Chapter 20: Recombinant DNA Technology, Lecture Notes Flashcards
1
Q
Modern techniques
A
Developed in 70’s
Virtual revolution in biotechnology
First commercial approval was 1982 for bacterial produced human insulin
Has led to an explosion of this industry
2
Q
Clone definition
A
Identical organisms
Identical cells
Identical molecules
Descended from a single ancestor
3
Q
Cloning
A
Produces many identical copies
Use for research
Use commercially
4
Q
Recombinant DNA….
What is it?
A
Combination of DNA molecules not naturally found together
Methods derived from nucleic acid biochemistry
5
Q
recombinant DNA technology
A
A collection of methods used to create DNA molecules by in vitro ligation of DNA from two different organisms, and the replication and recovery of such recombinant DNA molecules.
6
Q
Recombinant DNA method
A
Purify target DNA
Digest target DNA with enzymes (restriction endonucleases) that cut DNA at specific sequences
Join these fragments with other carrier DNA molecules (vectors)
Results in RECOMBINANT DNA molecule.
Recombinant DNA molecule is transferred into a host cell for replication.
Recombinant DNA passed on within population of host cells, making more copies.
Cloned DNA recovered, analyzed etc.
Cloned DNA within host cell can be transcribed, mRNA translated, gene product isolated (eg. insulin).
It’s hard to overestimate how this methodology has changed biological research
7
Q
restriction endonuclease
A
A bacterial nuclease that recognizes specific nucleotide sequences in a DNA molecule, often a palindrome, and cleaves or nicks the DNA at those sites.
Provides bacteria with a defense against invading viral DNA.
Restriction endonuclease are widely used in the construction of recombinant DNA molecules.
8
Q
palindrome
A
In genetics, a sequence of DNA base pairs that reads the same backward or forward. Because strands run antiparallel to one another in DNA, the base sequences on the two strands read the same backward and forward when read from the 5’ end. For example: 5’-GAATC-3’ 3’-CTTAAG-5’ .
Palindromic sequences are noteworthy as recognition and cleavage sites for restriction endonucleases.
9
Q
Recombination:
First Steps
A
DNA isolated from cells
Many methods are used to isolate nucleic acids
Fractionation methods separate nucleic acids from proteins and other cellular debris
Many simple commercial kits are (Qiagen) available for this today to extract DNA from countless sources
10
Q
DNA extracted with DNeasy tissue extraction kit (Qiagen Inc.)
A
Lanes 2, 4, 6, 8, 10, 12, 14: Undigested genomic DNA, green arrows
Lanes 1 & 15: DNA ladder, red arrows
11
Q
Specific DNA Digestion
A
The restriction enzyme EcoRI recognizes and binds to the palindromic nucleotide sequence GAATTC.
Cleavage of the DNA at this site produces complementary single-stranded tails.
These single-stranded tails anneal with single-stranded tails from other DNA fragments to form recombinant DNA molecules.
12
Q
Restriction Endonucleases (RE)
A
Each RE recognizes a specific sequence of DNA (recognition site), which is a palindrome (same sequence 5’->3’ on opposite strands)
13
Q
reannealing
A
Formation of double-stranded DNA molecules from denatured single strands.
14
Q
RE specificity, source, and naming
A
Some common restriction enzymes, with their recognition sequences, cutting sites, cleavage patterns, and sources.
Popular REs include EcoRi, Hindlll, and BamHI.
All of these RE create “sticky” ends.
All of these recognize 6 base sites: “6-cutters”
15
Q
RE recognizing 4 base sites:
“4-cutters”
A
Some, eg. TaqI, create “sticky” ends.
Some, eg. AluI & HaeIII, create “blunt” ends.
16
Q
Annealing allows recombinant DNA molecules to form by complementary base pairing.
The 2 strands are not covalently bonded until DNA ligase comes along to fill the gaps.
A
DNA from different sources is cleaved with EcoRI and mixed to allow annealing to form recombinant molecules. The enzyme DNA ligase then chemically bonds these annealed fragments into an intact recombinant DNA molecule.
17
Q
Fragment Ligation
A
Join the ends of DNA together
Complementary (“sticky”) ends will anneal with one another
2 different molecules cut with the same RE can anneal to one another
DNA ligase forms covalent links to form recombinant molecule
18
Q
Vectors
A
Used to carry DNA fragments
Used as a way to get genetic material in (a vector) to a host cell where the vector & fragment can be replicated
19
Q
Vector requirements
A
Ability to replicate itself and the DNA fragment it carries
RE sites in vector
Selectable marker (eg. antibiotic resistance) to distinguish hosts with and without vector
Easy to recover vector from host cells
20
Q
The plasmid pUC18 offers several advantages as a vector for cloning.
