Genetics and Mutations: Understanding DNA Polymorphisms and Their Impact on Organisms, Papers of Molecular biology

Download Genetics and Mutations: Understanding DNA Polymorphisms and Their Impact on Organisms and more Papers Molecular biology in PDF only on Docsity! September 28 • Mutation • In class exercise(not graded) • Superman paper introduction • Questions about superman paper • Tuesday In class exercise on superman paper A little review on chromatin Recombination – Read and look at movies on website Thursday-quiz on chromatin and superman paper Two key concepts • Structure of DNA allows for efficient inheritance and encoding of information • Alterations in DNA seq can have consequences to the organism because information is altered. 12.3 CENTRAL DOGma “% onl DNA Trane RNA raion Protein The central dogma for mutants/polymorphisms The minimal gene • ATG: OPEN READING FRAME ENCODING A VERY IMPORTANT PROTEIN WITH LOTS OF ESSENTIAL AMINO ACIDS:STOP CODON Nomenclature • Nonsense-change of an amino acid to a stop codon • Missense-change of one amino acid to another • Frameshift-addition or deletion of a base to change the reading frame • Null? Does every change in an amino acid kill the protein? The code protects against mutation • Many changes are silent • Many changes result in similar amino acids Does every mutation result in a change in amino acid sequence? CT (— ) | ‘ | t t t ' no active ' ' . . gene . only mutant gene is active present both genes are active (A) (B) (C) Figure 8-64. Molecular Biology of the Cell, 4th Edition. Tests for gene function Ponalonaus +“ selectable unique “barcode” g marker gene sequence yeast target gene x N\ \ < ——~ ee yeast chromosome | \____ yeast target gene x ——— HOMOLOGOUS RECOMBINATION target gene x replaced by selectable marker gene and associated “barcode” sequence (A) YEAST Figure 8-73 part 1 of 2. Molecular Biology of the Cell, 4th Edition. Knockouts and Knockins-yeast and mice transposable element target gene disrupting target gene : chromosome PCR primer based on transposable element sequence - ~4 PCR primer based on target gene PCR product detected on gel only if ger'g MSS transposable element has inserted in target gene of interest (B) ARABIDOPSIS AND DROSOPHILA Figure 8-73 part 2 of 2. Molecular Biology of the Cell, 4th Edition. Insertions and transgenes-Arabidopsis, Drosophila, Nematodes plasmid cloning vector inserted normal gene (A) AMM UHHeTcner eee neeeneneneeenttAt GAC LUN Ss ws “ Zany =; %, Uy COMM MMMM MMMM TT > STRAND SEPARATION cee ; synthetic oligonucleotide primer containing desired mutated sequence CTG (B) wit wi “G_cs STRAND COMPLETION BY DNA POLYMERASE AND DNA LIGASE (C) T See U INTRODUCTION INTO CELLS FOLLOWED BY REPLICATION AND SEGREGATION INTO DAUGHTER CELLS Figure 8-69 part 1 of 2. Molecular Biology of the Cell, 4th Edition.  (D) SC ———— le i Ss % CTT | TRANSCRIPTION 5 $$ G AC — 3’ | TRANSLATION —— Asp normal protein is made sa « ig BS Ze = z y s CMM | TRANSCRIPTION \——____— GCC — 3’ | TRANSLATION Ala~— protein with the single desired amino acid change is made Figure 8-69 part 2 of 2. Molecular Biology of the Cell, 4th Edition.  Developmental equivalence • The primordia are initially equivalent, and can adopt any of the 4 fates • But cells usually have one set fate Mutants: A-apetala-2 B-apetala-3 C-agamous Most of these mutants affect two whorls When all of the floral identity genes are missing, leaves are made instead of flowers Adenine and Cytosine are methylated HL CH; NH, N CH, N~ s Yr DS 4A oO we N N \ | 6. Methyladenine (m®a) 5-Methylcytosine (m®C) residue residue HJ _CHg N oy Ay N*-Methylcytosine (m4c) residue Adenine and Cytosine are methylated There are restriction enzymes that do not cut DNA when methylated at specific bases within the recognition sequence DNA methylation • Known to be important for X-inactivation, imprinting, methylation of CpG islands, etc 2 alleles of the same gene are tightly linked • Recombination between 2 alleles in the same gene should be rare Complementation test part 2 • If you can take a wild-type copy of a gene and “rescue” a mutant, the defective activity in the mutant is fixed by the wild-type gene product. • Remember back to the Griffiths and Avery experiments and what I asked you to think about The nature of mutation • Mutants are variants that have a mutation in a specific gene and are stable. • Alleles that have make no detectable protein are considered nulls or amorphs. • The reversion rate for this kind of mutation is rare. Bisulfite sequencing Bisulfite sequencing: C to U (T) But methyl C is nonreactive And will stay as a C Run bisulfite and regular Sequencing side by side Remember Ed Southern? Remember in situ hybridization? Anti-sense line • Expression of an RNA molecule complementary to an mRNA can in some situations, inhibit transcription of that gene and cause a phenotype – More on this in the second half of the class