these traits.
Project CRAH - Characterizing the genetic basis of desiccation resistance in
Drosophila species.
Supervisors: Dr. Charles Robin and Prof. Ary Hoffmann

Certain rainforest species of Drosophila have been shown to exhibit very little genetic
variation in the ability to withstand desiccation stress (Hoffmann et al 2003 Science
301:100). This lack of variation will limit the adaptability of these species to
environmental change and so it is important to understand why these species lack the
genetic variation. Our approach has been to identify genes underlying desiccation
resistance in the model organism Drosophila melanogaster and then examine the
molecular variation of these genes in the rainforest species. In this project, candidate
desiccation resistance genes will be verified using gene-specific association studies and
transgenic approaches. Then variation in the orthologs of these genes will be examined in
the rainforest species. In the masters version of this project, new candidate genes will be
sought by phenotyping a panel of 192 inbred Drosophila melanogaster lines and
performing a genome-wide association study.




CONTACT DETAILS
 Dr. Charles Robin
 Department of Genetics, The University of Melbourne
 Bio21 Institute
 Room 267
 Phone 8344 2349
 crobin@unimelb.edu.au




The following two projects (JJCR1-JJCR2) are offered in the Genomics and Systems
Biology Lab of Dr. Jeremy Jowett, at the Baker Heart and Diabetes Institute.
Charlie Robin will act as the Genetics Department „internal‟ supervisor. Please contact
Jeremy to discuss the projects and contact Charlie about supervisory arrangements etc.




Research Focus of Dr. Jowett's Research Laboratory
There is great diversity among the people you see and meet for example height,
complexion or eye colour, characteristics (or traits) that are easy to discern. There are
others that are of a more subtle nature and may not be immediately apparent like athletic
ability and intelligence. This diversity in humans also extends to our level of
predisposition for development of many common non-infectious diseases such as cancer,
type 2 diabetes and heart disease. In fact these diseases "run in families" (or are
heritable). Additionally, they are “complex” meaning that an individual‟s risk is
influenced not only by many genes combined together (polygenic) but also by the
environment, such as the types of food we eat and how much exercise we do.
The genomics and systems biology laboratory is working toward providing a better
understanding of the process that causes type 2 diabetes, obesity and related
cardiovascular disease, (such as heart failure) by identifying disease-predisposing genes,
their products and how those products work at a molecular level inside cells. We are
using the latest exciting advances in DNA sequencing and genetic variation detection
technology and combining this with gene expression analysis (genomics) in a systems
biology approach to discover these genes and their function. Understanding their
function and role in disease development will aid in the development of more accurate
diagnostic tests and lead to improved therapeutic drugs which will provide better
treatments than currently available for these diseases or even prevent them developing
altogether.




JJCR1 - Gene Discovery in Type 2 Diabetes.
Supervisors: Dr. Jeremy Jowett and Dr. Charles Robin

Changes in human behaviour and lifestyle over the last century have resulted in a
dramatic increase in the incidence of Type 2 Diabetes Mellitus (T2D) worldwide in part
driven by increased obesity. Once diagnosed, currently available therapies are inadequate
to control disease progression and there are few new therapeutics in the development
pipeline due to our limited understanding of the molecular pathogenesis. The complex
etiology of T2D involves both environmental and genetic risk factors. A substantial effort
has been made to identify the underlying genetic basis of familial predisposition,
however currently we have only discovered the basis of about 10% of the risk, so there is
a lot more to be identified.

This project will use next generation DNA sequence technology (NGS) to identify all
genetic variation in a specific region within a cohort which has been successfully piloted
in our laboratory. Comprehensive knowledge of all variation will allow full genetic
dissection of the segregating variants contributing to the linkage signal and guarantee
identification of the causative gene or genes. This project will focus on cataloguing
genetic variation in a large multi-generation family based cohort at chromosome 12q24, a
region that we have previously discovered to contain genes influencing blood glucose
levels (a measure upon which type 2 diabetes is diagnosed). Another component will
examine specific genes as candidates for their role in controlling glucose levels. Finally,
validation experiments using in vitro model systems of glucose homeostasis will also be
applied to confirm a role of the candidate genes. Ultimately these findings will translate
to an improved understanding of the molecular mechanisms of disease leading to novel
and more specific therapeutics to control disease progression in afflicted individuals.