A
Because of its small size, it accepts relatively large DNA fragments for cloning.
It replicates to a high copy number, and has a large number of restriction sites in the polylinker, located within a lacZ gene.
Bacteria carrying pUC18 produce blue colonies when grown on media containing Xgal.
DNA inserted into the polylinker site disrupts the lacZ gene; this results in white colonies and allows direct identification of colonies carrying cloned DNA inserts.
21
Q
First generation vectors
A
pUC vectors (5-10kb)
Derived from bacterial plasmids
Replicates 500X in host
Large number of RE sites (polylinker)
Selection system (ampicillin gene)
Insert discrimination system (lacZ gene)
22
Q
b-galactosidase
A
A bacterial enzyme, encoded by the lacZ gene, that converts lactose into galactose and glucose.
23
Q
LacZ insert identification
A
LacZ gene is part of bacterial Lac operon
Produces Beta-galatosidase that metabolizes lactose to produce glucose and galactose
Beta-galactosidase action results in a blue by-product when X-gal is present in the growth medium (color indicator)
24
Q
LacZ insert identification method
A
Grow bacteria with X-gal (5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside)
When intact, LacZ gene product results in blue colonies
However, if a DNA fragment is inserted in polylinker region, LacZ gene is inactivated
Result: no X-gal metabolism ==>WHITE COLONIES
25
Plasmid Cloining Overview
Plasmid vectors are isolated and cut with a restriction enzyme.
The DNA to be cloned is cut with the same restriction enzyme, producing a collection of fragments.
These fragments are spliced into the vector and transferred to a bacterial host for replication.
Bacterial cells carrying plasmids with DNA inserts are identified by growth on selective medium and isolated.
The cloned DNA is then recovered from the bacterial host for further analysis.
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Next vector system
Need to handle larger insert (10-15kb)
Bacterial virus (phage) vectors
Eg., M13, Lambda phage vectors
27
Lambda phage vector system
Central third of phage DNA expendable
Cut out central third of lambda
Leaves left arm, right arm, central region
Isolate arms, recombine with DNA fragment of interest, and ligate together
Introduce packaged ligated phage to host cell
Phage replicates in host, makes phage particles that contain inserts
Form plaques on bacterial lawns
Can isolate & use for further analysis
28
Phage lambda as a vector.
DNA is extracted from a preparation of lambda phage, and the central gene cluster is removed by treatment with a restriction enzyme.
The DNA to be cloned is cut with the same enzyme and ligated into the arms of the lambda chromosome.
The recombinant chromosome is then packaged into phage proteins to form a recombinant virus.
This virus infects bacterial cells and replicates its chromosome, including the DNA insert.
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Cosmids
Can carry up to 50kb inserts
Cosmid: part phage, part plasmid
Engineered vector
Ligate fragments into cosmid, package into protein heads, infect hosts
30
The cosmid pJB8 contains a:
bacterial origin of replication (ori),
a single cos site (cos),
an ampicillin resistance gene (amp, for selection of colonies that have taken up the cosmid),
and a region containing 4 restriction sites for cloning (BamHI, EcoRI, ClaI, and HindIII).
Because the vector is small (5.4 kb long), it can accept foreign DNA segments between 33 and 46 kb in length.
The cos site allows cosmids carrying large inserts to be packaged into lambda viral coat proteins as though they were viral chromosomes.
The viral coats carrying the cosmid can be used to infect a suitable bacterial host, and the vector, carrying a DNA insert, will be transferred into the host cell. Once inside, the ori sequence allows the cosmid to replicate as a bacterial plasmid.
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Bacterial artificial chromosomes (BACs)
Good for large fragments
Based on F(ertility) factor of bacteria
Clone up to 300kb
Multiple cloning sites, selective markers, promoters to express inserted genes
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A bacterial artificial chromosome (BAC) caption
The polylinker carries a number of unique restriction sites for the insertion of foreign DNA.
The arrows labeled T7 and Sp6 are promoter regions that allow expression of genes cloned between these regions.
33
Yeast artificial chromosomes (YACs)
Have telomeres, ori, and centromere
Joined to selectable markers, cluster of RE sites
Clone inserts 100kb to 1Mb
Important vector for genomic sequencing, eg. Human Genome Project
34
YAC
A cloning vector in the form of a yeast artificial chromosome, constructed using chromosomal components including telomeres (from a ciliate), and centromeres, origin of replication,and marker genes from yeast. YACs are used to clone long stretches of eukaryotic DNA.
