restriction endonucleases cut at specific sites & leave sticky ends
DESCRIPTION
RESTRICTION ENDONUCLEASES CUT AT SPECIFIC SITES & LEAVE STICKY ENDS. EcoR1 animation. Leave “sticky ends” that can be used to join DNA from different organisms. PLASMIDS. Small circular self replicating DNA molecule in bacteria separate from bacterial chromosome 2-30 genes - PowerPoint PPT PresentationTRANSCRIPT
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RESTRICTION ENDONUCLEASES CUT AT SPECIFIC SITES & LEAVE STICKY ENDS
EcoR1 animation
Leave “sticky ends” that can be used to join DNA from different organisms
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PLASMIDS
• Small circular self replicating DNA molecule in bacteria separate from bacterial chromosome
• 2-30 genes
• Often carry genes for antibiotic resistance or genetic recombination
• Can be exchanged between bacteria• Bacterial “sex” = conjugation (facilitated by F plasmids)
• Role in rapid evolution
• Method for spreading “antibiotic resistance”•R PLASMIDS
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PLASMIDS
• PLASMID MOVIE Part 2• LAB 6:• Cells can be made “competent” by using
calcium chloride and “heat shock” to change their cell walls - makes them better able to pick up plasmids;
• rapidly growing cells are made competent more easily
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Can be cut with restriction enzymes and used to incorporate foreign DNA into bacteria
Bacteria reproduce, copying the inserted gene along with plasmid
PLASMIDS & RECOMBINANT DNA
• Ti plasmid movie
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Green Fluorescent Protein(GFP)
• Genetic tool
• Originally from jellyfish
• Way to tell if gene has been incorporated
http://www.vet.upenn.edu/schoolresources/communications/publications/bellwether/61/stem_cells.html
http://mabryonline.org/blogs/larkin/GFP%5CGFP_aequorea_victoria-1.jpeg
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REVERSE TRANSCRIPTASE
• Found in RETROVIRUSES (EX: HIV)• Uses RNA message to make a DNA copy• Info flows in reverse RNA → DNA
• Can take eukaryotic RNA message after introns have been removed and change it into a DNA sequence to be read by bacteria (no RNA processing in prokaryotes)
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REVERSE TRANSCRIPTASE
http://biology200.gsu.edu/houghton/4564%20'04/figures/lecture%204/AAAreverse.jpg
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GENE CLONING
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http://biology200.gsu.edu/houghton/4564%20'04/figures/lecture%204/pcranimatie.gif
PCR movie
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Genetics of Viruses & Bacteria Chapter 18
http://www.awesomebackgrounds.com/s-energy-and-power.htm
Control of gene expression in Eukaryotes
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Bacteria• Bacteria review
– one-celled organisms– prokaryotes– reproduce by mitosis
• binary fission
– rapid growth• generation every ~20 minutes• 108 (100 million) colony overnight!
– dominant form of life on Earth– incredibly diverse
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Bacterial genome
• Single circular chromosome– haploid– naked DNA
• no histone proteins
– ~4 million base pairs• ~4300 genes• 1/1000 DNA in eukaryote
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No nucleus!• No nuclear membrane
– chromosome in cytoplasm– transcription & translation are coupled
together• no processing of mRNA
– no introns– but Central Dogma
still applies• use same
genetic code
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Binary fission• Replication of bacterial
chromosome• Asexual reproduction
– offspring genetically identical to parent
– where does variation come from?
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Variation in bacteria
• Sources of variation– spontaneous mutation– transformation
• plasmids• DNA fragments
– transduction– conjugation– transposons
bacteria shedding DNA
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Spontaneous mutation• Spontaneous mutation is a
significant source of variation in rapidly reproducing species
• Example: E. coli– human colon (large intestines)– 2 x 1010 (billion) new E. coli each day!– spontaneous mutations
• for 1 gene, only ~1 mutation in 10 million replications• each day, ~2,000 bacteria develop mutation in that gene• but consider all 4300 genes, then:
4300 x 2000 = 9 million mutations per day per human host!
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Transformation • Bacteria are opportunists
– pick up naked foreign DNA wherever it may be hanging out
• have surface transport proteins that are specialized for the uptake of naked DNA
– import bits of chromosomes from other bacteria
– incorporate the DNA bits into their own chromosome
• express new gene• form of recombination
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Swapping DNA
• Genetic recombination by trading DNA1 3 2
arg+trp-
arg-trp+
minimalmedia
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Plasmids • Plasmids
– small supplemental circles of DNA• 5000 - 20,000 base pairs• self-replicating
– carry extra genes• 2-30 genes
– can be exchanged between bacteria• bacterial sex!!• rapid evolution • antibiotic resistance
– can be imported from environment
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Plasmids This will beimportant!
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Plasmids & antibiotic resistance• Resistance is futile?
– 1st recognized in 1950s in Japan
– bacterial dysentery not responding to antibiotics
– worldwide problem now• resistant genes are
on plasmids that are swapped between bacteria
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Biotechnology
• Used to insert new genes into bacteria– example: pUC18
• engineered plasmid used in biotech
antibiotic resistance gene on plasmid is used as a selective agent
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Transduction
Phage viruses carry bacterial genes from one host to another
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Conjugation• Direct transfer of DNA between 2 bacterial cells
that are temporarily joined– results from presence of F plasmid with F factor
• F for “fertility” DNA
– E. coli “male” extends sex pilli, attaches to female bacterium
– cytoplasmic bridge allows transfer of DNA
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Any Questions??
