Advanced Genetics (3 credits) BI 399 Special Topics
Instructor
Office
Phone
Office Hours
E-mail
Spring 2009
Dr. Vince Buonaccorsi
VL1050
x3579
M/T/W/F 9-10AM
buonaccorsi@juniata.edu
Description:
Profound changes in technology occurring within the last few years are rapidly transforming our
view of molecular processes at scales ranging from extremely fine processing of single “gene”
transcripts to patterns that occur genome-wide. Our coverage will focus on investigation of
evolutionarily significant (both adaptive and maladaptive) genetic and genomic change in
eukaryotic populations in light of emerging technologies. Topics covered will include gene
ontology (classification), regulation of expression and development, processes affecting genetic and
genomic variation within and between populations, molecular genetic basis of simple and complex
phenotypes, finding the molecular basis of adaptive or disease traits in model and non-model
species, phylogenetic reconstruction, phylogeography and speciation. Case studies will be used to
challenge students’ understanding of conceptual material in context. Mathematical and conceptual
material are fully integrated and explained. Students will present two articles from the primary
literature and write an independent research proposal. Recent review articles, primary literature,
and webinars from leading researchers will be used to capture some of the excitement of the field.
CW. Prereqs, BI 105 and BI 106.
Learning Objectives:
a. Understand capabilities of emerging tools used in genetic analysis
b. Verbal fluency in basic principles and practices of genetics
c. Understand how geneticists study evolution at a variety of time scales
d. Independent mastery and creative thought in one area of genetics via writing research
proposal
Assessment:
Demonstrate understanding of material through two midterms (30%)
Presentations, case studies (25%)
Write a research proposal (45%)
Absence policy: Attendance is required. Arrive promptly. Late arrival is unprofessional. Acceptable
absence:
If Dr.'s note or written statement from Dean of Students, College approved athletic
activity, college approved field trip If you haven't already, inform me today about any absences, and
your makeup plans.
Withdrawal Policy:Students may withdraw from the course as described in the Pathfinder
(http://intranet.juniata.edu/policies/pathfinder), and may be permitted to withdraw from the course
until the last day for withdrawal allowed by the registrar. However, all students withdrawing from
the course must meet with the instructor to obtain the required signature.
Academic Integrity: Students are expected to comply with all academic integrity guidelines as
described in the Pathfinder (http://intranet.juniata.edu/policies/pathfinder).
Preliminary Schedule
Weeks 1& 2 Genetic variation and emerging technologies in genomics
Week 3
Gene structure and identification
Weeks 4&5 Gene expression and emerging technologies in transcriptomics
Midterm I
Week 6
Development
Weeks 7-10 Population variation in genes and genomes
Midterm II
Week 11
Finding the molecular basis of adaptive or disease traits; model, non-model spp
Week 12
Phylogenetics; phylogeography
Week 13
Speciation genetics
Week 14
Proposal presentations
Due Weds Week 4:
Due Weds Week 9:
Due Tues Week 13:
Case studies
Fine structure of a gene and consequences of mutation
Puget Sound and the Endangered Species Act
Analysis of human phylogeography
Tips for reading primary literature
Spring 2009
MAKE A LIST OF QUESTIONS YOU HAVE AS YOU READ (i.e. How did they assay the
enzyme level in those patients?)
1. Introduction
a. Do you understand the background material? If not, investigate further.
b. What hypothesis is being tested and why is it important?
2. Materials and Methods
a. Did the authors provide enough detail that someone else could replicate their data
independently?
3. Figures and figure legends
a. Can you follow the research the researchers’ thought process just by looking at the
figures?
b. Is there any information missing or incorrect in the figures or figure legends that is
confusing or invalidates the paper’s claims?
4. Results
a. Does the text support the figures and tables?
b. Does it make sense to you, or are you more confused than when you started?
5. Discussion
a. Do you understand the research claims they are making?
b. Why are their findings important?
c. Do you believe the authors’ claims?