35
Cloning into a yeast artificial chromosome
The synthetic chromosome contains telomere sequences (TEL), a centromere (CEN4) derived from yeast chromosome 4, and an origin of replication (ori).
These elements give the cloning vector the properties of a chromosome. TRP1, SUP4, and URA3 are yeast genes that are selectable markers for the left and right arms of the chromosome, respectively.
Within the SUP4 gene is a restriction site for the enzyme SnaB1.
Two BamH1 sites flank a spacer segment.
Cleavage with SnaB1 and BamH1 breaks the artificial chromosome into two arms.
The DNA to be cloned is treated with the same enzyme, producing a collection of fragments.
The arms and fragments are ligated together, and the artificial chromosome inserted into yeast host cells.
Because yeast chromosomes are large, the artificial chromosome accepts inserts in the million base pair range (Mb 5 megabase).
36
A Ti plasmid designed for cloning in plants
Segments of Ti DNA, including those necessary for opine synthesis and integration, are combined with bacterial segments that incorporate cloning sites and antibiotic resistance genes (kanR and tetR).
The vector also contains an origin of replication (ori) and a lambda cos site that permits recovery of cloned inserts from the host plant cell.
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Ti plasmid
A bacterial plasmid used as a vector to transfer foreign DNA to plant cells.
38
Gene Transfer in Eukaryotic Cells:
| Plant cell hosts
Done using bacterial plasmids as vectors
39
Gene Transfer in Eukaryotic Cells:
| Mammalian cell hosts
Encapsulation or endocytosis to transfer DNA to mammalian cell hosts
Micromanipulation
40
Cloning in Yeast cells
Eukaryotic, acts like bacteria for growth and manipulation
Extensively studied, entire genome seq.
To study function of eukaryotic proteins, need a eukaryotic system
Yeast considered safe
41
Transgenic definition
Plants or animals carrying a foreign gene are transgenic
42
transgenic organism
An organism whose genome has been modified by the introduction of external DNA sequences into the germ line.
43
Cell Free Cloning:
| PCR
Polymerase Chain Reaction
Cloning without need for host cell
Making many copies (millions)
44
polymerase chain reaction (PCR)
A method for amplifying DNA segments that depends on repeated cycles of denaturation, primer annealing, and DNA polymerase–directed DNA synthesis.
45
Kary Mullis
Developed PCR method
Parts known previously
Hard to overestimate the effect of PCR on how molecular genetics is done today
1993 Nobel Prize
46
Basic PCR Procedure
Denature target DNA
Anneal specific primers to flank DNA target of interest
Extend primers using Taq DNA polymerase
Repeat 25-40X
25 cycles results in 10^6 increase in target
47
Basic PCR Procedure:
| Taq DNA polymerase
Taq polymerase is key to PCR, derived from thermophilic bacteria Thermus aquaticus
A hot spring bacteria, the polymerase works at elevated temps: 68-72C
Taq polymerase survives high temperatures (eg. 95C)
48
PCR amplification
In the polymerase chain reaction, the target DNA is denatured into single strands; each strand is then annealed to a short, complementary primer.
The primers are synthetic oligonucleotides that are complementary to sequences flanking the region to be amplified.
DNA polymerase and nucleotides extend the primers in the 3' direction, using the single-stranded DNA as a template.
The result is a double-stranded DNA molecule with the primers incorporated into the newly synthesized strand.
In a second PCR cycle, the products of the first cycle are denatured into single strands, primers are annealed, and DNA polymerase then synthesizes new strands.
Repeated cycles can amplify the original DNA sequence by more than a millionfold.
49
PCR Advantages
FAST, no cell based cloning required
Done by programmable machine, essentially a glorified heating/cooling block
PCR is VERY sensitive, can work with only a few targets
Great for forensics, degraded DNA, etc
50
PCR Limitations
PCR is VERY sensitive, can lead to false positives due to contaminating DNA
Standard PCR requires knowledge of DNA sequences surrounding region of interest
51
Other uses of PCR
Identification of RE variants
Microsatellite analysis
Screening for genetic disorders
Diagnostic screening for infectious organisms
Forensics
Paleobiology
52
How do we monitor/test for H5N1? PCR of course!!