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Bacterial GeneticsRegulation of Gene Expression
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Bacterial metabolism• Bacteria need to respond quickly to
changes in their environment– if have enough of a product,
need to stop production• why? waste of energy to produce more• how? stop production of synthesis enzymes
– if find new food/energy source, need to utilize it quickly
• why? metabolism, growth, reproduction• how? start production of digestive enzymes
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Reminder: Regulation of metabolism• Feedback inhibition
– product acts as an allosteric inhibitor of 1st enzyme in tryptophan pathway
= inhibition-
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Another way to Regulate metabolism• Gene regulation
– block transcription of genes for all enzymes in tryptophan pathway
• saves energy by not wasting it on unnecessary protein synthesis
= inhibition-
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Gene regulation in bacteria• Control of gene expression enables
individual bacteria to adjust their metabolism to environmental change
• Cells vary amount of specific enzymes by regulating gene transcription– turn genes on or turn genes off
• ex. if you have enough tryptophan in your cell then you don’t need to make enzymes used to build tryptophan
– waste of energy– turn off genes which codes for enzymes
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So how can genes be turned off?
• First step in protein production?– transcription– stop RNA polymerase!
• Repressor protein– binds to DNA near promoter region blocking
RNA polymerase• binds to operator site on DNA• blocks transcription
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Genes grouped together • Operon
– genes grouped together with related functions • ex. enzymes in a synthesis pathway
– promoter = RNA polymerase binding site• single promoter controls transcription of all genes in operon• transcribed as 1 unit & a single mRNA is made
– operator = DNA binding site of regulator protein
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operatorpromoter
Repressor protein model
DNATATA
RNApolymerase
repressor
repressor repressor protein
Operon: operator, promoter & genes they control
serve as a model for gene regulation
gene1 gene2 gene3 gene4RNA
polymerase
Repressor protein turns off gene by blocking RNA polymerase binding site.
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operatorpromoter
Repressible operon: tryptophan
DNATATA
RNApolymerase
repressor
tryptophan
repressor repressor protein
repressortryptophan – repressor proteincomplex
Synthesis pathway modelWhen excess tryptophan is present, binds to tryp repressor protein & triggers repressor to bind to DNA
– blocks (represses) transcriptiongene1 gene2 gene3 gene4
RNApolymerase
conformational change in repressor protein!
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Tryptophan operonWhat happens when tryptophan is present?Don’t need to make tryptophan-building enzymes
Tryptophan binds allosterically to regulatory protein
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operatorpromoter
Inducible operon: lactose
DNATATARNA
polymerase
repressor repressor protein
repressorlactose – repressor proteincomplex
lactose
repressor gene1 gene2 gene3 gene4
Digestive pathway model When lactose is present, binds to lac repressor protein & triggers repressor to release DNA
– induces transcription
RNApolymerase
conformational change in repressor protein!
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Lactose operonWhat happens when lactose is present?Need to make lactose-digesting enzymes
Lactose binds allosterically to regulatory protein
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Jacob & Monod: lac Operon• Francois Jacob & Jacques Monod
– first to describe operon system– coined the phrase “operon”
1961 | 1965
Francois JacobJacques Monod
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Operon summary• Repressible operon
– usually functions in anabolic pathways• synthesizing end products
– when end product is present in excess,cell allocates resources to other uses
• Inducible operon – usually functions in catabolic pathways,
• digesting nutrients to simpler molecules
– produce enzymes only when nutrient is available• cell avoids making proteins that have nothing to do, cell
allocates resources to other uses
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Any Questions??
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Fred Sanger
1958 1980
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• TRANSFORMATION in bacteria
• TRANSDUCTION with viruses
• CONJUGATION - Bacteria “sex”
movie Conjugation
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Only a fraction of genes in a cell areexpressed (made into RNA) at any given time.
How does the cell decide which will be turned on and which will stay “silent”?
You already know about _____________ regions that show RNA polymerase where to start.
There are other ______________________ thatcontrol whether a gene is ON or OFF.
PROMOTER
REGULATORY SITES
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E. Coli lac operon
Group of genes that operate together are called an ________________OPERON
http://www.life.uiuc.edu/bio100/lectures/s97lects/16GeneControl/lac_operon_ind.GIF
Genes code for enzymes neededto digest lactose sugar.
Only needed if glucose is not available
See a MOVIE
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Most of time glucose is available so
lac operon is turned _____ by a____________ molecule that sits on aregulatory site next to the
promotercalled the ___________
REPRESSOR
OPERATOR
OFF
http://www.life.uiuc.edu/bio100/lectures/s97lects/16GeneControl/lac_operon_ind.GIF
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Cells need to get rid of the repressorand turn _____the lac genes to digestlactose instead.
The presence of lactose causes a change in the ____________ molecule so so it can’t bind the operator site.
REPRESSOR
ON
What if there’s NO GLUCOSE?
Image modified from: http://www.life.uiuc.edu/bio100/lectures/s97lects/16GeneControl/lac_operon_ind.GIF
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Cells turn genes ON & OFF as needed
Many genes are regulated by_____________ proteins that keep them turned off until needed.
Others use proteins that speed up_______________ or affect ___________________
REPRESSOR
transcription
protein synthesis
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EUKARYOTES are more COMPLEX Additional regulatory sequences:
1. ___________ regions upstream from promoters bind many different regulatory
proteins
2. __________ (TATATA or TATAAA)helps position RNA POLYMERASE
TATA box
Image by Riedell
ENHANCER
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DEVELOPMENT & DIFFERENTIATION
Gene regulation is also important in shaping way organisms develop
How does a zygote become a multi-cellular organism?
How does it know what kind of cell to be?
http://www.angelbabygifts.com/
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DEVELOPMENT & DIFFERENTIATION
Cells ________________ by turning different genes on and off.
BUT… How does a cell know where it is in the body? and what genes it should turn on? and when?
DIFFERENTIATE
http://www.ncu.edu.tw/~ls/graph/faculty_pictures/whole_time/SLC/SLC_lab-1.jpg