Outline

•  Resources, approaches, technologies, and tools used in functional genomics studies

–  Chemical, physical, and insertional mutagens induced mutations

–  Reverse genetics

–  Site-specific mutation

–  Forward genetics--molecular marker and gene mapping

–  Gene expression profiling Gene expression regulation ü Post gene expression regulation and RNA editing

ü Epigenetic modification

Induced-mutation is valuable resource to study gene function

•  Mutations are changes in the genetic sequence, and they are a main cause of diversity among organisms and individuals.

•  Induced mutations of a gene provide possibility to understand gene’s function, where other genes are same between wild type and mutant. This way, phenotype change can be associated with the gene.

Wild type Gene 1 Gene 2 Gene n

Mutant Gene 1 Gene 2 Gene n ✗  

Insertional mutagenesis

•  Insertional mutagenesis is the creation of mutations of DNA via incorporation of additional bases. Insertional mutations can occur naturally, mediated by bacteria, virus, and transposon.

•  Two inserational mutagens

–  Transfer DNA (T-DNA)

ü  How was T-DNA discovered?

ü  How to use T-DNA to create insertional mutations?

ü  How many mutation lines are needed in order to cover most genes?

–  Transposon

T-DNA inserts into plant genome from agrobacterium

Tzfira and Citovsky, Trends in cell biology 2002

T-DNA from agrobacterium insertion into plant genome

•  Tumor-promoting gene: Auxin and Cytokinin are plant hormones that enable the plant cell grow uncontrollably, thus forming the tumors

•  Biosynthetic genes: opine is amino acid derivatives used by the bacterium as a source of carbon and energy

T-DNA as insertional mutagenesis

•  Insertional mutations can be artificially created in the lab

•  The length of the insert is 17 kb in this case, causing loss of gene function

•  KanR gene confers bacterial kanamycin resistance, allowing selection of the transformed plants or tissues

Azpiroz-Leehan and Feldmann, Trend in genetics 1997

Agrobacterium-mediated transformation --Tissue culture approach

•  Regeneration of whole plants generally requires long term (weeks to months)

•  Time/labor intensive to construct large scale of mutational lines using tissue-culture method

•  https://www.youtube.com/watch?v=L7qnY_GqytM

kanamycin  

How many insertions and mutated lines are needed to saturate the genome?

•  Total number of insertion sites is function of size of gene (x kb) and size of genome

Ø  A 5-kb gene requires 110,000 T-DNA inserts to achieve 99% probability of being mutated

Ø  Median gene length of 2.1 kb in Arabidopsis requires 280,000 inserts

Ø  Average 1.5 inserts per line, ~186,000 lines are needed

p =1− (1− (x /120, 000))n

Krysan et al., Plant Cell 1999

Number of inserts per line inferred from segregation ratio of self progenies of the mutant

•  One insert in M1 plant, then segregation ratio of transformed plants (R) vs non-transformed plants (S) is 3:1

–  RR:RS:SS=1:2:1

–  R:S=3:1

•  Two inserts

–  R:S=??

•  Three inserts

–  R:S=??

Agrobacterium-mediated

transformation --Seed infection method

•  If a cell with inserts in a germinated seed forms reproductive tissues of the T1 plant, then some T2 seeds from the T1 plant have inserts

Forsthoefel et al., 1992

Agrobacterium-mediated transformation --Flora dip method

•  Dipping flowering plants in Agrobacterium that are suspended in a solution

•  Collect T2 seeds from the treated plants

•  Plant T2 seeds on medium containing kanamycin

•  Transformed Kan-resistant plants with green cotyledons can be identified

•  Kanamycin-resistant seedlings are transferred to soil and grow to mature to collect T3 seeds

Clough et al., The Plant Journal 1998

T-DNA insertion collections in Arabidopsis

Ins+tu+on   Popula+on  size  

Genotype   Reference  

Salk  Ins)tute     150,000   Columbia-­‐0   Alonso  et  al.,  2003  

Bielefeld  University,  Germany  

71,000   Columbia-­‐0   Kleinboel)ng  et  al.,  2012  

Syngenta   100,000   Columbia   Sessions  et  al.,  2002  

Characters of T-DNA insertional mutations

•  Large insertion causes loss of gene function.

•  Insertional mutations can not be generated for all genes. For genes required for life, insertion leads to lethal.

•  Low insertion rate, ~1.5 inserts per line

–  Gene with small size has low chance to get insertional mutation

•  T-DNA does not work for all species; for many plants of interest, no transformation methods are available.