Atypical Avian Influenza (H5N1)
Reverse transcription–polymerase chain reaction (RT-PCR) specific for H5 gene band (358 bp) of avian influenza H5N1 that was recovered from our patient from nasopharyngeal aspirates by using H5-1/H5-2 primer. Lane A, molecular standard; lane B, H5 band isolated from our patient (358 bp); lane C, negative control; lane D, positive control.
RT-PCR specific for H5 gene band (229 bp) of avian influenza (H5N1) that was recovered from our patient from nasopharyngeal aspiration by using H5-1456/H5-1685 primer. Lane A, molecular standard; lane B, positive control; lane C, H5 band isolated from our patient (229 bp).
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Take home point:
____ is one of the most versatile techniques in the modern molecular genetics laboratory
PCR is one of the most versatile techniques in the modern molecular genetics laboratory
54
Derivations of Basic PCR
Reverse Transcriptase PCR (RT-PCR):
Amplify from RNA
qPCR or “real time” PCR:
Quantitatively determine the amount of nucleic acid (eg., to determine number of organisms)
Nested or semi-nested PCR:
Second PCR amplification using one internal primer (semi-nested) or two internal primers (nested)
55
Gene Libraries
Clone a large region or the whole genome
At least 1 copy of all sequences in host genome
Performed using “host cloning” techniques
Choose vector so that fewest # of clones required to get the whole genome
56
Number of clones can be calculated
N=ln(1-P)/ln(1-f)
N= number of clones required
P=probability of recovering a particular sequence
f=fraction of the genome in each clone
57
Human genome example
Human genome: 3bil bases of DNA
Assume P=.99 (probability of getting a particular sequence = 99%)
Use Lambda vector (17kb/vector), therefore f=17000/3 x 10^9= 5.7 x 10^-6
N= ln(1-.99)/ln(1-5.7 x 10-6)
N=ln(0.01)/ln(0.9999943)
N= -4.605/-0.0000057
N=807,894
N= ~8.1 x 105 lambda clones to cover the human genome at 1 fold (1X) coverage
58
Two Alternative libraries
Chromosome Specific Library
cDNA Library
59
Each human chromosome has a unique profile, resulting from the absorption of two fluorescent dyes.
Based on differential fluorescence, chromosomes can be separated from each other by flow sorting.
60
Chromosome Specific Library
Useful to study single chromosomes
Can separate single human chromosomes
Has been done for each human chromosome
Some chromosomes (eg. yeast) can be separate by pulsed field gel electrophoresis and isolated, then cloned
61
Yeast chromosome III results
Found half the genes on yeast chromosome III were unique
Big surprise given how genetically well characterized yeast is
Also shows that sequencing of entire chromosomes or genomes can reveal more info than observed by classical mutational analysis
62
cDNA Library Methods
Almost all mRNAs have polyA tail
cDNA Method takes advantage of that
Poly dT primer added to isolate mRNAs
A complement synthesized by reverse transcriptase
Creates RNA-DNA helix
RNA digested with RNase H
DNA polI synthesizes complement DNA because of hairpin loop
S1 nuclease cleaves hairpin, get ds cDNA
Can then clone ds cDNA
Then have library of EXPRESSED genes
63
Producing cDNA from mRNA.
Because many eukaryotic mRNAs have a polyadenylated tail (A) of variable length at their 39-end, a short poly-dT oligonucleotide annealed to this tail serves as a primer for the enzyme reverse transcriptase.
Reverse transcriptase uses the mRNA as a template to synthesize a complementary DNA strand (cDNA) and forms an mRNA/cDNA double-stranded duplex.
The mRNA is digested with alkali treatment, or by the enzyme RNAse H.
The 39-end of the cDNA often folds back to form a hairpin loop.
The loop serves as a primer for DNA polymerase, which is used to synthesize the second DNA strand.
The S1 nuclease opens the hairpin loop; the result is a double-stranded cDNA molecule that can be cloned into a suitable vector, or used as a probe for library screening.
64
cDNA Library Methods
Creates RNA-DNA helix
RNA digested with RNase H
DNA polI synthesizes complement DNA because of hairpin loop
S1 nuclease cleaves hairpin, get ds cDNA
Can then clone ds cDNA
Then have library of EXPRESSED genes
65
Library Screening
Clones from library grown on nutrient plate
Replica plate made by transfer to filter
Transferred bacteria lysed, immobilized on filter, and DNA made single stranded
Colony DNA then screened with DNA probe (labeled in some way: radioactive, fluorescent)
Excess probe washed off filter
If radioactive probe used, place film on filter
Compare position of positive colony to original plate to determine colony of interest
Pick original colony, transfer to growth medium, analyze further
66
Screening a plasmid library to recover a cloned gene
The library, present in bacteria on Petri plates, is overlaid with a DNA binding filter, and colonies are transferred to the filter.
