Gene mutation origins and repair processes2

Review of some concepts from Chapter 11 Chromosome mutation This chart describes chromosome constitutions for a normally diploid animal An individual that is normally diploid and has only one chromosome set is called monoploid to distinguish it from individuals that are normally haploid The terms, monosomic, disomic and trisomic are used to describe aneuploid conditions Aneuploid: an individual organism whose chromosome number differs by part of a chromosome set The terms haploid, monoploid, diploid and triploid are used to describe multiples of the basic chromosome set Complementation: The production of a wild type phenotype when two different mutations are combined in a diploid A scientist has identified two new alleles she calls a1 and b1 a1 and b1are recessive alleles Both a1/ a1 and b1 / b1 mice have no fur She wants to figure out if a1 and b1 are alleles of the same gene a1/ a1 X b1 / b1 All progeny have fur Therefore a1 and b1 complement This indicates that a1 and b1 are alleles of different genes (99.9% of the time) a1 and b1are recessive alleles a1/ a1 X b1 / b1 a1 b+ a+ b1 a+ b1 X a1 b+ a1 b+ a+ b1 All progeny have fur a1 and b1 complement a1 and b1 are mutant alleles of different genes More on complementation Her labmate finds another recessive allele, c1 The phenotype for homozygous c1/c1 mice is that they have no fur He tests if c1complements a1 and b1 c1/c1 X a1/a1 : all progeny have fur c1/c1 X b1/b1 : no progeny have fur He concludes that c1 complements a1, but not b1 Therefore, c1 and b1 are alleles of the same gene (99.9% of time) c1 and b1 are in the same complementation group c1 is a recessive allele c1/c1 X b1/b1 : no progeny have fur c1 b1 X c1 b1 c1 No progeny have fur b1 c1 and b1 fail to complement; c1 and b1 are mutant alleles of the same gene Using deficiency/deletion chromosomes to map mutations red square indicates the region of chromosome deleted c1 df1 c1 df2 df1 fails to complement c1 , df2 complements c1 Therefore, c1 is contained in the region deleted in df1 More on Deletion/Deficiency Mapping pn (prune): only deletion 264-38 fails to complement fa (facet): all but 258-11 and 258-14 fail to complement pn/df 264-38 ;see pruned phenotype pn/ any of the other df; see wild type phenotype Therefore, pn contained in 2D4-3A2, or 3E1-3E2 fa is in 3C7 Mechanisms of Dosage Compensation mammals XX (female) XY (male) One X chromosome is inactive in females Called X inactivation The inactive X is called a Barr body C elegans (nematode) XX (hermaphrodite) XO (male) Both X chromosomes produce 1/2 the gene product (hypotranscription) in hermaphrodites as compared to males Hermaphrodites have both male and female internal genitalia and produce both eggs and sperm Drosophila XX (female) XY (male) The one male X chromosome is hypertranscribed Historical perspective on the discovery of X chromosome inactivation in mammals In 1953 Dr Mary Lyon made this observation about mouse coat color: only females of certain strains showed spotting or mottling, not males Hypothetical fictional example: Assume genes for blue and yellow coat are on the X yellow+ yellow blue + blue - In female mice some of the cells will have the maternal X and some will have the paternal X active Therefore, some cells will produce yellow fur and some blue Males will either have only the maternal X active; therefore no spotting Specific Locus Test: Want to identify new recessive mutations in gene c Genetic Screen vs Genetic Selection Types of genetic selection auxotroph: a strain that will proliferate only when the medium is supplemented with a specificsubstance not required by wild type Genetic screens • • Morphological Mutation Can be applied to any problem, depending upon ingenuity and resources Biochemical mutations – screening for auxotrophs from mutagenized prototrophs • Morphological mutations – change in shape or form • Lethal mutations – premature death – recessive lethals are more useful than dominant lethals that are difficult to maintain A geneticist can screen for a mutation affecting any phenotype As long as you can score the phenotype you can screen for mutations that affect the biological process • Conditional mutations – display wild-type under permissive (nonrestrictive) conditions – display mutant phenotype under restrictive conditions – e.g., temperature-sensitive mutations • Behavioral mutations Behavioral screen Temperature sensitive mutation: An example is a mutation in a protein required for cell division that becomes unstable at high temperature 18°C-no phenotype 29°C-cell division phenotype observed Modifier Screen •Secondary screens –search for mutations that alter mutant phenotype modifier mutations QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture Screen based on gene expression Enhancer A Enhancer B “Enhancer Trapping” Sort through the mutations identified Linkage mapping and complementation analysis Determining the type of allele generated • Both gain-of-function and loss-of-function can be dominant or recessive • Loss-of-function – partial or complete elimination of activity of gene’s encoded product • Gain-of-function – hypermorph: more gene activity – neomorph: novel gene activity A single gene can have both loss-of-function and gain-of-function alleles Remember can have different alleles of same gene Distinguishing between loss- and gain-of-function mutations Null Hypomorph Gain-of-function Neomorph not always true [...]