Chapter 20: Recombinant DNA Technology, Lecture Notes Flashcards Preview

Molecular Genetics 4500 > Chapter 20: Recombinant DNA Technology, Lecture Notes > Flashcards

Flashcards in Chapter 20: Recombinant DNA Technology, Lecture Notes Deck (129):
1

Modern techniques

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

Clone definition

Identical organisms
Identical cells
Identical molecules

Descended from a single ancestor

3

Cloning

Produces many identical copies
Use for research
Use commercially

4

Recombinant DNA....
What is it?

Combination of DNA molecules not naturally found together

Methods derived from nucleic acid biochemistry

5

recombinant DNA technology

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

Recombinant DNA method

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

restriction endonuclease

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

palindrome

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

Recombination:
First Steps

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

DNA extracted with DNeasy tissue extraction kit (Qiagen Inc.)

Lanes 2, 4, 6, 8, 10, 12, 14: Undigested genomic DNA, green arrows
Lanes 1 & 15: DNA ladder, red arrows

11

Specific DNA Digestion

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

Restriction Endonucleases (RE)

Each RE recognizes a specific sequence of DNA (recognition site), which is a palindrome (same sequence 5’->3’ on opposite strands)

13

reannealing

Formation of double-stranded DNA molecules from denatured single strands.

14

RE specificity, source, and naming

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

RE recognizing 4 base sites:
“4-cutters”

Some, eg. TaqI, create “sticky” ends.

Some, eg. AluI & HaeIII, create “blunt” ends.

16

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.

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

Fragment Ligation

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

Vectors

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

Vector requirements

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

The plasmid pUC18 offers several advantages as a vector for cloning.

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

First generation vectors

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

b-galactosidase

A bacterial enzyme, encoded by the lacZ gene, that converts lactose into galactose and glucose.

23

LacZ insert identification

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

LacZ insert identification method

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.

26

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.

29

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.

31

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

32

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.

37

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).

53

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

67

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!!

86

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.

116

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.

117

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.

118

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

119

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

120

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!

121

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

122

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

123

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

124

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.

125

Geneticists Advance Our Understanding of Molecular Processes Using a Model Organism: A Case Study in Mice

.....

126

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

127

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.

128

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.

129

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