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



FALL/WINTER

Name and Title: Amy A. Caudy, Associate Professor


TITLE OF RESEARCH PROJECT: Discovery and Characterization of Novel Enzymes using Metabolomics and Genetics



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.


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.


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%



FALL/WINTER

Name and Title: Dr. Alan Cochrane (Professor)


TITLE OF RESEARCH PROJECT: Regulation of HIV‐1/Adenovirus RNA Processing: Insights into novel therapeutics



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.


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.


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


299Y PROJECT DESCRIPTIONS 2018‐2019

FALL/WINTER

Name and Title: Barbara Funnell, Professor


TITLE OF RESEARCH PROJECT: Mechanisms of Plasmid Maintenance in Bacteria

Number of 299Y Spots: 1


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.


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.


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.



FALL/WINTER

Name and Title: Dr. Thomas Hurd, Assistant Professor


TITLE OF RESEARCH PROJECT: Determining how Deleterious Mitochondrial DNA Mutations are Eliminated



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).


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.



FALL/WINTER

Name and Title: Marc Meneghini, Associate Professor


TITLE OF RESEARCH PROJECT: Identification of New Substrates for the Set1/MLL Histone Methyltransferase.



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.


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.


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).



FALL/WINTER

Name and Title: William Navarre, Ph.D., Associate Professor


TITLE OF RESEARCH PROJECT: Analysis of Microbes Isolated from Laboratory Mice



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.


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.


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



FALL/WINTER

Name and Title: Aaron Reinke, Assistant Professor


TITLE OF RESEARCH PROJECT: Identification and Characterization of C. elegans Mutants that Provide Resistance to

Microsporidia Infection.



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.


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.


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


299Y PROJECT DESCRIPTIONS 2018‐2019

FALL/WINTER

Name and Title: Mei Zhen


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.


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.


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

10% Oral presentation at lab meeting before April 2017

20% lab competency

10% good recording of data

research opportunity program project … genetics... · defined roles for pips and their regulatory...

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