Exam 4: Lecture 8 Flashcards Preview

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Yeast 1- Hybrid Assay

-designed to identify specific DNA-protein interactions
-used to identify TF's bound to specific enhancer sequence


Yeast-1 Hybrid Assay Step 1

-design transcriptional reporter that contains enhancer element, core promoter, fragment, and reporter like lacZ
-construct integrated into yeast genome


Yeast-1 Hybrid Assay Step 2

-clone cDNAs that code for TF's into plasmids that contain GAL4 activation domain
-each plasmid will contain single cDNA
-when plasmid transcribed and translated will code for chimeric protein that contains GAL4 activation domain fused to individual TF
-entire plasmid library will contain 1000's of different TF containing plasmids, each fused to GAL4 AD


Yeast-1 Hybrid Assay Step 3

-yeast cells containing transcriptional reporter transformed with plasmid library
-each cell plated onto media and allowed to proliferate into colony
-cells treated with analog of lactose
-if TF-AD chimeric protein binds to enhancer it will activate expression of lacZ which will in turn cleave lactose analog


Cloning DNA Fragments

-involves digesting genomic DNA with restriction fragments and ligating them into plasmids which can then be transformed into bacterial cells
-genomic DNA and plasmids digested with same restriction enzyme so ends of genomic fragments and plasmid DNA will have same compatible ends
-linear fragments ligated together
-ligated plasmids can be transformed into bacterial cells
-each bacterial cell takes up single plasmid
-plasmid replicated independently of bacterial chromosome and at high levels
-allows for amplification of fragment of interest
-as bacterial cells replicate, form colonies on semi-soft agar medium


Cellular components/processes DNA Cloning takes advantage of

-restriction enzymes
-DNA ligase
-antibiotics and antibiotic resistance genes
-bacterial transformation
-DNA replication



-naturally occurring cellular pieces of DNA that are found in bacterial and some single-celled eukaryotes
-have own origin of replication (can replicated independently of bacterial chromosome)
-can replicate at higher frequency than bacterial chromosome (could be 1000's of copies in single cell)
-also carry antibiotic resistance genes that can be transferred during bacterial conjugation


Modified Plasmids

-in order to clone and amplify DNA fragments
-still contain origins of replication and antibiotic resistance genes
-contain cluster of restriction enzyme sites called multiple cloning site (MCS).


Cloning in Modified Plasmids

-DNA fragments that need to be cloned and amplified placed in MCS
-genomic DNA and plasmid must be digested with same restriction enzyme in order to have compatible or "sticky" ends
-enzymes that leave blunt ends can be used too and are glued into MCS with ligase


Role of Antibiotic Resistance Gene

-used as selectable marker to kill bacterial cells that fail to take up plasmid during transformation experiment
-if plasmid contains resistance gene against antibiotic X then the media must be treated with antibiotic X
-any bacterial cells that fail to take up plasmid will be killed by antibiotic
-if antibiotic and antibiotic resistance gene don't match, then all bacterial cells will be killed regardless of whether or not they have been transformed


Yeast-1 Hybrid Test Error

-prone to yield false positives
-second method must be used to confirm potential interactions suggested by Y1H assay
-one method is Electro Mobility Shift Assay (EMSA)


Electro Mobility Shift Assay (EMSA) Step 1

-generate radioactively labeled oligonucleotide that contains potential DNA binding site
-usually no longer than ~50 bp and will run to end of agrose gel


Electro Mobility Shift Assay (EMSA) Step 2

-purify TF of interest
-then mixed with radioactively labeled oligonucleotide
-mixture treated with chemical that crosslinks protein to DNA


Electro Mobility Shift Assay (EMSA) Interpretation of Results

-if TF of interest binds to oligonucleotide then total weight of protein-DNA complex will be larger than oligonucleotide alone and will run higher on gel
-if TF does not bind to oligonucleotide fragment will run at same size as control lane


Chromatin Immunoprecipitation (ChIP)

-allow for detection of protein-DNA binding on genome wide scale
-chromatin isolated from cells and digested with restriction enzyme or mechanically sheared
-smaller chromatin fragments are chemically cross-linked to preserve protein-DNA interactions
-chromatin passed through column that contains antibody that recognizes TF of interest
-using 2 different salt concentrations unbound fragments first removed and discarded while bound fragments separated and saved
-chromatin treated with chemical that releases TF from DNA fragments
-fragments can then be sequenced
-any sequence found in common from all fragments likely going to represent binding site of DNA binding protein of interest


Why Chromatin Immunoprecipitation?

-can determine binding site for TF
-can determine where in genome TF is bound by looking at sequences that are directly adjacent to TF binding site