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Exam II, Functional Genomics. 2009
a. Clearly the outcome of the C. elegans research experiment was very successful;
among other things, they were able to identify many genes of interest (gene
involved in embryo genesis, especially cell division) and design a favorable largescale screen for cell division genes which now represents an efficient discovery tool
to assign gene function. What was the significance/role of RNAi in this experiment?
b. How did they come to determine the optimal pool size for this large-scale study?
Describe what a knockout system is and the three knockout systems that were
talked about in class : Transponson tagging; homologous recombination; and the
Arabidopsis model developed by Dr Young (insertion of t-DNA).
If you were given a gene from the RNAi study involving C. elegans, and all you knew
about that gene is its sequence and that it produces a phenotype that disrupts
development, what would you do to figure out what the gene’s products are really
doing?
In the C. elegans experiments using RNAi to inactivate genes and establish those
required for cell division, under certain circumstances a phenotypic expression
could be the result of an unexpected gene. Explain the circumstances of this
occurrence and how to reduce the likelihood of this?
If using T-DNA as an insertional mutagen to knock-out a specific gene for
Arabidopsis, what variables are important to consider in efficiently ensuring that
sufficient T-DNA-transformed lines are created so that the gene of interested has
been targeted? Once you have created enough lines to target the gene, how is
screening efficiently performed?
What are some of the drawbacks to the RNA mediated interference (RNAi) approach
to discovering the function of novel genes? Why might further verification of an
observed phenotype from an inactivated gene be necessary? What was done to
avoid these false positives?
1. You are studying an human oncogene family and find a neighboring gene
HCCR-2 which is thought to cause breast cancer. You are sick and tired of
working on case studies and screening large numbers of patients diagnosed
with breast cancer and are thinking of entering the world of bioinformatics
using comparative genomic methods. Explain how you would set up your
experiment using the mouse model to determine whether HCCR-2 plays a role
in the development of breast cancer.
2. Why is Agrobacterium used to knockout genes in plants, and how is this
used to do genomic studies in plants?
How does the RNAi process work, and how was it used in C. elegans to do a
functional genomics approach to do a discovery based experiment of genes on
chromosome III?
What are miRNAs and siRNAs and how do they differ in function in their respective
pathways? How might the origins of these pathways differ?
8. A gene cluster is discovered on human chromosome 14 that is thought to
encode one or more proteins that interact with cancer-inducing cells in
epithelial tissue. These genes are termed cancer interacting genes (or CIG
genes). It is not known whether these proteins act to inhibit or promote the
action of these cells. Detail experiment(s) could be done in order to identify
this relationship?
Why did the double-stranded RNA associated with a mutant phenotype in C. elegans
embryos require further analysis and what analysis was performed?
In the RNAi article, the authors say that the fact that all 27 genes in the fidelity of
meiotic divisions class encode components of the translation machinery suggests
that efficient protein synthesis is required for successful meiotic divisions. They go
on to say “Whether this reflects a requirement for overall protein synthesis, or a
requirement for the synthesis of a specific subset of proteins needed for the meiotic
divisions remains to be determined.” Which do you think it is and how could you
determine it?
You have been trying to knock out a certain developmental gene in an organism by
site-directed mutagenesis but, for whatever reason, you are unable to yield the
expected results. What is the problem? How do combat it (or how do you knock out
that gene)? Give an example of a organism that causes you such grief.
There are several main knock out techniques we’ve learned so far. What are they
and state some advantages and disadvantages of each.
3. Looking at Fig 1. B (An Apolipoprotein Influencing Triglycerides in Humans
and Mice Revealed by Comparative Sequencing) explain how this plot shows
why APOVA was used on the experiment? What aided the researchers in
discovering whether or not this interval was transcribed? How did they test to
see if the function was conserved and what are the differences between these
techniques?
What are the benefits in using Ti-plasmids in plant genomics and how do they work?
How is it possible to check to see if the plasmid has properly been inserted? What
were some of the problems the researchers in the Modulation of Cell Proliferation by
Heterotrimeric G Protein in Arabidopsis had when using this technique?
7. How didCraig Venter showed that marine bacteriophages serve as a
reservoir for bacterial genes, often harboring genes that are advantageous to
the host. He also suggested that bacteriophages may serve as “genetic
laboratories” for their hosts. Considering the ecology and genetics of marine
bacteriophages, why are these viruses potentially good “genetic laboratories”
for bacteria?
