Using mutants to clone genes
Objectives
1.
What is positional cloning?
2.
What is insertional tagging?
3.
How can one confirm that the gene cloned is the same
one that is mutated to give the phenotype of interest?
Reading
• References:
• Westhoff et. al. Molecular Plant Development: from gene to
plant. Chapter 3: 52-65.
Positional (map-based) Cloning
Map based cloning is a dependable method of cloning a gene
using a mutant phenotype, molecular genetic markers and
genetic recombination.
This method is most easily done in organisms where the
necessary tools (genetic map, physical map and or sequence
of the genome) are available.
Positional (map-based) Cloning
1. Use the mutant phenotype and DNA-based genetic markers
of known position to map, using recombination, the gene of
interest to a site on a specific chromosome.
DNA-Based Genetic Markers
The genomes of two individuals of the same species are rarely identical and
can have many nucleotide differences between them.
These variations in DNA sequences often do not alter the function of a gene
but can be used as phenotypes in genetic mapping by detecting the
differences using:
1. PCR amplification (simple sequence-length polymorphism = SSLP)
2. a combination of both PCR amplification followed by restriction
endonuclease digestion (cleaved amplified polymorphic sequences
= CAPS).
Small Insertions and deletions (SSLP) in DNA sequence can be
identified using PCR and gel electrophoresis
PCR primers amplify this region
Chromosome of Individual #1
CTGGACTACTACGAGTTACC
GACCTGATGATGCTCAATGG
Chromosome of Individual #2
CTGTTACC
GACAATGG
These simple sequence length
polymorphisms SSLP can be used as
co-dominant markers for specific
positions on a chromosome.
Homozygote Homozygote Heterzygote
#1
#2
DNA single nucleotide polymorphism may be identified using CAPS
Chromosome of Individual #1
CTGGGAATTCTTACC
EcoR1 site
Chromosome of Individual #2
CTGGGAAGTCTTACC
Amplify by PCR
Ind #1
Restrict amplified fragments
with EcoR1 and separate on
an electrophoretic gel.
#1 x #2
F1
Ind #2
Mapping to DNA-Based Genetic Markers
The genomes of individuals form different populations of the same species differ in a
large number of SSLPs. This variation can be detected and used as genetic markers for
specific positions on chromosomes.
When two such individuals are crossed all the differences will segregate in the F2
progeny and can be mapped relative to one another or any novel phenotype in one of
the parents.
Eg. Arabidopsis populations from different parts of the world are called ecotypes:
Columbia (ecotype from southern US)(Col)
Landsberg erecta (ecotype from Germany)(Ler)
Small Insertions and deletions in DNA sequence can be
identified using PCR and gel electrophoresis
PCR primers amplify this region
Chromosome of Columbia (Col) ecotype
homozygous for SSLP allele at locus 8,
chromosome 1
CTGGACTACTACGAGTTACC
GACCTGATGATGCTCAATGG
Chromosome of Landsberg erecta (Ler)
ecotype homozygous for SSLP allele at
locus 8, chromosome 1
CTGTTACC
GACAATGG
These microsatellites (simple
sequence length polymorphisms
SSLP) can be used as co-dominant
markers for specific positions on a
chromosome.
Col SSLP 8
Ler SSLP 8
Heterzygote
SSLP markers can be mapped using
recombination just like genes
• In a cross Columbia and Landsberg erecta, the
resulting F1 progeny will be heterozygous at all SSLP
loci that were identified between the two:
SSLP 16C/SSLP 16L; SSLP 72C/SSLP 72L; SSLP 8C/SSLP 8L
Therefore in the F2 generation they can be mapped relative
to one another
SSLP markers can be mapped using
recombination just like genes
Chromosome 1 of Columbia ecotype showing SSLP markers
SSLP8C
SSLP83C
SSLP16C
SSLP14C
SSLP41C
Chromosome 1 of Landsberg erecta ecotype showing SSLP markers
SSLP8L
SSLP83L
SSLP16L
SSLP14L
SSLP41L
Arabidopsis genetic map showing the position of
SSLP markers.
1
SSLP
8
SSLP
83
SSLP
14
2
3
4
SSLP
4
SSLP
102
SSLP
25
SSLP
68
5
SSLP
24
SSLP
43
SSLP
95
SSLP
71
SSLP
39
Apetala2 mutant has flowers where the sepals
and petals are replaced by reproductive organs
Apetala2 mutant has flowers where the sepals
and petals are replaced by reproductive organs
• AP2 normal flowers > ap2 flowers
• AP2 protein is required to make sure that the proper
organs are made in the outer part of the flower.