Colonies on the filter are lysed, and the DNA denatured to single strands.
The filter is placed in a hybridization bag along with buffer and a labeled single-stranded DNA probe.
During incubation, the probe forms a double-stranded hybrid with complementary sequences on the filter.
The filter is removed from the bag and washed to remove excess probe.
Hybrids are detected by placing a piece of X-ray film over the filter and exposing it for a short time.
The film is developed, and hybridization events are visualized as spots on the film.
Colonies containing the insert that hybridized to the probe are identified from the orientation of the spots.
Cells are picked from this colony for growth and further analysis.
Grow bacteria culture from single positive colony
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Chromosome walking
Use series of probing experiments to move along region of chromosome
Result of experiment 1 becomes probe for experiment 2
Result of experiment 2 becomes probe for experiment 3, etc
68
In chromosome walking, the approximate location of the gene to be cloned is known.
A subcloned fragment of a linked adjacent sequence recovers overlapping clones from a genomic library.
This process of subcloning and probing the genomic library is repeated to recover overlapping clones until the gene in question has been reached.
69
Cloned sequence characterization
Restriction mapping
Use RE to determine location, distance, and order of RE sites in a clone
Use single and double digests
Construct map of RE locations based on results
70
Constructing a restriction map.
Samples of the 7.0-kb DNA fragments are digested with restriction enzymes: One sample is digested with HindIII, one with SalI, and one with both HindIII and SalI.
The resulting fragments are separated by gel electrophoresis.
The separated fragments are measured by comparing them with molecular-weight standards in an adjacent lane.
Cutting the DNA with HindIII generates two fragments: 0.8 kb and 6.2 kb.
Cutting with SalI produces two fragments: 1.2 kb and 5.8 kb.
Models are constructed to predict the fragment sizes generated by cutting with HindIII and with SalI.
Model 1 predicts that 0.4-, 0.8, and 5.8-kb fragments will result from cutting with both enzymes.
Model 2 predicts that 0.8-, 1.2-, and 5.0-kb fragments will result.
Comparing the predicted fragments with those observed on the gel indicates that model 1 is the correct restriction map.
71
Double digest mapping problem
Enzyme I: 350, 950
Enzyme II: 200, 1100
Enzyme I & II: 150, 200, 950
72
Double digest results
150bp, 200bp, & 950bp
73
RE site models based on ___ digests
RE site models based on single digests
74
Cloned sequence characterization
Restriction mapping
Nucleic acid blotting (Southern blotting):
DNA sequencing
75
The Southern blotting technique
Samples of the DNA to be probed are cut with restriction enzymes and the fragments separated by gel electrophoresis.
The pattern of fragments is visualized and photographed under ultraviolet illumination by staining the gel with ethidium bromide.
The gel is then placed on a sponge wick in contact with a buffer solution and covered with a DNA-binding filter.
Layers of paper towels or blotting paper are placed on top of the filter and held in place with a weight.
Capillary action draws the buffer through the gel, transferring the pattern of DNA fragments from the gel to the filter.
The DNA fragments on the filter are then denatured into single strands and hybridized with a labeled DNA probe.
The filter is washed to remove excess probe and overlaid with a piece of X-ray film for autoradiography.
The hybridized fragments show up as bands on the X-ray film.
76
Northern blotting
Screen mRNA to determine expression characteristics of a gene
77
Western blotting
Screen proteins bound to a filter
78
Nucleic acid blotting (Southern blotting):
Use the cloned fragment to further characterize gene of interest (narrow down location by screening RE digests with clone).
Screen related species.
79
DNA sequencing
Ultimate characterization of a clone
Determine complete primary sequence of the clone
80
DNA sequencing using the chain termination method.
1) A primer is annealed to a sequence adjacent to the DNA being sequenced (usually at the insertion site of a cloning vector).
(2) A reaction mixture is added to the primer–template combination. This includes DNA polymerase, the 4 dNTPs (one of which is radioactively labeled) and a small amount of one dideoxynucleotide. 4 tubes are used, each containing a different dideoxynucleotide (ddATP, ddCTP, etc.).
(3) During primer extension, the polymerase occasionally inserts a ddNTP instead of a dNTP, terminating the synthesis of the chain, because the ddNTP does not have the 39-OH group needed to attach the next nucleotide. In the figure, ddATP and the A inserted from this didexoynucleotide are indicated with an asterisk. Over the course of the reaction, all possible termination sites will have a ddNTP inserted.
