RESEARCH OPPORTUNITY PROGRAM PROJECT … Genetics... · defined roles for PIPs and their regulatory...
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Transcript of RESEARCH OPPORTUNITY PROGRAM PROJECT … Genetics... · defined roles for PIPs and their regulatory...
RESEARCH OPPORTUNITY PROGRAM
299Y/399Y PROJECT DESCRIPTIONS 2018‐2019
FALL/WINTER
Name and Title: Julie Brill, Professor
Department: Molecular Genetics
TITLE OF RESEARCH PROJECT: Genetics of Phosphatidylinositol Phosphate (PIP) Signaling in Drosophila
Number of 299Y Spots: 1 Number of 399Y Spots: 1
OBJECTIVES AND METHODOLOGY:
Eukaryotes use a class of membrane lipids called phosphatidylinositol phosphates (PIPs) as crucial signaling molecules
to promote cell morphogenesis. We have been studying the cellular and developmental roles of enzymes that
regulate PIP levels using the fruit fly Drosophila melanogaster as a model system. In our experiments, we have
defined roles for PIPs and their regulatory enzymes in cytokinesis, gametogenesis and organelle biogenesis. Our
recent studies suggest that specific isoforms, as well as subcellular and tissue‐specific distribution of these enzymes
are important for normal development. With the advent of powerful genetic tools such as CRISPR/Cas9, we are now
in a position to create site‐specific mutations and introduce fluorescent protein fusions and epitope tags into
endogenous Drosophila genes. This project will employ CRISPR/Cas9 to mutate and tag PIP pathway enzymes to
better assess their localization, function and regulation during fly development
DESCRIPTION OF STUDENT PARTICIPATION:
The student will be taught standard Drosophila molecular genetic techniques, including design and molecular cloning
of guide RNAs, Drosophila genetics (stock maintenance, fly anatomy, scoring of visible markers, genetic crosses, etc.),
preparation of Drosophila genomic DNA, PCR and sequence analysis. In addition, depending on the particular project,
the student may be taught tissue dissections, immunostaining and fluorescence microscopy. The student will keep a
detailed lab notebook of the experiments and organize the results for presentation. He or she will also be given
detailed topics relevant to the project to research in the literature.
MARKING SCHEME (assignments with weight and due date):
10% ‐ short written report on the project (Nov. 9)
10% ‐ presentation of the project to the lab (Nov. 30)
10% ‐ final presentation of the project and results to the lab (Apr. 11)
10% ‐ lab notebook evaluation (end of term)
30% ‐ lab performance (end of term)
30% ‐ final report (Apr. 18)
Project Code: MGY 1
RESEARCH OPPORTUNITY PROGRAM
299Y/399Y PROJECT DESCRIPTIONS 2018‐2019
FALL/WINTER
Name and Title: Amy A. Caudy, Associate Professor
Department: Molecular Genetics
TITLE OF RESEARCH PROJECT: Discovery and Characterization of Novel Enzymes using Metabolomics and Genetics
Number of 299Y Spots: 3 Number of 399Y Spots: 3
OBJECTIVES AND METHODOLOGY:
Every cell on earth processes nutrients to release energy and form the chemical compounds needed for cellular
maintenance and growth. The study of metabolism, how cells transform nutrients and produce energy, reaches back
hundreds of years but is now undergoing a revolution due to the availability of new methods. Recent advances in
analytical chemistry, particularly in small molecule mass spectrometry and 2D NMR (nuclear magnetic resonance)
have demonstrated a far more complex small‐molecule landscape than can be accounted for by current maps and
models.
Our group uses mass spectrometry to identify and quantitate the chemicals. Our group is working to identify
previously unknown metabolic pathways within cells by using mass spectrometry to measure the changes in
intracellular metabolites that result from the deletion of previously uncharacterized enzymes.
There are two tremendous gaps in our understanding of metabolism. First, there are many chemical reactions that
are known to occur as cells break down nutrients, yet we do not know the genes that enable these reactions. Second,
recent technological advances in mass spectrometry have detected hundreds of chemicals in cells that are not
predicted by the current knowledge of metabolism. My group has had success pursuing both types of questions (Cell.