JJCR2 - Systems Biology Approach Discovers New Genes Controlling
Cholesterol and Cardiovascular Disease - But How do they Work?
Supervisors: Dr. Jeremy Jowett and Dr. Charles Robin

Many epidemiological studies have shown that a low levels of "good cholesterol", high
density lipoprotein cholesterol (HDL-C) is a strong independent risk factor for
development of cardiovascular disease (CVD) including atherosclerosis that causes heart
attacks and strokes. Major influences on low HDL-C levels include obesity, smoking,
sedentary behaviour, type 2 diabetes and genetic factors. Current therapies raise HDL-C
levels ineffectively, therefore there is an urgent need for new HDL-C raising drugs. A full
understanding of the mechanisms responsible for the regulation of HDL-C levels will
facilitate the design of new drugs for at risk individuals.

We have used Genome Wide Association Study (GWAS) data and combined this with
global gene expression profiling analysis to discover a set of new genes that control
HDL-C levels. This project will investigate the mechanism of action linking these genes
to HDL-C homeostasis. Initially the biological consequences of modulation (over-
expression and knockdown) in tissue culture models of HDL-C production, remodelling
and catabolism will be investigated. Next the upstream genes that were found to control
the expression levels of these genes will be validated using functional assays (siRNA
knockdown/ vector over-expression). These validation studies in tissue culture and
animal model systems will increase our understanding of the HDL-C homeostasis
mechanisms and reveal multiple new potential therapeutic targets for development of
interventions. This will lead to significant improvements in clinical care strategies to
combat the rising incidence of cardiovascular disease and reduce its impact on morbidity
and mortality in the population.

CONTACT DETAILS
Departmental Supervisor (must be contacted in the first instance)
 Dr. Charles Robin
 Department of Genetics, The University of Melbourne
 Bio21 Institute
 Room 267
 Phone 8344 2349
 crobin@unimelb.edu.au

  Dr. Jeremy Jowett
  Lab Head, Genomics and Systems Biology
  Baker Heart and Diabetes Institute
  75 Commercial Road, Melbourne
  Phone: 8532 1775
  Mobile: 0408 561 613
  jeremy.jowett@bakeridi.edu.au
HOFFMANN GROUP
Project MTSAH1 - Alternative splicing and stress: exploring candidate
multi-transcript genes for thermal plasticity in Drosophila.
Supervisors: Dr Marina Telonis-Scott and Prof. Ary Hoffmann

Understanding an organisms‟ potential to adapt to a rapidly warming climate is important
to predict long term species survival. In Drosophila, thermotolerance can be enhanced
by prior heat exposure (heat hardening). These partly reversible, „plastic‟ changes may
be adaptive if they confer resistance to temperature extremes.

At the cellular level, phenotypic plasticity (the ability of an organism to change its
physiology in response to the environment) for heat resistance is only partially
understood, and remains an outstanding question of importance in biology given that
thermal extremes tolerated in nature largely depend on phenotypic plasticity.

We have identified new molecular mechanisms for thermotolerance in D. melanogaster,
in the form on alternatively spliced genes, i.e. where different pre-mRNA regions are
„spliced‟ together in different combinations to generate multiple, and often functionally
distinct transcripts. Alternative splicing can expand a cells transcript/protein repertoire in
a condition dependent manner, such as during stress.

We are interested in further exploring the stress-specific splicing patterns of candidate
genes manner following heat hardening to examine the contribution of different splicing
variants to phenotypic plasticity for thermotolerance.

An Honours or Masters project will contribute to this project by investigating some of the
following aspects:

    1. Running heat stress assays with heat hardening to elicit the plastic response in
       flies and different times during stress and recovery.
    2. Use real-time PCR to assess stress-specific splicing variation in candidate multi-
       transcript genes.
    3. Compare splicing patterns in other Drosophila species for a better understanding
       of the role of splicing in climatic adaptation.

Throughout this project, there will be an opportunity to gain experience in Drosophila
husbandry and phenotypic assays, data analysis, RNA extraction, and real-time PCR.




Project MTSAH2 - Characterizing candidate genes for desiccation
resistance in Drosophila using RNA interference.
Supervisors: Dr Marina Telonis-Scott and Prof. Ary Hoffmann
The ability to survive environmental stress and adapt to a changing climate is crucial for
long term species survival. Climatic stress such as survival to desiccation has been
widely studied in the insect model Drosophila, which have successfully colonized arid
habitats including deserts and high altitudes as well as tropical and temperate zones,
providing an excellent model to study adaptation to desiccation at the intra- and inter-
population level. By contrast, several rainforest restricted species demonstrate little
ability to evolve resistance to desiccation. So far, it is unclear why some species adapt
easily while others are limited by low adaptive variation for survival to low humidity
environments.

In order to learn more about the genetic mechanisms underlying desiccation survival and
thus why some species adapt easily while others are more limited, we have used new
methods to map allele frequency changes associated with desiccation resistance in D.
melanogaster. We currently have many candidate genes to explore further and are
primarily interested in studying the functional effect of these genes on survival to low
humidity.