4. In Functional genomic analysis of cell division in C. elegans using RNAi of
genes on chromosome III, the authors conducted a successful “fishing”
experiment and discovered novel genes. They were blessed with certain
computational and technological advances not available in the not-to-far
distant past. Outline a traditional forward approach to gene
annotation/function assignment (think Mendel and Morgan, not Venter).
What are the differences/advantages of the approach used in this paper?
Venter wanted to hedge his bets at being successful in sequencing the human
genome. The CSA approach was one way he did this. In this approach he aligned the
Celera reads to the PFP BACs in the ‘matcher’. This separated Celera unique reads
from those that matched BAC reads. The ‘combining assembler’ then formed
scaffolds from the matched Celera and BAC reads. These scaffolds were then
integrated with scaffolds built from the Celera unique reads by the ‘tiler’ to form
larger scaffolds called ‘components’. After all this work to align and build these
components, they were put through the WGA process.
A) What was the reason(s) for construction of these components?
B) What was the reason(s) for then putting them through the WGA process?
Reverse genetic studies utilizes gene knock out method to study gene functions.
Transposons, site direct mutagenesis, and t-DNA are tools to perform knock out
studies, and RNAi is used to “knock down” gene function. What are the advantages
and disadvantages of using RNAi method for doing gene knock out over the other
two methods?
You have recently joined a lab that is directed by a rather eccentric lead scientist who has
specialized his career in creatine kinase proteins (CK). These have been well
characterized are made up of two subunits, which can be of two types. M (muscle type) is
located on human chromosome 19, and B (brain type) located on human chromosome 14.
CK proteins are found with specific subunits and in specific tissues: brain tissue (CKBB), skeletal muscle tissue (CK-MM), and heart tissue (CK-MB). Your boss has long
suspected that each subunit is not actually a unique gene, but composed of duplicated,
tandem genes. Previous researchers have determined the sequence of orhtolgous mouse
DNA for both chromosomal regions and have compared the mouse and human
sequences.
A) The give you the VISTA graphical plot with the levels of homology between mouse and
human sequence, represented below. Is there any reason to believe that either/both of the
subunits have a tandem, duplicated gene? (Human sequence is represented on the x-axis,
% similarity to mouse genome on the y-axis)
Chromosome 14
Chromosome 19
B) If either of the genes have a potential paralogous gene, how could you attempt to
elucidate a function?
5. Craig Venter and his sailing scientists circumvented the world, they collected
water samples, filtered the water to collect the microbial fraction, and analyzed the
DNA extracted from the microbial fraction for viral genes. The metagenomic
assembly process was not specific to viruses however, and many scaffolds contained
bacterial fragments and possibly environmental DNA from unknown sources. From
amid the mess of the multi-organismal scaffolds, the global ocean sampling
expedition claims to have found not just viral proteins, but TWO classes of viral
proteins.
A) What were the two classes of viral proteins they found?
B) How did they verify that the proteins they found were truly from a viral
genome, and how did they classify the genes?
An article depicts two companies boasting having a next generation technique
significantly faster and cheaper than present next generation sequencing products.
Describe in detail the steps for the typical 454 next generation sequencing and compare
and contrast it with the opposing companies’ new technology claim.
Why are evolutionarily conserved sequences helpful when studying the human
genome?
The C. elegan’s paper pointed out two possible causes of false phenotypes when
using RNAi with dsDNA. Give one possible cause for error and how they avoided
this error in their experiment.
6. What are the differences and similarities between the three next gen
sequencing techniques we discussed as far as principles used? How does this
improve on old-fashioned sequencing?
(a) If you wanted to study the function of a particular human gene, what qualities of
the mouse genome allow us to study the function of human genes in mice? (b) Using
Fig 1 from “An Apolipoprotein Influencing Triglycerides in Humans and Mice
Revealed by Comparative Sequencing” as a model, please explain what the gene
would have to have to study it in mice? (c) Using your knowledge of transgenes, and
knock out experiments please explain what you can deduce from Fig. 2 about the
function of the APOAV gene.
Describe the type of PCR used by most NEXTGEN sequencers. What are its benefits
over conventional PCR sequencing?
In reference to the cell division in C. elegans paper, which used RNAi as a highthroughput genetic screen to identify genes required for cell division, what sort of
complications did the researchers run into concerning related genes, and how did
they correct this?
Describe how the apolipoprotein APOAV was discovered using sequence and EST
data, and how its function was determined?