2011 Jun 10;145(6):969‐80., Anal Chem. 2010 Apr 15;82(8):3212‐21.). We recently used these full scan mass
spectrometric approaches to discover a major route for the synthesis of ribose, a key building block for DNA and RNA
and are currently engaged in similar work understanding the synthesis of the redox carrier rhodoquinone.. This
project combines cutting edge mass spectrometry approaches with the tools of genetics and biochemistry to discover
the function of previously uncharacterized enzymes. We use budding yeast and the nematode worm C. elegans, for
much of our work. Both are genetically tractable, fast growing organisms. The majority of metabolic reactions can be
traced to the origins of life billions of years ago, so we then test our observations in yeast in mammalian cells to
compare the roles of new pathways. An area of particular interest is the intersection of these uncharacterized
metabolic pathways with the cell division cycle.
DESCRIPTION OF STUDENT PARTICIPATION:
The students will construct and design genetically engineered yeast and mammalian cell strains with targeted
changes in candidate enzymes. This will involve PCR, DNA sequencing, and other molecular biology techniques to
Project Code: MGY 2
create cells with desired characteristics. The students will be involved in the preparation, mass spectrometric
measurement, and mathematical analysis of the data. The data will be compared with existing models of metabolism
to identify the route of synthesis and degradation of novel compounds. For those students with the interest and
aptitude, there are opportunities for design and fabrication of custom lab hardware and software to further extend
our capabilities for high throughput metabolomics analysis.
MARKING SCHEME (assignments with weight and due date):
Oral examination on project background – scheduled within first 4 weeksh ‐ 20%
Participation in group meeting and presentations including ROP299 poster session in March 2019
(2 times over term): 20%
Lab work (accuracy of work, evaluations of lab notebook, performed biweekly): 30%
Final project report: Due – last day of term: 30%
RESEARCH OPPORTUNITY PROGRAM
299Y/399Y PROJECT DESCRIPTIONS 2018‐2019
FALL/WINTER
Name and Title: Dr. Alan Cochrane (Professor)
Department: Molecular Genetics
TITLE OF RESEARCH PROJECT: Regulation of HIV‐1/Adenovirus RNA Processing: Insights into novel therapeutics
Number of 299Y Spots: 1 Number of 399Y Spots: 1
OBJECTIVES AND METHODOLOGY:
Multiple mammalian viruses (HIV‐1, adenovirus, influenza, Herpes) are dependent upon the host cell for the
processing and expression of their mRNAs. Work by my group has identified a number of host factors whose altered
expression/function result in dramatic changes in viral RNA processing and inhibition of virus replication. To
complement these findings, my group has also identified multiple small molecules which a suppress replication of
multiple different viruses by inducing changes in host factor function. Greater understanding of how these small
molecules act will provide important insights into the design of novel therapeutics for the treatment of multiple viral
infections.
DESCRIPTION OF STUDENT PARTICIPATION:
The students will be involved in the screening and analysis of shRNAs to host factors or small molecules for their
effects on HIV‐1/adenovirus gene expression with particular focus on measuring the changes in viral RNA processing
that underlay the observed effects. Work will involve the use of mammalian cell lines to measure changes in viral
protein expression by western blot or ELISA followed by qRTPCR, RT‐PCR and in situ hybridization to quantitate the
accompanying changes in viral RNA levels and localization. To complement these findings, we also examine how
changes in activity of putative targets of the compounds (by overexpression or depletion using shRNAs) mimic the
effects of the compounds on viral RNA processing. Together, the information will provide a detailed understanding of
the cellular pathways involved in controlling virus replication as well as identify pathways common to multiple viruses
that will help in the development of pan anti‐virals.