6. List the questions you still have.
Resources
WEEK 1: Emerging technologies in genomics
Next Generation Sequencing, JM Rothberg, Leamon JH. 2008. The development and impact of
454 sequencing, Nature Biotechnology, 26 1117-1124
Into to massively parallel sequencing, Margulies et al. 2005 Genome sequencing in open
microfabricated high density picoliter reactors Nature. Sep 15;437(7057):376-80.
Korbel, JO et. al. 2007. Paired-end mapping reveals extensive structural variation in the human
genome. Science. 318:420-426.
WEEK 2: Gene definition, gene ontology, systems biology
Mark B. Gerstein, Can Bruce, Joel S. Rozowsky, et al. 2007. What is a gene, post-ENCODE?
History and updated definition. Genome Res. 17: 669-681
WEEKS 3: Gene structure and identification
Small RNAs, H. Grobhans & Filipowicz, W. 2008. Emerging world of small RNAs. Nature. 451:
414-416.
Pseudogenes, R. Sasidharan and M. Gerstein. 2008. Protein fossils live on as RNA. Nature
453:729-731.
Systems Biology, Westerhoff, HV Palsson BO. 2004. The evolution of molecular biology into
systems biology. Nature Biotechnology. 22, 1249-1252.
Gene ontology, http://www.geneontology.org/
WEEKS 4&5: Emerging technologies in transcriptomics
TF Modeling: Lemon B., Tijan, R. 2001. Orchestrated response: a symphony of TFs for gene
control. Genes and development. 4:2551-2569
RNAseq: JC Marioni, CE. Mason, SM Mane, et al . 2008 RNA-seq: An assessment of technical
reproducibility and comparison with gene expression arrays. Genome Res. 18: 1509-1517
Mardis, E. 2007. ChIP-seq: welcome to the new frontier. Nature Methods. 4:613-614.
Functional Ecology: Roelofs, D. Aarts MGM, SChat H, van Straalen NM. 2008. Functional
ecological genomics to demonstrate general and specific responses to abiotic stress. Functional
Ecology. 12, 8-18.
Functional Ecology: Vera, JC.et al. 2008. Rapid transcriptome characterization for a nonmodel
organism using 454 pyrosequencing. Mol. Ecol. 17. 1636- 1647.
WEEK6: Development
VJ Lynch and GP Wagner. 2008 Resurrecting the role of transcription factor change in
developmental evolution. Evolution. 62-9: 2131-2154
Morpholinos and RNAi: JD Moulton, Yan, Y. 2008. Using Morpholinos to Control Gene
Expression. Current Protocols in Molecular Biology. Curr Protoc. Mol Biol. 2008 Jul;Chapter
26:Unit 26.8
JT Streelman, CL Peichel, DM Parichy. 2007. Developmental genetics of adaptation in fishes: The
case for novelty. Annu. Rev. Ecol. Syst. 38:655-81.
WEEKS 7-10: Population variation in genes and genomes
Begun, D. et al. 2007. Population genomics: Whole-genome analysis of polymorphism and
divergence in Drosophila simulans. PLoS Biology 5:pg 2534-2559.
Kohn et al. 2006 Genomics and conservation genetics TREE,21,629-637
Frankham 2005 Genetics and extinction, Biological Conservation 126,131-140
Hansson, B. Westerberg.L. 2002. On correlation between heterozygostiy and fitness in natural
populations. Mol. Ecol. 11, 2467-2474
Gibson, G. 2002. Microarrays in ecology and evolution, a review. Mol. Ecol. 11, 17-24
Whitehead, A and Crawford, DL 2006. Variation within and among species in gene expression:
raw material for evolution. Mol. Ecol. 15, 1197-1211.
Week 11: Finding the molecular basis of adaptive or disease traits; model, non-model spp
Storz, J.F. 2005. Using genome scans of DNA polymorphism to infer adaptive population
divergence. Mol. Ecol. 14, 671-688.
Week 12: Phylogenetics; phylogeography
Knowles 2004. The burgeoning field of statistical phylogeography. J. Evol. Biol. 17, 1-10
Hewitt 2001. The genetic legacy of the Quaternary ice ages, Nature, 405,907-913 Hewitt 2001.