(4) The newly synthesized chains are removed from the template, and the mixture is placed on a gel. Chains terminating in A are loaded in the A lane, those ending in C are loaded in the C lane, and so forth.
81
Deoxynucleotide vs. Dideoxynucleotide
Difference: In the dideoxynucleotide, there's NO 3’ OH group ---->
No extension of polymerization
82
dideoxynucleotide
A nucleotide containing a deoxyribose sugar lacking a 3’ hydroxyl group.
It stops further chain elongation when incorporated into a growing polynucleotide and is used in the Sanger method of DNA sequencing.
83
In DNA sequencing using dideoxynucleotides labeled with fluorescent dyes, all 4 ddNTPs are added to the same tube, and during primer extension, all combinations of chains are produced.
The products of the reaction are added to a single lane on a gel, and the bands are read by a detector and imaging system.
This process is now automated, and robotic machines, such as those used in the Human Genome Project, sequence several hundred thousand nucleotides in a 24-hour period and then store and analyze the data automatically.
Each fragment differs by one base in size, and a different color associated with each ddNTP.
84
Automated DNA sequencing using fluorescent dyes, one for each base.
The separated bases are read in order along the axis from left to right.
85
Sequencing Advances!!
25,000,000 bases sequenced in 4 hours!!!
By 2009: 5,000,000,000 base per day!!
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Dissection of Gene Function:
| Analysis in Model Organisms
Geneticists Use Model Organisms That Are Genetically Tractable
87
Good model organism traits
Easy to grow
Short generation time
Produce abundant progeny
Readily mutagenized and crossed
88
Conservation of Function
Many gene sequences are conserved from yeast to higher vertebrates
Carry out similar functions in a wide range of organisms
Allows fundamentals of metabolism, development, and disease to be studied in simple lab organisms
Knowledge obtained applied to more complex eukaryotes.
89
Yeast as a model organism
Yeast (Saccharomyces cerevisiae) is a favored model organism
Alternating haploid and diploid phases in the yeast life cycle are particularly useful for genetic analysis
Yeast genome has been completely sequenced
Wide variety of mutants and deletion strains are available.
90
Drosophila as a Model Organism
- ease of growth
- size of its genome
- short generation time (~10 days)
- large fecundity (~3000 offspring in a lifetime)
- Moderate crossing over in females and No crossing over in males simplifies genetic analysis.
91
Drosophila genetics methods
Use balancer chromosomes with a dominant marker gene in Drosophila
Simplifies the genetics of this diploid organism.
Also exploit P element transposons as a genetic tool to introduce cloned genes and to generate mutants
92
P element
In Drosophila, a transposable DNA element responsible for hybrid dysgenesis.
93
Mice as model systems
Mice have similar body plans and stages of development as humans
Similar genome size and number of chromosomes to humans
Many human genes have homologs in mice.
Transgenic mice available:
-are created by microinjection of fertilized eggs with transgenic DNA.
Transgenic mice are used to determine the function of cloned human genes
Gene knockout and targeted gene replacement are important genetic dissection techniques in mice
94
Mice have drawbacks in comparison to yeast and Drosophila:
- more difficult to grow, cross, and mutagenize
| - large-scale genetic screens cannot be readily performed in mice.
95
Geneticists Dissect Gene Function Using _____
Geneticists Dissect Gene Function Using Mutations and Forward Genetics
96
Forward Genetics
Radiation tends to trigger null mutations due to chromosome breaks, deletions, translocations, and other major rearrangements.
UV light, ethyl methane sulfonate (EMS), and nitrosoguanidine cause single base-pair changes or small deletions and insertions, usually resulting in conditional mutations.
Transposons are also used for mutagenesis.
Mutants detected using a genetic screen
Visual examination of large numbers of mutagenized organisms looking for a mutant phenotype
Dominant lethal mutations are usually not detectable in genetic screens
Recessive mutations can be readily detected in yeast during the haploid phase
Recessive lethal mutations isolated in haploid yeast as conditional mutations through replica plating
Requires mating and crossing of the F1 progeny to be observed in obligately diploid organisms.
97
Forward Genetics:
| Random mutagenesis
Random mutagenesis: generate one mutation per genome so that only one gene product is disrupted in each organism
98
Replica plating technique
After mutagenesis, yeast cells are spread onto a plate containing growth medium.
Some cells from the colonies that grow from each single cell are transferred to sterile velvet.