MARKING SCHEME (assignments with weight and due date):
Oral exam on project plan: scheduled one week before drop/add date: 20%
Participation in group meeting and presentations (2 times over term): 20%
Lab work (evaluations of lab notebook, performed biweekly): 30%
Final project report: Due – last day of term: 30%
Project Code: MGY 3
RESEARCH OPPORTUNITY PROGRAM
299Y PROJECT DESCRIPTIONS 2018‐2019
FALL/WINTER
Name and Title: Barbara Funnell, Professor
Department: Molecular Genetics
TITLE OF RESEARCH PROJECT: Mechanisms of Plasmid Maintenance in Bacteria
Number of 299Y Spots: 1
OBJECTIVES AND METHODOLOGY:
All bacterial species carry extrachromosomal DNA elements called plasmids, which exist symbiotically with the
bacterial host. Plasmids carry genes beneficial to the host, such as resistance to antibiotics or pathogenesis genes. In
bacteria, two proteins, usually called ParA and ParB, act to promote proper segregation or “partition” of both
plasmid and cellular chromosomes. Using a combination of molecular biology, genetics, biochemistry, single‐
molecule analyses, and cell biology, we are studying the activity of the plasmid‐encoded ParA and ParB proteins, how
they dynamically localize plasmid DNA in the bacterial cell, and how they coordinate these activities with the cell
cycle. Genes encoding ParA/ParB‐like partition systems have been identified in almost all naturally occurring bacterial
plasmids and species examined. P1 plasmid maintenance in Escherichia coli serves as our simple and nonpathogenic
model for the maintenance of many plasmids in pathogenic bacteria; transmission and maintenance of such plasmids
in bacterial populations contribute significantly to the rapid spread of antibiotic resistance and virulence among
pathogenic bacterial species. We generate and use a large number of mutant versions of the P1 proteins, which are
blocked at different steps in the partition reaction. The objective of the student project is to create and/or analyze
one or two specific plasmid mutations, in order to define novel steps in the partition reaction as well as to better
understand how to inhibit it.
DESCRIPTION OF STUDENT PARTICIPATION:
The student will analyze one or two specific P1 partition mutations. The project will involve general molecular biology
and genetic techniques, such as mutagenesis, cloning, PCR, and DNA sequence analysis, and/or biochemical analyses
such as protein purification and enzyme assays. For example, he/she will construct specific mutants by site‐directed
mutagenesis, perform assays to test effects on plasmid stability inside cells using bacteriological techniques, or
biochemical tests such as DNA binding and protein‐protein interaction assays. The exact approach will depend on the
specific mutations to be examined, which will be determined in consultation with Dr. Funnell at the start of the
school year.
MARKING SCHEME (assignments with weight and due date):
10% midterm report (due Nov 12, 2018)
20% lab presentations (#1 in late Nov 2018; #2 in mid March 2019)
Project Code: MGY 4
30% final written report (due end of term 2019)
10% evaluation of lab notebooks (written work)
30% evaluation of lab work/participation (through weekly ~30 minute meetings with Dr. Funnell)
Student will be given marks for midterm, first lab presentation, and an assessment of lab work and notebook to date
before the drop date.
RESEARCH OPPORTUNITY PROGRAM
299Y/399Y PROJECT DESCRIPTIONS 2018‐2019
FALL/WINTER
Name and Title: Dr. Thomas Hurd, Assistant Professor
Department: Molecular Genetics
TITLE OF RESEARCH PROJECT: Determining how Deleterious Mitochondrial DNA Mutations are Eliminated
Number of 299Y Spots: 1 Number of 399Y Spots: 1
OBJECTIVES AND METHODOLOGY:
Background
Unusual among organelles, mitochondria have their own genomes, which encode a small number of essential
genes. Unlikely nuclear genes, we inherit these mitochondrial genes only from our mothers. Given the
importance of these genes, mothers have evolved mechanisms to ensure they pass on good, mutant‐free copies
to their progeny. Without such mechanisms deleterious mutations would accumulate from one generation to
the next ultimately causing the collapse of the species. Exactly what the molecular nature of these selection
mechanisms is remains obscure, despite their fundamental importance. In this proposal we seek to understand
how these selection mechanisms work on a molecular level.
The medical importance of these mechanisms is demonstrated by the damage caused later in life by mutations
in the mitochondrial genome. While a mother may succeed in ensuring we start life with good mitochondrial
genes, mutations nonetheless inevitably arise in those genes as we age. This causes disease in people, most
often neurological, affecting on the order of 1 in 4,300. By understanding how mothers prevent deleterious
mitochondrial genes from being inherited, we aim to develop strategies to eliminate these bad mutations and
the diseases they cause as we age.