We are studying how floral morphogenesis is controlled
during development and would like to determine what
kind of protein is encoded by AP2.
ie Which of the 30,000 Arabidopsis genes known by DNA
sequence (entire genome has been sequenced) is AP2.
Procedure For Mapping a Mutant Phenotype Relative to Defined
DNA Markers
X
Chromosome ?
ap2/ap2 (Col)
chromosome 1 SSLP 16C/SSLP 16C
chromosome 4 SSLP 72C/SSLP 72C
AP2/AP2 (Ler)
SSLP 16L/SSLP 16L
SSLP 72L/SSLP 72L
F1 AP2/ap2
SSLP 16C/SSLP 16L
SSLP 72C/SSLP 72L
F2
See how often the Columbia allele of the AP2 gene (ap2) segregates with the
Columbia alleles of SSLP 16; SSLP 72 and all other mapped SSLP loci.
Procedure For Mapping a Mutant Phenotype Relative to Defined
DNA Markers
ap2/ap2 (Col)
x
AP2/AP2 (Ler)
F1 AP2/ap2
Ap2 Mutants isolated from the F2, DNA extracted from each and tested for
different molecular markers.
Plant 1 Plant 2 Plant 3
Plant 4 Plant5
Plant 6
Plant 7 Plant 8
F2 ap2/ap2 ap2/ap2 ap2/ap2, ap2/ap2, ap2/ap2, ap2/ap2, ap2/ap2, ap2/ap2
What is the expected frequencies of the alleles for one molecular marker
in these F2 progeny assuming no linkage to AP2?
Col
SSLP8C
SSLP83C
SSLP16C
SSLP14C
SSLP41C
SSLP8L
SSLP83L
SSLP16L
SSLP14L
Ler
SSLP1L
SSLP genotypes in DNA sequence of the ap2 mutants can be
identified using PCR and gel electrophoresis
PCR primers amplify this region
Chromosome 1 of Columbia (Col) ecotype
homo-zygous for SSLP allele at locus 8,
Chromosome1 of Landsberg erecta (Ler)
ecotype homozygous for SSLP allele at locus 8,
These microsatellites (simple
sequence length polymorphisms
SSLP) can be used as co-dominant
markers for specific positions on a
chromosome.
CTGGACTACTACGAGTTACC
GACCTGATGATGCTCAATGG
CTGTTACC
GACAATGG
SSLP 8C
rSSLP 8L
Heterzygote
Procedure For Mapping a Mutant Phenotype Relative to Defined
DNA Markers
ap2/ap2 (Col)
x
AP2/AP2 (Ler)
F1 AP2/ap2
Ap2 Mutants isolated from the F2 and DNA extracted
F2 ap2/ap2, ap2/ap2, ap2/ap2, ap2/ap2, ap2/ap2, ap2/ap2, ap2/ap2, ap2/ap2
Chromosome #
SSLP 71 #4 C/L
SSLP 8 #1 C/C
SSLP 83 #1 C/C
SSLP 16 #1 C/C
C/C
C/C
C/C
C/L
L/L
C/C
L/C
L/C
C/L
C/C
C/C
L/L
C/L
C/L
C/C
C/L
L/L
C/C
C/C
L/L
C/L
C/C
C/C
C/C
Col
SSLP8C
SSLP83C
SSLP16C
SSLP14C
SSLP41C
SSLP8L
SSLP83L
SSLP16L
SSLP14L
SSLP41L
Ler
C/C
C/C
C/C
L/C
Chromosome of Columbia ecotype with ap2-1 mutation
SSLP8C
[ap2-1]
SSLP83C
SSLP16C
SSLP14C
SSLP41C
SSLP16L
SSLP14L
SSLP41L
Chromosome of Landsberg erecta
SSLP8L
AP2
SSLP83L
The ap2-1 mutant phenotype is found to segregate with
SSLP83C and SSLP8C but no others. The map distance from
each of these two to AP2 is 1/16 = 6.25 map units.
If there is 12.5 map units between SSLP8 SSLP83 the AP2 gene
must lie between these two SSLP sites.