Discovery of transposon •  Transposons are genetic

elements that can move within genomes by either “copy-and-paste” or “cut-and-paste” mechanisms mediated by an enzyme called transposase

•  First study of transposable elements was conducted in corn by McClintock in 1940s

•  In 1983, direct evidence was obtained for transposable elements

•  Later, transposon was widely identified and studied in many species

Transposable elements in Bacteria

•  Insertion Sequences (IS) –  Size range from 768 bp to 5,000 bp –  End with terminal inverted repeats

(IRs) of 9 to 41 bp

Russell, Peter J. IGenetics. San Francisco: Benjamin Cummings, 2010

Transposon can increase their copy number

Replication

Transposon can increase their copy number

Transposition to sister chromatid

Transposon can increase their copy number

Transposition from a replicated site to

an unreplicated site

Transposable elements contribute to genome size variation

Species Genome size (Mb)

Gene number

Transposable element content (%)

Rice 450 41,000 25

Sorghum 730 34,000 63

Maize 2,300 33,000 85

Barley 5,100 30,400 84

Example 1. Ac-Ds transposon to induce insertional mutations in Arabidopsis

Sandereson et al., Gene and development 1995; Page and Grossniklaus, 2002

•  Two transformations of parents

•  Many insertions in F2 progenies

•  Unlinked transpositions

•  Stabilize insert

Ac   Ds  

Example 2. A mini-transposon for insertional mutagenesis in microorganisms

•  The mini transposon does not contain the transposase gene. And it won’t keep moving around.

KanR  MmeI   MmeI  IR   IR  

Create a population of insertional mutations using mini-transposon in microorganisms

•  A gene disruption library is constructed by first transposing the mini-transposon into bacterial genomic DNA in vitro and then transforming a bacterial population with the transposed DNA

•  E.g., a transpose requiring only a TA dinucleotide at the insertion site

van Opijnen et al., Nature methods 2009

Physical mutagenesis

•  Insertional mutagenesis does not work for all species; for many plants of interest, no transformation methods are available.

•  Physical or chemical mutagens cause DNA breakage and other damages inducing mutations and work for all species.

•  Physical mutagens

–  Ionizing radiation, such as Fast Neutron

–  Non-ionizing radiation, such as UV

Physical mutagens induce DNA break damage

•  Ionizing radiations

–  Fast moving particles such as fast neutrons have sufficient energy to physically ‘punch holes’ in DNA directly

–  Other ionizing radiations like X-ray and gamma rays could cause DNA breakage

Double strand break repair

Fast neutron may induce deletion and insertion mutations Fast  neutron  

Dele+on  

Inser+on  

1   2   3  

1   3  

1   3  2  

Flow chart for constructing mutagenized population

Takeshi Saito et al. Plant Cell Physiol 2011;52:283-296

Example: fast neutron induced mutation in soybean

•  60,000 soybean seeds of cv M92-220 were irradiated with FNs

•  15,000 seeds were irradiated at each of the doses: 4, 8, 16, and 32 Gray units (Gy)

–  Optimizing mutation frequency is paramount and must be empirically determined: if it is too low, too many plants will be required to discover mutations in a target gene; if it is too high, viability and/or sterility is likely to be a problem.

•  20,000 M1 seeds, 5,000 from each dose, were planted and harvested by single-seed descent

Bolon  et  al.,  2011  Plant  Physiology  

•  Sequenced a fast-neutron-induced mutant population of 1504 lines in the model rice cultivar Kitaake at 45-fold coverage

•  Identified 91,513 mutations an average of 61 mutations per line

–  Including 43,483 single base substitutions (SBSs), 31,909 deletions, 7,929 insertions, 3,691 inversions, 4,436 translocations, and 65 tandem duplications

•  In total, the mutations affect 32,307 genes. Deletions affect the greatest number of genes, 27,614, accounting for 70% of the 32,307 genes

•  The average deletion size is 8.8 kb, deletions smaller than 100 bp account for nearly 90% of all deletions

Li et al., Plant Cell 2017

Chemical mutagens induce mutations

•  Chemical mutagen causes DNA damages (base modification, change, and loss) and primarily point mutations

•  Key questions

–  How do chemical mutagens induce point mutations?

–  How to use chemical mutagen to create mutants?