Speciation, hybrid zones, and phylogeography or seeing genes in space and time . Mol Ecol. 10,
537-550
Week 13: Speciation genetics
Noor, M Feder JL, 2006 Speciation genetics: evolving approaches. Nature reviews genetics 7:851861.
Paper: In order for you to learn in depth, a more advanced application or theory in genetics, each
of you will write a review paper/research proposal. Topics will need to be approved by myself.
Pick an area that is important to you and that you would like to learn a lot more about. The paper
should be 8-10 pages, (double spaced, font 12) excluding bibliography and figures/tables.
Choosing a Topic (by end of week 4)
Do not underestimate the importance of choosing a suitable topic. Ideally, it should be 1)
interesting to you; 2) not so broad that it is unmanageable, 3) not so narrow you can’t find enough
information on it; 4) not so difficult that you can’t fully understand it.
A common approach is to start with a broad topic and do some general reading about it,
gradually narrowing it down to a workable size. As you narrow your topic and become familiar
with the literature, you need to develop a sense of your main objectives. What question will your
paper address and from what perspective? Are you shifting toward a particular viewpoint or
conclusion that can serve as a main point, or thesis, for the paper? If you limit your scope and
define your goals early in the project, your reading and note taking will be more directed and your
time will be more productive.
Occasionally the first topic you choose may be too narrow and you will have to enlarge it or
shift to a different subject altogether. Allow time for false starts, delays, and topic changes.
Recognizing a good subject to write about requires thought and effort.
Annotated Bibliography (by end of week 6)
An annotated bibliography is a brief summary, or annotation, about each source (book, magazine,
journal, etc.). No web references are allowed. The annotation describes the content of the work so
that future reference to the entry by a researcher will provide essential data. I suggest using the
MEDLINE or ASFA database to find references.
When writing the annotation, provide enough information in approximately three to five sentences
for readers to obtain a comprehensive understanding of the source's purpose, content, and special
value. Be sure to use complete sentences and to avoid wordiness.
Process for Writing an Annotated Bibliography
1.List the completed bibliographical citation.
2.Explain the main purpose of the work.
3.Briefly describe the content.
4.Evaluate the relevance of the information.
5.Note any special features.
I expect to see at least 10 references in the annotated bibliography and at least 20 in the final paper.
RESEARCH PROPOSALS (outline and
rough draft due end of week 11, final draft by end of
term)
Research proposals have many traits in common with research papers: both introduce a scientific question
or hypothesis, both put a specific study in the broader context of existing research, and both are organized in
ways that reflect the logic of the scientific method. Of course, a research proposal is missing a RESULTS
section, along with the author’s analysis and interpretation of those results. Instead, it seeks to propose and
justify the author’s research plans-perhaps for a senior project, a summer internship, a graduate thesis, or, in
the case of professional biologists, new or continuing work in a specialized field.
There is no one standard format for a research proposal. Authors seeking funding from NSF or other
agencies must adhere to the precise guidelines laid down by those organizations; student authors should
follow their instructor’s or university’s requirements. Nevertheless, all research proposals involve some of
the same challenges and constraints. If you are presented with the opportunity to do research, the following
guidelines should help you in preparing your own proposal.
CREATE A SPECIFIC AND INFORMATIVE TITLE
A focused title suggests a well-thought-out project. Titles such as “A study of stream ecology” or “Nutrient
uptake in seaweeds” convey little sense of direction or purpose; compare them with more specific versions:
”The effect of open and close canopy on the diversity of macroinvertebrates in Morrisville Creek”; “Ammonia
uptake by Ulva curvata growing on three different substrates.” You may need to modify your title as you
proceed with the research, but a good working title is essential to a well-received proposal.