Positional Cloning
SSLP8C
AP2
SSLP83C
[ ]
SSLP8C
AP2
Genetic map
recombination
DNA from the AP2 locus
With 10 genes =
Genes in the AP2 locus
The Sequences For All Annotated Genes Are
Available
•
•
•
Sequence: AT1G30960.1
Date last modified 2003-05-27Name AT1G30960.1 Tair Accession Sequence:2015773GenBank Accession NM_102835Sequence
Length (bp) 1314 Sequence
•
1 ATGAAAGCTT TTAGATCTCT ACGTATACTA ATTTCCATCT CACGAACGAC
51 GACGAAGACA ACACCTCGTA ATCCCCATCA AGCACAAAAC TTTCTCCGCC
101 GATTTTACTC AGCGCAGCCG AATCTAGACG AACCCACTTC CATCAATGAA
151 GACGGATCAA GCAGCGACTC TGTTTTCGAT AGTAGTCAAT ACCCAATCGA
201 CGATTCCAAT GTAGATTCCG TGAAGAAGCC CAAGGAAGCA ACTTGGGATA
251 AAGGGTACAG AGAAAGAGTA AACAAAGCCT TCTTTGGAAA CTTGACAGAG
301 AAAGGTAAAG TGAAAGTTGC AGAAGAAGAG AGTTCTGAAG ATGATGAGGA
351 TAGTGTTGAT AGGTCAAGGA TTCTCGCTAA GGCTCTCTTA GAGGCTGCGT
401 TAGAGTCACC AGATGAAGAA CTTGGTGAAG GTGAAGTTAG AGAAGAAGAT
451 CAGAAGTCGC TTAATGTCGG CATCATCGGT CCACCTAATG CAGGAAAATC
501 TTCGCTGACT AATTTCATGG TTGGAACAAA GGTTGCTGCT GCTTCACGGA
551 AGACTAACAC GACGACACAT GAAGTGTTAG GAGTATTGAC AAAAGGAGAT
601 ACACAAGTCT GTTTCTTCGA TACTCCGGGT CTGATGCTGA AGAAAAGCGG
651 ATATGGTTAC AAAGACATCA AGGCTCGTGT GCAAAATGCT TGGACTTCTG
701 TTGACCTGTT TGATGTCCTC ATTGTTATGT TTGATGTCCA TAGGCATCTC
751 ATGAGTCCCG ATTCAAGAGT GGTACGCTTG ATCAAATACA TGGGAGAAGA
801 AGAAAATCCG AAACAAAAGC GCGTTTTATG TATGAACAAA GTTGATCTGG
851 TTGAGAAGAA AAAGGATCTA TTAAAGGTTG CTGAGGAGTT CCAAGATCTT
901 CCGGCATATG AAAGATACTT CATGATATCG GGACTTAAGG GATCAGGAGT
951 GAAAGATCTT TCCCAATACT TAATGGATCA GGCTGTTAAA AAACCATGGG
1001 AAGAAGATGC ATTCACGATG AGTGAAGAAG TCTTGAAGAA CATTTCTCTT
1051 GAAGTTGTTA GGGAGAGATT ACTAGACCAT GTCCATCAGG AAATACCATA
1101 TGGTCTGGAG CACCGTCTAG TGGACTGGAA AGAGCTGCGT GACGGGTCTC
1151 TTAGAATTGA ACAGCATCTC ATCACTCCTA AACTTAGCCA ACGCAAGATT
1201 CTTGTAGGCA AGGGCGGTTG CAAGATCGGG AGGATAGGAA TTGAGGCCAA
1251 TGAAGAACTC AGGAGAATAA TGAACCGCAA AGTTCATCTC ATTCTCCAGG
1301 TTAAGCTCAA GTGA Comments (shows only the most recent comments by
default)
Attribution type name datesubmitted_by AGI-TIGR 2001-03-06submitted_by GenBank 2002-08-20
General comments or questions: curator@arabidopsis.org
Seed or DNA stock questions (donations, availability, orders, etc): abrc@arabidopsis.org
Positional (map-based) Cloning
1. Use the mutant phenotype and DNA-based genetic markers
to map, using recombination, the gene of interest to a region
on a specific chromosome.
2. Examine the sequence of chromosomal DNA from that
region to determine the number of annotated genes.
3. Narrow down to correct gene using predicted function,
mutant allele sequence, complementation, expression
analysis etc.
Insertional Tagging
1.
Isolate mutant phenotype of interest from an insertional mutagenized
population of plants.
(Insertion DNA must be cloned: eg TDNA or Transposon).
2.
Check that the transposon or TDNA in the mutant segregates with the
mutant phenotype.
---The segregation of an insert can often be followed using the phenotype of
a gene encoded in the insert (eg Kanamycin resistance), a probe for the
insert or PCR primers that can amplify part of the insert.
---repetitive elements (eg. transposons) may complicate such an analysis.
Insertional Tagging
1.
Isolate mutant phenotype of interest from an insertional mutagenized
population of plants.
(Insertion DNA must be cloned: eg TDNA or Transposon).
2.
Check that the transposon or TDNA in the mutant segregates with the
mutant phenotype.
3.
Clone or amplify the chromosomal DNA at the site of insertion using the
known sequence of the TDNA or transposon.
