mutants and proteins fri may 9

mutants and proteins fri may 9 - May 9, 2008 Read syllabus,...

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Unformatted text preview: May 9, 2008 Read syllabus, go to web site, reviews tba for course materials 1. Syllabus 2. Lecture notes 3. Weekly discussion quizzes w answers 4. OID BruinCast links 5. Exams (old samples, w/o answers) 6. Exam keys Madame Bovary's Ovaries Watch NYT 5/5/08 Mapping the Diseasesome NYT 5/5/08 Mutants and proteins A gene's nucleotide sequence is colinear the amino acid sequence of the encoded polypeptide. Charles Yanofsky E. coli genes for a subunit of tyrptophan synthetase compared mutations within a gene to particular amino acid substitutions. Trp- mutants in trpA Fine structure recombination map Determined amino acid sequences of mutants Fig. 8.4 A codon is composed of more than one nucleotide. Different point mutations may affect same amino acid. Codon contains more than one nucleotide. Each nucleotide is part of only a single codon. Each point mutation altered only one amino acid. A codon is composed of three nucleotides and the starting point of each gene establishes a reading frame. studies of frameshift mutations in bacteriophage T4 rIIB gene Fig. 8.5 Most amino acids are specified by more than one codon. Phenotypic effect of frameshifts depends on if reading frame is restored. Fig. 8.6 Cracking the code: biochemical manipulations revealed which codons represent which amino acids. Understanding the system involves Crick's insight about mRNA The discovery of messenger RNAs, molecules for transporting genetic information Protein synthesis takes place in cytoplasm deduced from radioactive tagging of amino acids. RNA, an intermediate molecule made in nucleus and transports DNA information to cytoplasm 5' to 3' direction of mRNA corresponds to N-terminal-toC-terminal direction of polypeptide. One strand of DNA is a template. The other is an RNA-like strand. Nonsense codons cause termination of a polypeptide chain UAA (ocher), UAG (amber), and UGA (opal). transcription translation Fig. 8.9 1. Transcription 2. Translation Summary Codon consist of a triplet codon each of which specifies an amino acid. Code shows a 5' to 3' direction. Codons are nonoverlapping. Code includes three stop codons, UAA, UAG, and UGA that terminate translation. Code is degenerate. Fixed starting point establishes a reading frame. UAG in an initiation codon which specifies reading frame. 5'- 3' direction of mRNA corresponds with N-terminus to Cterminus of polypeptide. Mutaiton modify message encoded in sequence Frameshift mutations change reading frame. Missense mutations change codon of amino acid to another amino acid. Nonsense mutations change a codon for an amino acid to a stop codon. Do living cells construct polypeptides according to same rules as in vitro experiments? Studies of how mutations affect amino-acid composition of polypeptides encoded by a gene Missense mutations induced by mutagens should be single nucleotide substitutions and conform to the code. Fig. 8.10 a Proflavin treatment generates trp- mutants. Further treatment generates trp+ revertants. Single base insertion (trp-) and a deletion causes reversion (trp+). Fig. 8.10 b Fig. 8.28 Mutations in a gene's coding sequence can alter the gene product. Missense mutations replace one amino acid with another. Nonsense mutations change an amino-acid-specifying codon to a stop codon. Frameshift mutations result from the insertion or deletion of nucleotides within the coding sequence. Silent mutations do not alter amino acid specified. Genetic code is almost universal but not quite. All living organisms use same basic genetic code. Translational systems can use mRNA from another organism to generate protein. Comparisons of DNA and protein sequence reveal perfect correspondence between codons and amino acids among all organisms. Transcription RNA polymerase catalyzes transcription. Promoters signal RNA polymerase where to begin transcription. RNA polymerase adds nucleotides in 5' to 3' direction. Terminator sequences tell RNA when to stop transcription. Surprise! Unexpected! RNA splicing removes introns. Exons sequences found in a gene's DNA and mature mRNA (expressed regions) Introns sequences found in DNA but not in mRNA (intervening regions) Some eukaryotic genes have many introns. Dystrophin gene underlying Duchenne muscular dystrophy (DMD) is an extreme example of introns. Fig. 8.15 Alternative splicing Amplifies gene diversity (number) Different mRNAs (resulting in different gene products with different functions) can be produced by same primary transcript Fig. 8.18 Mutations outside of the coding sequence can also alter gene expression. Promoter sequences Termination signals Splice-acceptor and splice-donor sites Ribosome binding sites Fig. 8.28 b Loss-of-function alleles are usually recessive. Null or amorphic mutations are alleles that completely block the function of a protein. Hypomorphic mutations produce much less of a protein or a protein with weak but detectable function. Fig. 8.29 Rocket immunoelectrophoresis reveals the amount of xanthine dehydrogenase produced in flies with different genotypes. Null allele 1 and hypomorphic allele 2 are recessive to wildtype. Incomplete dominance arises when phenotype varies in proportion to the amount of protein. Rarely, loss-of-function mutations are dominant. 1. Haploinsufficiency one wild-type allele does not provide enough of a gene product Fig. 8.31 a Heterozygotes for the null mutation of the T locus in mice have short tails because they have an insufficient amount of protein to produce a wild-type tail. Rarely, loss-of-function mutations are dominant. 2. Dominant-negative mutations alleles of a gene encoding subunits of multimers that block the activity of subunits produced by normal alleles Fig. 8.31 b Kinky: A dominant-negative mutation in mice causing a kink in the tail Fig. 8.31 c Gain-of-function mutations are almost always dominant. Rare mutations that enhance a protein function or even confer a new activity on a protein Fig. 8.31 d Antennapedia is a neomorphic mutation causing ectopic expression of a leg-determining gene in structures that normally produce antennae. Nonsense suppression (a) Nonsense mutation that causes incomplete nonfunctional polypeptide (b) Nonsensesuppressing mutation causes addition of amino acid at stop codon allowing production of full length polypeptide. Fig. 8.32 How can things go wrong? Let us count the ways! Autosomal recessive Albinism Cystic fibrosis PKU Sickle cell anemia Tay Sachs Autosomal dominant Achondroplasia BRCA1, 2 Familial hypercholestrolemia Huntington Disease Osteogenesis imperfecta (Ehlers Danlos syndrome) X-linked recessive G6PD deficiency (favism) Hemophilia Muscular dystrophy (Duchenne type) X-linked domiant Fragile X syndrome How things go wrong: no one genetic profile Autosomal recessive autosomal dominant x-linked recessive x-linked dominant Loss of function Albinism biosynthetic enzyme loss BRCA1, 2 (incomplete penetrance) cancer with loss of heterozygous + allele Cystic fibrosis loss or inactivation of CFTR protein Familial hypercholestrolemia haplo-insufficient LDR loss; homozygote worse Fragile X syndrome poly CGG repeat failure to express protein unknown function G6PD deficiency (favism) loss of enzyme inactivating fava bean supplied drug Hemophilia blood clotting factor loss Muscular dystrophy (Duchenne type) muscle attachment protein loss PKU degradation enzyme loss Tay Sachs degradation enzyme loss Gain of function (activation or ectopic expression) Achondroplasia Gly380Arg in FGFR3 gene causes continuous activation of signal Neomorphic (new function) Huntington Disease poly CAG repeat to protein with poly Q stretch, misfolds Sickle cell anemia Glu6Val in beta globin causes multi-unit polymerization Dominant negative Osteogenesis imperfecta missense mutations inactivate homopolymer proteins ...
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