Using mutants to clone genes
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1 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?
2 Reading References: Westhoff et. al. Molecular Plant Development: from gene to plant. Chapter 3:
3 Positional (map-based) Cloning Map based cloning is a labour-intensive but dependable method of cloning a gene using a mutant phenotype, RFLP-like genetic markers and genetic recombination. This method is generally restricted to organisms where the necessary tools, RFLP genetic map, physical map and or sequence of the genome are available.
4 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.
5 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. restriction endonuclease digestion /DNA probes (restriction fragment-length polymorpism = RFLP) 2. PCR amplification (simple sequence-length polymorphism = SSLP) 3. a combination of both PCR amplification followed by restriction endonuclease digestion (cleaved amplified polymorphic sequences = CAPS).
6 Chromosome of Individual #1 Chromosome of Individual #2 CTGGGAATTCTTACC EcoR1 site Amplify by PCR CTGGGAAGTCTTACC Ind #1 Ind #2 #1 x #2 F1 Restrict amplified fragments with EcoR1 and separate on an electrophoretic gel.
7 Mapping to DNA-Based Genetic Markers The genomes of different varieties of Arabidopsis (ecotypes) differ in a large number of nucleotides. When these two varieties 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. Commonly used Arabidopsis ecotypes are: Columbia (Col) Landsberg erecta (Ler)
8 Chromosome of Columbia ecotype with crl1-1 mutation crl1-1 CAPS16 C CAPS83 C CAPS8 C CAPS134 C CAPS41 C Chromosome of Landsberg erecta CAPS16 L CAPS83 L CRL1 CAPS8 L CAPS134 L CAPS41 L The crl-1 mutant phenotype is found to segregate with CAPS83 C and CAPS8 C but no others. Therefore the CRL1 gene must lie between these two CAPS sites.
9 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.
10 Arabidosis Genome
11 Arabidopsis Chromosome 1, UFO locus markers BACs Annotated genes
12 AT1G sequence Sequence: AT1G Date last modified Name AT1G Tair Accession Sequence: GenBank 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 submitted_by GenBank General comments or questions: curator@arabidopsis.org Seed or DNA stock questions (donations, availability, orders, etc): abrc@arabidopsis.org
13 Arabidopsis Chromosome 1, UFO locus markers ( ) BACs Annotated genes
14 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.
15 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.
16 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.
17 Insertional Tagging P coding region Gene X P coding region Gene X with insert Chromosome with genes including the one with insert
18 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. 2. Ligate the DNA into circles and amplify using divergent insert primers (inverse PCR)
19 Inverse PCR ligate Amplify by PCR Clone into vector
20 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.
21 Insertional Tagging Clone into vector Use sequences from gene X to identify the wild type allele. P coding region Gene X
22 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.
23 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.
24 Connecting a cloned gene with a mutant phenotype Genotype of plants homozygous for different alleles of the CRL1 gene: CRL1/CRL1 crl1-1/crl1-1 crl1-2/crl1-2 -cloned a wild type gene, MYB83, encoding a transcription factor. Is MYB83 gene CRL1? Clone MYB83 from each of the three plants above by PCR amplification. If MYB83 is CRL1 then MYB83 from CRL1/CRL1 will have a wild type sequence. MYB83 from crl1-1/crl1-1 will have a mutation. MYB83 from crl1-2/crl1-2 will also have a mutation but different from that of crl1-1.
25 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, selffertilizing 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.
26 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.
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