Objectives
The goal of this research project is to identify genes and pathways necessary for the elimination of deleterious
mitochondrial DNA mutations.
Methodology
The students will assist in the development of a high‐throughput quantitative PCR (qPCR) assay to measure
wildtype and mutant mitochondrial DNA in a Drosophila melanogaster model. The students will then use this
assay and RNAi to knockdown genes one by one to determine which are necessary for the elimination of
deleterious mitochondrial DNA mutations. Lastly, if time‐permits, the students will validate the ‘hits’ from the
above RNAi screen by generating null mutations in candidate genes using CRISPR/Cas9.
Project Code: MGY 5
Suggested Reading
1. Stewart, J. B. & Larsson, N.‐G. Keeping mtDNA in shape between generations. PLoS Genet. 10, e1004670
(2014).
2. Hurd, T. R. et al. Long Oskar Controls Mitochondrial Inheritance in Drosophila melanogaster. Dev. Cell 39,
560–571 (2016).
DESCRIPTION OF STUDENT PARTICIPATION:
This project is ideally suited to motivated students interested in gaining research experience for graduate
school. The students will work under the direct supervision of Dr. Hurd and a graduate student in the lab. The
students will be expected to participate in all aspects of the research process including: reading appropriate
background literature; helping to design and plan experiments; conducting experiments on a semi‐independent
basis, with the expectation of increased independence as the project progresses; keeping accurate and thorough
records of their experimental work; and participating in regular (bi‐weekly) lab meetings.
The students will be exposed to a variety of genetic, biochemical and molecular biology methods that are widely
applicable to a range of experimental disciplines. These include:
1. Quantitative PCR
2. Gene knockdown using RNAi
3. Molecular cloning
4. Drosophila genetics/husbandry
5. CRISPR/Cas9
Additionally, students should expect to learn how to present their data as publication‐quality figures, and to
improve their ability to communicate their research clearly and concisely, both orally and in writing.
MARKING SCHEME (% of grade; due date):
1. Research Proposal (10%; due 2 weeks after project start): Students will prepare a 2‐4 page written
summary of their research project, which will include background, hypothesis, aims, methods, predicted
outcomes and significance.
2. Attendance, work ethic and participation in the lab and in meetings (20%; throughout)
3. Experimental lab work (20%; throughout): Students will be evaluated on their ability to conduct
experiments, and to analyze and interpret the data generated.
4. Lab notes and organization (20%; throughout): Students will be evaluated on their ability to keep organized
and detailed experimental records, which will include aims, methods, results and interpretation.
5. Final Project Presentation (15%; due mid‐July or mid‐October): Students will present their research
objectives, results and future directions to the lab (20 minutes), followed by a questions discussion period.
Students will be evaluated on their presentation skills and ability to answer questions related to their
research project.
6. Final Research Report (15%; due at noon on the last day of term): Students will provide a 5‐10 page written
report describing the background and rationale for the research project, experimental methods, results and
interpretation, conclusions and future directions.
RESEARCH OPPORTUNITY PROGRAM
299Y/399Y PROJECT DESCRIPTIONS 2018‐2019
FALL/WINTER
Name and Title: Marc Meneghini, Associate Professor
Department: Molecular Genetics
TITLE OF RESEARCH PROJECT: Identification of New Substrates for the Set1/MLL Histone Methyltransferase.
