![Page 1: Recombinant DNA-based molecular techniques · 2020. 6. 30. · Recombinant DNA-based molecular techniques Prof. Mamoun Ahram Summer 2020. Analysis of transcriptional regulatory sequences:](https://reader033.vdocument.in/reader033/viewer/2022051900/5fef727545fc8969c7553a32/html5/thumbnails/1.jpg)
Recombinant DNA-based molecular techniques
Prof. Mamoun AhramSummer 2020
![Page 2: Recombinant DNA-based molecular techniques · 2020. 6. 30. · Recombinant DNA-based molecular techniques Prof. Mamoun Ahram Summer 2020. Analysis of transcriptional regulatory sequences:](https://reader033.vdocument.in/reader033/viewer/2022051900/5fef727545fc8969c7553a32/html5/thumbnails/2.jpg)
Analysis of transcriptional regulatory sequences:Role of enzymes
![Page 3: Recombinant DNA-based molecular techniques · 2020. 6. 30. · Recombinant DNA-based molecular techniques Prof. Mamoun Ahram Summer 2020. Analysis of transcriptional regulatory sequences:](https://reader033.vdocument.in/reader033/viewer/2022051900/5fef727545fc8969c7553a32/html5/thumbnails/3.jpg)
What are transcriptional regulatory sequences?
![Page 4: Recombinant DNA-based molecular techniques · 2020. 6. 30. · Recombinant DNA-based molecular techniques Prof. Mamoun Ahram Summer 2020. Analysis of transcriptional regulatory sequences:](https://reader033.vdocument.in/reader033/viewer/2022051900/5fef727545fc8969c7553a32/html5/thumbnails/4.jpg)
Firefly luciferase
![Page 5: Recombinant DNA-based molecular techniques · 2020. 6. 30. · Recombinant DNA-based molecular techniques Prof. Mamoun Ahram Summer 2020. Analysis of transcriptional regulatory sequences:](https://reader033.vdocument.in/reader033/viewer/2022051900/5fef727545fc8969c7553a32/html5/thumbnails/5.jpg)
The purpose and procedures
Purpose: study the activity of a gene at certain conditions or elucidate the function of certain regions of the promoter
The promoter of the gene only is placed upstream of a “reporter gene” such as the luciferase gene in a plasmid, the plasmid is transfected (inserted) into the cells, and the expression level of luciferase (instead of the gene itself) is measured.
![Page 6: Recombinant DNA-based molecular techniques · 2020. 6. 30. · Recombinant DNA-based molecular techniques Prof. Mamoun Ahram Summer 2020. Analysis of transcriptional regulatory sequences:](https://reader033.vdocument.in/reader033/viewer/2022051900/5fef727545fc8969c7553a32/html5/thumbnails/6.jpg)
Example
No promoter (negative control)
A good promoter (positive control)
The complete promoter
The promoter with deleted regions
Repressor region Activating region Core
![Page 7: Recombinant DNA-based molecular techniques · 2020. 6. 30. · Recombinant DNA-based molecular techniques Prof. Mamoun Ahram Summer 2020. Analysis of transcriptional regulatory sequences:](https://reader033.vdocument.in/reader033/viewer/2022051900/5fef727545fc8969c7553a32/html5/thumbnails/7.jpg)
Protein-protein interaction
![Page 8: Recombinant DNA-based molecular techniques · 2020. 6. 30. · Recombinant DNA-based molecular techniques Prof. Mamoun Ahram Summer 2020. Analysis of transcriptional regulatory sequences:](https://reader033.vdocument.in/reader033/viewer/2022051900/5fef727545fc8969c7553a32/html5/thumbnails/8.jpg)
(Co)-Immunoprecipitation
Antibody molecules that target a specific protein are conjugated to special beads.
A mixture of cell proteins are added to the beads.
Only the protein of interest is precipitated as well as other proteins bound to it (co-precipitated).
![Page 9: Recombinant DNA-based molecular techniques · 2020. 6. 30. · Recombinant DNA-based molecular techniques Prof. Mamoun Ahram Summer 2020. Analysis of transcriptional regulatory sequences:](https://reader033.vdocument.in/reader033/viewer/2022051900/5fef727545fc8969c7553a32/html5/thumbnails/9.jpg)
What is a DNA library?