Insertional Tagging
P
P
coding region
Gene X
region
coding
Gene X with insert
Portion of a chromosome with genes including the one with insert
Insertional Tagging
Digest genomic DNA with
restriction endonuclease
Identify the fragment carrying the insert:
Eg. 1. Make a library and probe with the insertion sequences or
2. Ligate the DNA into circles and amplify using divergent
insert primers (inverse PCR)
Inverse PCR
ligate
Amplify by PCR
Clone into vector
Insertional Tagging
1.
Isolate mutant phenotype of interest from an insertional mutagenized
population of plants.
(Insertion DNA must be cloned: eg TDNA or Transposon).
2.
Check that the transposon or TDNA in the mutant segregates with the
mutant phenotype.
3.
Clone or amplify the chromosomal DNA at the site of insertion using the
known sequence of the TDNA or transposon.
4. Sequence the DNA flanking the TDNA or transposon from the mutant and
use the sequence to identify the wild type gene.
Insertional Tagging
Clone into vector
Use sequences from gene X to identify the wild type allele.
P
coding region
Gene X
Connecting a cloned gene with a mutant
phenotype
Despite the method of cloning, one must confirm that the gene cloned (X) is
the same gene that is mutated in mutant M (gene M).
1. Transgene complementation.
The wild type fragment carrying gene X should be able to complement the
recessive mutant M phenotype. This hypothesis can be tested by
transforming the homozygous mutant with the wild type gene to check if
it will restore the wild type phenotype.
Eg. Transform pea rr plants with the SBEI gene to see if the gene will
complement the mutant phenotype.
Connecting a cloned gene with a mutant
phenotype
1. Transgene complementation.
2. Sequence gene X from several mutants homozygous for different alleles of
gene M.
If the ‘M’ gene and X gene are the same then one should find a gene X
mutation in every M mutant. This hypothesis can be tested by sequencing
gene X from several M mutants each carrying a different allele of the gene
of interest.
Eg. Sequence the SBEI gene in several different ‘r’ pea mutants each
homozygous for a different r mutant allele. One should find a different
mutation in the SBEI gene in every such ‘r’ mutant.
Connecting a cloned gene with a mutant
phenotype
1. Transgene complementation.
2. Sequence gene X from several mutants homozygous for different alleles
of gene M.
3. Cosegregation analysis.
DNA-based markers (RFLP) identifying gene X should cosegregate with the
mutant phenotype M in genetic crosses. This hypothesis can be tested
by crossing mutant M to a wild type plant, self-fertilizing the F1
progeny to produce F2 progeny and scoring F2 plants for the mutant
phenotype and the gene X molecular marker.
Eg. Follow the segregation of an RFLP for the SBEI gene with the wrinkled
seed phenotype of rr.
Connecting a cloned gene with a mutant
phenotype
Genotype of plants homozygous for different alleles of the AP2 gene:
• AP2/AP2
ap2-1/ap2-1
ap2-2/ap2-2
-cloned a wild type gene, MYB83, encoding a transcription factor.
Is MYB83 gene AP2?
Clone MYB83 from each of the three plants above by PCR amplification. If
MYB83 is AP2 then
• MYB83 from AP2/AP2 will have a wild type sequence.
• MYB83 from ap2-1/ap2-1 will have a mutation.
• MYB83 from ap2-2/ap2-2 will also have a mutation but different from that
of ap2-1.
Connecting a cloned gene with a mutant
phenotype
1. Transgene complementation.
2. Sequence gene X from several mutants homozygous for
different alleles of gene M.
3. Cosegregation analysis.
4. Reverse genetics.
Identify mutant alleles of gene X using reverse genetics.
Mutations in gene X should have the same phenotype as
mutant M and fail to complement the M mutant phenotype.
Eg. A loss of function mutation in the SBEI gene should have a wrinkled
seed phenotype.
• A population of plants has been transformed with a fragment of
DNA carrying a gene that confers antibiotic resistance (resistance is
a dominant phenotype. One plant from the population has an ap2
mutant phenotype and fails to complement a known ap2 mutant.
You hypothesize that the transformed DNA has inserted into the
AP2 gene resulting in a loss of function mutation. If so you can use
the line to clone the AP2 gene.
•
To check whether the transformed DNA fragment is actually in the
AP2 gene you cross the new ap2 mutant from the transformed
population to wild type and select 30 ap2 mutants from the F2
population. Seed from each of the ap2 mutants is tested for
resistance to the antibiotic. One hundred percent of the seed from
27 plants was resistant to the antibiotic. Seed from the other 3
plants was 75% resistant and 25% sensitive to the antibiotic.
• Is the new ap2 mutant caused by an insertion of the transformed
DNA into the AP2 gene?