Bi Lecture 3 Loss-of-function (Ch. 4A) Monday, April 8, 13

Transcription

1 Bi Lecture 3 Loss-of-function (Ch. 4A)

2 Infer Gene activity from type of allele

3 Loss-of-Function alleles are Gold Standard If organism deficient in gene A fails to accomplish process B, then gene A is NECESSARY for process B

4 Types of Null Alleles Function Protein Polypeptide mrna Localization Folding, modification, assembly Translation Activity Null Protein Null DNA pre-mrna pre-mrna splicing, nuclear export RNA Null Transcription DNA Null

5 Alanine A Ala Arginine R Arg Asparagine N Asn Aspartic acid D Asp Cysteine C Cys Glutamine Q Gln Glutamic acid E Glu Glycine G Gly Histidine H His Isoleucine I Ile Leucine L Leu Lysine K Lys Methionine M Met Phenylalanine F Phe Proline P Pro Serine S Ser Threonine T Thr Tryptophan W Trp Tyrosine Y Tyr Valine V Val

6 Example of loss of function alleles A. wild-type human EGFR cdna cagttgggcacttttgaagatcattttctcagcctccagaggatgttcaataactgtgaggtggtccttgggaatttggaaattacctatgtgcagaggaattatgat Q L G T F E D H F L S L Q R M F N N C E V V L G N L E I T Y V Q R N Y D B. nucleotide substitution leading to a stop codon cagttgggcacttttgaagatcattttctcagcctccagaggatgttcaataactgtgaggtggtccttgggaatttggaaattacctaagtgcagaggaattatgat Q L G T F E D H F L S L Q R M F N N C E V V L G N L E I T - V Q R N Y D C. insertion of a nucleotide leading to a frameshift and truncation cagttggggcacttttgaagatcattttctcagcctccagaggatgttcaataactgtgaggtggtccttgggaatttggaaattacctatgtgcagaggaattatgat Q L G H F - R S F S Q P P E D V Q - L - G G P W E F G N Y L C A E E L -

7 A B C D E F G a+ b+ c+ d+ e+ deficiency a1 b+ c+ d+ e+ deficiency a+ b1 c+ d+ e+ deficiency a+ b+ c1 d+ e+ deficiency a+ b+ c+ d1 e+ deficiency a+ b+ c+ d+ e1 deficiency a+ b1 c+ d+ e+ wild type wild type B Phenotype C Phenotype wild type wild type wild type a+ b+ c1 d+ e+

8 Dosage analysis of hypomorphic alleles Genotype % WT phenotype Interpretation +/+ 100% wild-type control h1/+ 100% allele is recessive h1/h1 30% mutant control h1/df 15% allele is enhanced in trans to a null h1/h1/h1 60% increased copy number has a less severe phenotype

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10 RNAi: decreases gene function A sirna B C RISC mrna D cleaved mrna

11 A B Transcription Dicer sirna

12 RNAi test for complete loss of function genotype RNAi phenotype interpretation c+ / c+ none 0% blue control c1 / c1 none 60% blue control c1 / c1 against C 90% blue c1 is hypomorphic c2 / c2 none 60% blue control c2 / c2 against C 60% blue c2 consistent with null

13 allelic series can define loss of function phenotype genotype phenotype +/+ 100% j1/j1 95% j2/j2 70 j3/j3 40 j4/j4 20 j5/j5 10 j6/j6 3 j1/j5 50% j5/j6 5% j2/j6 36% j3/j4 30% j2/j5 40%

14 allelic series can define loss of function phenotype j+/j+ Proportion wild-type phenotype 1.0 j1/j1 0.8 j2/j2 0.6 j2/j j1/j5 j3/j4 j4/j4 j6/j6 j5/j6 j2/j6 j5/j5 activity j3/j3

15 L3 AC Anchor Cell Vulval Precursor Cells (VPCs) L AC L3 Molt AC F epidermal cells vulval cells

16 wild-type Vulval Precursor Cells (VPCs) Anchor Cell Vulvaless Multivulva

17 Allelic series can define loss of function phenotype: let-23 lethality

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19 Loss-of-function alleles can be constructed A X linear dsdna ATG TGA Y B X

20 A a1 a2 double strand break B strand invasion C displacement and DNA synthesis templated by intact strand D ligation??? E a2 a2 resolution, leaving red chromosome converted

21 Loss-of-function alleles can be constructed A X linear dsdna ATG TGA Y B X

22 why would there be no phenotype for a lf?

23 other methods: GENOME EDITING TALENs ZFNs Crispr inhibitors

24 A breakthrough in genome engineering (called genome editing) uses engineered site-specific DNA binding proteins that have nuclease activity. The key steps are: 1. introduction of a site-specific nuclease; 2. generation of a double-strand break; 3. repair of the break can introduce a mutation by a. non-homologous end joining, which is errorprone (for example, DNA is chewed back by endonucleases. b. Homology directed repair converts the allele that has been cut.

25 ZFNs (Zinc-Finger Nucleases) have a DNA-binding domain composed of zinc finger units, each of which binds three nucleotides. TALENs (Transcription Activator Like Effector Nucleases) have a DNA-binding domain from TAL effector proteins of a bacterial pathogen of plants. A 34 amino acid repeat unit in the protein specifies binding to a single basepair. There are two amino acids in the repeat that are critical for DNA binding specificity: Asn Gly (NG) recognizes T; His Asp (HD) recognizes C; Asn Ile (NI) recognizes A and AsnAsn (NN) recognizes G or A. The TALEN works as a dimer. The nuclease is Fok1. CRISPR. Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR- associated

26 TALENs A 34 amino acid unit NI NG HD NN A T C G B GTAGACCCAGACCTGT bp AGACCTGTAGACCTGT C Left TALEN Fok1 Fok1 Right TALEN

27 Cas9 PAM target DNA nt target 5...NNNNNNGNNNNNNNNNNNNNNNNNNGGNNNNN... NNNNN NNN N C NCNNNNNNNNNNNNNNNNNNC 5 GNNNNNNNNNNNNNNNNNNGUUUUAGAGCUA G A 3 GCCUGAUCGGAAUAAAAUU CGAU AA GAA sgrna