Cells are transferred from velvet onto two new plates.
The replica plates are incubated at either the permissive (23° C) temperature or the restrictive (36° C) temperature.
Colonies that do not grow at the restrictive temperature are selected.
These colonies have temperature-sensitive mutations.
99
complementation test
A genetic test to determine whether two mutations occur within the same gene (or cistron).
If two mutations are introduced into a cell simultaneously and produce a wild-type phenotype (i.e.,they complement each other), they are often nonallelic.
If a mutant phenotype is produced, the mutations are noncomplementing and are often allelic.
100
Complementation analysis to determine whether two wingless mutations in Drosophila are in separate genes or are within the same gene.
......
101
Forward Genetics:
Epistasis analysis
each mutant must have a slightly different phenotype
if the phenotype of a double mutant organism homozygous for both mutations is the same as one of the single mutants, that single mutation is likely to represent an earlier step in a pathway
102
Example of epistasis analysis for two genes that control steps in a biochemical pathway that converts a colorless coat pigment to yellow, and then to brown.
Wild-type coat color is brown. The double mutant (aabb) displays the same phenotype as the single mutant (aa). This shows that Gene A controls a step prior to the step controlled by Gene B.
103
Forward Genetics:
| Suppressor mutations
Suppressor mutations rescue the original mutant phenotype
104
Forward Genetics:
| Enhancer mutations
Enhancer mutations increase the intensity of the mutant phenotype
They can pinpoint additional genes in a pathway that may not have been identified in original genetic screens.
105
Forward Genetics:
| Complementation analysis
Complementation analysis is performed by mating two homozygous mutant strains
Examine the F1 progeny for the wild-type and mutant phenotypes
Technique works only for recessive mutations.
106
Forward Genetics:
| Selection
Selection to select rather than screen for the desired mutant
Goal of selection is to remove (by inhibition or killing) those organisms that do not display the relevant phenotype
Leaves only the desired mutants in the population
107
Forward Genetics:
| Recessive lethal mutations
Recessive lethal mutations isolated in haploid yeast as conditional mutations through replica plating
Observation of recessive lethal alleles in diploid organisms requires more intricate strategies
Heterozygotes will not display the recessive phenotype and homozygotes will die
108
Forward Genetics:
Genes identified by mutational analysis in yeast cloned through functional complementation
This approach cannot be used in Drosophila or mice.
Chromosome walking is used to clone a gene identified by a mutant phenotype.
109
Geneticists Dissect Gene Function Using _____
Geneticists Dissect Gene Function Using Genomics and Reverse Genetics
110
Reverse Genetics
Begins with a cloned wild-type gene or protein
Progresses to site-directed mutagenesis and phenotype analysis to understand gene function.
A protein suspected of involvement in a particular phenotype can be purified and sequenced
An oligonucleotide probe designed from the amino acid sequence is used to probe a genomic or cDNA library to obtain the gene that encodes the protein
111
Strategy used to clone the Factor VIII gene from a porcine cDNA library.
The purified Factor VIII protein was cleaved into peptides and the amino acid sequence of each peptide was determined. All possible codon combinations that could code for a five amino acid sequence were calculated, as well as the corresponding DNA sequences. A mixture of antisense oligonucleotides was synthesized for each possible DNA sequence that could encode the five amino acid peptide. This mixture was labeled (***) and used to probe a porcine cDNA library in order to select the porcine Factor VIII gene.
112
Reverse Genetics:
| An alternative approach
An alternative approach to cloning a gene beginning with a purified protein
use an antibody to that protein to screen a cDNA expression library
113
Reverse Genetics:
Candidate gene may be subjected to further genetic and biochemical analysis, such as.....
DNA database searching and sequence alignment with homologous DNA or protein sequences
analysis of the pattern of gene expression by in situ hybridization or northern blot analysis
analysis of protein expression by immunofluorescence staining
114
in situ hybridization
A cytological technique for pinpointing the chromosomal location of DNA sequences complementary to a given nucleic acid or polynucleotide.
115
northern blot
An analytic technique in which RNA molecules are separated by electrophoresis and transferred by capillary action to a nylon or nitrocellulose membrane.
Specific RNA molecules can then be identified by hybridization to a labeled nucleic acid probe.
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Southern blotting
Developed by Edwin Southern, a technique in which DNA fragments produced by restriction enzyme digestion are separated by electrophoresis and transferred by capillary action to a nylon or nitrocellulose membrane.
Specific DNA fragments can be identified by hybridization to a complementary radioactively labeled nucleic acid probe using the technique of autoradiography.