START WITH AN OVERVIEW OF THE GENERAL SCIENTIFIC ISSUE (3 to 5 pgs)
Before proposing your own study, you need to introduce the broader topic to which your work relates. Most
research proposals begin with an Introduction (or Background to the study) section, which presents a
theoretical context for the proposed investigation by summarizing and integrating the work of others in the
field. Doing a good job on this part involves a thorough literature search and careful, selective use of primary
sources, which are cited in your text and then listed later in a Literature Cited section. Your introductory
discussion should also reflect a sensitivity to the scientific background possessed by your readers. Typically,
you’ll be submitting your proposal to other scientists; however, if it will be judged by nonscientific readers (for
example, a multidisciplinary committee), then keep specialized vocabulary to a minimum, and use a less
technical style.
STATE THE AIMS OF YOUR STUDY AND PROVIDE A CLEAR RATIONALE (2 to 4 pgs)
After providing background material and highlighting major findings, you can then turn to important gaps in
our knowledge. What conflicts or controversies exist in the literature? What questions remain? Relate such
issues to your proposed study and its specific aims. What questions will you investigate? How will your
work enlarge, clarify, or complement existing knowledge of the subject? Discussion of your own objectives
can come at the end of the Introduction or Background section, or in a subsequent section.
SUMMARIZE YOUR METHODS (2 to 3 pgs)
Next explain specifically how the research will be conducted, what kinds of data you will collect, and how
those data will be analyzed. The level of detail, and thus the amount of space devoted to the Methods
section, will depend on the type of proposal you are submitting and the audience for whom it is intended.
Remember that even the most promising research question will fall flat if you can’t devise a practical and
scientifically valid way to investigate it. Think through your methods completely, and become thoroughly
familiar with the procedures used in studies similar to yours. If you want to convince your readers to commit
their time and/or money to your project, then it must sound feasible.
FOLLOW-UP EXPERIMENTS (0.5 pgs)
If your experiment is successful, briefly describe at least three more experiments or procedures that
could be done to take your project to the next level.
Guidelines for choosing a discussion article
1. I may suggest an article or you may choose an article for our class to read that is a primary
research article.
2. You will need to obtain and read review articles on your topic (as well as other primary
literature). Review articles, books, the internet, and other primary references are acceptable
resources for background information. On the day of your presentation, you should provide
me with a bibliography of sources you consulted in preparation for your presentation. You
must consult at least two sources (excluding the internet) in addition to the article you
assign.
3. Please choose an article for the class assignment that is not more than 5 years old.
4. The article should be approved by me a week before your presentation date. Then the PDF
file should be e-mailed to me so that I can post it on the shared drive (if applicable). If no
PDF file is available, please provide me with a hard copy so that I can put it on reserve in
the library.
Guidelines for presenting primary literature
Presentations of peer-reviewed journal articles should be in the following format that illustrates the
scientific method:
1. What is the question and why is it important?
2. What were the researchers trying to discover? (Hypotheses and goals)
3. What methods were used to pursue each goal?
4. What did they find regarding each goal? (The results)
5. What do the results mean? (interpretations of the data)
6. Conclusions / Discussion (include own thoughts and comments)
SIGNUP
12-Feb ______________
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26-Feb ______________
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12-Mar ______________
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Case Study 1
Case study 1. Fine structure of a gene and consequences of mutation
There are very few cases outside of Drosophila where the genetic mechanism of speciation has been
resolved. Prior characterization of divergence between two sympatrically distributed sister species within the
Sebastes species flock suggested that one locus demonstrated extremely high inter-specific divergence and
low diversity within one species. Within the Sebastes species complex, Sebastes carnatus and
Sebastes chrysomelas are the most closely related sister species, differing morphologically only in
coloration pattern. Prior genome scan analysis between the two species showed that one
microsatellite DNA locus was extremely divergent relative to expectations for single loci based on
mutation-drift equilibrium given the mean divergence level between species. This suggests that
natural selection has acted on polymorphism within nearby genes in the genome. Given very low
diversity within one species, the outlier microsatellite have been affected by a selective sweep,
which is caused when a polymorphism is under selection and population variability is lost in DNA
linked to the polymorphism. Sequencing of adjacent areas of the genome in S. chrysomelas was
performed through genome walking. For Case Study #1, your job will be to characterize the
sequence for genes, pseudogenes, and repetitive elements using bioinformatic tools, and think about
the function of the genes in the area and what phenotypes they may lead to when mutated.