–  Characters of the induced mutations

Spontaneous point mutation --DNA replication error

Russell, Peter J. IGenetics. San Francisco: Benjamin Cummings, 2010

Generate  SNP  G/A  

•  Spontaneous mutation rate is very low, 10-7-10-11 per gene per generation –  Natural DNA replication error is very low –  Mismatch repair system

Chemical mutagen - Alkylation

•  Add alkyl group (e.g., -CH3) onto a base of guanine, producing O6–alkylguanine

•  The methylated guanine pairs with thymine rather than cytosine, giving GC-to-AT transitions

•  Ethyl-methanesulfonate (EMS) and Methylmethane sulfonate (MMS) are alkylating agents

Chemical mutagen - Deamination •  Deamination of cytosine produces uracil, which is not a normal

base in DNA, although it is a normal base in RNA

•  Chemical mutagen induce deamination: Nitrous acid (HNO2)

Chemical mutagen - Deamination •  If the uracil is not replaced, an adenine will be incorporated into the

new DNA strand during replication, resulting in a CG-to-TA transition mutation

•  Deamination leads to mismatch directly

Flow chart for constructing mutagenized populations using EMS

Till et al. 2003 Genome Research

•  M1 seeds were sown in the greenhouse to harvest M2 seeds from single M1 plants

•  Ten M2 seeds were sown and grown as an M2 family

•  Each M2 plant was initially inspected for visible phenotypic alterations, and the M3 homozygous seeds from each mutant were also obtained

Mutation rate of chemical mutagens

•  The average mutation densities from diploid species are one mutation per 380 kb, whereas this drops dramatically to one mutation per 49 kb in tetraploids (durum wheat and tobacco) and one mutation per 32 kb in hexaploids (bread wheat and oat)

Type of point mutations

•  Synomymous mutation: has no change on the encoded amino acid.

•  Missense mutation: This type of mutation is a change in one DNA base pair that results in the substitution of one amino acid for another in the protein made by a gene. Conservative missense mutation changes a same type of another amino acid, which may not change structre of the protein and do not change its function. Nonconservative missense mutation changes to another type of amino acid.

•  Nonsense mutation: A nonsense mutation is also a change in one DNA base pair. Instead of substituting one amino acid for another, however, the altered DNA sequence prematurely signals the cell to stop building a protein. This type of mutation results in a shortened protein that may function improperly or not at all.

Types of point mutations

DNA codon table

Characters of chemical mutagen induced mutations

•  Chemical mutagens, such as ethyl-methanesulfonate (EMS), cause primarily point mutation or single nucleotide polymorphism (SNP)

•  Higher mutation rate allows for saturation to be achieved using relatively fewer individuals

-  Arabidopsis: 120 Mbp 240-1200 mutations/line

•  Mutation rate similar among species, beneficial to large genomes

-  Wheat: 17,000 Mbp 34,000-170,000 mutations/line

•  Provides allelic series, and not just knockouts, which can yield refined insights into gene function

Characters of the induced mutations

Mutagen Main characteristics

Biological agents (e.g., T-DNA and transposons)

ü  Not work for all species ü  Insertion of specific DNA sequence ü  Loss of function ü  Low efficiency, 1-3 mutations per line

Physical agents (Fast neutrons, X-rays, etc.)

ü  Works for all species ü  Break DNA and cause deletions ü  Wide range of mutations ü  Medium efficiency

Chemical agents (e.g., EMS)

ü  Works for all species ü  Wide range of mutations, mainly point mutations ü  High efficiency, hundreds to hundreds of thousands of

mutations per line

Alonso and Ecker, Nature Review Genetics, 2006

References 1.  Azpiroz-Leehan, R. and Feldmann, K.A., 1997. T-DNA insertion mutagenesis in Arabidopsis:

going back and forth. Trends in Genetics, 13(4), pp.152-156.

2.  Clough, S.J. and Bent, A.F., 1998. Floral dip: a simplified method for Agrobacterium‐mediated transformation of Arabidopsis thaliana. The plant journal, 16(6), pp.735-743.

3.  Feldmann, K.A. and Marks, M.D., 1987. Agrobacterium-mediated transformation of germinating seeds of Arabidopsis thaliana: a non-tissue culture approach. Molecular and General Genetics MGG, 208(1-2), pp.1-9.

4.  Krysan, P.J., Young, J.C. and Sussman, M.R., 1999. T-DNA as an insertional mutagen in Arabidopsis. The Plant Cell, 11(12), pp.2283-2290.

5.  Page, D.R. and Grossniklaus, U., 2002. The art and design of genetic screens: Arabidopsis thaliana. Nature Reviews Genetics, 3(2), p.124.

6.  van Opijnen, T., Bodi, K.L. and Camilli, A., 2009. Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms. Nature methods, 6(10), pp.767-772.