 In vivo gene silencing techniques such as RNA interference will allow the functional
study of our candidate genes on the desiccation phenotype. The GAL4/UAS system will
be used to target the RNAi to specific tissues/cells in the fly, and different stages of
desiccation and recovery will be assessed.


An Honours or Masters project will contribute to this project by investigating some of the
following aspects:

    1. Generate genetic crosses of RNAi lines for candidate genes and controls.
    2. Assess the effect of candidate gene knockdown on the desiccation survival
       phenotype between the different sexes and tissues.
    3. Perform real-time PCR to quantify the effect of the knockdown on transcript
       abundance.


Throughout this project, there will be an opportunity to gain experience in Drosophila
husbandry and phenotypic assays, RNAi experiments and analysis, RNA extraction, and
real-time PCR.

CONTACT DETAILS

Dr Marina Telonis-Scott
Bio21 Institute,
Department of Genetics,
University of Melbourne
Phone: 8344-2281
Email: marinats@unimelb.edu.au
Project RLAH1 - Investigating the roles of a signal transduction
regulator in adaptive wing size variation in Drosophila melanogaster.
Supervisors: Dr Ronald Lee and Prof. Ary Hoffmann

One of the great mysteries in evolutionary biology is: Why are animals bigger in the
cold? Many theories have been proposed to explain this phenomenon but little is known
about its underlying genetic factors. In Australia, body size of the vinegar fly, Drosophila
melanogaster, increases gradually from tropical Queensland to temperate Tasmania,
spanning a 2500 km latitudinal transect. A signal transduction regulator has recently been
implicated to influence genes differentially expressed between cline-end populations. The
current project aims to test if expression of this candidate regulator shows any
geographical pattern along the east coast. The second objective is to test for an
association between DNA polymorphisms around this gene and body size in a large fly
population. Outcomes of this project will provide important insights into the molecular
basis of adaptive shift.



Project RLAH2 - Investigating the roles of Hsp23 in adaptive cold
tolerance in Drosophila melanogaster.
Supervisors: Dr Ronald Lee and Prof. Ary Hoffmann

Temperature plays a crucial role in determining distribution and abundance of animals. In
insects and other ectothermic organisms, temperature simultaneously affects
physiological processes, biophysical structures, and metabolic activities as well as
developmental rates and growth. Molecular chaperones such as the heat shock proteins
(Hsps) are important for thermal adaptation. Recently, we have demonstrated that Hsp23
is necessary for flies to recover from cold stress. This project further investigates the
functional roles of Hsp23 in chill coma recovery using the transgenic GAL4/UAS RNAi
system. In addition, we test for a clinal pattern in chill coma recovery performance in D.
melanogaster populations along the east coast of Australia. Finally, we compare Hsp23
expression levels between the phenotypic extremes, to confirm its roles in adaptive cold
tolerance.


CONTACT DETAILS

Dr Ronald Lee
Room 266,
Bio21 Institute
Department of Genetics
University of Melbourne
Phone: 8344-2348
Email: ronaldl@unimelb.edu.au
Project NEAHAW - Genetic control of pest mosquitoes - Assessment of
strains of Aedes aegypti
Supervisors: Dr Nancy Endersby, Prof. Ary Hoffmann and Dr Andrew Weeks

CESAR is involved in local and international research on mosquitoes. Aedes aegypti is
the principal vector of dengue virus and is found in subtropical to tropical regions
throughout the world. There are 50 million cases of dengue fever each year, but no
specific antiviral remedy. Reduction of disease incidence currently depends on control of
the vector.

Genetic control methods are under development for mosquito vector populations
including Ae. aegypti. A method under investigation is the introduction of mosquitoes
infected with life-span reducing Wolbachia strains and strains with reduced vector
competency. These strains are intended to replace the natural population of Ae. aegypti
in the field and, thereby, reduce transmission of dengue fever. The modified strains must
be fit and able to compete with the natural population.

Target release sites will be chosen in north Queensland, Vietnam and Thailand.
Population dynamics and genetic structure of the natural population of Ae. aegypti at
these sites must be examined thoroughly in order to model the effect of release of the
modified mosquitoes.

An Honours or Masters project will build on our previous projects by investigating some
of the following aspects:

    1.   Use of genetic markers to investigate relatedness of mosquitoes within houses
    2.   Assessment of laboratory and field mosquitoes for differences in life history traits
    3.   Effects of laboratory adaptation on fitness of mass-reared Ae. aegypti
    4.   Investigation of effects of insecticides on Wolbachia strains of Ae. aegypti (adult
         and larval bioassays)

Throughout this project, there will be an opportunity to gain experience in DNA
extraction and manipulation, sequence analysis, PCR, microsatellite and EPIC marker
screening, population genetic analysis, insecticide bioassays and laboratory experiments
on live mosquitoes.