Case Study 1 Learning Objectives in Molecular and Transmission Genetics
Understand how cellular processes allow gene prediction from a genomic sequence.
Become acquainted with some common Bioinformatic tools.
Think about how molecular changes may translate into phenotypic changes.
1) Find the 20,000 sequenced nucleotides on the P-drive for this class. Do a “somewhat
similar” “BlastN” search on the NCBI website to find nt homology with the posted
sequence. Don’t trust a hit unless the probability that the hit was random or “E value” is
below 10-10. Based on the information you see, which regions can you identify a match for?
Describe no more than 5 hits per region if there are multiple available. Only include regions
of at least 50bp of homology. Find at least one function for each gene or pseudogene
identified.
2) Given the nt sequence, use GENSCAN to find exons and protein coding sequences.
http://genes.mit.edu/GENSCAN.html. Save the protein sequences in notepad and the pdf of
the predicted exons.
3) Do a “BlastP” search for protein homology using the protein sequences identified from
GNESCAN Don’t trust a hit unless the significance or “E value” is below 10-10. Based on
the information you see, what do you think each hit is?
a. Find at least one function for each gene identified.
b. Find the closest hits in Danio rerio for one of your genes by restricting the organism
search criteria in Blastp. Record the Locus Identifier and which chromosome in the
Danio genome the hit occurred. Pull up the “map viewer” on the Danio rerio
genome project website to identify the neighboring genes for one of your genes.
4) Where possible, highlight and label exons and UTRs for each gene using the GENSCAN
info and info available on eukaryotic consensus sequences from class. Highlight each gene
in a different color. Find and label conserved sequences with respect to parts of the gene
that direct cellular machinery to begin transcription and translation, as well as splice sites,
start and stop codons, and the polyadenylation site.
5)
a. Invent a rare missense mutation that causes loss of expression at one of the genes.
b. Describe some phenotypic effects that might be expected in that individual, based on
the function(s) you discovered above.
c. Would the mutation be more likely dominant or recessive? Explain your reasoning.
d. If this individual had a family had 10 children, how many would be expected to
express the phenotype associated with the rare mutation given your answers above?
e. Invent a transition in a non-coding region that achieves the same result as the
missense mutation above.
6) Invent a mutation in one of identified genes that would likely cause a dominant phenotype.
If this individual had a family had 10 children, what proportion would likely get the disease,
assuming the trait is rare.
Advanced Genetics Test1. Read and bring the following paper to class un-marked. Vera et al.
2008, Rapid transcriptome characterization for a nonmodel organism using 454 pyrosequencing.
Molecular Ecology (2008) 17, 1636–1647
1.
Describe the main goal and approach used in the study.
2.
How were samples prepared for 454 sequencing in this paper? In your answer, include the
method they used to avoid sequencing mainly rRNA (since that is the most abundant class of rRNA
in the cell)?
3.
Why did they generate plasmid cDNA libraries for Sanger sequencing?
4.
What is the point of Figure 2? How is it possible for a data point to have a y-axis value of
greater than 1?
5.
What is the point of Figure 4? What evidence suggests that they are looking at alternative
splicing of the same gene rather than different genes? Be specific.
6.
Explain what aspect of Figure 5b illustrates “marked differences in transcript levels of
microsporidia genes among individuals”?
7.
What was the point of designing microarrays from their data? In your answer, provide an
example of how these specific microarrays might be used.
8.
Describe 5’RACE and how the authors suggest it could be used.
9.
What was the criteria they used to determine a SNP? What did they want to avoid
confusing a SNP with?
10.
Randy Bennett is interested in characterizing the expression polymorphisms that lead to
differential eye development in two populations of amphipods, populations that develop either in
the presence or absence of predators. No genomic resources exist in amphipods. Explain to him
the benefits and limitations of the Vera et al. (2008) 454 approach for transcriptome analysis that
apply to his case.