A library can be created for DNA fragments just like book libraries.
You can have clones of bacteria each containing a specific piece of DNA.
You can save these clones in the freezer and take whichever clone you want to study.
http://www.sumanasinc.com/webcontent/animations/content/dnalibrary.html
9
![Page 10: Recombinant DNA-based molecular techniques · 2020. 6. 30. · Recombinant DNA-based molecular techniques Prof. Mamoun Ahram Summer 2020. Analysis of transcriptional regulatory sequences:](https://reader033.vdocument.in/reader033/viewer/2022051900/5fef727545fc8969c7553a32/html5/thumbnails/10.jpg)
Genomic vs. cDNA library
How?
![Page 11: Recombinant DNA-based molecular techniques · 2020. 6. 30. · Recombinant DNA-based molecular techniques Prof. Mamoun Ahram Summer 2020. Analysis of transcriptional regulatory sequences:](https://reader033.vdocument.in/reader033/viewer/2022051900/5fef727545fc8969c7553a32/html5/thumbnails/11.jpg)
Genomic vs. cDNA library
![Page 12: Recombinant DNA-based molecular techniques · 2020. 6. 30. · Recombinant DNA-based molecular techniques Prof. Mamoun Ahram Summer 2020. Analysis of transcriptional regulatory sequences:](https://reader033.vdocument.in/reader033/viewer/2022051900/5fef727545fc8969c7553a32/html5/thumbnails/12.jpg)
Yeast two-hybrid systemTaking advantage of domains
In yeast, an upstream activating sequence (UAS) exists.
UAS is controlled by a transcription factor that is made of two domains
A DNA-binding domain (BD)
An activating domain (AD) that is responsible for the activation of transcription.
Both must be close to each other in order to transcribe a reporter gene such the LacZ gene.
![Page 13: Recombinant DNA-based molecular techniques · 2020. 6. 30. · Recombinant DNA-based molecular techniques Prof. Mamoun Ahram Summer 2020. Analysis of transcriptional regulatory sequences:](https://reader033.vdocument.in/reader033/viewer/2022051900/5fef727545fc8969c7553a32/html5/thumbnails/13.jpg)
Why is the LacZ gene used?
Yeast cells are grown in the presence of a lactose analog called X-gal, which generates a blue product when cleaved.
When the LacZ gene is activated, beta-galactosidase is produced, which cleaves X-gal generating blue colonies.
X-gal
![Page 14: Recombinant DNA-based molecular techniques · 2020. 6. 30. · Recombinant DNA-based molecular techniques Prof. Mamoun Ahram Summer 2020. Analysis of transcriptional regulatory sequences:](https://reader033.vdocument.in/reader033/viewer/2022051900/5fef727545fc8969c7553a32/html5/thumbnails/14.jpg)
Cloning of hybrid proteins
In order to discover unknown proteins (Y) that interact with a known protein (X), the X gene is cloned so it is produced integrated with the DB domain and the unknown gene or genes are separately cloned so they are produced integrated with AD.
Both recombinant plasmids are transferred into yeast cells so all of them express the known gene-BD hybrid, but each one expresses a different unknown gene-AD hybrid.
![Page 15: Recombinant DNA-based molecular techniques · 2020. 6. 30. · Recombinant DNA-based molecular techniques Prof. Mamoun Ahram Summer 2020. Analysis of transcriptional regulatory sequences:](https://reader033.vdocument.in/reader033/viewer/2022051900/5fef727545fc8969c7553a32/html5/thumbnails/15.jpg)
The possibilities and outcome
Blue yeast colonies are picked and plasmids are isolated to identify the unknown genes/proteins that interact with the known gene/protein.
![Page 16: Recombinant DNA-based molecular techniques · 2020. 6. 30. · Recombinant DNA-based molecular techniques Prof. Mamoun Ahram Summer 2020. Analysis of transcriptional regulatory sequences:](https://reader033.vdocument.in/reader033/viewer/2022051900/5fef727545fc8969c7553a32/html5/thumbnails/16.jpg)