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Unformatted text preview: BIO 106 Fall 2009 Professor Owen 11/2/2009 Gene Combinations Combinations of a few transcription regulator proteins can generate many different cell types during development.
ECB3 8-19 Gene Expression ECB3 8-23 Abnormal Gene Expression
see the th eye?! Recombinant DNA Technology & Molecular Biology Techniques Biology Techniques ECB3 8-23 Humans Experiment with DNA Cloning a Gene into a Plasmid
A plasmid is a small circular DNA that can replicate inside li a bacterium. Cooper 4.16 Cells24-RecombDNA & Cell Cycle 1 1 BIO 106 Fall 2009 Professor Owen 11/2/2009 Cloning a Gene into a Plasmid Plasmids
• A plasmid, or “vector”, can be cut open and new doubledouble-stranded DNA added. • Usually the additional DNA includes genes for selection, such as antibiotic resistance.
amp=gene for ampicillin resistance (protein -lactamase) promoter includes upstream regulators & TATA Nucleotide sequences recognized by “Molecular the restriction endonuclease scissors” endonuclease amp
can also include a reporter gene (such as green fluorescent protein) plasmid vector YFG
YFG = “your favorite gene” A plasmid is a small circular DNA that can replicate inside a bacterium. ECB2 10-21 ORI=origin of replication (for bacteria to make copies) ORI Bacteria Restriction Restriction Endonucleases
Restriction endonucleases cleave DNA at specific nucleotide sequences. Some cut straight across, others create staggered “sticky” ends. The target sequences are often palindromic. We can manipulate bacteria to produce many constructed plasmids for experiments.
ECB2 10-22 a man, a plan, a canal, panama
Modified from Cooper 4.17 Palindromes
Go hang a salami, I'm a lasagna hog!
from Melanie Poole ’11 Expression of Cloned Genes Have more? Send them to me by email! Modified from Cooper 4.21 Cells24-RecombDNA & Cell Cycle 1 2 BIO 106 Fall 2009 Professor Owen 11/2/2009 DNA Sequencing (both –OH’s gone) DNA Sequencing Cooper 4.20 Dideoxynucleotides, which lack OH groups at 3’ end, terminate DNA synthesis. These molecules can have a fluorescent label attached to them. ECB3 10-20 DNA Sequencing DNA Sequencing DNA Sequencing Cooper animation 4.8 Cooper 4.20 The fluorescently labeled dideoxynucleotides, each ATGC with a different color, can be read by a laser as the DNA fragments separate on a gel. Automated sequencing uses fluorescent dyes to mark ECB3 10-22 each nucleotide. Southern Blotting
Southern blotting is used to detect specific DNA fragments using a labeled probe (usually DNA). Named after Edward M. Southern. If the technique to probe DNA that has been separated by size is called a “Southern”, what do you think a technique to probe mRNA should be called? Cooper 4.25 A. B. C. D. Northern Southern Eastern Western Cells24-RecombDNA & Cell Cycle 1 3 BIO 106 Fall 2009 Professor Owen 11/2/2009 DNA Microarrays
Review: Cell division is regulated by signals from outside the the cell ECB3 10-33 Able to simultaneously monitor the expression of hundreds or thousands of genes. ECB2 16-6 Protein Phosphorylation Review:
Protein phosphorylation is rapid and reversible
Protein kinases are ways to turn other proteins on/off. proteins on/off. Why a phosphate? Why not just destroy the protein? Phosphorylation is faster and requires less energy!
ECB2 16-15 Cell Cell Division
Four stages with two main events: 1) Duplicate DNA (synthesis) 2) Divide into two cells (cytokinesis)
Animation: Cooper 16.1 Cooper 16.1 ECB3 18-2 Cell Cycle Phases Using this diagram, which part of the cell cycle can be called “mitosis”? C D A B G1 and G2 stand for Gap The time before S phase (G1) and before M phase (G2) Cells24-RecombDNA & Cell Cycle 1 4 ...
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