CONTACT DETAILS

Dr. Nancy Endersby
Bio21 Institute,
Department of Genetics,
The University of Melbourne
Ph: 8344 2281
Email: nancye@unimelb.edu.au
CAMAKARIS GROUP
OVERALL RESEARCH PROGRAM---- Genetic and
molecular basis of copper homeostasis and human inherited
and acquired diseases involving disturbances in copper
homeostasis (eg. Alzheimer’s disease)
Copper is toxic to biological systems, yet it is an essential trace metal for aerobic life
being required by a number of key metalloenzymes. The paradox of an essential trace
metal being toxic to the cell in larger amounts can be resolved by the existence of specific
transport, storage and detoxification mechanisms. Maintaining a correct copper balance,
ie homeostasis, is critical.

There are a number of diseases due to acquired or inherited copper deficiency or copper
toxicity states. In addition, chronic marginal copper deficiency may be an important
cofactor in cardiovascular disease and osteoporosis. Disturbances in copper metabolism
are linked to several neurodegenerative diseases in humans including Alzheimer's
disease, prion disease (eg. mutant prion protein involved in "mad cow" disease) and
forms of motor neuron disease. We and other investigators have published evidence that
Amyloid Precursor Protein or a cleavage product, beta amyloid (which accumulates
in the Alzheimer‟s disease brain), is involved in copper homeostasis. Excess extracellular
copper bound to beta amyloid may result in functional copper deficiency leading to
oxidative stress and hence Alzheimer‟s disease brain pathology. We believe that copper
enters cells and is distributed within cells bound to carriers in a series of regulated steps
analogous to a metabolic pathway. Detoxification systems (some inducible) also exist to
cope with conditions of copper excess.

Our strategy is to use mutants in which copper metabolism is disturbed and “knockdown”
of candidate genes in order to dissect the pathways of copper metabolism and to
understand copper homeostasis and its regulation. Menkes disease is a potentially lethal
X-linked recessive disorder of copper metabolism in humans. The Menkes (MNK,
ATP7A) gene has been cloned and encodes a transmembrane Cu-translocating P-type
ATPase (ie: an ATP-driven Cu "pump"). MNK is a crucial copper transporter being
required for delivery of copper to several Cu-dependent enzymes for absorption of copper
from the gut, reabsorption of copper in the kidney and for transfer of copper across the
blood-brain barrier. MNK has recently been found to be involved in the mechanism of
resistance to important drugs used in cancer chemotherapy, and to be involved in cell
migration (a key process in cancer).

We discovered a novel system of regulation for metal transport proteins whereby the
ligand (Cu) induces the intracellular trafficking of its own transporter (MNK) from the
trans-Golgi network (TGN) to the plasma membrane (PM). At the TGN, MNK delivers
copper to copper-dependent enzymes in the secretory pathway. Trafficking involves
vesicles and we propose that the overall mechanism provides a swift way of eliminating
excess copper (copper is essential for a number of enzymes but excess copper is toxic and
cells must get rid of it). When copper drops to safe levels, vesicles containing MNK
protein recycle back to the TGN. Furthermore, we have found that in polarized epithelial
cells, when Cu levels increase, MNK traffics from the TGN and is targeted to the
basolateral membrane which is consistent with MNK functioning to pump copper from
the gut epithelial cells to the blood. We have recently discovered that MNK is
phosphorylated by kinases when copper is elevated. This is an important breakthrough, as
Cu-responsive kinase phosphorylation may provide signalling mechanisms, which are
involved in localisation/trafficking of MNK, and may be part of a more general signalling
response pathway(s) which allows cells to respond to changes in copper levels.

In collaborative studies, we are investigating the role of phosphorylation of metal binding
sites we have identified in ATP7A and in particular how phosphorylation modulates the
folding of these sites (and hence affinity for copper) using the exciting approach of
Supercomputer Molecular Dynamics Simulations.

We have also recently discovered copper-responsive trafficking of Amyloid Precursor
Protein of Alzheimer‟s disease. The unravelling of the mechanisms underlying this will
be important in understanding the role of copper in Alzheimer‟s disease and will inform
on approaches to therapeutics.

Project JC – Projects may be offered at a later date (watch this space).
Potential projects should be discussed with Prof. Jim Camakaris (contact details below)

Please also visit the Camakaris Lab Web page, which contains a publications list, at
http://www.genetics.unimelb.edu.au/research/camakaris/


Contact details:
Professor Jim Camakaris
Department of Genetics, The University of Melbourne
Room 2.12
Phone: 8344 5138
j.camakaris@unimelb.edu.au

				
DOCUMENT INFO