Remember: Final Draft of Posters Due at 10 am tomorrow! Today: More Gene Regulation Intro to Genetic Technology One last poster team meeting!? Remember: Final Draft of Posters Due at 10 am tomorrow!

Next Up: Regulating at the Level of Transcription!

Regulating Transcription Regulation is almost always based on initiation!

Regulating Transcription: Transcription Factors Transcription factors may be general (required for transcription of all genes) or specific, required for high level expression of particular genes)

Regulating Transcription: Enhancers and Activators

Evidence for Enhancers Michael R. Botchan and his colleagues have produced visual evidence of this model of enhancer action. They created an artificial DNA molecule and observed how it interacted with enhancers using an electron microscope

Regulating Transcription Some specific transcription factors function as repressors! Common Mechanisms: Block binding of activators Bind to their own control elements Recruit histone deacetylases

Combinatorial Control Numbers and diversity of control elements suggest that a particular combination of control elements regulates transcription.

Gene Clusters for Coordinated Control Eukaryotic genes may also be packaged together for regulation. But each has its own promoter and is individually transcribed. More often, genes in a pathway are scatted on multiple chromosomes, but respond to the same combination of control elements.

Post-Transcriptional Regulation

Post-Transcription: RNA Processing (This helps explain our embarrassingly small number of genes!)

Post-Transcription: mRNA Degradation Shortening the poly-A tail can trigger removal of the 5’ cap, followed by breakdown of the mRNA Hypothesis, TTP promotes mRNA instability. Prediction: In TTP knocked out, mRNA will… Figure: Effect of TTP deficiency on the stability of TNF-a mRNA in bone-marrow derived macrophages J. Blackshear, Biochem. Soc. Trans.. (2001) 30, (945–952)

Post-Transcription: RNA Interference (RNAi) Experimental Observation: Injecting dsRNA into a cell can silence the corresponding gene! http://www.nature.com/focus/rnai/animations/animation/animation.htm

Post-Transcription: Initiation of Translation Regulatory proteins can block attachment of ribosomes Lack of poly-A tails blocks translation Block may be global (all translation; i.e. egg cells, dormant plants)

Post-Transcription: Protein Processing and Degradation Many proteins must be cleaved or modified to become active. Proteins may also be labeled for destruction by attaching a small protein, ubiquitin, that attracts proteosomes

Genome Regulation and Cancer Viruses may carry oncogenes (genes causing cancer) Oncogenes are similar to proto-oncogenes (genes involved in normal cell division) in our genome

A Multistep Model for Cancer

Next Up: DNA Technology What’s interesting, exciting, frightening? Capture your thoughts: Question 1

DNA Tools: Restriction Enzymes What do Restriction Enzymes do? Where do they cut (and why)??

DNA Tools: Restriction Enzymes

DNA Tools: Gel Electorphoresis You Try! Question 2

DNA Tools: Southern Blots You Try! Question 3

DNA Tools: Western and Northern Blots What can a Northern or Western Blot tell us that a Southern can’t?? You Try: Question 4

DNA Tools: Fingerprints DNA Fingerprints can be made from highly variable regions (polymorphisms). Two are commonly used: Single Nucleotide Polymorphisms (SNPs) and Short Tandem Repeats (STRs)

DNA Tools: Fingerprints You Try! Question 5

Building Recombinant DNA

DNA Tools: Vectors Good Vectors: Replicate Independently Provide Recognition Sequences for Restriction Enzymes Include a Reporter Gene Are Small

Where do you get DNA to Clone?? DNA (Gene) Libraries? Are all these clones useful to you?

How Could You Get a Library of Expressed, Edited Genes?!?

DNA Tools: Knock-Outs

DNA Tools: Antisense RNA

DNA Tools: Micro-arrays Let’s watch! http://www.bio.davidson.edu/Courses/genomics/chip/chip.html

Applications: Medically Useful Proteins

Applications: Medically Useful Proteins

Applications: Pharming Webster and Pete; Photo BBC

Applications: Agriculture

Genetic Technology: DNA Sequencing Why would we want to sequence DNA?? What enzymes/systems might be helpful?

DNA Sequencing, Step 1

DNA Sequencing, Step 2

DNA Sequencing, Step 2 Normal nucleotide Dideoxy-nucleotide

DNA Sequencing, Step 3

Automated DNA Sequencing

DNA Sequencing: The Results

Applications: Creating Life?! Let’s Watch! http://www.ted.com/index.php/talks/view/id/227 Capture your thoughts! Question 6