... Reverse genetics Forward genetics Designing a genetic screen (forward genetics) Different mutagens give rise to different DNA changes (chapter 10) Genes have different mutational target sizes (forward genetics) Directed Mutations: You start with a gene and want to know the gene s mutant phenotype (reverse genetics) Gene knockouts are generated by homologous recombination Site directed mutagenesis (reverse... common ancestor How do geneticists study gene function? Disrupt the gene and analyze the resulting phenotype Forward genetics: Classical approach to genetic analysis Genes are first identified by their mutant phenotype and mutant alleles, and then subsequently cloned and analyzed Reverse genetics: Scientist begins with cloned gene and sequence information He/she introduces engineered mutations into the... mutation in TGF-β gene TGF- β gene distribution in human genome What does TGF-β protein do? How can we study the function of TGF-β? Can study in model systems like Drosophila melanogaster, Caenorhabditis elegans, Saccharomyces cerevisiae, and Mus musculus Scientist have completed genome sequence, have tools to map genes, and introduce genes into genome Genes are conserved Organisms have genes from a common... in diploids, both dominant and recessive alleles can be identified in the F1 Specific Locus Test: Want to identify new recessive mutations in gene c Genetic Screen vs Genetic Selection Types of genetic selection auxotroph: a strain that will proliferate only when the medium is supplemented with a specificsubstance not required by wild type Genetic screens • • Morphological Mutation Can be applied to... ingenuity and resources Biochemical mutations – screening for auxotrophs from mutagenized prototrophs • Morphological mutations – change in shape or form • Lethal mutations – premature death – recessive lethals are more useful than dominant lethals that are difficult to maintain A geneticist can screen for a mutation affecting any phenotype As long as you can score the phenotype you can screen for mutations... introduces double-stranded RNA (dsRNA) into cell that is homologous to a gene/ transcript dsRNA is cleaved into small RNAs (siRNAS) Enzyme called Dicer These serve as templates for the RNAi pathway directing cleavage of the mRNA via RISC complex Cells have endogenous small RNAs that can also silence genes (miRNAs) RNAi will phenocopy a mutation at a DNA locus Chemical Genetics Somatic vs Germline mutation In... deletions and duplications microarray/ DNA Chip DNA from wild type Cy3 DNA from mutant Cy5 A Mutant has a deletion (chr 1) B Mutant has a duplication (chr 4) Cy5/Cy3=1 Cy5/Cy3=.5 Cy5/Cy3=2 normal chromosome deletion duplication mutant/wild type Cy5/Cy3=15 tandem duplication Chapter 12 Mutational Dissection Internal organ placement in normal girl and girl affected with situs inversus Caused by mutation. .. directed mutagenesis (reverse genetics) Shown here is a scheme for mutating a gene cloned into a circular plasmid An example of when you want to perform site directed mutagenesis clone a gene that shares sequence identity to a DNA binding protein ADRTSVCGNSVTNPIL AGRTSVTILNSVTNRA Amino acid sequence of known hypothetical DNA binding protein Amino acid sequence of your new cloned gene arrow indicates residue... binding With site directed mutagenesis can determine if that residue is important for DNA binding in the product of the gene you cloned Antisense RNA-translation of RNA inhibited mRNA is considered sense strand; antisense is complementary strand phenocopy: mimicking a mutant phenotype by manipulating something in the interior environment of the cell RNAi: Short double-stranded RNA molecules direct an... screen for mutations that affect the biological process • Conditional mutations – display wild-type under permissive (nonrestrictive) conditions – display mutant phenotype under restrictive conditions – e.g., temperature-sensitive mutations • Behavioral mutations Behavioral screen Temperature sensitive mutation: An example is a mutation in a protein required for cell division that becomes unstable ... mutational target sizes (forward genetics) Directed Mutations: You start with a gene and want to know the gene s mutant phenotype (reverse genetics) Gene knockouts are generated by homologous recombination... study gene function? Disrupt the gene and analyze the resulting phenotype Forward genetics: Classical approach to genetic analysis Genes are first identified by their mutant phenotype and mutant... cerevisiae, and Mus musculus Scientist have completed genome sequence, have tools to map genes, and introduce genes into genome Genes are conserved Organisms have genes from a common ancestor How geneticists