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a+ --> a- mutation (forward mutation) a- --> a+ reverse mutation (reversion) Presence of pink pigment Four-o’clock plants Incomplete dominance the term used to describe the general case in which the phenotype of a heterozygote is intermediate between those of the two homozygotes, on some quantitative scale of measurement +/- Point mutations at the molecular level Purine replaced by a different purine; pyrimidine replaced by a different pyrimidine: TRANSITIONS Purine replaced by a pyrimidine; pyrimidine replaced by a purin: TRANSVERSIONS Animation ed.9: 9.2&9.17 TRANSLATION Animation ed.9: 9.2&9.17 TRANSLATION Now, insertions and deletions of base pairs: Selection of auxotrophs by filter enrichment Forward mutation-A mutation that converts a wild-type allele into a mutant allele Reverse mutation-The production of a wild-type gene from a mutant gene Equivalent reversion UCC (Ser) forward Wild type CGC (Arg, basic) forward Wild type Intragenic suppressor UGC (Cys) Mutant reverse AGC (Ser) Wild type CCC (Proline) reverse CAC (His, basic) Mutant Pseudo-wild type Intragenic suppressor Intergenic suppressor Intergenic suppressor Nonsense suppressor Chromosome transmission fidelity (Ctf) assay non-essential Chromosome Fragment M SUP11 ade2-101 kar3D sic1D rad50D xrs2D WT ade2-101 CIN mutant ade2-101 ade1-101 Animation ed9: 9.19a Nonsense mutation Animation ed9: 9.19b Nonsense suppressor Animation 9.19c Nonsense suppression rodns and suppressor-tRNA together give WT phenotype Regulatory Coding Using genomic sequence to find a specific gene When doing GENETIC mapping, Molecular Markers can be used as a locus Single Nucleotide Polymorphisms (SNPs) AACGTCATCG vs. AACGTTATCG Microsatellites (variable # of short repeats) CGCGCG vs. CGCGCGCGCG vs. CGCG Restriction Fragment Length Polymorphism (RFLP) SNP leading to a loss/gain of a restriction cut site When doing GENETIC mapping, Molecular Markers can be used as a locus Almost all SNPs, Microsatellites, etc. are SILENT, and there are millions of them They are mile-markers, not destinations! ,אבני דרך !ולא יעדים A specific gene, the breast cancer gene BRCA1 was found By using the genomic map at increasing levels of resolution Is there linkage between a mutant gene/phenotype and a SNP? SNP1 SNP1’ USE standard genetic mapping technique, with SNP alternative sequences as “phenotype” ..ACGTC.. B= bad hair, Dominant B 2’ / b 2 ..ACGCC.. SNP2 SNP2’ ..GCTAA.. ..GCAAA.. SNP3 SNP3’ ..GTAAC.. ..GTCAC.. X B/b 1/1’ 2/2’ 3/3’ b/b 1/1 2/2 3/3 B/b 1/1’ 25% 2/2’ 47% 3/3’ 25% B/b 1/1 25% 2/2 3% 3/3 b/b 1/1’ 25% 2/2’ 3% 3/3’ 25% 25% 2/2 47% 3/3 25% b/b 1/1 25% SO…B is 6 cM from SNP2, and is unlinked to SNP 1 or 3 Is there linkage between a mutant gene/phenotype and a SNP? USE standard genetic mapping technique, with SNP alternative sequences as “phenotype” ..ACGTC.. B= bad hair, Dominant SNP1 SNP1’ ..ACGCC.. SNP2 SNP2’ ..GCTAA.. ..GCAAA.. SNP3 SNP3’ ..GTAAC.. ..GTCAC.. X B/b 1/1’ 2/2’ 3/3’ b/b 1/1 2/2 3/3 We have the ENTIRE genome sequence of mouse, so we know where the SNPs are Now-do this while checking the sequence of THOUSANDS of SNPs SO…B is 6 cM from SNP2, and is unlinked to SNP 1 or 3 The logic of creating sequence map of the genome Physical maps are maps of the order, overlap, and orientation of physically isolated pieces of the genome-in other words, maps of the distribution of the cloned genomic DNA from genomic clone libraries Part of the automated production line of a major human genome sequencing center A specific gene can be found in the genomic sequence by matching linkage and cytological maps with the Genome sequence CGCGCG vs. CGCGCGCGCG vs. CGCG Complementation groups Mutagenesis Plate to select for phenotype of interest Complementation groups First, we need to catalogue our mutants to complementation groups (Total of 138 mutants were isolated in the original CTF screen). Mutant#1 Mutant#2 Diploid x x Mate x x Diploid still shows CTF phenotype Mutant#1 and Mutant#2 are mutated in the same gene Same complementation group Complementation groups Mutant#3 x Mutant#4 x Mate Diploid x x Diploid dont show CTF phenotype Mutant#3 and Mutant#4 are mutated in different genes Different complementation groups Complementation groups Chromosome Transmission Fidelity (ctf) Mutants 138 Total # of mutant isolates: 101 37 19 Complementation Groups 37 Undesignated (single member) Estimated total # of genes represented ~ 50 ctf genes WOBBLE A situation in which the third nucleotide of an anticodon (at the 5’ end) can form two alignments. This third nucleotide can form hydrogen bonds not only with its Normal complementary nucleotide in the third position but Also with different nucleotide in the position. I stands for inosine, one of the rare bases found in tRNA, often in anticodon MESSAGE The genetic code is said to be degenerate because in many cases more then one codon is assigned to a single amino acid, and, in addition, several codons can pair with more then on anticodon (wobble)