Number of 299Y Spots: 1 Number of 399Y Spots: 1
OBJECTIVES AND METHODOLOGY:
Methylation of histone H3 on lysine‐4 (H3K4me) is conserved from yeast to human, and is one of the most
prominently studied chromatin modifications impacting transcriptional control and epigenetic inheritance. In the
budding yeast Saccharomyces cerevisiae, Set1 is the sole H3K4 methyltransferase. In humans, mutations in Set1
orthologs belonging to the MLL family cause devastating forms of leukemia and are associated with many other
cancers. Substrates for the Set1/MLL family outside of H3K4 are unknown however, and given the ancient
evolutionary origin of this protein family it seems likely that others exist. We have completed a synthetic dosage
lethality screen (SDL) screen to find new candidate substrates of Set1. SDL screens identify overexpressed yeast
proteins that cause reduced cell fitness specifically in a strain that has a gene of interest deleted. When the deleted
gene encodes a protein controlling a post‐translational modification, frequent screen hits have proven to be direct
targets of the modification controlled by the deleted gene. We have identified dozens of proteins that cause reduced
growth on strains lacking SET1 (set1∆). Each of these represents a candidate new target of Set1 methylation. The
objective will be to systematically confirm the screen hits with independent experiments assessing the growth of
wild‐type (WT) and set1∆ strains overexpressing different proteins using plasmid‐based inducible expression.
Confirmed screen hits will be focused on for more detailed follow up studies, eventually leading to LC‐MS/MS to
identify methylated lysine residues on the top candidates.
DESCRIPTION OF STUDENT PARTICIPATION:
The student will be responsible for preparing plasmids and transforming them into a battery of strains including WT,
set1∆, and strains with other mutations in SET1 and/or H3K4. These strains will then be used in “spotting assays” to
evaluate the growth characteristics under conditions that induce over‐expression from the selected plasmids.
Following collection of this data, selected proteins will be chosen for follow‐up molecular and genetic analysis.
MARKING SCHEME (assignments with weight and due date):
25%: Weekly reading assignments and group meetings.
This component will include, but not be restricted to, participation in weekly group meetings and journal
clubs.
50%: Weekly progress evaluations.
Project Code: MGY 6
The student will be expected to keep a notebook and discuss progress and anticipated experiments on a
weekly basis. Meetings with the supervisor will be arranged to go over the progress.
25%: Final written report / poster presentation.
The student will provide a final 2‐page written report and participate in a poster session to present their
results (either in the MolGen summer program poster session or in the undergraduate research forum).
RESEARCH OPPORTUNITY PROGRAM
299Y/399Y PROJECT DESCRIPTIONS 2018‐2019
FALL/WINTER
Name and Title: William Navarre, Ph.D., Associate Professor
Department: Molecular Genetics
TITLE OF RESEARCH PROJECT: Analysis of Microbes Isolated from Laboratory Mice
Number of 299Y Spots: 3 Number of 399Y Spots: 1
OBJECTIVES AND METHODOLOGY:
The bacteria associated with animals (microbiota) remain largely uncharacterized. There are 100s of bacterial
species in the microbiota of any animal, each with its own growth requirements and its own effect on its animal host
and the other microbes it interacts with. Our lab has isolated dozens of novel bacterial species from laboratory mice
and are characterizing these microbes for their metabolic capacity and their ability to either compete with or
collaborate with other microbes we have isolated.
DESCRIPTION OF STUDENT PARTICIPATION:
Students will learn basic microbial cultivation techniques, DNA purification, and each will be given a set of microbes
to characterize through standard metabolic assays. Students will assess their microbes for their ability to inhibit or
enhance the growth of other microbes in co‐culturing experiments. CRISPR systems of the bacteria will be examined
by genome sequencing and their ability to resist bacterial viruses (bacteriophages) will be assessed. One project will
involve genome sequencing and bioinformatics.
MARKING SCHEME (assignments with weight and due date):
Mid‐term report: 20% of final grade – due at end of term – last day before break for exams (early December)
Notebook: 15% of final grade ‐ Notebook and progress will be assessed monthly by the professor and student
advisor. Final mark given at end of second semester.
3 activity exercises in second term:
10%: detailed career goal statement – due end of third week of winter term.
10%: detailed write up of a lab protocol including references – due end of week after reading week
15%: sample grant writing exercise (1000 words) – due Friday, eighth week of winter term.
Final report: 30% of final grade – due during exam period and based on revision of sample grant proposal and
summary of results.
Project Code: MGY 7
RESEARCH OPPORTUNITY PROGRAM
299Y/399Y PROJECT DESCRIPTIONS 2018‐2019
FALL/WINTER
Name and Title: Aaron Reinke, Assistant Professor
Department: Molecular Genetics
TITLE OF RESEARCH PROJECT: Identification and Characterization of C. elegans Mutants that Provide Resistance to
Microsporidia Infection.