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In-situ hybridization of mRNA
In situ hybridization of whole mouse embryos, showing distribution of the Hoxc11 mRNA during development.
RNA is detectable as dark blue staining, near the posterior end of the embryo.
Head of the embryo is at left; tail at right.
Mouse embryos at 10.5 (A), 11.5 (B) and 12.5 (C) days of gestation, showing Hoxc11 mRNA concentrated in hindlimbs, vertebrae, and cells that will later form kidney and reproductive organs.
These data suggest that the Hoxc11 gene product is involved in the early development of these structures.
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Immunofluorescence staining of the developing mouse eye, showing the location of the Pitx2 protein.
The two right panels show mouse embryonic eyes, stained with a DNA-specific stain that denotes the nucleus of each cell.
The two left panels are the corresponding immunofluorescence stained samples, viewed with a fluorescence microscope.
Embryonic stages are 11.5 days (a) and 15.5 days (b).
The pattern of Pitx2 protein expression is consistent with the Pitx2 gene’s proposed role in early eye development. C: cornea, i: iris, pm: periocular mesenchyme.
Black hair is recessive to agouti
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Reverse Genetics:
| Targeted gene knockouts
Targeted gene knockouts involve deletion or disruption of a specific gene or protein to study the resultant phenotype
Methods have been developed for targeted gene knockout in both yeast and mice
Goal is to replace homozygous wild type locus with homozygous mutant locus
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Creating a knockout mouse.
(a) Inserting the knockout gene into embryonic stem (ES) cells. ES cells are derived from an agouti (brown) mouse.
(b) Creating the knockout mouse strain from knockout ES cells.
ES cells can be reinjected into mouse embryos and will populate all the tissues. An embryo can be created solely from ES cells.
If randomly integrated, the product of the tk gene preferentially phosphorylates ganciclovir (a nucleoside analog), inhibiting replication--->dead!
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Reverse Genetics:
| Targeted gene replacement
involves in vivo substitution of a gene containing a specific site-directed mutation for a wild-type version of the gene
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Oligonucleotide-mediated site-directed mutagenesis.
Oligonucleotide mediated site-directed mutagenesis
Select for each of these in a number of ways and use in further expts
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DNA Microarrays
Used to examine the expression of thousands of genes simultaneously.
Microarrays can be used to compare the pattern of gene expression in two or more tissues
- at different times during development
- in cells from normal versus diseased tissue
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ChIP-chip
(Chromatin-ImmunoPrecipitation-microarray chip)
Genome-wide screen for transcription factor binding sites using microarrays.
Genome-wide screen for transcription factor binding sites. The top line shows various proteins bound to their DNA binding sites. One protein is tagged with a protein fragment (HA) that can be recognized by a specific antibody. Formaldehyde is added to tissues or cultured cells to crosslink these proteins to DNA. Then, the DNA is extracted from the cells and sheared into small fragments. The antibody that recognizes the HA tag is added to the mixture and the antibody, with its protein/DNA fragment, is pulled out of the mixture (immunoprecipitation). The DNA is purified, labeled with a fluorescent dye. Labeled DNA is then hybridized to a DNA microarray. A positive hybridization signal indicates a DNA sequence that is bound by the HA-tagged protein.
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Geneticists Advance Our Understanding of Molecular Processes Using a Model Organism: A Case Study in Mice
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Amyotrophic Lateral Sclerosis (ALS)
Lou Gehrig’s disease
Progressive motor neuron degeneration, presently incurable
1-2 cases/100000/year worldwide
~10% of ALS cases familial, inherited in Dominant manner
Cause of some of these cases determined
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SOD1 gene
SuperOxide Dismutase 1 (SOD1) Dominant mutant found in 20% of familial ALS disease cases.
SOD1: ANTIoxidant enzyme, converts free radicals, which protects cells from these damaging byproducts of respiration.
Single amino acid substitution from glycine to alanine at amino acid position 93: G93A
Cloned human G93A used for transgenic mice studies.
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ALS in mice
The transgenic SOD mice also provide insights into potential therapies for ALS.
In some cases, drug therapies have been tested in transgenic SOD mice first
Drug therapies then have moved to clinical trials in humans.
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ALS in mice
The transgenic SOD mice also provide insights into potential therapies for ALS.
In some cases, drug therapies have been tested in transgenic SOD mice first
Drug therapies then have moved to clinical trials in humans.
Gene therapy in transgenic SOD mice with the IGF-1 gene has shown promise
