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 MarkersThe 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).

Chromosome of Individual #1

PCR primers amplify this region

Homozygote #1

Small Insertions and deletions (SSLP) in DNA sequence can be identified using PCR and gel electrophoresis

Chromosome of Individual #2

These simple sequence length polymorphisms SSLP can be used as co-dominant markers for specific positions on a chromosome.

Homozygote #2

Heterzygote

CTGGACTACTACGAGTTACCGACCTGATGATGCTCAATGG

CTGTTACCGACAATGG

Chromosome of Individual #2

CTGGGAATTCTTACC

Chromosome of Individual #1

CTGGGAAGTCTTACCAmplify by PCR

Restrict amplified fragments with EcoR1 and separate on an electrophoretic gel.

EcoR1 site

Ind #1 Ind #2#1 x #2 F1

DNA single nucleotide polymorphism may be identified using CAPS

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)

PCR primers amplify this region

Col SSLP 8

Small Insertions and deletions in DNA sequence can be identified using PCR and gel electrophoresis

These microsatellites (simple sequence length polymorphisms SSLP) can be used as co-dominant markers for specific positions on a chromosome.

Ler SSLP 8

Heterzygote

CTGTTACCGACAATGG

Chromosome of Columbia (Col) ecotype homozygous for SSLP allele at locus 8, chromosome 1

Chromosome of Landsberg erecta (Ler) ecotype homozygous for SSLP allele at locus 8, chromosome 1

CTGGACTACTACGAGTTACCGACCTGATGATGCTCAATGG

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

Chromosome 1 of Landsberg erecta ecotype showing SSLP markers

Chromosome 1 of Columbia ecotype showing SSLP markers

SSLP14LSSLP16L

SSLP41LSSLP83LSSLP8L

SSLP8C SSLP83C

SSLP41CSSLP16C SSLP14C

SSLP markers can be mapped using recombination just like genes

Arabidopsis genetic map showing the position of SSLP markers.

1 2 34 5

SSLP 8SSLP 83

SSLP 14

SSLP 25

SSLP 68

SSLP 102

SSLP 43SSLP 95

SSLP 24

SSLP 71

SSLP 4

SSLP 39

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.

Apetala2 mutant has flowers where the sepals and petals are replaced by reproductive organs

Procedure For Mapping a Mutant Phenotype Relative to Defined DNA Markers

Chromosome ? ap2/ap2 (Col) AP2/AP2 (Ler)chromosome 1 SSLP 16C/SSLP 16C SSLP 16L/SSLP 16L

chromosome 4 SSLP 72C/SSLP 72C 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.

X

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 8F2 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?

Ler

ColSSLP8C SSLP83C

SSLP41C

SSLP14LSSLP16L

SSLP1LSSLP83LSSLP8L

SSLP16C SSLP14C

PCR primers amplify this region

SSLP 8C

SSLP genotypes in DNA sequence of the ap2 mutants can be identified using PCR and gel electrophoresis

These microsatellites (simple sequence length polymorphisms SSLP) can be used as co-dominant markers for specific positions on a chromosome.

rSSLP 8L

Heterzygote

CTGTTACCGACAATGG

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,

CTGGACTACTACGAGTTACCGACCTGATGATGCTCAATGG

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

SSLP 71 #4 C/L C/C L/L C/L C/L L/L C/L C/C

SSLP 83 #1 C/C C/C L/C C/C C/C C/C C/C C/CSSLP 8 #1 C/C C/C C/C C/C C/L C/C C/C C/C

SSLP 16 #1 C/C C/L L/C L/L C/L L/L C/C L/C

Chromosome #

Ler

ColSSLP8C SSLP83C

SSLP41C

SSLP14LSSLP16L

SSLP41LSSLP83LSSLP8L

SSLP16C SSLP14C

Chromosome of Landsberg erecta

Chromosome of Columbia ecotype with ap2-1 mutation

ap2-1

AP2

SSLP8C SSLP83C

SSLP41C

SSLP14LSSLP16L

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.

SSLP41LSSLP83LSSLP8L

SSLP16C SSLP14C

[ ]

Positional Cloning

AP2

[ ]AP2

DNA from the AP2 locusWith 10 genes =

Genetic maprecombination

SSLP8C

SSLP8C

Genes in the AP2 locus

SSLP83C

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 AAACCATGGG1001 AAGAAGATGC ATTCACGATG AGTGAAGAAG TCTTGAAGAA CATTTCTCTT1051 GAAGTTGTTA GGGAGAGATT ACTAGACCAT GTCCATCAGG AAATACCATA1101 TGGTCTGGAG CACCGTCTAG TGGACTGGAA AGAGCTGCGT GACGGGTCTC1151 TTAGAATTGA ACAGCATCTC ATCACTCCTA AACTTAGCCA ACGCAAGATT1201 CTTGTAGGCA AGGGCGGTTG CAAGATCGGG AGGATAGGAA TTGAGGCCAA1251 TGAAGAACTC AGGAGAATAA TGAACCGCAA AGTTCATCTC ATTCTCCAGG1301 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.orgSeed 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 coding region

P coding regionGene X

Gene X with insert

Portion of a chromosome with genes including the one with insert

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)

Insertional Tagging

Inverse PCR

ligate

Clone into vector

Amplify by PCR

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 regionGene 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?