Number of 299Y Spots: 1 Number of 399Y Spots: 1
OBJECTIVES AND METHODOLOGY:
Pathogenic microbes are extremely widespread and cause much death and disease. Obligate intracellular pathogens
propagate exclusively inside host cells and include the causative agents of influenza, chlamydia, and malaria. Our
group is interested in understanding how intracellular pathogens can exploit hosts for their own benefit and how
hosts are able to defend against these types of pathogens. To tackle this problem, we have been studying a naturally
coevolved system of tractable Caenorhabditis nematodes hosts and Nematocida microsporidian pathogens.
Microsporidia are a phylum of obligate intracellular eukaryotic pathogens that can specifically infect a diverse range
of different hosts, including a number of species that cause death and disease in humans and agriculturally important
animal species. Using this system, we will identify mutations in C. elegans that provide resistance against
microsporidia infection. Though the characterization of these resistant mutants and the identification of the genes
responsible for the resistance, we will uncover the molecular mechanisms of how hosts can become resistant to
intracellular pathogen infection.
DESCRIPTION OF STUDENT PARTICIPATION:
Students will carry out experiments under the supervision of Dr. Reinke and experienced lab members, learning how
to manipulate and perform assays with C. elegans. Students will learn additional skills during this project including
forward and reverse genetic techniques, PCR and microscopy.
MARKING SCHEME (assignments with weight and due date):
Laboratory work and notebook‐assessed throughout the project (30%).
Presentation of project goals and results at lab meeting once during fall and once during winter term (40%).
Final written research report due April 12th 2019 (30%).
Project Code: MGY 8
RESEARCH OPPORTUNITY PROGRAM
299Y PROJECT DESCRIPTIONS 2018‐2019
FALL/WINTER
Name and Title: Mei Zhen
Department: Molecular Genetics
TITLE OF RESEARCH PROJECT: Using the Caenorhabditis elegans Nervous System to Investigate Neuronal Circuit
Development and Function
Number of 299Y Spots: 2
OBJECTIVES AND METHODOLOGY: How do neuronal circuits develop and remodel as animals progress from birth to
adulthood? How do they function robustly to help an animal respond appropriately to complex cues? These are
challenging questions to answer in humans, with 80‐100 billion neurons in the adult brain. In order to fully
understand how neuronal circuits work to regulate behaviours we need to know how the circuits are wired, and
how circuit activity is modulated. The fundamental processes and the rules that govern neuronal circuit formation
and function are well conserved between humans and the small nematode worm, Caenorhabditis elegans. C.
elegans larvae are born with 220 neurons, increasing to 302 neurons by the time the worm reaches adulthood. We
have been using serial‐section electron microscopy (EM) and computational approaches to map the entire
nervous system of young larval animals at synaptic resolution. This work will result in an unprecedented dataset
consisting of full reconstruction of the nervous systems of multiple animals, at multiple developmental stages. This
connectome reconstruction is being complemented by behavioural, genetic, calcium imaging and optogenetic
approaches to investigate the function of neuronal circuits in live animals. The insights from this project are
being used to further our understanding of how a nervous system forms and remodels across development, and how
neuronal circuits work together to mediate complex behaviours.
DESCRIPTION OF STUDENT PARTICIPATION:
The successful students will have the opportunity to:
1. Assist tracing neurons and identifying, and processing electron microscopy data
2. Assist with using the electron microscope to take high resolution images of the nervous system
3. Use molecular biology/ behavioural/ calcium imaging/ optogenetic techniques to investigate the function
and development of the nervous system.
MARKING SCHEME (assignments with weight and due date):
10% ‐ 2‐4 page report due 12 Nov 2018 (double spaced, including review of relevant literature, rationale of
project, hypothesis, methods, references in standard journal format)
40% ‐ final report due April 2019 (5‐10 pages, double spaced). Introduction, aim/hypothesis, methods,
o results, discussion, references (standard journal format).
10% participation in weekly lab meetings (duration of project)
Project Code: MGY 9