MICR 302 Lecture 16: MICR 302 Lecture 16
1 May 2018
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Lecture 16
PART 2 - SYSTEMS BIOLOGY AND NETWORKS
Systems Biology:
-The study of an organism as an integrated and interacting network of genes, proteins,
molecules, and biochemical reactions
-requires “systematic data collection” for all molecules in a cell
examples:
1. comprehensive study of the mRNAs
EX: Transcription of mRNA measured by Northern Blot
-this study looked at the pheromone pathways —> detection via change in the genes the
needed to fuse membranes (Fus1)
-done via Northern Blot
-based on radioactive ss nucleic acid probe which hybridizes to RNA due to base pairing
and can be resolved on the gel —> use complementary sequence to detect every mRNA in
yeast present and look for specific one
-do this ONE gene at a time (requires an a priori hypothesis)
Mechanism:
1. take a culture and expose it to mating pheromones —> within minutes of addition you can
detect the transcirpt
Global Monitoring of mRNA levels
—> MICROARRAY
-allows us to look at all transcripts inside the cell
MECHANISM:
1. take experimental and control cultures and add the pheromone to both (treat identically)
2. isolate the mRNA and purify
-this is easy to do because eukaryotic transcripts have a polyA tail —> use a polyT stitched
nucleotide to fish those out
-can colour label the RNAs through a reverse transcriptase reaction and capture the mRNAs
3. use an oligo-DT primer to anneal and extend and make a copy of every PolyA transcript
(include fluorescent nucleotide)
ABOUT:
-microarray is a competitive hybridization
-mix equimolar amounts of red and green RNA in tube, take that population of mixed RNA and
allow them to hybridize in the microarray
-essentially this is a northern blot for each spot
-at each spot there is a single stranded nucleic probe that represents a feature from
transcription (usually a CDA)
RESULTS:
-due to the competitive nature, if the gene did NOT change there will be an equal amount of
red:green —> cohybridization shows up as yellow
-spots where there theres more red or green means its unregulated
-control RNA = more = down regulated)
find more resources at oneclass.com
find more resources at oneclass.com
Microarrays measure thousands of genes in parallel:
-this is only possible because we know the genome and have a list of RNA we want to query
-this gives us a list of the genes that CHANGE in response to factor
-red = upregulated, green = downregulated.
Microarrays provide a global view of mating response:
-we are seeing how transcripts are made at the SYSTEMS level and we can apply a similar
approach to any biological system in response to factors of choice
EXAMPLE:
-we saw that genes such as Fus1 (fusing mated cells) and Fus3 (MAPK, positive feedback
loop to amplify) were red = UPREGULATED
-we saw that HHF1 (histone) and RNR1 (ribonucleotide reduction, prepares d NTPs) were
down regulated
-you don't want to continue DNA replication —> arrest at G1
Confirmation of the key role of Ste12
STUDY:
-wt yeast exposed to 50 nM pheromone
-genes towards the right of the graph were unregulated (FIG1), genes towards the left were
down regulated (HHF1)
-Y axis was years strain missing Ste12 …. no response without it.
-confirms Ste12 is responsible for changes
Hierarchical Clustering to visualize “big data”
EX: paper from 2000
-first approach using microarray to take apart a signal transduction pathway
-they did 46 separate microarrays where they titrated in pheromones to look at the responses
over time
EXAMPLE DATA POINT:
—> Far1 knockout
-when exposed to pheromone up regulation works fine BUT the genes do not go down for
cycle arrest
-if you do not have a CDKinhibitor pathway it will turn on but it cant engage in cell cycle arrest.
KEY: can compare experimental cables together to identify key components of signal pathways
Systematic Characterization of Proteins
-in terms of nucleic acids, there is high specify for the transcript of interest and you only need
to make a single strand complement to the transcript of interest (ex: 10 of interest = 10 dif
probes)
-proteins are MORE CHALLENING: need a different type of technology (simple, cost effective)
goal: measure all proteins at the same time
challenge: detecting 1000s of proteins in parallel is far more complex than detecting 1000s of
mRNAs
SOLUTIONS:
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
The study of an organism as an integrated and interacting network of genes, proteins, molecules, and biochemical reactions. Requires systematic data collection for all molecules in a cell examples: comprehensive study of the mrnas. Ex: transcription of mrna measured by northern blot. This study looked at the pheromone pathways > detection via change in the genes the needed to fuse membranes (fus1) Based on radioactive ss nucleic acid probe which hybridizes to rna due to base pairing and can be resolved on the gel > use complementary sequence to detect every mrna in yeast present and look for speci c one. Do this one gene at a time (requires an a priori hypothesis) 1. take a culture and expose it to mating pheromones > within minutes of addition you can detect the transcirpt. Allows us to look at all transcripts inside the cell.
